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เกี่ยวกับ “Bacteriocin”
1. AU3631593 - 05.10.1993
BACTERIOCIN FROM PEDIOCOCCUS CEREVISIAE
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=AU3631593
Inventor(s):
SOLINGEN PIETER VAN (--); STARK JACOBUS (--); LANGEVELD PIETER
CORNELIS (--)
Applicant(s):
GIST BROCADES NV (--)
IP Class 4 Digits: A23L; C12P; A23C
IP Class:
A23L3/3571; C12P21/00; A23C19/11
Application Number:
AU19930036315D (19930305)
Priority Number: EP19920200643 (19920305); EP19920203724 (19921212); WO1993EP00514
(19930305)
Family: AU3631593
1/1006
2. AU4708596 - 14.10.1999
NOVEL BACTERIOCIN PISCICOLIN 126
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=AU4708596
Inventor(s):
WETTENHALL RICHARD EDWARD HUGH (--); DAVIDSON BARRIE ERNEST (--);
HILLIER ALAN JAMES (--); HARMARK KIM (--); JACK RALPH WILSON (--); HICKEY MALCOLM
WAYNE (--); COVENTRY JOHN (--); WAN JASON (--)
Applicant(s): UNIV MELBOURNE (--); COMMW SCIENT IND RES ORG (--); FOOD AND
PACKAGING CENTRE MANA (--)
IP Class 4 Digits: C12N; C07K; C12P
IP Class:
C12N1/20; C12P21/02; C12N15/31; C07K14/195
Application Number:
AU19960047085 (19960222)
Priority Number: AU1995PN01310 (19950222); WO1996AU00096 (19960222); AU19960047085
(19960222)
Family: AU711557
2/1006
3. AU4968893 - 03.03.1994
BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=AU4968893
Inventor(s):
HOLO HELGE (NO); NES INGOLF FIGVED (NO); NISSEN-MEYER JON (NO)
Applicant(s): DZIEGLEWSKA HANNA EVA (GB); NORWEGIAN DAIRIES ASS (NO); HOLO
HELGE (NO); NES INGOLF FIGVED (NO); NISSEN MEYER JON (NO)
IP Class 4 Digits: C12N; C07K; C12P; A23C; A01K
IP Class:
C12N1/20; C12P21/02; C12N15/74; C12N15/31; A23C9/123; C07K13/00;
A23C19/032; A01K67/00
E Class: A23C19/11; C07K14/315; C07K14/195; A23C9/13E; C12N1/20
Application Number:
WO1993GB01799 (19930824)
Priority Number: GB19920017953 (19920824)
Family: AU4968893
Cited Document(s):
WO9119802
Abstract:
THE PRESENT INVENTION PROVIDES A POLYPEPTIDE HAVING OR INCLUDING AN AMINO ACID
SEQUENCE SUBSTANTIALLY CORRESPONDING TO ALL OR A PORTION OF THE AMINO ACID
SEQUENCE SET OUT IN FIGURE 1 (SEQ ID NO: 1) AND DERIVATIVES AND FRAGMENTS THEREOF
HAVING BACTERIOCIN AND/OR BACTERIOCIN IMMUNITY ACTIVITY.Description:
BACTERIOCIN
3/1006
The present invention relates to a novel bacteriocin, its isolation, synthesis and use.
Bacteriocins are peptides or proteins released by bacteria which show bactericidal activity towards
both the producing strain and/or other bacteria.
The producing organism will generally carry a gene coding for an immunity factor which provides
resistance to the bacteriocin, and the producing organisms are usually only affected by their own
bacteriocin at high cellular concentrations.
Due to their potential use as antibacterial agents, bacteriocins have been the subject of intensive
research. In recent years there has in particular been considerable interest in bacteriocins isolated
from lactic acid bacteria (LAB), in view of their potential utility in the food and brewing industries, in
particular the dairy industry.
Thus for example, their selective bactericidal activity renders LAB bacteriocins suitable for use in
cheese or yoghurt manufacture or in beer or distillery fermentations.
The LAB bacteriocins appear to be structurally quite different from other bacteriocins eg. the colicins
of Eschericia coli. LAB bacteriocins are usually small peptides, seldom containing more than 60
amino acids, while colicins are proteins of 300-800 amino acids.
Based on their structure, LAB bacteriocins may be divided into two groups. The first group contains
the so-called lantibiotics, which have been known for a long time and include in particular the known
bacteriocin nisin, (see for example Gross, et al J. Am. Chem. Soc.
93: 4634-4635, 1971 and Hurst, Adv. Appl. Microbiol. 27: 85-123, 1981). Lantibiotics consist of a
polypeptide chain in which certain amino acids have been post translationally modified. The
modified amino acids include lanthionine and methyllanthionine, and their precursors dehydroalanine
and dehydrobutyrine. Among the antibiotics, nisin is by far the most studied although three new LAB
antibiotics have recently been purified and characterised (Mrtvedt et al., J. Gen.
Microbiol. 136: 1601-1607, 1990 and Appl. Environ.
Microbiol 37: 1829-1834, 1991; Piard et al., Appl.
4/1006
Environ. Microbiol. 58: 279-284, 1992).
The second group of LAB bacteriocins contains those that consist of one short unmodified
polypeptide chain, such as lactococcin A (Holo et al., J. Bacteriol, 173: 3879-3887, 1991; Van
Belkum et al., Appl. Environ.
Microbiol. 57: 492-498, 1991), leucoccin A-UAL 187 (Hastings et al., J. Bacteriol. 173: 7491-7500,
1991), lactacin F (Murlane et al., J. Bacteriol. 173: 17791788, 1991 and Appl. Environ. Microbiol 57:
114-121, 1991), pediocin PA-1, sakecin P, and curvacin A. These bacteriocins contain between 35
and 60 amino acid residues, and have a high isoelectric point, often above 10. Many of these
bacteriocins share significant amino acid sequence homology over relatively large regions,
suggesting that these regions may be of importance for activity. Lactococcin A, which does not
share any apparent amino acid sequence homology with other isolated LAB bacteriocins, has been
shown to induce cell death by permeabilizing the membrane of susceptible cells (Van Belkum, et al.,
J. Bacteriol. 173: 7934-7941, 1992).
The antimicrobial activity of the bacteriocins that have so far been studied is due to the action of a
single peptide. We have now found, however, a novel lactococcal bacteriocin, which we have termed
lactococcin G, whose activity depends on the complementary action of two distinct peptides.
The novel bacteriocin of the invention has been isolated from Lactococcus lactis and purified and
bacteriocin activity was found to be associated with two peptides, designated a and p, the a peptide
of which may appear in two forms, a1 and a2, which are identical and may differ only in
conformation. A corresponding immunity factor has also been identified.
In one aspect the present invention thus provides a polypeptide having or including the amino acid
sequence substantially corresponding to all or a portion of the amino acid sequence set out in Figure
1 (SEQ ID NO: 1) and derivatives and fragments thereof having bacteriocin and/or bacteriocin
immunity activity.
Preferably, the said amino acid sequence substantially corresponds to the sub-sequences identified
as lag A, lag B and lag C in Figure 1.
Peptides a and p are believed to be expressed in a "pro" form which is processed to the mature a or
P peptide.
5/1006
According to a further aspect the present invention also provides a polypeptide having or including
the amino acid sequence a1 and a2 (SEQ ID NO: 2):
N Gly Thr Trp Asp Asp Ile Gly Gln Gly
Ile Gly Arg Val Ala Tyr Trp Val Gly
Lys Ala- Met Gly Asn Met Ser Asp Val
Asn Gln Ala Ser Arg Ile Asn Arg Lys
Lys Lys His C
and/or p (SEQ ID NO: 3):
N Lys Lys Trp Gly Trp Leu Ala Trp Val
Asp Pro Ala Tyr Glu Phe Ile Lys Gly
Phe Gly Lys Gly Ala Ile Lys Glu Gly
Asn Lys Asp Lys Trp Lys Asn Ile C and derivatives and fragments thereof having bacteriocin activity.
In a still further aspect, the invention also provides a polypeptide having or including the amino acid
sequence
Leu Phe Asn Asn Ile Val Val Phe Ile
Asn Phe Leu Ser Phe Val Phe Ile Leu
Val Gly Val Asp Ile Lys Tyr Asn Asp
Asn Arg Ile Lys Ile Val His Val Thr
Phe Phe Ile Ser Phe Ile Leu Val Met
Leu Thr Ser Leu Ile Ser His Asn Ser
Ile Ala Tyr Ser Leu Ser Gln Ile Leu
Glu Ile Leu Cys Ile Ile Cys Ile Leu
Leu Leu Phe Tyr Ile Leu Lys Lys Thr
Asn Ser Leu Ser Asn Arg Ala Asn Val
Val Phe Ile Ile Phe Ile Val Thr Gln
Val Ile Ile Ile Ile Asn Gln Leu Phe
Ile Arg (SEQ ID NO: 4) and derivatives and fragments thereof having bacteriocin immunity factor
activity.
Viewed from another aspect, the invention also provide a bacteriocin comprising peptide chains a
and p as designated above or combinations of active fragments or derivatives thereof and having or
including the amino acid sequences set out above. In such combinations, peptide a as designated
herein may be either peptide a1 or a2 or a mixture of the two.
6/1006
The terms "bacteriocin activity" and "active" are used to denote activity in inhibiting the growth of
bacterial species, for example a lactococcus test organism. Bacteriocins may kill or inhibit the growth
of bacteria by a number of mechanisms including lysis and it is not intended to limit to any particular
type of bactericidal activity. Active polypeptides and derivatives or fragments include those which,
whilst on their own do not exhibit bacteriocin activity, contribute to such activity when combined with
the complementary (a or P) peptide or its fragment or derivative.
The term "bacteriocin immunity factor activity" denotes activity in providing immunity to the
bacteriocin of the invention.
Derivative sequences included within the scope of the invention include functionally-equivalent
sequences modified by single or multiple amino acid substitution, addition and/or deletion and also
sequences where the amino acids have been chemically modified, including by glycosylation or
deglycosylation. By "functionally equivalent" is meant amino acid sequences having essentially
equivalent bacteriocin activity. Such functionally equivalent derivatives may occur as natural
biological variations or may be prepared using known techniques, for example functionally
equivalent recombinant polypeptides may be prepared using the known techniques of site-directed
mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of amino acids.
As mentioned above, modification of the amino acid sequences to obtain functionally-equivalent
derivative sequences may be amino acid substitution, as long as the activity of the polypeptide is not
affected. Thus for example, an amino acid may be replaced by another which preserves the
physicochemical character of the polypeptide eg. in terms of charge density,
hydrophilicity/hydrophobicity, size and configuration.
For example A may be replace by G or vice versa, V by A,
L or G; K by R; S by T or vice versa; E by D or vice versa; and Q by N or vice versa.
Generally, the substituting amino acid has similar properties eg. hydrophobicity, hydrophilicity,
electronegativity, bulky side chains etc. to the amino acid being replaced.
"Addition" derivatives include amino and/or carboxyl terminal fusions, for example by addition of
amino acid sequences of up to 300 eg. up to 200 or 100 residues, as well as intrasequence
insertions of single or multiple amino acids.
7/1006
Insertional amino acid sequence derivatives are those in which one or more amino acid residues are
introduced into a predetermined site in the protein although random insertion is also possible with
suitable screening of the resulting product. Deletional variants are characterised by the removal of
one or more amino acids from the sequence. Preferably, deletions or insertions are made in adjacent
pairs eg. a deletion of two residues or insertion of two molecules. In all cases the proviso is that the
modification preserves the activity of the polypeptide.
Derivative sequences falling within the scope of the invention may thus include for example amino
acid sequences having at least 60%, eg. at least 70% or 80% sequence homology with the
sequences of peptides art, a2 or p set out above. It should be noted however that functionallyequivalent derivative peptides may exhibit overall sequence homology below the given figures, but
may still fall within the scope of the present invention where they have conserved regions of
homology.
Bacteriocin activity, as mentioned above, requires the complementary action of the a and p
peptides, a1 being more effective when combined with p, than a2 and the novel bacteriocin as
provided according to the invention preferably comprises both a and p peptides.
In tests on Lactococcus Lactis subsp. Lactis 1403 indicator cells, the concentrations of peptides CLi
and p which inhibited cell growth by 50% were found to be 0.15 and 0.02 nM when the
complementing peptide was present in excess. When neither was in excess the concentrations were
respectively 0.3 and 0.04 nM. It thus appears that roughly 8 times more of a1 peptide is needed, and
whilst not wishing to be bound by theory, it is possible that a and p peptides interact in an
approximately 8 to 1 ratio in effecting bacteriocin activity.
A further aspect of the invention thus includes bacteriocin comprising peptides a and p or
fragments or derivatives thereof in a ratio of 5-10 to 1, preferably 7-9 to 1, especially 8 to 1
respectively.
As judged by its amino acid sequence, peptide a1 has an isoelectric point of 10.9, extinction
coefficient of 1.3 x 10-4 M-1 cm1, and a molecular weight of 4,346 (39 amino acid residues long).
Similarly, peptide p has an isoelectric point of 10.4, extinction coefficient of 2.4 x 104 M-1, and a
molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for a1
and p, respectively, were obtained by mass spectrometry. The N-terminal half of both the a and p
8/1006
peptides may form amphiphilic a-helices, suggesting that the peptides are pore-forming toxins that
create cell membrane channels through a "barrel-stave" mechanism and the novel bacteriocin of the
invention may exert its bactericidal effect in this manner.The C-terminal half of both peptides consists
largely of polar amino acids.
Peptides a1 and a2 are believed to represent different forms of the same peptide. Amino acid
sequence determination suggests that the amino acid sequences for peptides a1 and a2 are
identical and the two peptides were separated by their different behaviour during purification,
particularly in reverse phase chromatography.In particular upon rechromatography of purified a1 on
a reverse phase column, a certain proportion eluted as a2, suggesting that peptide a2 derives from
a1 Peptides a1 and a2 thus appear to represent the same gene product, but may differ in their
configuration in a manner which results in a2 having a slightly lower affinity for the reverse phase
column, and reduced bacteriocin activity when combined with p, than peptide a1.
A further aspect of the invention provides a composition comprising bacteriocin according to the
present invention, together with at least one of a carrier, and/or diluent, or excipient.
Such compositions may have a number of uses, particularly in microbiological processes and the
carrier, diluent or excipient may be any such conventional material, chosen according to the
proposed end use, for example a sterile liquid medium, buffer etc.
The novel bacteriocin of the invention may be used in industrial processes in which microbial
species eg.
lactobacteria such as lactococcus, are employed, for example in cheese and yoghurt manufacture.
The bacteriocin of the invention may be employed in fully or partially purified form or directly on the
culture supernatant. The latter may be advantageous in certain circumstances, for example when the
bacteriocin is to be used to selectively kill clostridia, as described below.
It may in certain cases be desirable to kill or arrest the growth of lactobacterial species for example,
lactococcus in cheese ripening, and the new bacteriocin may thus be of particular application in the
production of cheese,-or other diary products such as yoghurt.
9/1006
Alternatively, the bacteriocin may be used to kill undesired or contaminating bacterial cells in
various preparations. Such cells may be lactobacteria or they may be other bacterial strains, for
example species of
Bacillus (eg. B. Cereus) or Clostridium eg. C.
tvrobutvricum.
Thus, for example since certain bacteria, for example some Gram-negative bacteria, may be
resistant to bacteriocins, negative selection is possible by using the bacteriocin according to the
invention to remove certain cells, for example clostridia or strains of
L.lactis or other lactobacteria, from mixed cell populations e.g. in starter cultures for fermentation.
Where the productive strain of L.lactis is used as the sole or principle organism in an industrial
process such as cheese or yoghurt production, addition of the bacteriocin of the invention to the
starter culture serves to eliminate foreign organisms and may be effective against, for example,
spore forming clostridia or unwanted strains of L.lactis, or other lactobacteria.
The bacteriocin may advantageously be added to a cheese or yoghurt fermentation at a relatively
late stage, after lactic acid, protease and flavour production by the L.lactis organism has already
taken place.
By keeping the productive strain pure, either in the starter culture or in the milk or other medium,
uniformity of production can be improved.
The bacteriocin may also be used to kill selectively strains of lactic acid producing bacteria in beer
and distillery fermentations, since these are attributed in the literature to be the major cause of
spoilage in unpasteurised beers and give rise to the greatest proportion of infections during
fermentation.
Other bacterial species than clostridia or L.lactis may also present problems in the spoiling of foods
during processing or manufacture. For example, B.
cereus present in foods such as rice may lead to food poisoning.
10/1006
Strains of clostridium in particular are known to cause a problem in contaminating food
manufacturing processes and may lead for example to the spoiling of cheese. At present, clostridial
contamination is dealt with either by treatment with nitrates,- which are presently recognised to have
a number of disadvantage, notably from the toxicity point of view or using lysozyme which is not
generally effective. The use of bacteriocin according to the invention thus presents a considerable
advance over such prior art methods.This is an area of considerable commercial and economic
importance and a further aspect of the invention provides use of the novel bacteriocin of the
invention in selectively killing undesired or contaminating strains of bacteria, eg. lactic acid bacteria,
Clostridia, or Bacillus species in microbiological processes such as fermentation (eg. ethanol
fermentation) or food manufacturing processes, eg. in dairy processes such as cheese or yoghurt
production.
The invention also includes starter cultures of microorganisms containing the bacteriocin as an
inhibitor of contaminating bacterial eg. lactococcus or clostridia species. Such microorganisms may,
for example, be strains of L.lactis resistant to the bacteriocin eg. the producing organism, so that
only unwanted microorganisms are removed from the starter culture, or yeasts of use in beer or
distillery fermentations. Such starter cultures will normally be in lyophilised form.
Further uses of the novel bacteriocin include the production of cell wall preparations or for liberation
of nucleic acid material.
The novel bacteriocin of the invention may be isolated from cultures of Lactococcus lactis strain
LMG 2081 by fractionation of the growth medium whereby fractions enriched in the bacteriocin are
collected.
Known fractionation techniques may be applied to obtain the bacteriocin in electrophoretic purity.
Thus for example the organism may be grown in a suitable culture medium eg. M17 broth (oxoid)
and the supernatent subjected to fractional precipitation eg. with ammonium sulphate followed by
chromatography eg. a combination of ion exchange, hydrophobic and reverse phase
chromatography.
We have found in particular that the novel bacteriocin may be purified to homogeneity by a simple 4
step purification procedure which includes ammonium sulphate precipitation, followed by cation
exchange, octyl sepharose and reverse phase chromatography. Using this procedure up to a 7000fold increase in specific activity may be obtained.
11/1006
A further aspect of the invention thus includes a method of isolation of bacteriocin according to the
invention, wherein a culture of a microorganism expressing said bacteriocin is subjected to
fractionation whereby fractions enriched in said bacteriocin are collected. Preferably, in such a
method, the expressing organism is L.lactis strain LMG 2081.
Nucleic acid molecules comprising a nucleotide sequence encoding the novel bacteriocin or its
component peptides and/or its corresponding immunity factor (which provides resistance against
self-destruction in the producing strain) respectively form further aspects of the invention.
The region of the L. Lactis LMG 2081 genome coding for the novel bacteriocin of the invention and
its immunity factor has been identified and sequenced. In particular, we have identified and cloned
an operon which includes genes coding for the bacteriocin component peptides in their pro form as
well as for a further protein believed to be the immunity factor providing resistance against selfdestruction by the bacteriocin. The sequence of the operon is shown in
Figure 1 (SEQ ID NO: 5), which also shows the corresponding predicted amino acid sequence (SEQ
ID NO: 1). The putative promoter region and ribosome binding site are indicated.The gene
(designated lag a) coding for the pro-sequence of the a peptide (designated lag A) appears to run
from nucleotide 536 and a vertical arrow indicates where the peptide is cleaved between the amino
acids corresponding to nucleotides 580-581 to give the mature a peptide.
Translation of the p pro-sequence '(designated lag b for the gene and lag B for the pro-peptide)
starts at nucleotide 746 and again a vertical arrow indicates where cleavage occurs at the amino
acids corresponding to between nucleotides 820-821 to give the mature p peptide. Downstream of
the above-mentioned sequence is a sequence comprising a single long open reading frame, starting
at nucleotide 1027 and encoding a putative polypeptide of 110 amino acids, starting with LFNN and
ending with LFIR, believed to be the immunity factor (designated lag C).
The genes designated lag d and lag e are believed to code for proteins (designated lag D and lag E)
that are involved in the secretion of the mature a and p peptides; the lag d and lag e sequences
show homology to genes with such functions in other systems.
Accordingly, a further aspect of the invention provides a nucleic acid molecule comprising a
nucleotide sequence which encodes a bacteriocin, its component peptides and/or its corresponding
immunity factor, or a fragment thereof, substantially corresponding to all or a portion of the nucleotide
12/1006
sequence as shown in Figure 1 (SEQ ID NO: 5) or a sequence which is degenerate or substantially
homologous with or which hybridises with any such sequence.
Such nucleic acid molecules may be single or double stranded DNA, cDNA or RNA, preferably
DNA and include degenerate, substantially homologous, and hybridising sequences which are
capable of coding for the bacteriocin concerned. "Substantially homologous" as used herein includes
sequences displaying at least 60%, preferably at least 70% or 80% sequence homology and also
functionally-equivalent allelic variants and related sequences modified by single or multiple base
substitution, addition and/or deletion. By "functionally-equivalent" is meant nucleotide sequences
encoding polypeptides having essentially equivalent bacteriocin activity.
Nucleic acid sequences which hybridise with the sequence shown in Figure 1 (SEQ ID NO: 5) or
any substantially homologous or functionally-equivalent sequences as defined above are also
included within the scope of the invention. "Hybridisation" as used herein defines those sequences
binding under non-stringent conditions (eg. 6 x SSC 50 formamide at room temperature) and
washed under conditions of low stringency (eg. 2 x SSC, room temperature, more preferably 2 x SSC,
42"C) or conditions of higher stringency (eg. 2 x SSC, 65or) (where SSC = 0.15M Nacl, 0.015M
sodium citrate, PH7.2).Generally speaking, sequences which hybridise under conditions of high
stringency are included within the scope of the invention, as are sequences which, but for the
degeneracy of the code, would hybridise under high stringency conditions.
Derivative nucleotide sequences capable of encoding bacteriocin or bacteriocin derivatives
according to the invention may be obtained by using conventional methods well known in the art.
These include site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or
ligation of nucleic acids.
Bacteriocin according to the invention may be prepared in recombinant form by expression in a host
cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly
defined above, operatively linked to an expression control sequence, or a recombinant DNA cloning
vehicle or vector containing such a recombinant DNA molecule.
Appropriate recombinant DNA techniques are well known in the art and are described for example
by
Sambrook et al., 1989, (Molecular Cloning, a laboratory manual, 2nd Edition, Cold Spring Harbour
Press).
13/1006
The bacteriocin so expressed may be a fusion polypeptide comprising all or a portion of the
bacteriocin according to the invention and an additional polypeptide coded for by the DNA of the
recombinant molecule fused thereto. This may for example by p- galactosidase, glutathione-Stransferase, or any of the other polypeptides commonly employed in fusion proteins in the art.
Other aspects of the invention thus include cloning and expression vectors containing nucleic acid
molecules according to the invention coding for the bacteriocin and/or for the immunity factor.
Expression vectors appropriate to L. lactis are preferred. Such expression vectors include
appropriate control sequences such as for example translational (eg. start and stop codes) and
transcriptional control elements (eg. promoter-operator regions, ribosomal binding sites, termination
stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
The invention also includes transformed or transfected prokaryotic or eukaryotic host cells, or
transgenic organisms containing a nucleic acid molecule according to the invention as defined
above. Such host cells may for example be transformed strains of lactic acid bacteria eg. strains of L.
lactis.
Also included within the scope of the invention are methods for preparing the polypeptides of the
invention comprising culturing a host cell containing a nucleic acid molecule as defined above under
cdnditions whereby said polypeptide is expressed and recovering said polypeptide then produced.
The nucleic acid coding for the new bacteriocin and/or immunity factor may be incorporated into any
convenient cloning vector for amplification and into an expression vector for transformation of host
microorganisms such as L. lactis, for example cloning vector pIL253 (Simon and Chopin, Biochimie
70, 1988, 59566). Growth under suitable culture conditions will provide the bacteriocin in the growth
medium, from which it can be isolated by the techniques described above.
Furthermore, host cells such as strains of lactic acid bacteria eg. L. lactis may be transformed with
multiple copies of a plasmid or other vector containing the required nucleic acid sequence to provide
an improved strain giving rise to enhanced production of the bacteriocin. Such improved strains may
provide more rapid killing and hence accelerated cheese ripening when used in cheese manufacture.
In particular, the strain of L.lactis which produces the bacteriocin may be provided with such multiple
copies of the vector; this will thus be able to proliferate without premature destruction by the
bacteriocin.
14/1006
The new bacteriocin may also be prepared by chemical synthesis, for example using solid phase
synthesis, advantageously using a polypeptide synthesis apparatus, as commercially available. In
such a synthesis, active side chain groupings (e.g. amino or carboxyl groups) of the respective
amino acids will be protected and the final step will be deprotection and/or removal from the inert
support to which the polypeptide is attached during synthesis.
In building up the peptide chains, one can in principle start either at the C-terminal or the
N-terminal although only the C-terminal starting procedure is in common use.
Thus, one can start at the C-terminal by reaction of a suitable derivative of, for example histidine with
a suitable protected derivative of leucine. The histidine derivative will have a free amino group while
the other reactant will have either a free or activated carboxyl group and a protected amino group.
After coupling, the intermediate may be purified for example by chromatography, and then
selectively
N-deprotected to permit addition of a further
N-protected and free or activated amino acid residue.
This procedure is continued until the required amino acid sequence is completed.
Carboxylic acid activating substituents which may, for example, be employed include symmetrical
or mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2,4,5,
trichlorophenyl- ester,
N-hydroxybenzotriazole ester (OBt), N-hydroxysuccinimidylester (OSu) or pentafluorophenylester
(OPFP).
The coupling of free amino and carboxyl groups may, for example, be effected using
dicyclohexylcarbodiimide (DCC). Another coupling agent which may, for example, be employed is
N-ethoxycarbonyl-2-ethoxy-1,2dihydro-quinoline (EEDQ).
In general it is convenient to effect the coupling reactions at low temperatures, for example, -20 C
up to ambient temperature, conveniently in a suitable solvent system, for example, tetrahydro- furan,
dioxan, dimethylformamide, methylene chloride or a mixture of these solvents.
15/1006
It may be more convenient to carry out the synthesis on a solid phase resin support.
Chloromethylated polystyrene (cross-linked with 1% divinyl benzene) is one useful type of support; in
this case the synthesis will start the C-terminal, for example by coupling N-protected histidine to the
support.
A number of suitable solid phase techniques are described by Eric Atherton, Christopher J. Logan,
and
Robert C. Sheppard, J. Chem. Soc. Perkin I, 538-46 (1981); James P. Tam, Foe S. Tjoeng, and R. B,
Merrifield J. Am. Chem. Soc. 102, 6117-27 (1980); James
P. Tam, Richard D. Dimarchi and R. B. Merrifield Int. J.
Peptide Protein Res 16 412-25 (1980); Manfred Mutter and
Dieter Bellof, Helvetica Chimica Acta 67 2009-16 (1984).
A wide choice of protecting groups for amino acids are known and are exemplified in Schrdder, E.,
and
LUbke, K., The Peptides, Vols. 1 and 2,. Academic Press,
New York and London, 1965 and 1966; Pettit, G.R.,
Synthetic Peptides, Vols. 1-4, Van Nostrand, Reinhold,
New York 1970, 1971, 1975 and 1976; Houben-Weyl,
Methoden der Organischen Chemie, Synthese von Peptiden,
Band 15, Georg Thieme Verlag Stuttgart, NY, 1983; The
Peptides, Analysis, synthesis, biology 1-7, Ed: Erhard
Gross, Johannes Meienhofer, Academic Press, NY, San
Fransisco, London; Solid phase peptide synthesis 2nd ed., John M. Stewart, Janis D. Young, Pierce
Chemical
Company.
Thus, for example amine protecting groups which may be employed include protecting groups
which may be employed include protecting groups such as carbobenzoxy (Z-), t-butoxycarbonyl
(Boc-), 4-methoxy-2,3,6trimethyl-benzene sulphonyl (Mtr-), and 9-fluorenylmethoxycarbonyl (Fmoc-).
It will be appreciated that when the peptide is built up from the
C-terminal end, an amine protecting group will be present on the amino group of each new residue
added and will need to be removed selectively prior to the next coupling step. One particularly useful
16/1006
group for such temporary amine protection is the Fmoc group which can be removed selectively by
treatment with piperidine in an organic solvent.
Carboxyl protecting groups which may, for example be employed include readily cleaved ester
groups such as benzyl (-OBZ1), p-nitrobenzyl (-ONB), or t-butyl (-tOBu) as well as the coupling on
solid supports, for example methyl groups linked to polystyrene.
It will be appreciated that a wide range of other such groups exists as, for example, detailed in the
above-mentioned literature references, and the use of all such groups in the hereinbefore described
processes fall within the scope of the present invention.
A wide range of procedures exists for removing amine- and carboxyl-protecting groups. These must,
however, be consistent with the synthetic strategy employed. The side chain protecting groups must
be stable to the conditions used to remove the temporary a-amino protecting groups prior to the next
coupling step.
Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be
removed simultaneously by acid treatment, for example with trifluoro acetic acid.
A further aspect of the invention provides a process for the preparation of bacteriocin polypeptides
according to the invention in which a corresponding protected or immobilised polypeptide is
subjected to deprotection or removal from a solid support.
The invention will now be described in more detail in the following non-limiting Example with
reference to the following Figures in which:
Figure 2 shows reverse phase chromatography of lactococcin G (fraction IV). (A) The optical density
profile (---------) and propanol gradient (---- --- --).
(B) Bacteriocin activity without complementation (........), with complementation with a1 ( (--------.------),
and with complementation with p (----O----). The amount applied on the column represents that
obtained from approximately 2 liter culture;
Figure 3 shows reverse phase chromatography of (A) a2 (B) p, and (C) a1* The optical density
profile (----------) ) and propanol gradient (---- --- --). The bacteriocin activity without complementation
(........) and with complementation (----0----) with (B) a1 or with (A and C) P .The amount applied on
the column represents that obtained from approximately 2 liter culture;
17/1006
Figure 4 shows the amino acid sequence of a1, a2 and P (SEQ ID NOS: 2 and 3). N and C
indicates the, respectively, N- and C- terminal ends of the peptide;
Figure 5 shows the amount of ; 1 and p which in combination inhibited growth of the indicator strain
by 50%;
Figure 6 shows an Edmundson a-helical wheel representation of the amphiphilical region in (A) a1
and (B) P. For an the amphiphilical region shown starts with residue number 3 and ends with
number 27, for p it starts with residue number 8 and ends with number 25.
The shaded areas indicate non-polar residues, whereas the light areas indicate polar residues.
Examnle 1
MATERIALS AND METHODS
Bacterial strains and media
The bacteriocin producer was strain Lactococcus lactis LMG 2081, obtained from J. Narvhus,
Agricultural
University, As, Norway. The indicator organism used in the bacteriocin assay was lactococcus lactis
subsp.
lactis IL 1403 (Chopin, et al., Plasmid, 11: 260-263, 1984.) Both strains were grown at 30"C in M17
broth (Oxoid) without lactose, but supplemented with 0.4t (wt/vol) glucose. The M17 broth was also
supplemented with Tween 80 to a final concentration of 0.1% (vol/vol) when culturing strain LMG
2081 for the production of bacteriocin.
Bacteriocin assay
The bacteriocin was quantified in a microtiter plate assay system (Gels, et awl., Appl. Environ.
Microbiol. 45: 205-211, 1983). Each well of the microtiter plate contained 200 1 of M17 broth
(supplemented with 0.4% (wt/vol) glucose and 0.1% (vol/vol) Tween 80), bacteriocin fractions at twofold dilutions, and the indicator organism, Lactococcus lactis subsp. lactis IL 1403 (A600 = 0.1). The
microtiter plate cultures were incubated 3 hours at 30"C, after which growth inhibition of the indicator
organism was measured spectrophotometrically at 500 nm b using a Dynatech Microplate Reader.
One bacteriocin unit (BU) was defined as the amount of bacteriocin which inhibited growth of the
indicator organism by 50% (50% of the turbidity of the control culture without bacteriocin).
Bacteriocin purification
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All the purification steps were performed at room temperature, and all the chromatographic
equipment, was obtained from Pharmacia-LKB Biotechnology (Uppsala,
Sweden). The bacteriocin was purified from 2-liter cultures of Lactococcus lactis LMG 2081 grown to
the late exponential/early stationary phase. The cells were removed by centrifugation at 4,000 x g for
15 min at 4"C, and 400 g of ammonium sulfate per liter culture supernatant was added. The protein
precipitate was pelleted by centrifugation at 7,000 x g for 20 min and solubilised in 20 mM sodium
phosphate buffer, pH 5.7 (buffer A, 250 ml per 2 liter culture) (fraction I).
Fraction I was applied to a flow rate of about 10 ml/min to a 7 ml S-Sepharose Fast Flow cation
exchange column equilibrated with buffer A. After subsequently washing the column with 20 ml of
buffer A, the bacteriocin was eluted from the column with 40 ml 1 M NaCl in buffer A (fraction II).
Ammonium sulfate was added to fraction
II to a final concentration of 10% (wt/vol), after which the fraction was applied at a flow rate of about 4
ml/min to a 2 ml Octyl-Sepharose CL-4B column equilibrated with 10% (wt/vol) ammonium sulfate in
buffer A. The column was then washed with 8 ml of buffer A, after which the bacteriocin activity was
eluted from the column with 10 ml 70% (vol/vol) ethanol and 30% buffer A (fraction III). Fraction III
was diluted to 50 ml with H20 containing 0.1% (vol/vol) trifluoroacetic acid (TFA) and subsequently
applied to a
C2/C10 reverse-phase column, PepRPC HR 5/5, equilibrated with 2-propanol/H2O (10 : 90),
containing 0.1% TFA. The bacteriocin was eluted with a linear gradient ranging from 30 to 5v o 2propanol containing 0.1% TFA (fraction
IV). The Bacteriocin peptides eluting from the reverse phase column (fraction IV) were in some cases
diluted 45-fold with H20 containing 0.1% TFA and rechromatographed on the reverse phase column.
Purified bacteriocin was stored in 50-60% 2-propanol and or ethanol containing 0.1% TFA at -20 C.
Mass spectroscopy analysis
Mass spectroscoopy analysis of peptides was performed using the Biolon Mass Analyser (Applied
Biosystem, Sweden) as described earlier by Sorensen et al., Biomed. Environ. Mass Spectrom. 19:
713-720, 1990.
Peptide fractions were dissolved in 50-100 1 TFA containing 20% acetonitrile. From each fraction, 5
1 were loaded to a target and data accumulated for 10 min at 16 kV.
Amino acid sequencing
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The amino acid sequence was determined by Edman degradation using an Applied Biosystems
(Foster City,
Calif.) 477A automatic sequencer with an online 120A phenylthiohydantoin amino acid analyser.
RESULTS
Purification of bacteriocin
Initial screening showed that Lactococcus lactis
LMG 2081 produced antagonistic activity towards various
LAB and a number of different Clostridia. This strain produced bacteriocin constitutively during
growth, although maxium activity was found in the culture medium at the very end of the exponential
or early stationary phase of growth. The activity started to decrease after a few hours in the stationary
phase. The stability of the bacteriocin in the culture depended on the bacteriocin producing strain.
With other strains which produced the same bacteriocin, maximum activity was found in the middle
of the exponential phase, whereas little if any activity was found towards the end of this growth
phase.This may be due to the cell envelopeassociated preteinase produced by these strains, as this
proteinase rapidly degrades the bacteriocin when present in the culture media.
The purification scheme developed for isolating the bacteriocin for sequencing is shown in Table 1.
TABLE 1. Purification of Lactococcin G
Vol Total Total activity Sp actb Increase Yield
Fraction (ml) A280a (BU) sp actb (%)
Culture supernatant 2000 67.000 30 x 106 450 1 100
Ammonium sulfate precipittion (fraction I) 250 1,050 17 x 106 16,000 35 57
Binding to cation exchanger (fraction II) 40 5.8 6 x 106 1 x 106 2,200 20
Binding to Octyl
Sepharose (fraction III) 10 2.5 6 x 106 2.5 x 106 5,600 20
Reverse phase chromatography (fraction IV) 1 1.5 5 x 106 3 x 106 6,700 17 a Total A280 is the
absorbance at 280 mm multiplied by the volume in ml.
b Specific activity is bacteriocin units (BU) divided by the absorbance at 280 nm.
Tween 80 (final concentration of 0.1% (vol/vol) was added to the culture medium before ammonium
sulfate precipitation in order to obtain binding of the bacteriocin to the cation exchanger for
20/1006
bacteriocin activity, as this increased the sensitivity of the assay 2-10 fold. The bacteriocin was
concentrated 8-fold from the culture media by ammonium sulfate precipitation.
This resulted in a 30 to 40-fold increase in the specific activity and a recovery of about 60% of the
activity (Table 1, fraction 1). By subsequently binding the bacteriocin in fraction 1 to a cation
exchanger and eluting it with 1 M Nail, an increase in the specific activity of more than 2,000 was
obtained (Table 1, fraction II). From this stage and on the yield remained at about 20% (Table 1). The
specific activity increased 5,000-6,000-fold after binding the bacteriocin in fraction II to OctylSepharose and eluting it with 70% ethanol (Table 1, fraction III). When fraction III was applied to the
reverse phase column and eluted with a steep propanol gradient (6/my), the bacteriocin activity
coeluted with an absorbance peak at about 40% propanol (results not shown).This absorbance peak,
however, did not appear to be entirely homogeneous, as two shoulders could be discerned on both
sides of the main peak.
Bacteriocin activity depends on the complementary action of two peptides
Upon rechromatography of fraction IV, this time eluting the bacteriocin activity using a shallow
propanol gradient (0.5%/ml), 4 absorbance peaks were obtained (Fig. 2). The last 3 of these peaks
were termed a2 ss and a1 in the order of which they eluted together with the a1 absorbance peak,
but the total activity was greatly reduced compared to that which was applied to the column (Fig. 2).
However, upon adding an aliquot of the fraction containing an to each of the column fractions, there
was a complete recovery of bacteriocin activity in the fraction containing p (Fig.
1). Similarly there was a complete recovery of bacteriocin activity in the fraction containing a1 and to
a lesser extent in the fraction containing az, when an aliquot of ss was added to each fraction (Fig.
2).
Each peptide a, a2 and B was purified to homogeneity by rechromatography on the reverse phase
column (Fig.
3). Whereas relatively little activity was seen when each peptide was assayed for bacteriocin activity
alone, the activity was recovered when the ss peptide was complemented with the a peptide, and to
a lesser extent with the a peptide (Fig. 3). No additional increase in the bacteriocin activity was seen
upon adding the a2 peptide to fractions containing both the B and at peptides. Thus the
complementary action of the two peptides, an a and a ss peptide appeared to be necessary to
obtain bacteriocin activity.
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A small amount of ss which presumably had not been entirely separated from a1 on the previous
reverse phase column, was detected upon rechromatography of a1 (Fig. 3
C). A small optical density peak apparently due to a2 as well as a minor peak eluting ahead of a2 similarly to the optical density peak which eluted slightly ahead of 22 in Fig. 2 - were also detected
(Fig. 3 C). It is unlikely that the presence of these two latter peaks was due to incomplete separation
from a1 on the previous reverse phase column, since they did not contaminate the ss preparation to
the same extent (Fig. 3 B). Upon rechromatography of purified a1 after storage in 50% propanol for 3
months at -20 C, 30-40% of the peptides eluted as expected for a1, whereas 30-40% eluted similarly
to a2 in Fig 2. As much as 10% of purified a1 eluted similarly to a2 upon rechromatography after
storage for 24 h. This suggests that a2 and the component which eluted ahead of a2 were derived
from a1.
Mass spectroscopy analysis of the a and ss peptides
In order to determine the molecular weights of a1 and B and confirm their purity after the final
reverse phase chromatography step (Fig. 3), these peptides were analyzed by plasma disorption
mass spectroscopy. Single peaks were observed with molecular weights of 4376 and 4109 for CL
and B, respectively.
Amino acid sequence of the a and ss peptides
The complete amino acid sequence of the a1 and a2 (39 amino acid residues) and B (35 amino
acid residues) peptides (SEQ ID NOS: 2 and 3) are shown in Fig. 4. It appears from the sequences
that a2 is identical to a1 (Fig. 4), consistent with the apparent formation of a2 peptides upon
rechromatography of a1 on the reverse phase column.
Relative amounts of a and ss to obtain bacteriocin activity
The concentrations of a1 and B which in combination inhibited growth of the indicator organism by
50% were determined, and the results were plotted as an isobologram (Fig. 5). When a1 was in
excess (greater than 1.3 nM, 0.25 pmoles/well), the presence of B at a concentration of 0.02 nM
(0.04 pmoles/well), resulted in 50% growth inhibition (Fig. 5). Similarly, with an excess of ss (greater
than 0.45 nM (0.03 pmoles/well), the presence of a1 at a concentration of about 0.15 nM (0.03
pmoles/well) resulted in a 50% growth inhibition (Fig. 5). Thus in order to obtain 50% growth inhibition
in the presence of an excess of the complementary peptide, 7-8-fold more a1 than B was
needed.When neither a1 nor B was in excess, the concentrations which resulted in 50% growth
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inhibition were 0.3 nM (0.06 pmoles/well) for a1 and 0.04 nM (0.008 pmoles/well) for ss (Fig. 5).
Again there was 7-8-fold more of a1 than B.
The concentrations which inhibited growth by 50% appeared to be invariant to the number of target
cells present (within a 30fold range in the cell number), as the same concentrations of a1 and B
resulted in 50% growth-inhibition irrespective of whether the cell density was such that the A was
0.01 or 0.3.
summary
Three optical density peaks associated with bacteriocin activity were obtained upon reverse phase
chromatography in the final purification step. The peptides associated with the 3 optical density
peaks were termed a1, a2 and B.
The bacteriocin activity was due to the complementary action of an a and the B peptide. In
combination with the ss peptide, a1 gave a mugh higher bacteriocin activity than a2. Upon
rechromatography of purified a1 on a reverse phase column, some of it eluted as expected for a2.
This suggests that a1 and a2 may in fact be the same peptide, but that they differ in their
configuration in a manner which results in a2 having a slightly lower affinity to the reverse phase
column and reduced activity when combined with B than a1. This view was supported by the amino
acid sequencing data. The a2 peptide was sequenced and this sequence appeared to be identical
to the corresponding sequence of a1.
As judged by amino acid sequencing, a1 contained 39 amino acid residues and its molecular
weight should be 4346. A molecular weight of 4376 was obtained by mass spectrometry indicating
that the peptide is not grossly modified. Judging from its sequence, ss contains 35 amino acid
residues and its molecular weight should be 4110. This is in good agreement with the molecular
weight of 4109 obtained by mass spectrometry, indicating that this peptide is not modified. From the
amino acid sequence, the isoelectric point and extinction coefficient of a1 were calculated to be
10.9 and 1.3 x 104 M-1 cm1, respectively.For the ss peptide the isoelectric point and extinction
coefficient were calculated to be 10.4 and 2.4 x 104 M1 cml respectively.
The amino acid sequence of both a and ss is such that these peptides are likely to be pore-forming
toxins that create cell membrane channels through a "barrelstave" mechanism, and thus produce an
ionic imbalance in the cell (See Ojcius et al, TIBS, 16 225-229, 1991). A region in a1 starting with
23/1006
amino acid residue number 3 and ending with residue number 27 may form an amphiphilic a-helix,
as is evidence when this sequence is displayed on an Edmundson a-helical wheel (Fig. 6A).
The polar amino acids are found almost completely on one side of the a-helix, whereas the nonpolar
residues are found on the opposite side of the helix (Fig. 6A). The amphiphilic distribution of the
amino acids in this region is nearly perfect, the only exception being glycine (residue number 9)
which appears on the hydrophobic side (Fig. 6A). However, glycine may be considered to be
relatively neutral with respect to its hydrophilic/hydrophobic character. Moreover, the substitution of
one amino acid by one of an opposite hydrophobicity may not represent an intolerable disruption of a
peptide's amphiphilic character (see
Ojcius, Supra).The 25 amino acids long amphiphilic region in a1 may allow peptide-monomers to
oligomerize into membrane-spanning pores in such a manner that the non-polar side of the a-helix
faces the membrane lipids, whereas the polar side faces towards the center of the pore (see Ojcius
(Supra) and Lear et al, Science 240:1177-1181, 1988). The amphiphilic region in a1 should be long
enough to span a membrane, as a minimum of about 20 residues are needed to form a
membranespanning a-helix. Ten of the 12 C-terminal amino acid residues, starting with position 28
and ending with 39, in a1 are polar, of which the last 5 are basic (Arg-Lys
Lys-Lys-His-COOH).In this connection it is interesting to note that lactococcin A and lactocin S,
bacteriocins produced by Lactococcus lactis subsp cremoris and
Lactobacillus sake. respectively, also contain a basic
C-terminus, the last two C-terminal amino acids being histidine for both of these bacteriocins, (Holo
(Supra), Mrvedt (Supra)). It seems likely that the 11 amino acid residues long polar C-terminal region
in a1 does not penetrate the membrane. One may speculate that its function might be to recognise
bacteriocin-binding sites on the target cells and thereby create a local high concentration of
bacteriocin monomers on the outside of the cell membrance. This high concentration near the
membrane could then induce oligomerization of the monomers into transmembrane pores.Another
possible function of the polar C-terminal region might be to stabilize a correct peptide-configuration
in a hydrophilic environment. The first two N-terminal amino acid residues in a1 are polar, and may
possibly be the part of the pore which is located inside the cell.
The situation is similar for the 13-peptide. An amphiphilic a-helix may be formed in the region
starting with amino acid residue number 8 and ending with residue number 25 (Fig 6 B). In this
region there are two exceptions to a perfect amphiphilic amino acid distribution: glycine (residue 22)
which appears on the hydrophobic side and isoleucine (residue 24) which appears on the
hydrophilic side. The former should not be a major problem, due to the neutral character of glycine.
24/1006
one would expect that proline at position 11 would cause a break or bend in the a-helix structure of
the amphiphilic region. The region spans 18 amino acid residues, which may be somewhat less than
required to span the cell membrane. However, in front of the amphiphilic region there are 5 nonpolar
amino acids starting with residue number 3 and ending with number 7 which presumably also will be
part of the transmembrane region. The hydrophilic amino acid, lysine as found at positions 1 and 2
at the N-terminus; and only 2 of 10residues starting with position 26, in the C-terminal part of the
molecule are hydrophobic. Similar to ar, one might expect that these regions will be located outside
the membrane, the long polar C-terminal region being on the cell's outside. Although other lactic acid
bacteria-produced bacteriocins that have been sequenced do not appear to have such a marked
amphiphilic distribution of amino acids as a and B such a distribution is also seen in the C-terminal
part of lactococcin A and Lactocin S.There is evidence that lactococcin A, permeabilizes target cell
membranes (Van
Belkum (Supra) as also appears to be the case for nisin (Sahl et al., Arch Microbiol 149: 120-124,
1987).
The concentrations of a1 and ss which inhibited growth of the indicator cells by 50% were
respectively 0.15 and 0.02 nM when the complementing peptide was present in excess. When
neither was in excess the concentrations were, respectively, 0.3 and 0.04 nM.
Thus 7-8-fold more of a1 than B was needed. If the two peptides associate to the target cells with
equal efficiency, this ration may reflect that a1 and ss interact in an approximately 8 to 1 ratio, for
instance in pore formation. The presence of approximately 40 ss molecules per target cell together
with an excess of a1 is enough to induce 50% growth inhibition. The number of ss molecules that
interact with a target cell may possibly be even smaller, since the concentration of bacteriocin which
inhibited growth by 50% appeared to be invariant to the number of target cells present within a 30fold range.
Example 2
Cloning and sequencing of the bacteriocin (lactococcin G (LcnG)) was carried out as follows.
Chromosomal DNA was purified from Lactococcus lactis LMG 2081 according to standard methods,
and digested with
SpeI. SpeI fragments were fractionated by agarose gel electrophoresis, and DNA fragments of 3-7
kbp were isolated using Magic Minipreps (Promega). A sub-genomic library was constructed by
ligating the fragments with
25/1006
SpeI digested and phosphatase treated lambda ZAP II arms (stratagene). The clone containing the
bacteriocin (LcnG) structural gene and immunity gene was isolated by screening the library with a
degenerate oligonucleotide probe deduced from the amino acid sequence of LcnG (Nissen-Meyer et
al.). The hybridization was carried out according to standard protocols (Molecular cloning,
Eds; Sambrook, et al., supra). Sequences were determined by the chain termination method (Sanger
et al.) using sequenase (United States Biochemical Corp.).
The sequence obtained is shown in Figure 1 (SEQ ID NO: 5). Claims:
CLAIMS
1. A polypeptide having or including an amino acid sequence substantially corresponding to all or a
portion of the amino acid sequence set out in Figure 1 (SEQ ID
NO: 1) and derivatives and fragments thereof having bacteriocin and/or bacteriocin immunity activity.
2. A polypeptide as claimed in claim 1 having or including the amino acid sequence a1 and a2 (SEQ
ID NO: 2):
N Gly Thr Trp Asp Asp Ile Gly Gln Gly
Ile Gly Arg Val Ala Tyr Trp Val Gly
Lys Ala Met Gly Asn Met Ser Asp Val
Asn Gln Ala Ser Arg Ile Asn Arg Lys
Lys Lys His C
and/or p (SEQ ID NO: 3):
N Lys Lys Trp Gly Trp Leu Ala Trp Val
Asp Pro Ala Tyr Glu Phe Ile Lys Gly
Phe Gly Lys Gly Ala Ile Lys Glu Gly
Asn Lys Asp Lys Trp Lys Asn Ile C and derivatives and fragments thereof having bacteriocin activity.
3. A polypeptide having or including the amino acid sequence
Leu Phe Asn Asn Ile Val Val Phe Ile
Asn Phe Leu Ser Phe Val Phe Ile Leu
Val Gly Val Asp Ile Lys Tyr Asn Asp
26/1006
Asn Arg Ile Lys Ile Val His Val Thr
Phe Phe Ile Ser Phe Ile Leu Val Met
Leu Thr Ser Leu Ile Ser His Asn Ser
Ile Ala Tyr Ser Leu Ser Gln Ile Leu
Glu Ile Leu Cys Ile Ile Cys Ile Leu
Leu Leu Phe Tyr Ile Leu Lys Lys Thr
Asn Ser Leu Ser Asn Arg Ala Asn Val
Val Phe Ile Ile Phe Ile Val Thr Gln
Val Ile Ile Ile Ile Asn Gln Leu Phe
Ile Arg (SEQ ID NO: 4) and derivatives and fragments thereof having bacteriocin immunity factor
activity.
4. A bacteriocin comprising polypeptides a and p or fragments or derivatives thereof as defined in
claim 2.
5. A bacteriocin as claimed in claim 4 comprising polypeptides a and p, or fragments or derivatives
thereof, in a ratio of 5-10 to 1 respectively.
6. A bacteriocin as claimed in claim 5 comprising polypeptides a and p, or fragments or derivatives
thereof, in a ratio of 8 to 1 respectively.
7. A composition comprising a bacteriocin as claimed in any one of claims 4 to 6 and/or a
bacteriocin immunity factor as claimed in claim 3 together with at least one of a carrier, and/or diluent,
or excipient.
8. Use of a bacteriocin as claimed in any one of claims 4 to 6 in selectively killing undesired or
contaminating strains of bacteria in microbiological or food manufacturing processes.
9. A starter culture of microorganisms for use in a microbiological process, comprising a bacteriocin
as claimed in any one of claims 4 to 6, said microorganisms being resistant to said bacteriocin.
10. A method of cheese or yoghurt production in which a bacteriocin as claimed in any one of claims
4 to 6 is added to effect lysis of lactic acid bacteria.
27/1006
11. A method of isolation of a bacteriocin and/or a bacteriocin immunity factor as claimed in any one
of claims 3 to 6 wherein a culture of a microorganism expressing said bacteriocin and/or immunity
factor is subjected to fractionation whereby fractions enriched in said bacteriocin and/or immunity
factor are collected.
12. A method as claimed in claim 11 wherein the microorganism is Lactococcus lactis strain LMG
2081.
13. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide or its
derivatives or fragments as claimed in any one of claims 1 to 3.
14. A nucleic acid molecule comprising a nucleotide sequence which encodes a bacteriocin, its
component peptides and/or its corresponding immunity factor, or a fragment thereof, substantially
corresponding to all or a portion of the nucleotide sequence as shown in Figure 1 (SEQ ID NO: 5) or
a sequence which is degenerate or substantially homologous with or which hybridises with any such
sequence.
15. An expression or cloning vector comprising a nucleic acid molecule as claimed in claim 13 or
claim 14.
16. A host cell or transgenic organism containing a nucleic acid molecule as claimed in claim 13 or
claim 14.
17. A host cell as claimed in claim 16 being a lactic acid bacterium.
18. A method for preparing a polypeptide, derivative or fragment as claimed in claim 1, comprising
culturing a host cell as defined in claim 16 or claim 17 under conditions whereby said polypeptide is
expressed and recovering said polypeptide, derivative or fragment thus produced.
19. A process for the preparation of bacteriocin and/or bacteriocin immunity factor polypeptides as
claimed in any one of claims 1 to 3 in which a corresponding protected or immobilised polypeptide is
subjected to deprotection or removal from a solid support.
28/1006
4. AU638616 - 08.07.1992
CLONED GENE ENCODING FOR BACTERIOCIN FROM PEDIOCOCCUS ACIDILACTICI
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=AU638616
Inventor(s):
HENDERSON JAMES T (US); MARUGG JOHN D (NL); VANDENBERGH PETER A
(US); LEDEBOER ADRIANUS M (NL)
Applicant(s):
QUEST INT (NL)
IP Class 4 Digits: C12N; A23L; C07K
IP Class:
C12N15/31; A23L3/3571; C07K15/00
E Class: A23L3/3571; C07K14/195
Application Number:
EP19910122124 (19911223)
Priority Number: US19900635965 (19901231)
Family: AU638616
Equivalent:
AU8820091; CA2056086; DE69125921D; DE69125921T; DK493779T; ES2102381T;
JP2618148B2; JP7067652
Cited Document(s):
EP0406545; EP0453719; EP0326062; WO8706264; EP0293547
Abstract:
ISOLATION AND IDENTIFICATION OF A GENE ENCODING FOR A BACTERIOCIN PRECURSOR IN
PEDIOCOCCUS ACIDILACTICI, CLONING OF THE GENE IN A VECTOR PLASMID AND
TRANSFORMATION TO BACTERIA IS DESCRIBED. THE BACTERIOCIN IS PARTICULARLY USEFUL
FOR INHIBITING LISTERIA IN FOOD PRODUCTS.Description:
29/1006
BACKGROUND OF THE INVENTION
(l) SUMMARY OF THE INVENTION
The present invention relates to a sequenced gene encoding for a bacteriocin in Pediococcus
acidilactici and in particular to a gene that is essential for the production of the functional bacteriocin,
called hereafter helper protein, and to the cloned gene in a vector which is transformed into a
bacterium. In particular, the present invention relates to a sequenced gene encoding for a
bacteriocin derived from a plasmid in Pediococcus acidilactici.
(2) Prior Art
The pediococci are a diverse group of Gram-positive homofermentative lactic acid bacteria often
found as saphrophytes on vegetable material (Gonzalez, C. F., and B. S. Kunka, Appl. Environ.
Microbiol. 53:2534-2538 (l987); and Mundt, J. O., W. G. Beattie, and F. R. Wieland, J. Bacteriol.
98:938-942 (l969)) Commercially, pediococci are used in the fermentation of vegetables (Pederson,
Bacteriol. Rev. l3:225-232 (l949) and meats (Smith, J. L., and S. A. Palumbo, J. Food Prot. 46:997l006 (l983)).
Some strains of P. pentosaceus, P. cerevisiae and P. acidilactici have been found to contain resident
plasmids although the roles of most of these remain unknown (Gonzalez, C. F., and B. S. Kunka,
Appl. Environ. Microbiol. 46:8l-89 (l983); Graham, D. C., and L. L. McKay, Appl. Environ. Microbiol.
50:532-534 (l985); and Raccach, M., CRC Crit. Rev. Microbiol. l4:29l-309 (l987)). The association of
raffinose fermentation and plasmid DNA has been reported (Gonzalez, C. F., and B. S. Kunka, Appl.
Environ. Microbiol. 5l:l05-l09 (l986)), as has been the ability of P. acidilactici to ferment sucrose
(Gonzalez, C. F. and B. S. Kunka, Appl. Environ. Microbiol 53:2534-2538 (l987)). Moreover, there
have been several reports which associate the production of bacteriocins with host plasmid DNA
(Daeschel, M. A., and T. R. Klaenhammer, Appl. Environ. Microbiol. 5l:l538-l54l (l985); Gonzalez, C.F.,
30/1006
and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (l987); Graham, D. C., and L. . McKay, Appl.
Environ. Microbiol. 50:532-534 (l985); and Bhunia et al, J. Applied Bact. 65:26l-268 (l988)). It was
shown by Gonzalez, C. F. and B. S. Kunka (Appl. Environ. Microbiol. 53:2534-2538 (l987)) that
bacteriocin production was encoded by a 9.0 kbp plasmid pSRQll in P. acidilactici PACl.0. Further
work (Pucci, M. P., E. R. Vedamuthu, B. S. Kunka and P. A. Vandenbergh, Appl. Environ. Microbiol.
54:2349-2353 (l988)) demonstrated that the bacteriocin of P. acidilactici PACl.0 was active against a
wide spectrum of gram positive lactic acid bacteria, and also against Listeria monocytogenes. This
anti-listerial activity was observed in broth and on agar plates, as well as in some dairy
products.Inhibition of L. monocytogenes by this bacteriocin, PA-l, has also been noted in fermented
semi-dry sausage (Berry, E. D., M. B. Liewen, R. W. Mandigo and R. W. Huthine, J. Food Protection
53, l94-l97 (l990)) and fresh meat (Nielsen, J. W., J. S. Dickson and J. D. Crouse, Appl. Environ.
Microbiol 56, 2l42-2l45 (l990)). The cloning of genes for the production of the bacteriocin has not
been described and this would be useful for producing bacteriocin in significant quantities in genera
unrelated to Pediococcus, or enhancing production in the pediococci.
Cloned Gram-positive genes for different unrelated proteins have been shown to express in
Escherichia coli (Gilmore, M. S., Curr. Top. Microbiol. Immunol. ll8:2l9-234 (l985); Rogeson, J. P., R.
G. Barletta, and R. Curtiss III, J. Bacteriol. l53:2ll-22l (l983); and Smorawinska, M., J. C. Hsu, J. B.
Hansen, E. K. Jagusztyn-Krynicka, Y. H. Abiko, and R. Curtiss III, J. Bacteriol. l53:l095-l097 (l983)).
OBJECTS
It is therefore an object of the present invention to provide the sequenced gene for the bacteriocin
and its essential helper protein(s), which are used as vectors that can be transferred to other
microorganisms that contain the genetic information of these genes in such a way that the functional
bacteriocin is produced by these new hosts. Such microorganisms are particularly in the genera
Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia, Bacillus and
yeasts. These and other objects will become increasingly apparent by reference to the following
description and the drawings.
IN THE DRAWINGS
31/1006
Figure l shows a restriction endonuclease site map of pSRQll. P. acidilactici PACl.0 plasmid pSRQll is
9.0 kbp and contains the gene for PA-l bacteriocin.
Figures 2A and 2B show restriction endonuclease site maps of pSRQll.l and pSRQll.2, respectively.
Both plasmids are l4.8 kbp and contain erythromycin resistance (ery) genes at the locations
indicated. The E. coli origin of replication (ori) and the remaining part of the chloramphenicol
resistance (cml) gene are shown. Numbered triangles ( INCREMENT ) indicate areas of each
plasmid which had been subsequently deleted.
Figure 3A shows a restriction endonuclease site map of pSRQ220. Plasmid pSRQ220 is 9.3 kbp and
is a chimera of Escherichia coli plasmid pBR322 and PACl.0 plasmid pSRQll digested with EcoRI
and SalI and ligated together. The Escherichia coli origin of replication (ori) and the ampicillin
resistance (amp) gene are indicated. The 5.6 kbp EcoRI-SalI fragment is indicated by the open box.
Figure 3B shows a physical map of the 5.6 kbp EcoRI-SalI fragment from pSRQ220. The horizontal
arrows denote open reading frames discussed hereinafter (ORF 1, ORF 2, and ORF 3). The
horizontal lines, indicated by numbered triangles ( INCREMENT 1, INCREMENT 2 and INCREMENT
3), represent three deletions present in plasmids pUR5204 ( INCREMENT 1), pSRQ220.2
( INCREMENT 2), and pSRQ11.13 ( INCREMENT 3), respectively.
Figure 4 shows the nucleotide sequence of the 5.6 kbp EcoRI-SalI insert from pSRQ220. The derived
amino acid sequences of ORF1, ORF2, and ORF3 are also shown. The arrow indicates the start of
the mature PA-1 bacteriocin. The TAG termination codons are denoted with an asterisk (*).
Figure 5A shows a coomassie stained 5-22% acrylamide SDS-PAGE gel of purified PA-1. a = 66000,
b = 45000, c = 36000, d = 29000, e = 24000, f = 20100, g = 14200, h = 6500 Daltons. Standards a
through g are MW-SDS-70L (Sigma), standard h is aprotinin (Sigma).
Figure 5B shows an unstained gel overlayed with a lawn of Pediococcus pentosaceus FBB63
indicator cells. Inhibition zone (large arrow) is apparent. 1 = 110000, 2 = 84000, 3 = 47000, 4 =
33000, 5 = 24000, 6 = 16000 Daltons. Prestained standards (Biorad) were used.
GENERAL DESCRIPTION
32/1006
The present invention relates to a nucleotide sequence as given in Figure 4 and derivatives thereof
which produces a bacteriocin precursor.
The present invention further relates to a vector containing a nucleotide sequence containing ORF 1
as described in Figure 4 maintained in a bacterium in which the nucleotide sequence is preceded by
a promoter system and followed by a terminator sequence both functional in the bacterium and
express a bacteriocin encoded by the nucleotide sequence in the bacterium.
The nucleotide sequence of the present invention can be maintained in a vector which operates in
various bacteria or yeasts. All that is required is that the microorganisms express the bacteriocin.
The DNA encoding the bacteriocin can be replicated by means of a polymerase chain reaction as
described in Chemical Engineering News, pages 36-46, October l, l990 and in other references. The
appropriate 3' and 5' terminal regions of the DNA encoding the bacteriocin can be used as primers
defining the region to be replicated.
The gene segment is preferably derived from Pediococcus acidilactici NRRL-B-l8050 also known
herein as pACl.0, which is deposited with the Northern Regional Research Laboratory in Peoria,
Illinois under the Budapest Treaty. The genes involved in bacteriocin activity are carried on a 9.0 kbp
plasmid designated herein as pSRQll. A DNA segment (SalI to EcoRI; 5.6 kbp) is ligated in purified
farm in a vector plasmid pBR322 and called pSRQ220. This plasmid is transformed to Escherichia
coli NRRL-B-l8429 and deposited at the same depository under the Budapest Treaty.
U.S. Patent No. 4,883,673 which is assigned to a common assignee describes the isolation of a
bacteriocin from Pediococcus acidilactici NRRL-B-l8050 which inhibits various bacteria. A plasmid in
this strain was disclosed to encode for the bacteriocin. The bacteriocin was described to be useful in
foods to inhibit bacterial spoilage. U.S. Patent No. 4,929,445, assigned to a common assignee,
describes a method of using the bacteriocin to inhibit Listeria monocytogenes which produces a
severe illness in humans. The plasmid pSRQll was described as the source of the bacteriocin. The
usefulness of the bacteriocin is well established.
SPECIFIC DESCRIPTION
33/1006
The following Examples show the steps in sequencing the gene encoding for the bacteriocin.
Bacterial strains and media. The bacterial strains used are listed in Table 1. Pediococcus spp. were
routinely maintained on MRS agar (Difco Laboratories, Detroit, MI). Escherichia coli strains were
routinely carried on Lennox L agar (Gibco/BRL, Gaithersburg, Md.). Escherichia coli strains were
also grown on modified MRS agar (no citrate or acetate) or in M9 medium (Maniatis, T., E. F. Fritsch,
and J. Sambrook, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY (l982)) supplemented with l% yeast extract (Oxoid, Ltd., Basingstoke, Hampshire,
U.K.) and l% Hy Case TM (Sheffield Products, Norwich, NY) for bacteriocin assays. Selective
antibiotic concentrations were as follows: ampicillin, 25 ug/ml; tetracycline, l0 ug/ml; erythromycin, 50
ug/ml; and chloramphenicol, 25 ug/ml. All antibiotics were purchased from Sigma Chemical Co.,
St.Louis, MO.
Bacteriocin assays. Production of bacteriocin was assayed as previously described (Gonzalez, C. F.,
and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)). Strains were patched on MRS agar
or modified MRS agar for Escherichia coli and incubated at 35 DEG C for l8 hours. The plates were
then overlaid with soft agar (0.8%) seeded with indicator cells. Isolates which produced a clear,
defined zone of inhibition were considered as bacteriocin producers.
One arbitrary unit (AU) of bacteriocin was defined as 5 microliters of the highest dilution of culture
supernatant yielding a definite zone of growth inhibition on the indicator lawn. The titer was
expressed as the reciprocal of the highest dilution showing inhibition.
Isolation and analysis of plasmid DNA. Covalently closed circular plasmid DNA was isolated from
Escherichia coli by the method of Clewell and Helinski (Clewell, D. B., and D. R. Helinski,
Biochemistry 9:4428-4440 (l970)). Escherichia coli strains were screened for plasmid content as
previously described (Macrina, F. L., J. A. Tobian, K. R. Jones, R. P. Evans, and D. B. Clewell, Gene
l9:345-353 (l982)). Pediococcus plasmid DNA was obtained by a scaled up modification of the
LeBlanc and Lee procedure (LeBlanc, D. J., and L. N. Lee, J. Bacteriol. 140:1112-1115 (1979)) as
described by Gonzalez and Kunka (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol.
46:81-89 (1983)). Plasmid DNA and restriction endonuclease digests were analyzed by agarose gel
electrophoresis on 0.8% agarose (Bethesda Research Laboratories, Inc., Gaithersburg, MD) slab
gels.Size standards were Escherichia coli V517 (Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J.
Ayers, and S. McCowen, Plasmid 1:417-420 (1978)) for undigested plasmid DNA and HindIII -
34/1006
digested bacteriophage lambda DNA (Bethesda Research Laboratories) for restriction endonuclease
- cleaved plasmid DNA.
DNA enzymology. Restriction endonuclease digestions were performed in low-, medium-, or high-salt
buffers, as recommended by Maniatis et al. (Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).
Restriction enzymes were obtained from Bethesda Research Laboratories. DNA ligation reactions
were carried out with T4 DNA ligase (Bethesda Research Laboratories) at 4 DEG C for 18 hours
according to conditions recommended by the manufacturer.
Bacterial transformations. Escherichia coli was transformed by the CaCl2 heat shock method
(Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1982)) with cells harvested at an optical density at 660 nm of 0.2 to 0.3.
Purification of PA-1. Cultural supernatant was neutralized to pH 6.0 with sodium hydroxide prior to
gel filtration. A 450 ml aliquot of neutralized supernatant was applied to a 5 cm X 55 cm column
(Pharmacia) containing one liter of Spectra/Gel AcA 202 (Spectrum) gel filtration resin which had
been equilibrated with 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0. Activity was
eluted using the same buffer. Active fractions were pooled and applied to a 2.5 cm x 90 cm CMSepharose column equilibrated with .05 M MES, pH 6.0. Activity was eluted with a linear gradient
to .05 M MES containing l M sodium chloride, pH 6.0. Active fractions were pooled and dialyzed
against a l0 fold excess of water using l000 Da molecular weight cut-off dialysis tubing (Spectra-Por
6, Spectrum).Dialysate volume was reduced l2 fold by applying the dialysis tubing directly to solid 20
KDa polyethylene glycol (Carbowax, Union Carbide) and was then further reduced 3.5 fold by
vacuum centrifugation (Speed-Vac, Savant). Concentrated PA-l was applied to a l.0 cm x 25 cm Cl8
reversed-phase column (Vydac) equilibrated with 0.l% aqueous trifluoroacetic acid. Activity was
eluted with a linear gradient to 45% acetonitrile over 30 minutes at l.5 ml/min. Active fractions were
determined by directly spotting aliquots of column effluent on MRS plates overlaid with soft agar
containing indicator cells. Active fractions were dried by vacuum centrifugation and stored at -20
DEG C. Specific activity is defined as AU per milligram protein. Protein analyses were performed
using the BCA protein assay kit (Pierce) using directions supplied with the kit.
Example l
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Restriction endonuclease map of pSRQll. The genes involved in bacteriocin PA-l activity were
previously shown to be associated with the presence of a 9.0 kilobase plasmid, designated pSRQll
(Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (l987)). Plasmid pSRQll
was digested with a number of restriction endonucleases to generate the restriction site map shown
in Figure l. The plasmid contained several unique sites including EcoRI, NdeI, XbaI, SalI, and SstI.
Other restriction enzymes which cleaved the plasmid were ClaI, HindIII, PvuII, and EcoRV. The
following restriction sites were not found on pSRQll: AvaI, BamHI, SphI, NruI, PstI, and BglII.
Expression of PA-1 bacteriocin in E. coli. Plasmid pSRQ11 was digested with EcoRI and cloned into
the EcoRI site on plasmid pVA891 (Macrina, F. L., et al., Gene 25:145-150 (1983)), which contains an
erythromycin resistance marker expressed in both Escherichia coli and streptococci. Recombinant
plasmids were obtained with pSRQ11 inserted in both orientations and were designated pSRQ11.1
and pSRQ11.2 as shown in Figure 2. These Escherichia coli strains were assayed for expression of
the PA-1 bacteriocin as previously described (Gonzalez, C. F., and B. S. Kunka, Appl. Environ.
Microbiol. 53:2534-2538 (1987)). The strains were grown on modified MRS medium and overlaid with
Pediococcus pentosaceus FBB63 indicator strain.Escherichia coli strains containing pSRQ11.1 and
pSRQ11.2 both produced zones of inhibition in the indicator lawn while the control Escherichia coli
V850 strains showed no zone of inhibition (Table 2).
Table 2. Plasmids derived from pSRQ11 The plasmid pSRQll was also cloned in the unique EcoRI
site of the E. coli-Streptococcus shuttle plasmid pSA3. The resulting clone was called pSRQl6l. When
the E. coli V850 strain carrying pSRQl6l (Table 2) was grown overnight in M9 medium supplemented
with l% yeast extract and l% Hy Case, the filter sterilized culture supernatant yielded approximately
400 AU/ml of the bacteriocin PA-l. This observation indicated that E. coli V850 (pSRQl6l) was
producing and excreting PA-l into the media. Also, other E. coli strains were transformed with the
plasmid pSRQl6l and observed to produce PA-l.From this data, it was concluded that a gene
fragment encoding bacteriocin PA-l from P. acidilactici PAC 1.0 can be expressed and is functional
in an E. coli host strain.
Example 3
Deletion derivative analysis of pSRQ11 subclones.
36/1006
In order to localize the region encoding the PA-1 gene(s), SalI and PvuII deletion derivatives of
pSRQ11.1 and pSRQ11.2 were obtained (Figure 2). The SalI deletion of pSRQ11.1 retained activity
while the PvuII deletion derivatives displayed no zones of inhibition against the indicator strain (Table
2). Both the PvuII and SalI deletion derivatives of pSRQ11.2 expressed no PA-1 activity (Table 2).
These data suggested that the bacteriocin gene was located on the approximately 5.6 kbp EcoRISalI fragment of pSRQ11.1 as shown in Figure 2A. This 5.6 kbp EcoRI-SalI fragment then was
subcloned into the EcoRI and SalI restriction sites on the Escherichia coli plasmid pBR322 (Bolivar et
al., Gene 2:95-113 (1977)), and the resulting chimeric plasmid was designated pSRQ220 (Figure 3A).
The Escherichia coli strain containing pSRQ220 was assayed and found to express bacteriocin
activity.Two additional deletion derivatives of pSRQ220, i.e., a plasmid derivative lacking a 2.7 kbp
HindIII fragment and a plasmid derivative lacking a 1.3 kbp HindIII-SalI fragment (Figure 3B), were
assayed and both found to be negative for PA-1 activity. Also the following deletion derivatives were
obtained: pSRQ210, which consisted of the pSRQ11, XbaI-SalI fragment cloned into E. coli vector
pACYC184 (Chang, A. C. Y., et al., J. Bacteriol. 134:1141-1156 (1978)), and pSRQ211, which
consisted of pSRQ11 HindIII fragment c (from map coordinates 1.5 to 4.2, Figure 1) also cloned into
pACYC184. Neither of these two strains expressed PA-1 activity.Together with the bacteriocin PA-1
negative PvuII and ClaI deletion derivatives (Figures 2A, and 3B (Table 2)), these results show that
several genes, or one very long gene (or operon), present on the 5.6 kbp EcoRI-SalI fragment, are
responsible for PA-1 activity.
Example 4
Insertional inactivation of bacteriocin PA-l production.
Since the XbaI restriction site is unique on both pSRQll and pSRQ220 and lies within the region
involved in PA-l production, it was chosen as a site to insert a foreign DNA fragment and interrupt
transcription of the bacteriocin gene. Plasmid pACYCl84, approximately 4 kbp in size and also
containing a single XbaI site, was cloned into the XbaI site on pSRQ220. The strain containing the
resulting recombinant plasmid, pSRQ22l, was assayed for PA-l activity and proved negative (Table
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2). When the pACYCl84 insert was removed by XbaI digestion, followed by religation, resulting in
pSRQ 22l.l, activity was once again restored. Another construct where the XbaI-EcoRI fragment of
pSRQ220 was replaced by the XbaI-EcoRI fragment of pACYCl84 also was negative for bacteriocin
activity (Table 2).
Example 5
Nucleotide sequence analysis of pSRQ220.
The DNA sequence of the 5.6 kbp SalI-EcoRI DNA fragment, as present on plasmid pSRQ220, was
established by the Sanger dideoxy chain termination procedure (Sanger, F., Nicklen, S., and Coulson,
A. R., Proc. Natl. Acad. Sci. USA, 74:5463-3967 (l977)) with the modifications as described by Biggin
et al (Biggin, M.D. et al., Proc. Natl. Acad. Sci. USA, 80:3963-3965 (l983)), using alpha-S-dATP (2000
Ci/mmol) and Klenow enzyme (Amersham), ddNTP's (Pharmacia-PL Biochemicals) and dNTP's
(Boehringer). The sequencing reaction products were separated on a denaturing polyacrylamide gel
with a buffer gradient as described by Biggin et al. (Biggin, M. D. et al., Proc. Natl. Acad. Sci. USA,
80:3963-3965 (l983)). Purified, double-stranded plasmid DNA of pSRQ220 served as template in the
sequence reaction, following the procedure described by Hattori and Sakaki (Hattori, M., and Sakaki,
Y., Anal.Biochem. 152: 232-238 (l986)). Deoxy-oligonucleotide primers were synthesized on a DNAsynthesizer (Applied Biosystems 380A) using the Phosphoamidit technique (Barone, A. D. et al.,
Nucleic Acid Research, 12:4051-4061 (1984)).
The DNA sequence when translated in all possible reading frames revealed at least three open
reading frames (Fig. 4). The first open reading frame (ORF 1) encodes a protein which consists of 62
amino acid residues followed by a TAG stop codon (Fig. 4). The second open reading frame (ORF 2),
positioned just downstream of ORF 1, codes for a protein which consists of 112 amino acid residues
followed by a TAG stop codon (Fig. 4). Further downstream the third open reading frame (ORF 3)
predicts a protein consisting of 724 amino acid residues with a TAG stop codon (Fig. 4).
ORF 1 encodes a protein of 62 amino acids of which amino acid residues 19 to 62 correspond
entirely with the amino acid sequence of a protein, which was isolated from P. acidilactici NRRL-B-
38/1006
18050 called bacteriocin PA-1, and which, when separated on a polyacrylamide gel, inhibited P.
pentosaceus FBB-63 effectively in an overlay experiment which is the subject of U.S. application
Serial No. 514,102 (Fig. 4, and Fig. 5). This proves that ORF 1 encodes a precursor of bacteriocin
PA-1, containing an 18 amino acid N-terminal peptide which is cleaved off during the process of
synthesis or excretion.
Both the PvuII deletion derivative pSRQ11.13 and the HindIII deletion derivative pSRQ220.2 (Table 2;
Figure 3B) result in a loss of PA-1 bacteriocin activity. As these deletions disturb both ORF 2 and
ORF 3, or ORF 3 only, but not the PA-1 bacteriocin encoding gene (ORF 1), it can be concluded that
also the presence of either ORF 2 or ORF 3, or both is necessary for PA-1 bacteriocin activity.
Example 6
Site-specific mutagenesis of genes involved in PA-l bacteriocin production.
The specific role in PA-l bacteriocin production of each of the open reading frames was determined
by introduction of frameshift mutations in the various genes.
Plasmid pSRQ220 contains two sites for the restriction enzyme BalI. One is situated in the pBR332part of the plasmid, whereas the other is positioned within ORF l which encodes the PA-l bacteriocin
(Figure 3A, and 3B). A frameshift mutation in ORF l was introduced by insertion of a double-stranded
oligonucleotide linker fragment with the sequence 5'-TGCATGGATCCTGATC-3' into this BalI-site.
Plasmid pSRQ220 was therefore partially digested with BalI, generating linear blunt-ended DNA
molecules. This was achieved by incubation of the plasmid DNA in a restriction buffer for a short time
period using only low amounts of the restriction enzyme. The linker fragment was added and allowed
to ligate with the BalI-treated vector DNA.Insertion of the linker fragment disrupts the BalI site, but
introduces a new and unique BamHI site into the plasmid, that was used for identification of the
desired mutant. After transformation of the ligation mixture, plasmid DNA was isolated from the
transformants and screened for the presence of a BamHI site, concomitant with the loss of a BalI site.
In this way plasmid pUR52l7 was identified which carried the desired linker insertion within ORF l.
Introduction of the mutation was confirmed by determination of the nucleotide sequence around the
39/1006
restriction site of the mutant. E. coli cells containing pUR52l7 were assayed for PA-l bacteriocin
activity and found to have lost this property. This result is in good agreement with the previous
obtained deletion data and it again proves that the presence of ORF l is essential for PA-l
activity.Restriction enzyme HindIII has only two restriction sites in pSRQ220, one of which is
positioned in ORF 2, while the other is positioned in ORF 3 (Figure 3B). These sites were therefore
well suited for introduction of mutations in these genes. Plasmid pSRQ220 was partially digested with
HindIII, as described above. To fill in the 3'-restriction ends Klenow enzyme and a mixture of the four
dNTP's (A, T, G, C, 1mM each) were added to the DNA-sample, followed by incubation at 37 DEG C
for 30 minutes. After ligation for 16 hours at 15 DEG C the DNA-mixture was transformed to E. coli
294. Plasmid DNA was isolated from the transformants and screened for the loss of the HindIII
restriction sites by digesting with HindIII. Introduction of the mutations was confirmed by
determination of the nucleotide sequence around the restriction site of each mutant.In this way
plasmid pUR5206 which carried a mutation at the HindIII site in ORF 2, and plasmid pUR5205 which
carried a mutation at the HindIII site in ORF 3 were identified. E. coli cells containing pUR5206 were
assayed and found to express PA-1 bacteriocin activity, whereas E. coli cells containing pUR5205
were negative for PA-1 bacteriocin activity. From these data it can be concluded that, besides the
presence of the PA-1 bacteriocin gene (ORF 1), also the presence of an intact ORF 3 is needed for
PA-1 bacteriocin activity. The function of ORF 2 is not known. Although E. coli cells containing
pUR5206 are able to produce bacteriocin PA-1 activity, it cannot be ruled out that ORF 2 is involved
in the secretion or processing of bacteriocin PA-1. From the nucleotide sequence analysis some
other tentative open reading frames can be deduced (data not shown). Therefore it is possible that
other information is present on the 5.6 kbp EcoRI-SalI fragment which is also needed for PA-1
bacteriocin activity.
It is intended that the foregoing description be only illustrative of the present invention and the
present invention is limited only by the hereinafter appended claims.
Claims:
1. A nucleotide sequence or an RNA sequence corresponding to the nucleotide sequence which can
be isolated from a strain belonging to the genus Pediococcus, and having a nucleotide sequence
containing the genes for both a bacteriocin precursor and at least one protein essential for obtaining
a functional active bacteriocin.
40/1006
2. A nucleotide sequence according to Claim 1, in which the Pediococcus is Pediococcus acidilactici.
3. A nucleotide sequence according to Claim 1, in which the Pediococcus is Pediococcus acidilactici
NRRL-B-18050.
4. A nucleotide according to Claim 1 containing three open reading frames ORF1, ORF2 and ORF3
as given in Figure 4, and derived from the plasmid pSRQ11.
5. A nucleotide sequence according to Claim 1 containing a 5.6 kbp EcoRI-SalI DNA fragment of
plasmid pSRQ11 as given in Figure 4.
6.A nucleotide sequence according to Claim 1 also containing transcriptional and translational
initiation and termination sequences of open reading frames as given in Figure 4.
7. A nucleotide sequence encoding a bacteriocin precursor, said nucleotide sequence being
selected from the group consisting of the nucleotide sequence given as ORFl in Figure 4, and
modifications thereof that encode a protein still having the capability of being converted into an
active bacteriocin.
8. A bacteriocin precursor having an amino acid sequence as given in Figure 4 that corresponds to
ORFl, and modifications thereof that still have the capability of being converted into an active
bacteriocin.
9.A vector, that can be stably maintained in a host microorganism, which vector can be maintained
as a plasmid or can integrate into a chromosome of the host microorganism, comprising a nucleotide
sequence according to Claim l as a recombinant DNA factor in the vector.
10. A vector according to Claim 9, in which the nucleotide sequence contains open reading frames
ORFl and ORF3, and optionally ORF 2 as given in Figure 4.
11. A vector according to Claim 9 containing the 5.6 kbp EcoRI-SalI fragment as given in Figure 4.
12. A vector according to Claim 9, which comprises modified versions of any of ORF1, ORF2 and
ORF3 as given in Figure 4, which either encode the polypeptides encoded by the ORF1, ORF2 and
41/1006
ORF3, or encode modified polypeptides that still have similar activity as those encoded by the ORF1,
ORF2 or ORF3.
13.A vector according to Claim 9 containing a nucleotide sequence containing ORF1, and optionally
ORF2 or ORF3 or both, as given in Figure 4, in which the ORFs are under control of one or more
promoter systems functional in said host microorganism and at least after a most down stream ORF a
terminator sequence is present, wherein the ORF2 can have a promoter system, optionally followed
by a terminator sequence, or ORF1 can form part of an operon containing ORF2 or ORF3, or ORF2
and ORF3 together.
14. A microorganism transformed by introducing the vector of Claim 9, capable of producing a
bacteriocin.
15. A microorganism transformed by introducing the vector of Claim 10, capable of producing a
bacteriocin.
16. A microorganism transformed by introducing the vector of Claim 11, capable of producing a
bacteriocin.
17.A microorganism transformed by introducing the vector of Claim 12, capable of producing a
bacteriocin.
18. A microorganism transformed by introducing the vector of Claim 13, capable of producing a
bacteriocin.
19. A microorganism according to Claim 14 selected from the group consisting of the genera
Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia and yeasts.
20. The vector of Claim 9 that replicates or is stably maintained, in the microorganism selected from
the group consisting of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus,
Pediococcus, Escherichia and yeasts.
21. The vector of Claim 9 which replicates or is stably maintained, in the microorganism which
expresses the bacteriocin.
42/1006
22. A nucleotide sequence encoding a protein essential for obtaining a functional active bacteriocin,
said nucleotide sequence having either the nucleotide sequence given as ORF3 in Figure 4, or a
modification thereof that encodes a protein still having the capability of assisting in production of an
active bacteriocin.
23. A polynucleotide encoding a protein, said polynucleotide having the nucleotide sequence of
ORF2 as given in Figure 4, or a modification thereof that encodes a protein still having the capability
of the protein encoded by ORF2.
24. The nucleotide sequence of Claim l derived by a polymerase chain reaction method.
25. A nucleotide sequence as given in Figure 4 and derivatives thereof which produce a bacteriocin
precursor.
43/1006
5. AU648463 - 22.12.1993
YOGURT PRODUCT CONTAINING BACTERIOCIN FROM PEDIOCOCCUS ADILACTICI
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=AU648463
Inventor(s):
VEDAMUTHU EBENEZER R (US)
Applicant(s):
QUEST INT (NL)
IP Class 4 Digits: A23C
IP Class:
A23C9/123; A23C9/158
E Class: A23C9/123E; A23C9/158B
Application Number:
EP19930106910 (19930428)
Priority Number: US19920898543 (19920615)
Family: AU648463
Equivalent:
AU3830293; CA2098079
Cited Document(s):
EP0360290; US5096718; EP0293547
Abstract:
A METHOD FOR PRODUCING A YOGURT PRODUCT CONTAINING A BACTERIOCIN IS
DESCRIBED. IN THE PREFERRED METHOD, A MILK BASED MEDIUM IS CULTURED WITH
PEDIOCOCCUS ACIDILACTICI TO PRODUCE THE BACTERIOCIN, THE PEDIOCOCCUS
ACIDILACTICI IS THEN HEAT INACTIVATED AND FINALLY A YOGURT CULTURE IS ADDED TO
THE MEDIUM WITH THE BACTERIOCIN AND CULTURED TO PRODUCE THE YOGURT PRODUCT.
THE YOGURT PRODUCT CAN BE DRIED, EITHER BY LYOPHILIZATION OR SPRAY-DRYING OR
OTHER MEANS, PREFERABLY TO A POWDER, FOR USE IN VARIOUS FOODS.Description:
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BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a yogurt product with increased shelf life containing a bacteriocin
derived from a Pediococcus acidilactici. A preferred method for producing the yogurt product is by
fermenting a milk based medium with Pediococcus acidilactici (PA), heating the medium to terminate
growth of the Pediococcus acidilactici and fermenting the first fermentate after the heating with a
yogurt culture to produce the yogurt product which contains bacteriocin active against undesirable
flora.
(2) Prior Art
Yogurt products are well known and are produced by equal parts by cell count of Lactobacillus
bulgaricus and Streptococcus thermophilus and are described, for instance, in U.S. Patent Nos.
3,420,742 to Farr and 4,339,464 to Vedamuthu.
There are numerous foods which use yogurt as an ingredient in fluid or dried form. Some examples
are salad dressings, sauces, baking mixtures, and other food systems requiring a tart, clean, lactic
acid flavoring. There are a few dried yogurt powders available in the marketplace.
All the fluid and dried yogurt products currently available are made out of dairy bases which have
either been acidified by direct addition of edible acids (primarily lactic acid and delta gluconolactone for coagulation and acidity) or fermented using a yogurt starter bacteria. The
fermented products in addition to dairy ingredients, contain dead, injured and live cells of yogurt
bacteria and their metabolic by-products. Those products made by direct acidification will contain
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the acid(s) used for acidification. Dried yogurt powders can contain any neutralizing agent used to
adjust the pH to the desirable range for good or acceptable dehydration of the product.
The usefulness of fluid and rehydrated yogurt products is in providing the specific textural and flavor
attributes desired in the food system. Their addition to food systems is not intended to provide a
barrier against spoilage or pathogenic bacteria which may gain entry into the food system. Acidity, if
present, can provide very limited bacterial inhibition; however, neutralization limits this inhibition. It
would be highly desirable to provide bacterial inhibition in the yogurt product.
Pediococcus acidilactici is known to produce a bacteriocin which has a broad spectrum against
spoilage bacteria. Such bacteriocins are described in U.S. Patent No. 4,929,445 to Vandenbergh et
al; U.S. application Serial No. 07/375,344; and U.S. Patent No. 4,883,673 to Gonzalez et al.
Broad spectrum bacteriocins tend to retard the growth of yogurt cultures. This is true of nisin. Thus
nisin has to be blended into the final product, thereby producing a significant risk of contamination of
the final product.
It would be desirable if the bacteriocin could be introduced into the milk based medium used to
produce the yogurt product. In this manner, the bacteriocin could protect the product as it was
produced. The problem is that any acids or the like in excess generated by the bacteriocin
producing cultures can inhibit the yogurt cultures.
Pediococcus acidilactici is used in meat fermentations. Generally it is not used commercially for milk
fermentations because it grows poorly on milk based media. A Pediococcus which grows well in a
milk based medium is needed.
OBJECTS
It is therefore an object of the present invention to provide a method whereby the bacteriocin is
introduced into the milk based media prior to introducing the yogurt culture. Further, it is an object of
the present invention to provide a fermentation step for providing the bacteriocin in the yogurt
product. Further still, it is an object of the present invention to provide a Pediococcus acidilactici
culture which grows well in a milk based medium. Further still, it is an object of the present invention
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to provide a method for producing a yogurt product which is simple and economical. These and
other objects will become increasingly apparent by reference to the following description.
GENERAL DESCRIPTION
The present invention relates to a method for producing a yogurt product which comprises: providing
a bacteriocin in a milk based medium by fermenting the medium; and providing yogurt flavors in the
fermented medium.
The present invention further relates to a method for producing a yogurt product which comprises:
providing a bacteriocin from Pediococcus acidilactici in a milk based medium; and fermenting the
milk based medium containing the bacteriocin with a yogurt culture to produce the yogurt product
containing the bacteriocin, which provides inhibition of undesirable bacterial growth in the yogurt
product.
In particular, the present invention relates to a method for producing a yogurt product which
comprises: fermenting a milk based medium with a bacteriocin producing Pediococcus acidilactici to
produce a first fermentate containing the bacteriocin; heating the medium to terminate the growth of
the Pediococcus acidilactici; and fermenting the first fermentate after the heating with a yogurt
culture to produce the yogurt product containing the bacteriocin in an amount which provides
inhibition of undesirable bacterial growth in the yogurt product.
Further, the present invention relates to a yogurt product which comprises: a milk based medium
containing yogurt flavors; and a bacteriocin produced by a Pediococcus acidilactici in the medium in
an amount which promotes inhibition of undesirable bacterial growth.
Finally, the present invention relates to a yogurt product which comprises: a milk based medium
containing fermentates from fermentation by a yogurt culture; and a bacteriocin produced by a
Pediococcus acidilactici in an amount which provides inhibition of undesirable bacterial growth. The
product can contain live yogurt cultures.
This invention relates to a method which provides fluid or dried yogurt products containing
bacteriocins from Pediococcus acidilactici as barriers to spoilage and/or pathogenic flora, which are
47/1006
gram-positive. In yogurt, listeria and other low temperature lactobacilli are especially important
undesirable bacteria.
A fluid or dried yogurt product is provided containing a biologically derived Pediococcus acidilactici
bacteriocin active against spoilage and/or pathogenic flora, preferably developed in situ in a milk
based medium by using a single-stage or two-stage fermentation. The microorganisms used in
developing the yogurt product are cultures traditionally used in such fermentations.
The preferred strain is Pediococcus acidilactici NRRL-B-18925. This strain has been deposited under
the Budapest Treaty with the Northern Regional Research Laboratory (NRRL) in Peoria, Illinois. This
strain is particularly effective in producing the bacteriocin in a milk based medium. There are
numerous other Pediococcus acidilactici strains which are available from the American Type Culture
Collection or the NRRL which can be grown in milk to produce strains which effectively grow in milk
based media.
Preferably the milk based medium is fermented between 25 DEG and 45 DEG C to produce the
bacteriocin. The medium includes non-fat milk solids, a carbohydrate source and milk hydrolyzates
which provide proteins and amino acids for growth of the Pediococcus acidilactici (PA). The milk
solids are not actively metabolized by the Pediococcus acidilactici (PA) and are impregnated with
the bacteriocin. The milk solids are preferably present in an amount between 0.1 and no more than
5.0 % by weight in the milk based medium to avoid precipitation on further processing. The
fermented medium after production of the bacteriocin is neutralized to a pH between about 5.2 and
6.3 to provide a suitable medium for the yogurt culture. The medium is heated to about 65 to 80 DEG
C to terminate growth of the Pediococcus acidilactici.
Generally the heat treated Pediococcus acidilactici fermented medium is fortified with nonfat milk
solids in an amount between about 5 and 15 percent depending upon the amount in the milk based
medium used for the Pediococcus acidilactici fermentation. The fortified medium is then fermented
with the yogurt culture to produce the yogurt product. Preferably, this second stage fermentation is at
about 35 DEG to 43 DEG C.
The yogurt product can also be produced by direct acidulation. This method is not preferred.
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The yogurt product is preferably dried to a powder to make it easier to ship for use as an additive to
salad dressings and the like. Preferably spray drying at about 87 DEG to 102 DEG C is used since a
powder is produced directly. The product may also be lyophilized.
The product contains 100 to 10,000 AU per gram of the bacteriocin. The unit "AU" is defined as 5
microliters of the highest dilution of the yogurt product yielding a zone of growth inhibition with a lawn
of a gram positive bacteria (Pediococcus pentosaceus FBB-63) on an agar plate.
SPECIFIC DESCRIPTION
EXAMPLE 1
This example describes a two-stage fermentation using a bacteriocin producing strain and a yogurt
culture. The first-stage fermentation involved the use of a strain of Pediococcus acidilactici which
produced the bacteriocin PA-1 in situ in a milk based medium. Using the same medium, a secondstage fermentation using a yogurt starter culture was performed to obtain yogurt containing the
bacteriocin. The yogurt was lyophilized as such, or after pH was adjusted to 5.5. The yogurt product
had the typical tartness and "green" flavor associated with yogurt along with live yogurt bacteria and
the bacteriocin Most importantly and unexpectedly, the liquid and the dried yogurt products had an
equivalent or only slightly lower titer of the bacteriocin PA-1 found immediately after the initial
fermentation by the Pediococcus acidilactici.Thus, one of the technological problems that had to be
overcome related to the prevention of extreme precipitation and wheying off experienced when the
original milk medium was heat-treated to destroy the pediococci such that the second fermentation
could proceed unhindered.
The first step was to obtain a strain of Pediococcus acidilactici that would produce bacteriocin PA-1
in milk. Strain PAC 10.0 (Pediococcus acidilactici NRRL-B-18925) produced the maximum titer of
bacteriocin (pediocin) in milk fortified with 0.5% yeast extract and 1.0% glucose by volume. Because
yeast extract does not qualify as a milk derived ingredient, milk fortified with various milk-derived
hydrolysates was used. Of the various milk protein hydrolysates tested, EDAMIN K at 1.0% by
volume level gave the maximum titer of bacteriocin when PAC 10.0 was used. EDAMIN K is a
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hydrolysate of whey proteins made by Sheffield Products, Norwich, NY. Strain PAC 10.0 produced
between 800 and 1600 AU/ml of bacteriocin when cultured in reconstituted 11% nonfat milk solids
fortified with 1.0% glucose and 1.0% EDAMIN K, all reconstituted weight per volume.Lower levels of
the whey protein derivative did not give equivalent titer of bacteriocin. Higher levels failed to boost
the bacteriocin titer. Milk fortified at 1.0% by weight per volume each of glucose and EDAMIN K was
selected for use.
When the milk system cultured for pediocin production was heat-treated for the second-stage yogurt
fermentation, excessive precipitation of the acid-denatured milk protein occurred. The system was
thus unsuitable for yogurt fermentation. Adjustment of the pH of the cultured bacteriocin-containing
mix before heat-treatment also failed to alleviate the precipitation problem. It was found that the
bacteriocin titer remained unchanged even when the nonfat solids level in the mix was dropped to
1.0% from 11% (weight per volume) which was unexpected.When strain PAC 10.0 was initially
cultured in a menstruum made up of a mix containing 1.0% each of nonfat milk solids, glucose and
EDAMIN K, and subsequently adjusted to pH 6.3 followed by fortification with 11 to 12% nonfat milk
solids (weight per volume) and was heat-treated at 65 DEG -80 DEG C for 60 minutes with either
intermittent or constant agitation, there was very little or no precipitation and the bacteriocin
remained active. The resulting mix was suitable for yogurt fermentation. The mix, after reducing the
temperature, was inoculated with 1.0% by volume yogurt starter (FARGO TM 404 available from
Quest International, Inc., 1833 57th Street, P. O. Box 3917, Sarasota, Florida 34230) and incubated at
35 DEG C for 16 to 18 hours. At the end of incubation, the yogurt product was cooled, and analyzed
for pH, bacteriocin titer, and organoleptic quality. The major advantage achieved in performing a
two-stage fermentation in the same menstruum was that no dilution of bacteriocin titer occurred. If
the two fermentations were to be separately done and then mixed together to obtain yogurt
containing pediocin, there would be a dilution of bacteriocin titer. Also, the two-stage method allowed
the development of a unique procedure for application in milk fermentations to produce a yogurt
product.
Table 1 shows the bacteriocin level at various stages of the fermentation. The yogurt product had an
acceptable level of bacteriocin for inhibiting undesirable bacteria.
EXAMPLE 2
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In this Example, the bacteriocin as a purified chemical was added to the milk based medium prior to
adding the yogurt culture. The bacteriocin was added to a level of 800 AU/ml. The second stage
fermentation was then performed as in Example 1. The resultant fermentate retained the original titer.
EXAMPLE 3
The application of bacteriocin-containing yogurt powder was tested in a frozen yogurt system. A total
volume of 300 ml mix for frozen yogurt was made. An unfermented base mix was made separately
and combined with sufficient amount of fully fermented yogurt mix such that the final acidity of the
combined mix was 0.3% as lactic acid.The proportions worked out to be a mixture of 255 parts of the
base mix and 45 parts of fully fermented yogurt.
Columns=2
Title: Composition of the base mix:
40% Cream25 parts
Whole Milk201 parts
Non-fat dry Milk12 parts
Sugar45 parts
80% Corn Sweetener24 parts
Stabilizer-Emulsifier2 parts
Columns=2
Title: Composition of yogurt mix:
Skim milk 0.2% milk fat97 parts
Non-fat dry milk3 parts
The two mixes were separately heat treated at 100 DEG C for 5 minutes. The yogurt mix was
cultured with a yogurt starter at 35 DEG C overnight. The base mix was stored at 5 DEG C. The
following morning, the final mix was made by combining the required proportions of the base mix
(255 parts) and the yogurt (45 parts).After uniform mixing of the two components, the mix was
divided into 100 ml portions into 3 separate sterile screw-cap Erlenmeyer flasks. They were labeled 1,
2 and 3. To flask 1, two grams of non-fat milk powder was added and mixed in. To flask 2, two grams
of previously made bacteriocin-containing yogurt powder of Example 1 was added and mixed in. The
yogurt powder contained 4000 AU/gm. Hence, the contents of flask 2, had an equivalent of 80 AU/ml
of the bacteriocin. To flask 3, two grams of non-fat milk powder was added. The three flasks were
51/1006
placed in a boiling water-bath for 10 minutes, and cooled in a bath of tap water. When the flasks
cooled down to room temperature, flask 3 was isolated from the other two.To flasks 1 and 2, a
suitable dilution (1 x 10) of an overnight culture of Listeria monocytogenes LM04 was added to
provide approximately 1 x 10 cells of the pathogen per ml of the mix. The three flasks were emptied
into three separate previously labeled wide-mouth plastic containers. The containers were closed
with suitable lids and placed in a -20 DEG C freezer.
After 4 days and 15 days, each container was removed and a sufficient portion of the frozen material
chipped out into three separate sterile snap-cap plastic tubes. The containers were returned to the
freezer after sampling. The samples were thawed in a 37 DEG C water-bath and immediately after
thawing were plated on "OXOID" Listeria Selective Agar (Unipath Ltd., Basingstoke, Hampshire,
England). On this agar, Listeria form dark grey or black colonies with dark grey or black zones. Other
flora are inhibited on this medium. Suitable sample portions or dilutions were spread-plated to get
accurate counts.
Addition of yogurt powder containing the bacteriocin so as to give 80 AU/ml of the frozen yogurt mix
reduced the population of added listeria from 1.2 x 10/ml to 1.0/ml within 4 days of freezing. After 15
days, none could be found in the mix containing the yogurt powder. The inoculated control
maintained the numbers of added listeria throughout the storage.
It is intended that the foregoing specification be only illustrative of the present invention and that the
present invention be limited to the hereinafter appended claims. Claims:
1. A method for producing a yogurt product which comprises:
(a) providing a bacteriocin from Pediococcus acidilactici in a milk based medium; and
(b) fermenting the milk based medium containing the bacteriocin with a yogurt culture to produce
the yogurt product containing the bacteriocin which provides inhibition of undesirable bacterial
growth in the yogurt product.
2.A method for producing a yogurt product which comprises:
(a) fermenting a milk based medium with a bacteriocin producing Pediococcus acidilactici to
produce a first fermentate containing the bacteriocin;
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(b) heating the medium to terminate the growth of the Pediococcus acidilactici; and
(c) fermenting the first fermentate after the heating with a yogurt culture to produce the yogurt
product containing the bacteriocin in an amount which provides inhibition of undesirable bacterial
growth in the yogurt product.
3. The method of Claim 2 wherein the bacterium is Pediococcus acidilactici.
4. The method of Claim 2 wherein the bacterium is Pediococcus acidilactici deposited as NRRL-B18925.
5. The method of Claim 2 wherein the product is dried to a powder.
6.The method of Claim 2 wherein the fermentation in step (a) is at a temperature between about 25
DEG and 45 DEG C.
7. The method of Claim 2 wherein the fermentation in step (b) is at a temperature between about 35
DEG and 43 DEG C.
8. The method of Claim 2 wherein the bacterium is Pediococcus acidilactici, wherein the fermentation
in step (a) is at a temperature of 25 DEG to 45 DEG C and in step (b) at 35 DEG to 43 DEG C.
9. The method of Claim 8 wherein the Pediococcus acidilactici is deposited as NRRL-B-18925.
10. The method of Claim 9 wherein the drying is at a temperature of up to about 87 DEG to 102 DEG
C.
11. The method of Claim 10 wherein the drying is by spray drying.
12. The method of Claim 10 wherein the drying is by lyophilizing.
13.The method of Claim 2 wherein the milk based medium contains between about 0.1 to 5 percent
milk in step (a) and then is fortified with milk powder after step (b) in order to provide enough milk
solids for producing the yogurt culture.
14. The method of Claim 13 wherein the milk is a non-fat dry milk powder.
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15. A yogurt product which comprises:
(a) a milk based medium containing yogurt flavors; and
(b) a bacteriocin produced by a Pediococcus acidilactici in the medium in an amount which
promotes inhibition of undesirable bacterial growth.
16. A yogurt product which comprises:
(a) a milk based medium containing fermentates from fermentation by a yogurt culture; and
(b) a bacteriocin produced by a Pediococcus acidilactici in an amount which provides inhibition of
undesirable bacterial growth.
17. The product of Claim 16 wherein the bacteriocin is produced by fermenting the milk based
medium with the Pediococcus acidilactici.
18. The product of Claim 16 wherein the Pediococcus acidilactici is deposited as NRRL-B-18925.
19. The product of Claim 16 in dry form.
20. The product of Claim 19 in powdered form.
21. The product of Claim 17 wherein the bacteriocin is present in an amount between about 100 and
10,000 AU per gram of the yogurt product.
22. The product of Claim 13 wherein the yogurt acidity is imparted by lactic acid and deltagluconolactone.
23. A method for producing a yogurt product which comprises:
(a) providing a bacteriocin in a milk based medium by fermenting the medium; and
(b) providing yogurt flavors in the fermented medium.
24. The method of Claim 23 wherein the yogurt acidity is imparted by lactic acid and deltagluconolactone.
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6. CN1513981 - 21.07.2004
RICE WINE LACTOBACILLUS STRAIN FOR PRODUCING BACTERIOCIN AND ITS USE METHOD
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=CN1513981
Inventor(s):
LI ZONGJUN (CN); JIANG HANHU (CN); DONG MINGSHENG (CN)
Applicant(s):
NANJING UNIV OF AGRICULTURE (CN)
IP Class 4 Digits: C12N; A23L
IP Class:
C12N1/20; A23L1/31; A23L1/314
Application Number:
CN20030132323 (20030814)
Priority Number: CN20030132323 (20030814)
Family: CN1513981
55/1006
7. EP0424484 - 25.12.1990
NOVEL BACTERIOCIN COMPOSITIONS FOR USE AS ENHANCED BROAD RANGE BACTERICIDES
AND METHODS OF PREVENTING AND TREATING MICROBIAL INFECTION
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0424484
Inventor(s):
BLACKBURN PETER (US); GUSIK SARA-ANN (US); POLAK JUNE (US); RUBINO
STEPHEN D (US)
Applicant(s):
NEW YORK HEALTH RES INST (US)
IP Class 4 Digits: A61K
IP Class:
A61K37/02; A61K37/54
E Class: A01N63/02+M; A61K38/16B
Application Number:
US19890317627 (19890301)
Priority Number: US19890317627 (19890301)
Family: WO9009739
Equivalent:
AU5285090; AU618714; CA2028140; CZ279273; DE69011460D; DE69011460T;
DK424484T; FI103861B; HU217574; HU55607; IE64710; IE900721L; IL93527; JP2984744B2;
JP3504864T; KR145738; NO304721B; NO904729; NZ232700; PL163940B; PL284095; RU2048151;
WO9009739; ZA9001499
Abstract:
BROAD RANGE BACTERIOCIN COMPOSITIONS ARE PROVIDED. THE COMPOSITIONS CAN BE
DISSOLVED OR SUSPENDED IN A SUITABLE SOLVENT OR MATRIX AND ARE MORE ACTIVE
TOWARDS A BROADER RANGE OF BACTERIA THAN ARE ANY OF THE COMPONENT PARTS. THE
DISSOLVED OR SUSPENDED COMPOSITIONS CONSTITUTE ENHANCED BROAD RANGE
BACTERICIDES. THE COMPOSITIONS INCLUDE LYSOSTAPHIN AND A LANTHIONINE
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CONTAINING PEPTIDE BACTERIOCIN; LYSOSTAPHIN, A LANTHIONINE CONTAINING PEPTIDE
BACTERIOCIN AND A CHELATING AGENT; AND LYSOSTAPHIN, A LANTHIONINE CONTAINING
PEPTIDE, A CHELATING AGENT AND A SURFACTANT. EACH COMPONENT IS PRESENT IN THE
ENHANCED BROAD RANGE BACTERICIDE IN SUFFICIENT AMOUNT SUCH THAT THE
BACTERICIDE IS MORE EFFECTIVE AGAINST STAPHYLOCOCCI THAN IS LYSOSTAPHIN ALONE
AND IS MORE EFFECTIVE AT TREATING AND PREVENTING A BROAD RANGE OF MICROBIAL
INFECTIONS. METHODS OF TREATING BACTERIAL INFECTIONS USING SAID COMPOSITIONS
AND BACTERICIDES ARE PROVIDED.Description:
BACKGROUND OF THE INVENTION
This application relates to bacteriocin compositions for use as enhanced broad range bactericides
and methods of preventing and treating microbial infection.
Bacteriocins such as lysostaphin and nisin are proteins produced by bacteria that inhibit the growth
of and sometimes kill bacteria closely related to the species of their origin. Lysostaphin is a
bacteriocin that lyses and kills practically all known species of Staphylococcus, but is inactive
against bacteria of other genera. Lysostaphin, isolated from culture filtrates of Staphylococcus
simulans (NRRL B-2628) grown according to published references, is an endopeptidase which
cleaves the polyglycine cross-links of the peptidoglycan found in the cell walls of Staphylococcus.
Cultures of S. simulans grown under conditions which induce the production of lysostaphin are
immune to the bacteriocin while the same cultures grown under conditions whereby lysostaphin is
not produced are sensitive to the bacteriocin.
Lysostaphin is a naturally occurring bacteriocin secreted by a single known strain of S. simulans
originally isolated and named Staphylococcus staphylolyticus by Schindler and Schuhardt. The
production of lysostaphin by S. staphylolyticus has been described previously in U.S. Pat. No.
3,278,378 issued Oct. 11, 1966 and in Proceedings of the National Academy of Sciences, 51:414421 (1964). The single organism S. staphylolyticus (NRRL B-2628) which produced lysostaphin was
recently identified as a biovar of S. simulans by Sloan et al., Int. J. System. Bacteriol., 32:170-174
(1982). Since the name S. staphylolyticus is not on the Approved List of Bacterial Names, the
organism producing lysostaphin has been redesignated as S. simulans.
Previously it was shown that the action of lysostaphin can be potentiated by penicillin and other
antibiotics. See copending U.S. application No. 188,183 to Blackburn et al. filed Apr. 28, 1988.
57/1006
Nisan, although sometimes referred to as a peptide antibiotic is more properly referred to as a
bacteriocin. Nisin is produced in nature by various strains of the bacterium Streptococcus lactis. It is
a food preservative used to inhibit the outgrowth of spores of certain species of Gram positive bacilli,
including those arising from strains of Clostridium known to be responsible for Botulism food
poisoning. A summary of nisin's properties appears in Hurst, Advances in Applied Microbiology,
27:85-123 (1981). The publication describes what is generally known about nisin. Nisin, produced by
Streptococcus lactis, is commercially available as an impure preparation, Nisaplin.TM., (Aplin &
Barret Ltd., Dorset, England)
Nisin belongs to the class of peptides containing lanthionine. Also included in that class are subtilin,
epidermin, cinnamycin, duramycin, ancovenin, and Pep 5. These bacteriocin peptides are each
produced by different microorganisms. However, subtilin obtained from certain cultures of B. subtilis,
and epidermin obtained from certain cultures of Staphylococcus epidermidis, have molecular
structures very similar to that of nisin, Hurst, pp. 85-86; and Schnell et al. Nature 333:276-278.
Structurally similar, lanthionine containing peptide bacteriocins are believed to be effective in place
of nisin in the present invention.
Nisin has been applied effectively as a preservative in processed cheese, and dairy products. The
use of nisin in processed cheese products has been the subject of recent patents. See U.S. Pat. Nos.
4,584,199 and 4,597,972. The use of nisin to inhibit the outgrowth of certain Gram positive bacterial
spores has been well documented. See Taylor, U.S. Pat. No. 5,584,199, and Taylor, U.S. Pat. No.
4,597,972, Tsai and Sandin, "Conjugal Transfer of Nisin Plasmid Genes from Streptococcus lactis
7962 to Leuconostoc dextranicum 181", Applied and Environmental Microbiology, p. 352 (1987); "A
Natural Preservative", Food Engineering International, pp. 37-38 (1987); "Focus on Nisin", Food
Manufacture, p. 63 (1987). Nisin is sometimes found naturally-occurring in low concentration in milk
and cheese, and is believed to be completely non-toxic and non-allergenic to humans. Nisin has
recently been recognized as safe by the FDA as a direct food ingredient in pasteurized cheese
spread, pasteurized processed cheese spread and pasteurized or pasteurized processed cheese
spread with fruits, vegetables, or meats. As nisin is proteinaceous, any residues in ingested foods
are quickly degraded by digestive enzymes.
The general acceptance of nisin as a food preservative has been limited by the teaching that, as a
bacteriocin, the activity of nisin was restricted to include only those Gram positive bacteria closely
related to the bacterial species of its origin. Furthermore, nisin has not previously been shown to
have bactericidal activity towards Gram negative bacteria. Since food contamination and spoilage
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result from a diversity of Gram positive and Gram negative bacteria, it is not surprising, therefore, that
nisin has received only limited acceptance as a food preservative. Moreover, because of the
heretofore restricted activity of nisin as a bacteriocin, its uses as such outside of the food area have
not been indicated.
It has recently been demonstrated that a composition comprising nisin and non-bactericidal agents
such as chelating agents and surfactants has bactericidal activity towards a wide range of Gram
negative bacterial species and enhanced activity towards a broad range of Gram positive bacterial
species. For instance Gram negative bacteria shown to be sensitive to the enhanced bactericide are
Salmonella typhimirium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Bacterioides gingivalis and Actinobacillus actinomycetescomitans. Gram positive bacteria shown to
be sensitive to the enhanced bactericides are Staphylococcus aureus, Streptococcus mutans,
Listeria monocytogenes, Streptococcus agalactiae and coryneform bacteria. See copending
Blackburn et al., U.S. patent application entitled Nisin Compositions For Use as Enhanced, Broad
Range Bactericides which is a continuation-in-part of U.S. patent application Ser. No. 209,861 filed
June 22, 1988 which is hereby incorporated herein by reference.
SUMMARY OF THE INVENTION
It has now been found that the activity of bacteriocins such as lysostaphin and nisin can be
surprisingly enhanced and the overall range and speed of their activity can be increased by
combining the two bacteriocins. The properties of the novel bacteriocin compositions containing
lysostaphin and nisin should also be further enhanced by the addition of chelating agents and/or
surfactants which enhance and broaden the range of nisin and lysostaphin activity.
All the novel bacteriocin compositions of this invention comprise lysostaphin and nisin (herein
"composition"). The bacteriocin composition becomes an enhanced broad range bactericide
(hereinafter "bactericide") on being dissolved or suspended in a suitable carrier for example a
solvent or suitable liquid, solid, or colloidal matrix. The novel bactericides contain lysostaphin in an
amount sufficient to be effective as a bactericide towards Staphylococcus, and nisin is present in an
amount sufficient to enhance the bactericidal effect of lysostaphin toward Staphylococci. Other
compositions comprise lysostaphin, nisin, and a chelating agent and may also contain a surfactant.
This composition in a carrier yields a novel bactericide wherein the lysostaphin and nisin are present
in the same concentration range as in the lysostaphin/nisin composition and the chelating agent is
present in an amount sufficient to enhance the bactericidal effect of nisin against both Gram positive
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and Gram negative bacteria. A still further composition comprises lysostaphin, nisin, and a surfactant.
This composition in a carrier yields a novel bactericide wherein the surfactant is present in an amount
sufficient to enhance the bactericidal effect of nisin and lysostaphin against Gram positive bacteria.
The compositions can be used directly or in carriers for treatment and prevention of bacterial
contamination and infection by dissolving the composition in a suitable solvent or suspending in a
suitable matrix and applying it to an affected area or by adding it to another composition to combat
and prevent infection.
Most chemical disinfectants are too corrosive or otherwise too toxic to be used in foods and many
medical applications, the majority of antibiotics act too slowly to be useful as disinfectants, and are
not permitted in foods because of the risk of acquired antibiotic resistance that would attend such
use. The novel bactericides are non-corrosive, non-toxic, suitable for use in foods and on open
wounds, effective against antibiotic resistant bacteria and act rapidly against dividing or non-dividing
bacteria, so as to be useful also as a disinfectant.
The compositions or the bactericides can be incorporated into ointments or coatings for the
treatment of infections, wound dressings or surgical implants and other medications such as nasal
instillations, oral rinses, disinfectant scrubs, wipes or lotions. The bactericides can be used for
cleaning medical instruments and the like and in circumstances where environmental disinfection is
desired but where chemical germicidals are precluded because of the risks of corrosive or otherwise
toxic residues. The broad range bactericides are particularly suited for food related uses such as
treatment of meat, especially poultry, eggs, cheese and fish or food packaging and handling
equipment, and for the control and prevention of contamination of raw ingredients, processed foods
and beverages by bacterial pathogens and other microbial spoilage organisms.
Unlike the activity of most broad spectrum germicidals which is compromised by the presence of
complex organic matter, the bacteriocin compositions and bactericides of the present invention are
effective in the presence of organic matter, such as milk or serum.
DETAILED DESCRIPTION OF INVENTION
The compositions of the claimed invention comprise lysostaphin and nisin, lysostaphin, nisin and a
chelating agent, or lysostaphin, nisin, a chelating agent and a surfactant. To provide enhanced
broad range bactericides, the compositions are dissolved in a suitable solvent or suspended in a
60/1006
suitable matrix. Compositions comprising lysostaphin, nisin, a chelating agent and/or a surfactant,
dissolved in a suitable carrier for example an aqueous solvent or buffer or suspended in a suitable
matrix, are believed to have broad range rapid bactericidal activity against both Gram positive and
Gram negative bacteria.
Preferably the composition is dissolved in a liquid carrier or suspended in a liquid, colloidal or
polymeric matrix such that lysostaphin is present in the bactericide in the range of 0.1 to
100 .mu.g/ml and is enhanced by the presence of the bacteriocin nisin in the range of 0.1 to
300 .mu.g/ml and the resulting bactericide is significantly more bactericidal towards Staphylococcus
than lysostaphin alone. The total bactericidal activity of such a novel bactericide is believed to be
further potentiated and effective against a broader range of both Gram negative and Gram positive
bacterial species when the nisin in the bactericide is enhanced by a chelating agent as taught by
copending application to Blackburn et al. entitled Nisin Compositions For Use as Enhanced, Broad
Range Bactericides. The combination of lysostaphin, nisin and a chelating agent should also attain
further broad range bactericidal activity by the addition of a surfactant as also taught by the
Blackburn et al. application.
For example nisin is activated and enhanced toward a broad range of Gram positive bacteria by a
chelating agent such as EDTA in the range of 0.1 to 20.0 mM. In the presence of EDTA, nisin has
bactericidal activity against Gram negative organisms and its activity against Gram positive bacteria
is enhanced and active over a wider pH range and towards a broader range of Gram positive
bacteria. In addition the presence of a surfactant in the range of 0.01% to 1.0% in the bactericide
improves the effectiveness of the nisin towards Gram positive bacteria. Suitable nonionic surfactants
include, but are not limited to polyoxyalkylphenols (e.g. Triton X-100), polyoxyalkylsorbitans (e.g.
Tweens), and glycerides (e.g. monolaurin and dioleates). Suitable ionic surfactants include, but are
not limited to emulsifiers, fatty acids, quaternary compounds and anionic surfactants (e.g. sodium
dodecyl sulphate) and amphoteric surfactants, for example, cocamidopropyl betaine.
Suitable carriers for the bactericides of the present invention include but are not limited to generally
recognized aqueous buffers. Suitable matrices for suspension of the novel compositions of the
present invention include but are not limited to organic solvents, colloidal suspension and polymers
compatable with the bactericide.
Lysostaphin used in the invention can be produced by fermentation techniques wherein S. simulans
is grown in liquid culture. Such fermentation techniques are described in U.S. Pat. No. 3,278,378 and
61/1006
in Proceedings of the National Academy of Sciences, 51:414-421 (1964). Various improvements in
the production of lysostaphin by fermentation techniques have also been made as documented in
U.S. Pat. Nos. 3,398,056, and 3,594,284. The latter two references disclose improvements in culture
medium and inoculation techniques whereby the production of lysostaphin by fermentation can be
accelerated and improved.
In addition, lysostaphin can be produced by recombinant microorganisms, including strains of
Escherichia coli, Bacillus subtilus, and Bacillus sphaericus. A method for obtaining lysostaphin from
microorganisms transformed by recombinant plasmids encoding the gene for lysostaphin is fully
disclosed in U.S. patent application No. 034,464, which is a continuation-in-part of U.S. patent
application No. 852,407. Both applications are incorporated herein by reference. Preferably, the
lysostaphin is obtained from B. sphaericus strain 00, containing a recombinant plasmid which directs
the synthesis of lysostaphin. This provides for production of high levels of lysostaphin substantially
free from staphylococcal immunogenic contaminants and facile lysostaphin purification since the
lysostaphin accumulates directly in the growth medium. B. sphaericus transformants containing
plasmids pBC16-lL or pROJ6649-IL have been found to be particularly suited for this purpose,
although other strains are also useful as a source of lysostaphin. These plasmids are fully described
in the above-mentioned copending applications.
Produced by S. simulans during exponential growth, lysostaphin is first secreted as an inactive
precursor that is processed extracellularly to the mature active bacteriocin by a protease produced
in the stationary growth phase. In contrast to the natural production of lysostaphin, lysostaphin
produced by a recombinant strain of B. sphaericus as described in U.S. patent application No.
034,464, accumulates extracellularly as the mature active protein during the exponential growth
phase.
Nisin can be obtained commercially as an impure preparation, Nisaplin.TM. from Aplin & Barrett, Ltd.,
Dorset, England, and can be obtained by isolating naturally-occurring nisin from cultures of
Streptococcus lactis and then concentrating the nisin by known methods. There are also reported
methods for producing nisin using altered strains of Streptococcus. See Gonzalez, et al. U.S. Pat. No.
4,716,115 issued Dec. 29, 1987. It should also be possible to produce nisin by recombinant DNA.
Nisin is a member of the family of lanthionine containing bacteriocins. It is believed that, due to the
structural similarity, other lanthionine containing bacteriocins will be equally as effective as nisin in
combination with lysostaphin.
62/1006
The following non-limiting examples will further illustrate the invention and demonstrate the
effectiveness of the new enhanced broad range bactericides. It is believed that since the degree and
range of nisin activity are also enhanced by chelating agents, the compositions of lysostaphin, nisin
and a chelating agent will also yield novel bactericides with enhanced bactericidal activity compared
to compositions of lysostaphin and nisin alone.
All tests in the following examples were performed at 37 DEG C. The efficacy of the enhanced broad
range bactericides was determined by assaying bactericidal activity as measured by the percent
bacterial survival after treatment with the bactericide. Generally, after incubation of a 10@7 cell per
ml suspension of target species with the novel bactericide for specified lengths of time, bacteria
were collected by centrifugation for 2 minutes. The bacterial pellet was washed free of the
bactericide with a rescue buffer, termed herein Phage buffer (50 mM Tris-HCl buffer pH 7.8, 1 mM
MgSO4, 4 mM CaCl2, 0.1M NaCl, and 0.1% gelatin), resuspended and serially diluted into Phage
buffer, and 100 .mu.l of the suspended bacteria were spread on nutrient agar plates. Surviving
bacteria were determined by scoring colony forming units (CFU) after incubation for 24-48 hours at
37 DEG C. An effective bactericide according to this invention is one which allows less than 0.1% of
the initial viable count of the bacteria to survive.
EXAMPLE 1
Lysostaphin and Nisin
Staphylococcus aureus cells were suspended and incubated in milk at 37 DEG C. for 2 hours with
various concentrations of lysostaphin, nisin, or a combination of lysostaphin and nisin in the milk. The
bactericidal efficacy of the bactericides was estimated by determining the percent survival of
bacteria as described above. The results of such an experiment are given in Table 1.
TABLE 1
______________________________________
Bactericidal Activity of Lysostaphin, Nisin,
and Their Combinations Towards Stachylococcus aureus
Lysostaphin
Nisin .mu.g/ml
.mu.g/ml 0 0.2 0.5 1.0 2.0 4.0
______________________________________
63/1006
% survival 2 hr@a
0 100 45 33 9 2.5 0.5
0.5
0.1 43 0.7 2.6 0.15
0.04 0.004
5.6
<10@-3
1.0 <10@-3
<10@-4
-- -- <10@-4
-______________________________________
@a Initial viable counts: 5 .times. 10@7 cfu/ml.
Nisin alone in milk has little practical bactericidal activity towards Staphylococci. Lysostaphin alone
in milk is bactericidal towards S. aureus and can produce more than a five log reduction in viable
cells at a concentration of 1.0 .mu.g/ml. Lysostaphin, when combined with nisin in the milk, provides
a composition which is a novel bactericide whereby the bactericidal activity of the bactericide is
significantly and surprisingly superior to that of either bacteriocin alone and is more active than their
anticipated additive effects. This is best illustrated at a limiting lysostaphin concentration
(0.1 .mu.g/ml) shown in Table 1. Thus, when the application of lysostaphin is limited by its available
activity, a bacteriocin composition comprising lysostaphin with nisin in a suitable carrier such as milk
in this example can be expected to provide an enhanced broad range bactericide.
EXAMPLE 2
Lysostaphin+Nisin+EDTA+Surfactant
The data in Table 2 illustrate the novel bactericide potency of a composition comprising lysostaphin,
nisin, EDTA, and monoglyceride surfactant towards S. aureus and S. algalactiae in milk, a complex
food medium. Previously, it was shown that low concentrations of EDTA potentiate the activity of nisin
while higher concentrations of EDTA inhibited the activity of nisin, see the copending application to
Blackburn, et al. In milk, higher concentrations of EDTA are less inhibitory to the bactericidal activity
of the bacteriocin composition.
TABLE 2
______________________________________
64/1006
Bactericidal Activity of Lysostaphin, Nisin,
EDTA, and Monoglyceride in milk at 37 DEG C. towards
Staphylococcus aureus and Streptococcus agalactiae
0.23 L 0.1 L
1.0 N 1.0 N
Species 0.1% ML 1.0% ML Control@c
______________________________________
% Survival 2 hr
S. agalactiae@b
0.0001@E
0.0007@E
100
(McDonald)
S. aureus@a
0.004@E
0.002@E
100
(Newbould)
______________________________________
N = Nisin .mu.g/ml; L = Lysostaphin .mu.g/ml; ML = monolaurin
E = contained 50 mM EDTA
a = S. aureus initial viable count: 8.1 .times. 10@7 cells/ml
b = S. agalactiae initial viable count: 6.6 .times. 10@7 cells/ml
c = no bacteriocin or monoglyceride
Claims:
We claim:
1. A composition comprising lysostaphin and a lanthionine containing bacteriocin.
2. The composition as defined in claim 1 wherein the lanthionine containing bacteriocin is selected
from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5.
3. A composition as defined in claim 1 additionally comprising a chelating agent.
4. A composition as defined in claim 3 comprising a surfactant.
65/1006
5. The composition as defined in claim 3 wherein the chelating agent is selected from the group
consisting of alkyldiamine tetraacetates, CaEDTA, Na2 CaEDTA, EGTA and citrate.
6. The composition as defined in claim 5 wherein the alkyldiamine tetraacetate is EDTA.
7. The composition as defined in claim 4 or 23 wherein the surfactant is selected from the group
consisting of Tritons, Tweens, glycerides, emulsifiers, fatty acids, quaternary compounds,
amphoteric and anionic surfactants.
8. An enhanced broad range bactericide comprising a carrier, lysostaphin and a lanthionine
containing bacteriocin.
9. The enhanced broad range bactericide as defined in claim 8 wherein the lanthionine containing
bacteriocin is selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin,
ancovenin and Pep 5.
10. An enhanced broad range bactericide as defined in claim 8 comprising a chelating agent.
11. An enhanced broad range bactericide as defined in claim 8 comprising a surfactant.
12. The enhanced broad range bactericide as defined in claim 8 wherein the lysostaphin and the
lanthionine containing bacteriocin are present in sufficient quantities such that the bactericide has
enhanced activity against staphylococci and Gram positive bacteria.
13. The enhanced broad range bactericide as defined in claim 10 wherein the lysostaphin, the
lanthionine containing bacteriocin and the chelating agent are present in quantities such that the
bactericide has enhanced activity against staphylococci and against at least one of the bacteria from
the group consisting of Gram negative and Gram positive bacteria.
14. The enhanced broad range bactericide as defined in claim 11 or 24 wherein the surfactant is
present in an amount sufficient such that bactericide has enhanced activity against staphylococci
and against at least one of the group consisting of Gram negative and Gram positive bacteria.
66/1006
15. The enhanced broad range bactericide as defined in claim 10 wherein the chelating agent is
selected from the group consisting of alkyldiamine tetraacetates, EGTA and citrate.
16. The enhanced broad range bactericide as defined in claim 15 wherein the alkyldiamine
tetraacetate is EDTA.
17. The enhanced broad range bactericide as defined in claim 11 wherein the surfactant is selected
from the group consisting of Tritons, Tweens, glycerides, fatty acids, emulsifiers, quaternary
compounds, amphoteric and anionic surfactants.
18. The enhanced broad range bactericide as defined in claim 13 wherein the Gram negative
bacterial target is selected from the group consisting of Salmonella typhimurium, Escherichia coli,
Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides gingivalis and Actinobacillus
actinomycetescomitans.
19. The enhanced broad range bactericide as defined in claim 13 wherein the Gram positive
bacterial target is selected from the group consisting of spore forming bacilli, Staphylococcus aureus,
Streptococcus mutans, Listeria monocytogenes, Streptococcus agalactiae, and cornyeform bacteria.
20. The enhanced broad range bactericide as defined in claim 9 wherein the effective concentration
of lysostaphin is between about 0.1 to 100 .mu.g/ml and the concentration of the nisin is between
about 0.1 to 300 .mu.g/ml.
21. The enhanced broad range bactericide as defined in claim 10 wherein the concentration of
lysostaphin is between about 0.1 to 100 .mu.g/ml, the concentration of the lanthionine containing
bacteriocin is between about 0.1 to 300 .mu.g/ml and the concentration of chelating agent is
between about 0.1 mM and 20 mM.
22. The enhanced broad range bactericide as defined in claim 11 wherein the concentration of
surfactant is between about 0.01% and 1.0% of the final volume.
23. A composition as defined in claim 1 comprising a surfactant.
24. An enhanced broad range bactericide as defined in claim 10 comprising a surfactant.
67/1006
8. EP0545911 - 09.06.1993
LANTHIONINE-CONTAINING BACTERIOCIN COMPOSITIONS FOR USE AS BACTERICIDES
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0545911
Inventor(s):
BLACKBURN PETER (US); GUSIK SARA-ANN (US); POLAK JUNE (US); RUBINO
STEPHEN D (US)
Applicant(s):
APPLIED MICROBIOLOGY INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K37/02
E Class: A23L3/3463; A23C19/11; A23L3/3463A; A01N37/46+M; A01N63/02+M; A61K7/16D13;
A61K8/64; A61Q11/00; A23C9/158B; A61K8/37C; A61K8/41L; A61K8/44
Application Number:
EP19930200152 (19890616)
Priority Number: CS19890006897 (19891206); EP19890907595 (19890616); US19880209861
(19880622); US19890317626 (19890301)
Family: EP0545911
Abstract:
COMPOSITIONS COMPRISING A LANTHIONINE-CONTAINING BACTERIOCIN SUCH AS NISIN
AND A SURFACTANT, ESPECIALLY A NONIONIC ONE, ARE USEFUL AS BACTERICIDES,
ESPECIALLY AGAINST GRAM POSITIVE BACTERIA. THEY HAVE BOTH FOOD AND MEDICINAL
APPLICATIONS. TO AVOID OVERLAP OF CLAIM WITH THE PARENT APPLICATION, CHELATING
AGENTS ARE EXCLUDED FROM THE CLAIMED COMPOSITIONS AND USES.Description:
68/1006
Nisin is a polypeptide with antimicrobial properties which is produced in nature by various strains of
the bacterium Streptococcus lactis. It is a known food preservative which inhibits the outgrowth of
spores of certain species of Gram positive Bacilli.
Although sometimes mistakenly and imprecisely referred to as an antibiotic, nisin is more correctly
classified as a bacteriocin, i.e., a proteinaceous substance produced by bacteria and which has
antibacterial activity only towards species closely related to the species of its origin. Nisin is a
naturally-occurring preservative found in low concentration in milk and cheese, and is believed to be
completely non-toxic and non-allergenic to humans.
Nisin has recently been recognized as safe by the FDA as a direct food ingredient in pasteurized
cheese spread, pasteurized processed cheese spread, and pasteurized or pasteurized processed
cheese spread with fruits, vegetables, or meats. Furthermore, since it is a polypeptide, any nisin
residues remaining in foods are quickly digested.
A summary of nisin's properties appears in Hurst, A., Advances in Applied Microbiology 27:85-123
(1981). This publication describes what is generally known about nisin. Nisin, produced by
Streptococcus lactis, is available commercially as an impure preparation, Nisaplin TM , from Aplin &
Barrett Ltd., Dorset, England and can be obtained by isolating naturally-occurring nisin from cultures
of Streptococcus lactis and then concentrating the nisin according to known methods. There are also
reported methods for producing nisin using altered strains of Streptococcus. See Gonzalez et al., US
Pat. No. 4,716,115, issued December 29, 1987. It should also be possible to produce nisin by
recombinant DNA technology.
Nisin has been applied effectively as a preservative in dairy products, such as processed cheese,
cream and milk. The use of nisin in processed cheese products has been the subject of recent
patents. See US Pat. Nos. 4,584,199 and 4,597,972. The use of nisin to inhibit the growth of certain
Gram positive bacteria has been well documented. However, its complete success and acceptance
as a food preservative has heretofore been hampered by the belief that nisin was ineffective against
Gram negative and many Gram positive bacteria. Gram negative bacteria are almost always present
in conjunction with Gram positive bacteria and are a major source of food spoilage and
contamination. See Taylor, US Pat. No. 5,584,199, issued April 22, 1986 and Taylor, US Pat.No.
4,597,972, issued July 1, 1986; Tsai and Sandine, "Conjugal Transfer of Nisin Plasmid Genes from
Streptococcus Lactis 7962 to Leuconostoc Dextranicum 181, Applied and Environmental
69/1006
Microbiology, Feb. 1987, p. 352; "A Natural Preservative", Food Engineering Int'l, May 1987, pp. 3738; "Focus on Nisin", Food Manufacture, March 1987, p. 63.
It has now been found that in the presence of surfactant alone, nisin has enhanced activity against
Gram positive bacteria.
The parent application relates to compositions of enhanced bactericidal activity comprising a
lanthionine-containing bacteriocin and a chelating agent. These compositions can also contain a
surfactant.
It has now been found that in the presence of surfactant alone, nisin has enhanced activity against
Gram positive bacteria.
This invention provides bacteriocin compositions of nisin or other, lanthionine containing bacteriocins,
in combination with surfactants. The invention further provides the compositions dissolved or
suspended in a suitable carrier to yield enhanced broad range bactericides.
In the present invention, surfactants, valuable as cleansing agents, suitable for combination with nisin
include, but are not limited to, the nonionic surfactants Tweens, Tritons, and glycerides, ionic
surfactants such as fatty acids, quaternary compounds, anionic surfactants and amphoteric
surfactants such as cocamidopropyl betaine and emulsifiers.
Since Gram positive and Gram negative bacteria are almost always found together in foods, the
effectiveness of the nisin compositions towards Gram negative bacteria such as Salmonella
typhimurium, Escherichia Coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides
gingivalis, Actinobacillus actinomycetescomitans, and other Gram negative pathogens and Gram
positive bacteria will be of great use. The bactericides are particularly suited for the control and
prevention of contamination of raw ingredients, processed foods and beverages by bacterial
pathogens and other microbial spoilage organisms. Potential food related uses include treatment of
meats, especially poultry, eggs, cheese and fish and treatment of food packaging and handling
equipment.Further uses include as food preservative, such as in processed cheese, cream, milk,
dairy products and in cleaning poultry, fish, meats, vegetables, and dairy and food processing
equipment. The use of the nisin compositions should not be limited to food related used and the nisin
compositions should be useful in any situation in which there is a need or desire to eliminate Gram
negative and Gram positive bacteria.
70/1006
The compositions can be dissolved in a suitable carrier for example an aqueous solvent or buffer or
suspended in any suitable liquid, colloidal or polymeric matrix to create bactericides. Such
compositions preferably contain from 0.01 to 0.2 percent of the surfactant. The compositions or
bactericides can be incorporated into ointments or coatings for medicinal uses such as the treatment
of infections, wound dressings or surgical implants and as a broad spectrum disinfectant for skin or
oral rinses, disinfectant scrubs, wipes or lotions. The bactericides can be used for cleaning medical
instruments, in pre-operative surgical scrubs and the like. The bactericides are particularly useful in
circumstances where environmental disinfection is desired but where chemical germicidals are
precluded because of the risks of corrosive or otherwise toxic residues.
Unlike the activity of most broad spectrum germicidals which is compromised by the presence of
complex organic matter, the compositions of the present invention are effective as bactericides in the
presence of organic matter, such as milk or serum.
Nisin belongs to the class of peptide bacteriocins containing lanthionine. Also included among that
class are subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5. These bacteriocin
peptides are each produced by different microorganisms. However, subtilin obtained from certain
cultures of Bacillus subtilis, and epidermin obtained from certain cultures of Staphylococcus
epidermidis, have been found to have molecular structures very similar to that of nisin (see Hurst, pp.
85-86, and Schnell et al., Nature, 333:276-278). It is therefore believed that because of the molecular
similarities, other lanthionine-containing peptide bacteriocins will be equally as effective as nisin in
combination with non-ionic surfactants in eliminating Gram negative and Gram positive bacterial
contaminations.
The effectiveness of the nisin, and by extension other lanthionine-containing peptide bacteriocin,
compositions as bactericides against Gram negative bacteria is surprising, since the prior art
generally teaches away from this activity of nisin.
In order to demonstrate the superior and unexpected rapid activity of the composition containing
nisin and various surfactants against Gram positive bacteria, a number of experiments were
conducted with the bactericides. These experiments are meant as illustration and are not intended to
limit this invention. It is to be expected that other, lanthionine-containing peptide bacteriocins would
be effective substitutes for nisin.
71/1006
All tests in the following examples were performed at 37 DEG C. The efficacy of the enhanced broad
range bactericides was determined by assaying bactericidal activity as measured by the percent
bacterial survival after treatment with the bactericide. Generally, after incubation of a 10 cell per ml
suspension of target species with the novel bactericide for specified lengths of time, bacteria were
collected by centrifugation for 2 minutes. The bacterial pellet was washed free of the bactericide with
a rescue buffer, termed herein Phage buffer (50mM Tris-HCl buffer pH 7.8, 1mM MgSO4, 4mM
CaCl2, 0.1 M NaCl, and 0.1% gelatin), resuspended and serially diluted into Phage buffer, and 100ml
of the suspended bacteria were spread on nutrient agar plates. Surviving bacteria were determined
by scoring colony forming units (CFU) after incubation for 24-48 hours at 37 DEG C.An effective
bactericide according to this invention is one which allows less than 0.1% of the initial viable count of
the bacteria to survive.
Example 1
Nisin and Surfactant (monolaurin) Activity Against Gram Positive Bacteria
The bactericidal activity of nisin can be significantly enhanced when combined with a surfactant
alone. This is best illustrated at a limiting nisin concentration (0.2 mu g/ml) as shown in Table A. At
concentrations up to 0.1%, the food grade surfactant monolaurin has little significant bactericidal
activity towards Streptococcus agalactiae in the complex medium milk. Nisin, at concentrations up to
0.2 mu g.ml, likewise does not exhibit significant bactericidal activity in milk. However, the
combination of the two agents, 0.1% monolaurin and nisin 0.2 g/ml, is extremely potent towards S.
agalactiae. This bactericide is over 100 times more active than what would be expected for the
additive effect and 10,000 times more active than either of the components individually. Thus, when
the application of nisin is limited by its available activity, a bactericide comprising nisin with a
surfactant can be expected to be more useful.
Example 2
72/1006
Nisin and Surfactant (glyceride monooleate) Activity Against Gram positive Bacteria
An example of where the application of nisin is limited by its available activity is illustrated by the
data in Table B. Although nisin is bactericidal towards L. monocytogenes, the data in Table B
demonstrate that in a complex medium like milk the available nisin activity towards this organism is
restricted. However, the bactericide comprised of nisin with the glyceride monooleate is effective in
milk towards this foodborne pathogen, even though monooleate by itself had no bactericidal activity
towards this organism. Claims:
1. A bactericidal composition comprising a lanthionine-containing bacteriocin, characterized in that it
includes a surfactant, but excludes a chelating agent.
2. A composition according to Claim 1, characterized in that the surfactant is non-ionic.
3. A composition according to Claim 1, characterized in that the surfactant is monolaurin or glyceride
monooleate.
4. A composition according to Claim 1, characterized in that the surfactant is selected from the group
consisting of Tritons, Tweens, glycerides, fatty acids, emulsifiers, quaternary compounds,
amphoteric and anionic surfactants.
5. A composition according to any preceding Claim, characterized in that the lanthionine-containing
bacteriocin is nisin or subtilin.
6. A composition according to any preceding Claim, characterized in that it further comprises a
carrier.
7.A composition according to Claim 6, characterized in that the concentration of surfactant is from
0.01 to 1.0 percent.
73/1006
8. A composition according to Claim 6 or 7, characterized in that the carrier is an aqueous solvent or
buffer.
9. Use, other than in surgery or therapy, in combination of (1) a lanthionine-containing bacteriocin
and (2) a surfactant, in the absence of a chelating agent, as a bactericide effective against Gram
positive bacteria.
10. Use according to Claim 9 for preserving food.
11. Use according to Claim 10 for preserving dairy products.
12. Use according to Claim 11 for preserving milk.
13. Use according to Claim 9 for cleaning poultry, fish, meats vegetables and dairy and food
processing equipment.
14. A composition according to any one of Claims 1 to 8 for medicinal use.
15.A composition according to Claim 14 for use in the treatment of infections, wound dressings or
surgical implants.
16. A composition according to Claim 14 for use as a broad spectrum disinfectant for skin or oral
rinses, disinfectant scrubs, wipes or lotions.
17. An enhanced broad range bactericide comprising a carrier, a lanthionine-containing bacteriocin
and a surfactant, but not containing a chelating agent.
18. A bactericide according to Claim 17, wherein the surfactant is selected from the group consisting
of Tritons, Tweens, glycerides, fatty acids, emulsifiers, quaternary compounds, amphoteric and
anionic surfactants and is present in an amount sufficient such that the bactericide has enhanced
effectiveness against at least one of the bacteria from the group consisting of Gram negative and
Gram positive bacteria.
19. A bactericide according to Claim 18, wherein the concentration of surfactant is between about
0.01 and 1.0 percent.
74/1006
20. Each and every portion of the description and Claims of the parent Application No. 89 907 595.6
insofar as relates to compositions comprising a lanthioninecontaining bacteriocin and a surfactant or
the conjoint use of said bacteriocin and surfactant as a bactericide, but excluding subject matter
claimed in the allowed Claims of said application.
75/1006
9. EP0573768 - 15.12.1993
MULTIPLE BACTERIOCIN PRODUCING LACTOCOCCUS AND COMPOSITIONS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0573768
Inventor(s):
PETER A (US)
VEDAMUTHU EBENEZER R (US); HENDERSON JAMES T (US); VANDENBERGH
Applicant(s):
QUEST INT (NL)
IP Class 4 Digits: C12N; A23L; C07K; C12P; C12R
IP Class:
C12P21/02; C12N1/21; C12N15/31; A23L3/3571; C12R1/46; C07K7/10
E Class: C07K14/315; A23L3/3571
Application Number:
EP19930106911 (19930428)
Priority Number: US19920880003 (19920508)
Family: EP0573768
Equivalent:
DE69319503D; DE69319503T; DK573768T; JP2106448C; JP6098761; JP8024568B
Cited Document(s):
EP0446619; EP0137869
76/1006
Abstract:
A GROUP N LACTOCOCCUS WHICH PRODUCES TWO OR MORE BACTERIOCINS FROM
DIFFERENT LACTOCOCCUS ARE DESCRIBED. LACTOCOCCUS LACTIS SUBSPECIES LACTIS
18922 WITH DNA ENCODING TWO BACTERIOCINS FROM DIFFERENT LACTOCOCCUS IS
PARTICULARLY DISCLOSED FOR USE IN FOODS TO INHIBIT LACTOBACILLUS CASEI AND
OTHER FOOD CONTAMINANTS. BACTERIOCIN CONTAINING COMPOSITIONS, BACTERIAL
MIXTURES AND METHOD OF USE IN FOODS ARE ALSO DESCRIBED.Description:
Cross-Reference to Related Applications
The present application is a continuation-in-part of U.S. Application Serial No. 07/840,503, filed
February 24, 1992, which is a continuation-in-part of Serial No. 07/492,969, filed March 13, 1990, now
abandoned, and application Serial No. 07/721,774, filed July 1, 1991.
Background of the Invention
77/1006
(1) Field of the Invention
The present invention relates to Lactococcus which produce at least two bacteriocins and to
compositions incorporating the bacteriocins. In particular, the present invention relates to
Lactococcus which contain DNA transferred from a donor Lactococcus which provides a bacteriocin
along with a bacteriocin from a recipient Lactococcus strain.
(2) Prior Art
The genus Lactococcus includes dairy lactic streptococci that belong to the Lancefield serological
group N. The species involved are Lactococcus lactis subsp. lactis, Lactococcus lactis subsp.
cremoris, and citrate-fermenting Lactococcus lactis subsp. lactis biovar diacetylactis (Schleifer, K. H.,
FEMS Microbiol. Rev. 46(3): 201-203 (1987)). The aforementioned species are traditionally used in
dairy fermentations and are "generally regarded as safe" (GRAS) by the United States Department of
Agriculture.
Certain strains of group N Lactococcus spp. produce proteinaceous antagonistic substances
against closely related bacteria called bacteriocins (Klaenhammer, T. R., Biochimie 60(3): 337-349
(1988)). Most of these bacteriocins have a narrow spectrum of activity either antagonistic to other
strains within the species or to certain strains in closely related species. Nisin, a bacteriocin
produced by certain strains of Lactococcus lactis subsp. lactis, however, has a wider spectrum of
activity affecting other Gram-positive bacteria including the spore-forming Clostridia and Bacillus spp.
(Delves-Broughton., Food Technol. 44(11):100-112, 117 (1990)).
Because of the traditional use of the dairy lactococci in various food fermentations (Gilliland, S. E.,
Bacterial Starter Cultures for Foods. CRC Press Inc., Boca Raton, FL pages 5 to 23, (1985)), their
GRAS status and their non-pathogenicity, bacteriocins produced by these bacteria can be safely
used in food systems (either produced in situ in the food system by adding the live cultures or added
to food systems as cell-free purified or semi-purified or crude preparations) to extend the shelf life or
to provide a margin of safety against potential food-borne pathogens. The utility of such bacteriocinproducing strains could be particularly increased if the bacteriocin-producing potential of the strains
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is broadened by introducing into such strains genetic information for different types of bacteriocins
from closely related strains or species.
A transfer of genetic information can be achieved by conjugation, transformation (including
electrotransformation), and transduction. The problem is generally, that the bacteriocins are
antagonistic to the recipient Lactococcus and the recipients are either killed or the transfer does not
take place.
OBJECTS
It is therefore an object of the present invention to provide Lactococcus strains which encode at least
two bacteriocins and which are useful in food fermentations. Further, it is an object of the present
invention to provide compositions and a method of use incorporating the bacteriocins. Further still, it
is an object of the present invention to provide compositions which are safe and economical. These
and other objects will become increasingly apparent by reference to the following description and
the drawings.
IN THE DRAWINGS
Figure 1 is a schematic diagram showing the steps in mating of plasmid pSRQ400 into a recipient
Lactococcus strain which is preferred.
GENERAL DESCRIPTION
The present invention relates to a group N Lactococcus sp. selected from the group consisting of
Lactococcus lactis, Lactococcus cremoris and Lactococcus lactis biovar diacetylactis and
containing DNA from Lactococcus lactis NRRL-B-18809 (LLA 2.0) encoding a bacteriocin, wherein
the Lactococcus sp. encodes the bacteriocin from the Lactococcus lactis NRRL-B-18809 along with
a different bacteriocin from a strain which is a parent to the Lactococcus sp.
79/1006
In particular, the present invention relates to a method for preserving a food against Lactobacillus
casei growth present as a contaminant in the food which comprises: providing in the food cells of a
group N Lactococcus sp. selected from the group consisting of Lactococcus lactis, Lactococcus
cremoris and Lactococcus lactis biovar diacetylactis and containing DNA from Lactococcus lactis
NRRL-B-18809 encoding a bacteriocin, wherein the Lactococcus sp. encodes the bacteriocin from
the Lactococcus lactis NRRL-B-18809 along with a different bacteriocin from a strain which is a
parent to the Lactococcus sp., wherein the Lactococcus sp. strain is provided in the food and
wherein the cell numbers are sufficient to inhibit Lactobacillus casei.
Further still, the present invention relates to a composition which comprises in admixture (A) a first
bacteriocin produced by Lactococcus lactis NRRL-B-18809 and (B) a second bacteriocin produced
by Lactococcus NRRL-B-18535, wherein the ratio by weight of (A) to (B) is between about 25 to 1
and 1 to 25, and wherein the composition inhibits Lactobacillus casei and other bacteria which can
be present as contaminants in a food.
The present invention also relates to a method for preserving a food against growth of Lactobacillus
casei and other bacteria which can be present as contaminants which comprises providing a
composition which comprises in admixture (A) a first bacteriocin produced by Lactococcus lactis
NRRL-B-18809 and (B) a second bacteriocin produced by Lactococcus NRRL-B-18535, in amounts
sufficient to inhibit the Lactobacillus casei and the other bacteria present as contaminants in the food.
The present invention further relates to a method for preserving a food against Lactobacillus
bulgaricus growth present as a contaminant in the food which comprises providing in the food cells
of a Lactococcus lactis which produces a nisin-like bacteriocin and cells of Lactococcus lactis
NRRL-B-18809 in an amount sufficient to inhibit the Lactobacillus casei.
Finally the present invention relates to a bacterial composition which comprises (A) cells of a
Lactococcus lactis which produces a nisin-like bacteriocin and (B) cells of Lactococcus lactis NRRLB-18809 in an amount sufficient to inhibit a Lactobacillus casei and other contaminant bacteria in a
food.
The donor strain is Lactococcus lactis NRRL-B-18809, also known as LLA 2.0, which produces a
unique bacteriocin. The strain contains plasmid pSRQ400 which encodes the bacteriocin. The
preferred recipient strain is Lactococcus lactis NRRL-B-18535, also known as LLA 1.2, which
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produces a nisin-like bacteriocin. The preferred transconjugant strain is Lactococcus lactis NRRL-B18922. These strains are on deposit with the Northern Regional Research Laboratory in Peoria,
Illinois under the Budapest Treaty. Other strains which can be used as recipients are Lactococcus
cremoris and Lactococcus lactis biovar diacetylactis which produce a bacteriocin, although they are
not preferred.
The bacteriocin produced by Lactococcus lactis NRRL-B-18809, as the donor strain (LLA 2.0), has
the formula The strain LLA 2.0 is described in detail in U.S. Application Serial No. 07/721,774. It
produces a unique bacteriocin. The bacteriocin produced by Lactococcus lactis NRRL-B-18535 as a
recipient strain is nisin-like, although differing from nisin in one amino acid. This bacteriocin is
described in detail in U.S. application Serial No. 07/492,969. The exact chemical and physical
structure of this bacteriocin is uncertain.
The bacteriocins are provided together in foods. They can be provided by using a single
transconjugant strain producing both bacteriocins or by two strains producing the bacteriocins
individually. The Lactococcus sp. can also be combined together and introduced into the food.
The Lactococcus are grown in a conventional growth media to express the bacteriocin(s) and can be
frozen or lyophilized. The bacteriocin(s) can be isolated from the growth media and used in the food.
They can also be preserved in lyophilized or frozen form for shipment prior to use.
Where the bacteriocins are to be produced, the preferred media are those described in application
Serial Nos. 07/492,969 and 07/721,774 for Lactococcus lactis NRRL-B-18809 and NRRL-B-18535.
The most preferred media was MRS Lactobacillus Broth (Difco, Detroit MI). The bacteriocin was
isolated from the growth media and can be concentrated as described in these applications using
ultrafiltration and the like.
The mixture of bacteriocins are used in the foods in a ratio of the bacteriocin from LLA 2.0 and from
LLA 1.2 in a ratio of 1 to 25 and 25 to 1 by weight. Preferably the ratio is 1 to 20 and 1 to 25 by weight
of LLA 2.0 to LLA 1.2. The composition can be supplemented with various food grade fillers, such as
starch, dextrose and the like to provide bulk.
SPECIFIC DESCRIPTION.
81/1006
The method for producing the preferred transconjugant strain of Lactococcus lactis producing two (2)
bacteriocins is described hereinafter in Example 1. Conjugation was used since the plasmid
transferred was too large for transformation or transduction. The plasmid can be reduced in size to
accomplish DNA transfer by these latter methods; however conjugation is the easiest method.
Example 1 shows the isolation of the transconjugant strain and the testing of the strain.Example 2
shows the use of the transconjugant strain in various foods.
Example 1
Conjugative transfer of pSRQ400
The object of this Example 1 was to establish that (1) the resident plasmid pSRQ400 (69 Kb)
encoding for lactose fermentation and bacteriocin production (LL-2), could be introduced into
another closely-related strain; (2) the transfer of pSRQ400 from Lactococcus lactis LLA 2.0 would be
accomplished by conjugation (mating). The recipient is (a) insensitive to bacteriocins produced by
LLA2.0; (b) is not inhibitory to LLA 2.0; (c) has antibiotic markers to select against the donor LLA 2.0;
(d) is lactose negative (Lac), so that transfer of pSRQ 400 can be easily followed; and (e) is a related
strain (i.e.) within the same species or genus.
The strain used was Lactococcus lactis LLA 1.0, which could be passed through suitable gradations
of desired antibiotics to obtain a spontaneous resistant mutant. On testing against LLA 2.0, it was
found that the strain was insensitive to bacteriocins (LL-2A and LL-2B) produced by LLA 2.0. Also
LLA 2.0 was not inhibited by LLA-1, which produced a bacteriocin called LL-1. Additionally, strain
LLA-1 was lactose negative (Lac). Because of these favorable traits LLA-1 was chosen as a recipient.
It was important at the outset to establish suitable screening procedures other than Lac phenotype to
select for and confirm presumptive transconjugants. Other distinguishing characteristics between
LLA-1 and LLA-2 had to be established. To achieve this end, a two-pronged approach was used.
(1) Screen LLA-1.0 and LLA-2.0 against a previously isolated phage, which was virulent for LLA 1.0.
Screening showed that phage lla-1, was specific for LLA-1.0 and did not infect LLA 2.0.
(2) Screen LLA-1.0 and LLA-2.0 against several possible indicators and find out if LLA-2.0 inhibited
a strain that is not inhibited by LLA 1.0. Earlier work had shown that colonies of LLA-2.0 inhibited
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Lactobacillus casei 842 (NRRL-B-15972), while LLA 1.0 failed to show a similar inhibition. These
characteristics could be used for non-selective marker analysis of possible transconjugants.
To further facilitate easy screening of Lac colonies appearing on selective agar plating of mating
mixtures, it was decided to select a spontaneous mutant of LLA-1.0 resistant to these drugs.
Selection of Str derivative of LLA 1.0
Columns=5
Head Col 1: [Str]/ml
Head Col 2: [Str]/5 ml
Head Col 3: ml BMG
Head Col 4: Str soln
Head Col 5:Total vol
005.00.05.0
5254.750.25 (D)5.0
201004.90.1 (C)5.0
502504.750.25 (C)5.0
1005004.500.50 (C)5.0
50025004.750.25 (B)5.0
100050004.500.50 (B)5.0
150075004.250.75 (B)5.0
2000100004.001.00 (B)5.0
Make up stock sol.= 100,000 mu g/ml (Soln. A) Filter sterilize. BMG = Basel Medium with Glucose.
1/10 dil A = 10,000 mu g/ml (Soln. B) Use sterile distilled water for dilution.
1/10 dil B = 1,000 mu g/ml (Soln. C).
1/10 dil C = 100 mu g/ml (Soln. D).
str = Streptomycin
Culture LLA-1.0 was step-wise transferred through these solutions and a resistant isolate (Str ) was
purified and frozen. It was given the designation LLA 1.1. This strain was then used to develop a
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double-drug resistant mutant by exposure to fusidic acid. The isolated LLA 1.1 was sensitive to
phage lla-1.
To get a Fus marker on LLA 1.1, Basel medium with glucose (BMG) agar was dispersed in 20 ml
volumes into sterile screw-cap tubes.
5 mg of fusidic acid was weighed out, and dissolved in 5.0 ml methanol to give 1 mg (1000 mu
g)/ml-Soln A, it was diluted 1/10 to give Soln. B.
Columns=3
Head Col 1: [Fusidic]/ml
Head Col 2: [Fusidic]/20 ml
Head Col 3: ml Fusidic Stock
1 mu g20 mu g0.2 ml of B
5 mu g100 mu g0.1 ml of A
10 mu g200 mu g0.2 ml of A
20 mu g400 mu g0.4 ml of A
Agar mixed with the drug was poured into sterile petri plates and allowed to solidify. The plates were
dried of the surface moisture.
One tenth ml (0.1 ml) of an 18 hour culture of LLA 1.1 in BMG broth was spread on plates containing
1 mu g/ml and 5 mu g/ml fusidic acid. Colonies appearing on one of these plates were streaked
onto plates containing higher concentration of the drug. By such a procedure a derivative resistant to
20 mu g/ml of fusidic acid was obtained. This derivative was designated as LLA 1.2.
The strategy was to plate the mating mixtures on basal medium with lactose, bromocresol purple a
pH indicator (BCP) and streptomycin. Lac colonies from mating plates were screened on Fus plates
to select for transconjugants.
Microorganisms: The spontaneous streptomycin-and fusidic acid-resistant, lactose-negative,
plasmid-free, potent bacteriocin-producing Lactococcus lactis subsp. lactis strain designated as
LLA 1.2 was used as the recipient in mating experiments. This strain produced a nisin-like
bacteriocin. The donor was another strain of Lactococcus lactis subsp. lactis designated as LLA 2.0.
The donor strain, LLA 2.0, produced two related bacteriocins, one of which was believed to be a
degradation product of the other. Strain LLA 2.0 contained the single plasmid (pSRQ400) 69.0 Kb in
size. Curing of this plasmid resulted in the loss of lactose-fermenting ability and bacteriocin
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production by LLA 2.0. Strain LLA 1.2 had no inhibitory action against LLA 2.0 and vice
versa.Furthermore, colonies of LLA 2.0 were very inhibitory to Lactobacillus casei subsp. casei 842,
while LLA 1.2 had no effect against this lactobacilli. Lactobacillus casei can be contaminants in
foods or may be added to control molds.
A specific bacteriophage, designated lla-1, which was virulent for LLA 1.2, but was inactive against
LLA 2.0, was used to test for authentic transconjugants in mating experiments. Activity against
Lactobacillus casei subsp. casei 842 was an additional test used in the selection of transconjugants.
The microorganisms used and their characteristics are listed in Table 1. Media: Strain LLA 1.2 as
routinely propagated in Basal Medium with glucose (BMG). Strain LLA 2.0 was cultured in Basal
Medium with lactose (BML). The compositions of these media are described by Gonzalez and Kunka
(Gonzalez, C. F. and B. S. Kunka., Appl. Environ. Microbiol. 46:81-89 (1983)). For solid media 1.5%
agar was added to the respective broths.For selective plating of mating mixtures, BML-agar
containing 0.008% bromocresol purple (BCP) as a pH indicator, and 1000 micrograms/ml
streptomycin as selective agent was used. Lactose-positive colonies on BML(BCP)-Str plates were
counter selected on BML(BCP)-Fus plates to eliminate spontaneous Str mutants of the donor. To test
for phage-sensitivity a soft-agar overlay, seeded with the isolate was spotted with a high titer lysate of
phage lla-1. The media used for this test were solid and semisolid BMG agar fortified with 0.02%
calcium chloride.
Bacteriocin Assay: Titer of bacteriocin in cell-free supernatants of cultures was determined on
supernatants passed through 0.45 mu m filter. The indicator used was Pediococcus pentosaceus
FBB63C. Filtered supernatants and two-fold serial dilutions of the filtrates were spotted at 5 microliter
volumes on moisture-free MRS-soft agar overlays of the indicator as described by Gonzalez and
Kunka (Gonzalez., C. F. and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)). One
activity unit (AU) is defined as the reciprocal of the highest dilution yielding a definite inhibition zone
on the indicator lawn.
For demonstrating bacteriocin-production by colonies, individual colonies were transferred to
moisture-free surface of buffered MRS-agar plates (MRS agar containing 1.9% of sodium beta glycerophosphate) with sterile toothpicks such that colonial growth from the transfers measured 1-2
mm in diameter. The transfers were made such that the developing colonies were separated from
one another by at least 2 cm. After colonies developed, a layer of soft MRS-agar seeded with
Lactobacillus casei subsp. casei 842 was poured. After incubating overnight at 35 DEG C, plates
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were examined for inhibition of the indicator. A wide zone (>/= 1 cm) of clearing with crisp margins
was scored positive for bacteriocin production.
Culture Mating Procedure: Agar surface mating procedure was used.The mating mixtures (Donor to
Recipient ratio of 1:2 and 1:4) and donor and recipient controls were spread on the surface of BMGagar. A set of five plates for each experimental variable was used. Each plate was spread with 0.2 ml
of the sample. Plates were incubated overnight at 32 DEG C, and the cells were harvested by
washing the agar surface with sterile phosphate buffer, pH 7.0, using 2.0 ml per plate. The total of
approximately 10 ml. per sample from each set of five plates were pooled together, and the cells
sedimented by centrifugation. After washing with 10 ml of phosphate buffer, the cells were
resuspended in 1.1 ml buffer. The cells were then spread on five BML(BCP)-Str plates at 0.2 ml per
plate. Plates were incubated in a Gas Pak Jar with the hydrogen-carbon dioxide generating pouch at
32 DEG C for 48 hours. At the end of the incubation period, the plates were examined.
Colony Screening: All lactose-positive, yellow colonies from the plates containing mating mixtures
were short-streaked on BML(BCP)-Fus plates to select against spontaneous Str donor colonies.
Presumptive transconjugants from the BML(BCP)-Fus plates were screened for inhibitory activity
against L. casei subsp. casei 842. Those exhibiting antagonistic activity, were streaked for single
colony isolation (purification step) and retested for activity against strain 842. Colonies positive for
antagonistic activity were tested for sensitivity to phage lla-1. Colonies that were sensitive to the
phage were grown in broth and stocked.
Plasmid DNA Isolation and Electrophoresis: Procedures described by Gonzalez and Kunka
(Gonzalez, C. F. and B. S. Kunka, App.. Environ.Microbiol. 46: 81-89 (1983)) were used for the
propagation of cells, cell-lysis, plasmid DNA isolation, electrophoretic separation and eithidium
bromide staining of gels.
Purification of bacteriocin activity of transconjugant LLA 1.2 (pGK41) T1 (Lab designation 302):
Two liters of MRS broth (Difco, Detroit, Michigan) were inoculated at 1% with an 8 hour old culture of
strain 302 (propagated in MRS broth) and were grown statically at 32 DEG C for 24 hours. Cells were
removed by centrifugation at 16,300 x g for 15 minutes at 4 DEG C. The supernatant was filtered
using a Minitan tangential filtration apparatus (Millipore, Bedford, Massachusetts) equipped with a
0.2 mu m pore size polyvinylidene difluoride (PVDF) membrane. Bacteriocin production was
assayed as previously described.
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Filtrate from tangential filtration was concentrated approximately 6-fold using a spiral-wound
cellulose-based S1Y3 ultrafiltration cartridge with a 1 ft surface area and a 3000 dalton molecular
weight cutoff (Amicon, Danvers, MA). Concentration was performed at 4 DEG C using a peristaltic
pump (Cole-Parmer, Chicago, Illinois) to maintain a 20 lb/in differential across the membrane.
A 350 ml aliquot of concentrated supernatant was applied to a 10 cm x 20 cm column (1.57 liters) of
DEAE-650M anion exchange resin (Toso-Haas, Philadelphia, PA) equilibrated with 0.1 M sodium
acetate buffer, pH 4.0 at a flow rate of 110 ml/min. Absorbance of the eluent at 280 nanometers was
monitored and eluent was collected from the first increase from baseline absorbance until baseline
absorbance was again reached. The eluent volume was 3150 ml and the activity was 1600 AU/ml.
The entire volume of eluent from anion exchange chromatography was applied to a 10 cm x 35 cm
column (2.75 liters) of CM-650M cation exchange resin (Toso-Haas, Philadelphia, PA) which had
been equilibrated against .1 M sodium acetate buffer, pH 4.0. Activity was eluted using the same
buffer containing 1 M sodium chloride at pH 4.0. Eluent was collected from the first increase in
conductivity from baseline conductivity (0.159 micro Siemans). Collection was terminated when
absorbance at 280 nanometers returned to baseline absorbance. The eluent volume was 2000 ml
and the activity was 400 units/ml.
The eluent from cation exchange chromatography was concentrated approximately 10-fold by
ultrafiltration using an S1Y3 cartridge (Amicon Danvers, MA) until 100 ml remained. Sodium chloride
content was then reduced approximately 125-fold by diafiltering three times with 400 ml deionized
water and then concentrating to about 100 ml again. The cartridge was emptied and then washed
with 100 ml deionized water. The concentrate was combined with the wash solution to obtain 230 ml
bacteriocin with 3200 AU/ml activity.
Volume of the bacteriocin concentrate was further reduced using vacuum centrifugation (Savant,
Farmingdale, NY) until 16 ml remained with an activity of 64000 AU/ml. Aliquots of this concentrate
were applied to a 2.5 cm x 25 cm ODS column (Vydac, Hisperia, CA) equilibrated with 0.1 %
trifluoracetic acid (TFA) in water. Activity was eluted using a gradient which typically used a linear
change over 30 minutes from 20% to 40% acetonitrile (AcCN) containing 0.1% trifluoroacetic acid. A
flow rate of 10 ml/min was used. Fractions were collected at 0.5 minute intervals and activity was
located in the chromatogram by directly spotting 5 mu l from each fraction onto an FBB63C indicator
plate.Protein elution was monitored using a UV detector at 230 nanometers wavelength.
87/1006
Characterization of bacteriocins: Comparisons were done of the activities of purified LL-1, LL-2A and
LL-2B from the parent strains and the isolated 302A and 302B from the transconjugant strain. These
comparisons were done on a 0.45cm x 25 cm C18 analytical column (Vydac, Hisperia, California).
RESULTS
Initial mating experiments revealed that the donor produced too many Str colonies and, because of
this, it was very difficult to select colonies for screening. To avoid this problem, an erythromycinresistance marker (Ery) was introduced into the donor by electroporating the shuttle vector pGK41. In
later mating experiments, selective plating of control and mating mixtures were made on BML(BCP)Str Ery plates. On these plates no breakthrough of donor colonies was seen.
Mating mixtures plated on the erythromycin-containing agar produced very weakly lactose-positive
(Lac) colonies. All such colonies were screened on fusidic acid-containing medium and colonies
showing ready growth on that medium were checked for activity against strain 842. Those that were
positive for inhibition against strain 842 were tested for susceptibility to the specific phage.
Two colonies satisfied all the screening criteria. This represented a transfer frequency of 2.0 x 10 per
donor cell input, as shown in Table 2. Table 2. Frequency of transfer of Bac phenotype between
Lactococcus lactis subsp. lactis LLA 2.0 and Lactococcus lactis subsp. lactis LLA 1.2 (pGK 41).
Columns=4
Head Col 1: Donor
Head Col 2: Recipient
Head Col 3: Transfer Frequency
Head Col 4: Transconjugants
LLA 2.0LLA 1.22.0 x 10LLA 1.2 (pGK 42) T1
(pGK 41)LLA 1.2 (pGK 41) T2
Laboratory designation 302.
88/1006
The two isolates, however, lost their Lac phenotype but retained the inhibitory activity against strain
842 during the screening process. Examination of their plasmid content showed the lack of any
plasmid in both the strains.
Further testing of the transconjugants consisted of the analysis of the bacteriocin(s) produced by
one of the strains. Purification of bacteriocin(s) produced by one of the transconjugants, LLA 1.2
(pGK41) T1 (laboratory designation 302) was done primarily to verify the presence of two distinct
bacteriocins. The strains appeared to be identical.
Elution times of purified LL-1, LL-2A and LL-2B from the parent strains were determined separately. A
30 minute 20-40% AcCN gradient elutes LL-2A at 26.5', LL-2B at 37' and LL-1 at 35'. The purified
302 concentrate using the 20-40% AcCN gradient revealed two areas of activity, one at 26.5' and the
other between 38 and 40'. Only the second of these areas was associated with a defined peak. The
active fractions were isolated and named 302A and 302B, respectively. Further runs were completed
using this method. At completion, five separate HPLC runs were performed, each of the heart cuts
(determined by association with a peak) were brought up to 0.5 ml with dH2O, titered and analyzed
for protein concentration. These pure fractions were stored at -20 DEG C.
To compare the various purified bacteriocin entities obtained through purification, mixtures were
made combining 302B with equivalent amounts of LL-1 or LL-2B to determine which of the
bacteriocins would coelute with 302B. Previously, it had been found that LL-1 and LL-2B, though
eluting within a very close range, resolved as two peaks using a 0-45% AcCN gradient. Equal
amounts of LL-2B and 302B also showed elution of two peaks indicating that two separate
bacteriocins were present. In contrast, equal amounts of LL-1 and 302B eluted only a single peak.
Thus, 302B appears to be identical to LL-1. In a similar manner, 302A was compared to LL-2A. On
an analytical scale (0-45% AcCN, 30') 302A and LL-2A elute at 27.1'.The results are shown in Tables
3 and 4.
Id=Table 4 Columns=3 Typical HPLC elution times of bacteriocins from strains LLA-1.2, LLA-2.0 and
302.
Head Col 1: Bacteriocin
Head Col 2: 20-40% AcCN 30'
Head Col 3: 0-45% AcCN 30' Analytical Scale
LL-2A26.5'27.7'
302A26.5'27.1'
LL-2B37'31.5'
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LL-135'31.1'
302B38'31.1'
Note:
Mixing equal amounts LL-1 and LL-2B yields two peaks.
Mixing equal amounts 302B and LL-1 yields a single peak.
Mixing equal amounts 302B and LL-2B yields two peaks.
Mixing equal amounts LL-2A and 302A yields a single peak.
The mating did result in expression of two different bacteriocin activities, one appearing to be LL-1
and the other comparable to LL-2A, the weaker of the two bacteriocins associated with LLA 2.0
bacteriocin production. Thus, the Bac genes from LLA 2.0 have been successfully integrated into the
LLA 1.2 chromosome with partial phenotypic expression evident by production of the bacteriocin LL2A.
The bacteriocin LL-2B was not detected in purified isolates from 302 medium. Purified LL-2B isolated
from LLA 2.0 medium has a single peak in the HPLC chromatogram and will, over time, form a
second entity which elutes at the same position as LL-2A. Purified LL-2A seems stable over time
since it continues to appear as a single peak in the chromatogram. From these results the conclusion
can be reached that LL-2A is related to LL-2B and that the relationship is such that either LL-2A is a
product of irreversible alteration of the molecular structure of LL-2B, or that LL-2A represents a slowly
forming but much more stable conformation of LL-2B. The presence of LL-2A in isolates of 302
medium is, therefore, interpreted as indirect evidence of the presence of the parent LL-2B protein at
some prior point, even though it was not detected at the time of final analysis.
Inhibition of Lactobacillus casei 842 was tested using each of the purified bacteriocins LL-1, LL-2A,
LL-2B, 302A and 302B. Previous tests with whole cells showed inhibition when the lines LLA 2.0
(produces LL-2) or 302 (produces LL-1 and LL-2) were present but not when the line LLA 1.2
(produces LL-1) was present. In contrast, on spotting 5 mu l of 6400 AU/ml purified bacteriocin, LL-1,
LL-2B, and 302B inhibited the growth of L. casei 842, while LL-2A and 302A did not. Evidently, the
302 cell line is more effective in its anti-bacterial activity toward L. casei 842 than the LLA 1.2 cell line.
According to inhibition studies involving purified bacteriocins, only the LL-1 component of 302 cell
bacteriocin production is active against L. casei 842.From this it was concluded that genetic
modifications as a result of the mating experiment have increased the effectiveness of LL-1 mediated
activity against L. casei 842 compared to LLA 1.2 cells. The results are shown in Table 5.
90/1006
Id=Table 5 Columns=4 Activity of cellular and purified bacteriocins from strains LLA 1.2, LLA 2.0
and 302.
Head Col 1 to 2: Inhibition
by Whole cells
Head Col 3 to 4: Inhibition
by Purified Bacteriocins
LLA 1.2noLL-1yes
LLA 2.0yesLL-2Ano
LL-2Byes
302yes302Ano
302Byes
Inhibition of Lactobacillus casei strain 842.
Bacteriocins adjusted to 6400 Au/ml titer.
Example 2
The suppressive activity of Lactococcus lactis ssp. lactis NRRL-B-18922 against Lactobacillus casei
842 was tested in a sour cream dip system, which can also be extended to fermented milk-based
salad dressings. This system was specifically chosen because the microorganisms in question could
be used in these systems as supplementary shelf-life extending agents. Lactobacillus casei 842 has
previously been shown to inhibit molds (U.S. Patent No. 4,956,177) to King et al. This mold-inhibiting
lactobacilli, however, can produce excessive amounts of lactic acid in milk-based dip and dressing
mixtures and cause too much souring and phase-separation because of wheying-off. Hence, there is
a need for regulating acid-production and growth of L. casei 842 in such systems without affecting its
mold-suppressing activity. Because the transconjugant NRRL-B-18922 inhibits L. casei 842, their
interaction in a sour cream dip system was tested in this example.
91/1006
Procedure: Fresh sour cream was purchased from the store. One hundred gram portions of the sour
cream were weighed into four screw-cap jars. To each portion, 5.0 grams of "Ranch Flavor" spice mix
were added and mixed thoroughly with a spatula. The jars were labeled 1, 2, 3 and 4. Jar 1 served
as the control without any microbial additive. To jar 2, sufficient dilution of a fresh milk culture of L.
casei 842 was added such that a count of 1 x 10 to 1 x 10 cfu/g was available. To jar 3, a dilution of a
fresh culture of Lactococcus lactis ssp. lactis NRRL-B-18922 was added such that a count of 1 x 10
to 1 x 10 cfu/gm was present. To jar 4, L. casei 842 at the same level as in jar 2 and NRRL-B-18922
at the same level as in jar 3 were added as a mixture. All the jars were thoroughly mixed with four
separate spatulas.The L. casei 842 strain used in this example contained a chromosomal Rifamycinresistance marker. The transconjugant NRRL-B-18922 contained an Erythromycin-resistance plasmid.
The marked strains allowed selective enumeration of the two strains in the presence of other flora in
the sour cream with spice mixture as well as in the mixture containing both strains, namely, as in jar 4.
Counts of the added bacteria were determined immediately after uniform incorporation. For counting
NRRL-B-18922, APT-agar containing 5 micrograms/ml of Erythromycin was used. To count L. casei
842, MRS-agar with 200 micrograms/ml of Rifamycin was used. The samples were held at room
temperature for a week, and examined every day for visual appearance, aroma and odor. At the end
of the week, counts were made and the pH was measured.
Results:
Columns=8 Visible Mold Growth:
Head Col 1: Jar.No.
Head Col 2 to 8: Days at room temperature
SubHead Col 1:
SubHead Col 2: 1
SubHead Col 3: 2
SubHead Col 4: 3
SubHead Col 5: 4
SubHead Col 6: 5
SubHead Col 7: 6
SubHead Col 8: 7
1---++++++++
92/1006
2------3---++++++++
4------- = Negative; + = Slight; ++ = Large patches
Columns=8 Aroma and Odor:
Head Col 1: Jar No.
Head Col 2 to 8: Days at room temperature
SubHead Col 1:
SubHead Col 2: 1
SubHead Col 3: 2
SubHead Col 4: 3
SubHead Col 5: 4
SubHead Col 6: 5
SubHead Col 7: 6
SubHead Col 8: 7
1GFMMMMM
2GGGSSS+S+
3GGMMMMM
4GGGGGGG
G = Good, normal "ranch" odor and aroma
F = Flat, lack of acid odor and aroma
M = Moldy, musty odor
S = Sour smell
S+ = Extremely sour odor
The pH of jars 1 and 3 were not taken at the end of 7 days because of visible mold growth. The pH of
jar 2, which was excessively sour smelling, was 3.8. The pH of jar 4, which maintained the good
"ranch" aroma, was 4.0.
Conclusions:
93/1006
The addition of L. casei by itself controlled mold growth but resulted in excessive acid accumulation
when the "Ranch Dip" was held at room temperature for 7 days. Wtihout any additions or with only
NRRL-B-18922, mold contamination from the spice mixture grew unchecked and affected the visual
appearance, and imparted a moldy, musty odor to the dip. In the presence of NRRL-B-18922, L.
casei 842 inhibited the mold from developing. At the same time, the inhibitory activity of the
lactococci against the lactobacilli prevented the unrestricted increase in the number of the
lactobacilli thus controlling accumulation of excessive lactic acid. The visual appearance, aroma and
odor of jar 4 was normal.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
1. A group N Lactococcus sp. selected from the group consisting Lactococcus lactis, Lactococcus
cremoris and Lactococcus lactis biovar diacetylactis and containing DNA from Lactococcus lactis
NRRL-B-18809 (LLA 2.0) encoding a bacteriocin, wherein the Lactococcus sp. encodes the
bacteriocin from the Lactococcus lactis NRRL-B-18809 along with a different bacteriocin from a
strain which is a parent to the Lactococcus sp.
2. Lactococcus lactis subspecies lactis NRRL-B-18922.
3. A Lactococcus lactis subspecies lactis containing DNA encoding two bacteriocins, one from
Lactococcus lactis NRRL-B-18809 as a donor strain and one from Lactococcus lactis NRRL-B-18535
as a recipient strain.
4.A method for preserving a food against Lactobacillus bulgaricus growth present as a contaminant
in the food which comprises:
providing in the food cells of a group N Lactococcus sp. selected from the group consisting of
subspecies Lactococcus lactis, Lactococcus cremoris and Lactococcus lactis biovar diacetylactis
and containing DNA from Lactococcus lactis NRRL-B-18809 encoding a bacteriocin, wherein the
Lactococcus sp. encodes the bacteriocin from the Lactococcus lactis NRRL-B-18809 along with a
94/1006
different bacteriocin from a strain which is a parent to the Lactococcus sp., wherein the Lactococcus
sp. is provided in the food and wherein the cell numbers are sufficient to inhibit Lactobacillus casei.
5. The method of Claim 4 wherein the transconjugant is Lactococcus lactis subspecies lactis NRRLB-18922.
6.The method of Claim 4 wherein the Lactococcus contains DNA encoding two bacteriocins, one
from Lactococcus lactis NRRL-B-18809 as a donor strain and one from Lactococcus lactis NRRL-B18535 as a recipient strain.
7. A composition which comprises in admixture (A) a first bacteriocin produced by Lactococcus
lactis NRRL-B-18809 and (B) a second bacteriocin produced by Lactococcus NRRL-B-18535,
wherein the ratio by weight of (A) to (B) is between about 25 to 1 and 1 to 25, and wherein the
composition inhibits Lactobacillus casei and other bacteria which can be present as contaminants in
a food.
8. The composition of Claim 7 as a liquid.
9. The composition of Claim 7 as a powder.
10.A method for preserving a food against growth of Lactobacillus casei and other bacteria which
can be present as contaminants which comprises providing a composition which comprises in
admixture (A) a first bacteriocin produced by Lactococcus lactis NRRL-B-18809 and (B) a second
bacteriocin produced by Lactobacillus NRRL-B-18535, in amounts sufficient to inhibit the
Lactococcus casei and the other bacteria present as contaminants in the food.
11. The method of Claim 10 wherein the composition is a liquid.
12. The method of Claim 10 wherein the composition is a powder.
13. The method of Claim 10 wherein the ratio by weight of (A) to (B) is between about 25 to 1 and 1
to 25.
95/1006
14.A composition of bacteriocins in admixture which inhibit Lactobacillus casei and other bacteria
which can occur in foods which comprises a nisin-like bacteriocin and another bacteriocin having the
formula:
15. The composition of Claim 14 wherein the nisin-like bacteriocin is produced by Lactococcus lactis
NRRL-B-18809.
16. A method for preserving a food against Lactobacillus casei growth present as a contaminant in
the food which comprises:
providing in the food (A) cells of a Lactococcus lactis which produces a nisin-like bacteriocin and
(B) cells of Lactococcus lactis NRRL-B-18809 in an amount sufficient to inhibit the Lactobacillus
casei and other contaminant bacteria.
17. The method of Claim 16 wherein the Lactococcus lactis which produces the nisin-like bacteriocin
is Lactococcus lactis NRRL-B-18535.
18. A bacterial composition which comprises:
(a) cells of a Lactobacillus lactis which produces a nisin-like bacteriocin and cells of Lactococcus
lactis NRRL-B-18809 in an amount sufficient to inhibit a Lactobacillus bulgaricus in a food.
19. The method of Claim 18 wherein the Lactococcus lactis which produces the nisin-like bacteriocin
is Lactococcus lactis NRRL-B-18535.
96/1006
10. EP0589893 - 24.09.1996
PHARMACEUTICAL COMPOSITIONS AGAINST GASTRIC DISORDERS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0589893
Inventor(s):
EDWARDS PETER J (GB); MORWOOD KIM (GB)
Applicant(s):
APPLIED MICROBIOLOGY INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K38/00; A61K31/14
E Class: A61K38/16B; A61K9/20H4B; A61K9/20H6F; A61K9/28H6B2; A61K38/04
Application Number:
US19930129134 (19931214)
Priority Number: GB19910008129 (19910415); GB19910014149 (19910701); WO1992GB00656
(19920410)
Family: WO9218143
Equivalent:
AU1581192; AU665188; CA2108455; DE69213010D; DE69213010T; DK589893T;
IE921193; JP3334879B2; JP7503451T; WO9218143
Abstract:
PCT NO. PCT/GB92/00656 SEC. 371 DATE DEC. 14, 1993 SEC. 102(E) DATE DEC. 14, 1993 PCT
FILED APR. 10, 1992 PCT PUB. NO. WO92/18143 PCT PUB. DATE OCT. 29, 1992THE INVENTION
CONCERNS PHARMACEUTICAL COMPOSITIONS FOR USE IN THE TREATMENT OF GASTRIC
DISORDERS ASSOCIATED WITH HELICOBACTER PYLORI COMPRISING A BACTERIOCIN
ANTIMICROBIAL AGENT OPTIONALLY COADMINISTERED WITH A RELEASE-DELAYING
SUBSTANCE.Description:
97/1006
The present invention relates to pharmaceutical compositions of antimicrobial agents active against
Helicobacter pylori and their use in the treatment of gastrointestinal disorders associated with
Helicobacter pylori infection.
Helicobacter pylori (formerly known as Campylobacter pyloridis) is a spiral-shaped Gram-negative
organism which appears to live beneath the mucus layer of the stomach. Many recent studies have
shown an association between the presence of H. pylori in the gastric mucosa and histologically
confirmed gastritis.
In the light of these results, it has been suggested that the organism may be a pathogen which
causes, or at least exacerbates, gastritis, and may also be important in the aetiology of peptic
triceration. Reviews on the state of the art include those by C.A.M. McNulty in J. Infection, 1986, 13,
107-113, and by C. S. Goodwin et al. in J. Clin. Pathol., 1986, 39, 353-365.
H.pylori is known to be susceptible to a large number of antimicrobial agents in vitro. Furthermore,
several workers have shown that treatment of gastritis with antimicrobial agents, for example .beta.lactam antibiotics such as amoxycillin, or bismuth salts, leads to eradication of the associated
H.pylori organism in vivo.
Bacteriocins are peptide antimicrobial agents defined as proteinaceous substances produced by
bacteria which have antimicrobial activity only against species closely related to the species of origin.
An example of a bacteriocin which has found commercial application is nisin. Nisin is a lanthocin
comprising the atypical amino acid lanthionine. Nisin is a polypeptide having antibacterial properties
which is produced naturally by various strains of the bacterium Streptococcus lactis. It is found at low
concentration in milk and cheese. It is used as a food preservative to inhibit the outgrowth of spores
of certain species of Gram positive bacteria. See U.S. Pat. Nos. 5,584,199 and 4,597,972 (Taylor).
Nisin is recognised by the FDA as a direct food ingredient. It is considered non-toxic and nonallergenic to humans and, as a proteinaceous material, any residues in ingested foods are quickly
degraded by digestive enzymes. A summary of nisin's properties is to be found in Advances in
Applied Microbiology, 27, 85-123, (1981).
Recently, a purified form of nisin has been made available by Applied Microbiology Inc. under the
trade name AMBICIN N. It has been suggested for use in a variety of applications, for example for
use in oral care.
98/1006
Nisin, in common with other bacteriocin antimicrobial agents, is generally regarded as ineffective
against Gram negative bacteria, other than in the presence of non-bacterial enhancers such as
chelating agents and surfactants. See PCT Publication No. WO 89/12399 (Blackburn).
Surprisingly, it has now been found that the group of bacteriocin peptide antimicrobial agents is
efficacious in the in vitro eradication of the Gram negative organism Helicobacter pylori and therefore
has potential utility in the treatment of H.pylori mediated gastric disorders in mammals.
Accordingly, the present invention provides the use of a bacteriocin antimicrobial agent for the
manufacture of a medicament for the treatment of gastric disorders associated with H.pylori.
Suitable bacteriocin antimicrobial agents include nisin, gramicidin and tyrothricin including
derivatives and purified forms of bacteriocins such as Ambicin N.
The invention also includes a method of treatment of gastric disorders associated with Helicobacter
pylori in mammals which method comprises the administration to a mammal in need of treatment of
an effective amount of a bacteriocin antimicrobial agent.
A pharmaceutical composition for use in the treatment of gastric disorders associated with
Helicobacter pylori, comprising a bacteriocin antimicrobial agent and a pharmaceutically acceptable
carrier also forms part of the present invention.
It has been found that treatment of H. pylori associated gastritis with antimicrobial agents given by a
conventional oral dosing regimen may require a prolonged course of therapy to be effective.
Furthermore, follow-up of patients cleared of H.pylori infection by antimicrobial treatment has shown
that relapse (rather than reinfection) can be a problem.
In another aspect of the invention, substances having activity against H. pylori may be formulated as
gastric controlled release compositions, more especially as compositions which prolong residence
time of the antimicrobial agent within the stomach.
Bioadhesive materials have received considerable attention as platforms for controlled drug deliver.
They can be targetted, to specific drug administration sites, prolong the residence time and ensure
99/1006
an optimal contact with the absorbing surface. Many different types of bioadhesive materials, both
natural and synthetic, can be used in the design of controlled drug delivery systems.
Sucralfate, a basic aluminium sulphate sucrose complex, is an ulcer-preventing agent having antipepsin and antiadd properties. It has gastric muco-adherent properties such that when administered
orally it reacts with gastric juice to form a sticky paste which protects the mucosa by coating, and
also binds to ulcer-affected sites. The preparation and use of sucralfate is described in for example
U. S. Pat. No. 3432489 and "The Merck Index" 11th edition (1989) p1400 entry No 8853.
EP-A-0 403 048 (Warner-Lambert) describes medicated compositions comprising sucralfate and a
therapeutically effective amount of a medicament which is a) substantially water insoluble, or b) a
mixture of a water-soluble medicament and a release-delaying material which on admixture forms a
substantially water-insoluble medicament. The specification identifies gastro-unstable medicaments,
including peptides, as suitable compounds for formulation with sucralfate. Gastro-unstable
medicaments are defined in EP-A 0 403 048 as compounds which are degraded in the gastric fluids
of the stomach. Surprisingly, bacteriocin peptide antimicrobial agents have been found to remain
stable under the acid conditions prevailing in the stomach; they do not therefore fall within the class
of gastro-unstable peptides as defined by Warner-Lambert.
U.S. Pat. No. 4,615 697 (Robinson et al.) discloses a controlled release composition comprising an
effective amount of a treating agent, which may be a medicament, and a bioadhesive material which
is a water-swellable and water-insoluble, fibrous, cross-linked carboxy-functional polymer. The
controlled release compositions are described as adhering to the skin or the mucous membranes in
the presence of water.
In a further aspect, the present invention relies on the co-formulation or co-administration of a
bacteriocin antimicrobial agent which is active against Helicobacter pylori with one or more
substances capable of providing a controlled release of the antimicrobial agent in the stomach. In
particular, the present invention relies on the incorporation of a bacteriocin antimicrobial agent which
is active against H.pylori into the sticky paste formed by a muco adherent, for example sucralfate, in
situ, providing a sustained release or prolonged retention of the antimicrobial agent in the stomach,
so as to overcome, or at least mitigate, the disadvantages associated with conventionally formulated
antimicrobial agents and provide an effective treatment for H.pylori infections of the gastric and
duodenal mucosa in humans and domestic animals.
100/1006
Accordingly, the present invention provides pharmaceutical compositions comprising one or more
bacteriocin antimicrobial agents effective against H. pylori organisms, and one or more substances
providing a sustained release and/or prolonged retention of the bacteriocin antimicrobial agent in the
stomach.
The present invention extends to these compositions for use in therapy and to the use of these
compositions in the manufacture of a medicament for the treatment of gastric disorders associated
with Helicobacter pylori.
The antimicrobial agent may for example be co-formulated, suitably by intimate admixture, with a
muco-adherent or bioadhesive substance to form a bioadhesive complex. Such a complex confers
the additional benefit of locally targetting the antimicrobial agent to the mucus layer of the stomach
wall.
Bioadhesive materials suitable for use in compositions of the present invention include materials,
both natural and synthetic, which are capable of adhering to biological surfaces such as mucus
membranes. Examples of bioadhesive materials include natural gums and plant extracts and
synthetic materials such as sucralfate, cellulose derivatives, acrylic acid and methacrylic acid
derivatives, for example cross-linked acrylic and methacrylic acid copolymers available under the
Trade Names CARBOPOL and POLYCARBOPHIL.
Antimicrobial agents effective against H. pylori may alternatively be formulated to produce a floating
alginate raft within the stomach. Such formulations may include solid and liquid dosage forms, and
may be prepared according to processes known to persons skilled in the art.
Controlled release dosage forms, for example beadlets or granules, optionally encapsulated or
compressed to form tablets, also form part of the invention. Advantageously, beadlets or granules
are coated, layered, or form an intimate, homogeneous matrix with release-delaying materials. Such
dosage forms may be prepared using conventional techniques known in the art.
The bacteriocin antimicrobial agent is present in compositions of the invention in an appropriate
amount to provide an effective dose, which will depend on the pharmacological properties of the
antimicrobial agent employed. Normally a single dose used to treat an adult human will provide up to
20 g, generally 0.5 to 4.0 g of antimicrobial agent.
101/1006
Suitably the composition of the invention comprises from 0.1 to 5 parts by weight of antimicrobial
agent per part by weight of bioadhesive agent or muco-adherent, more preferably from 0.5 to 1 part
of antimicrobial agent per part of bioadhesive agent or muco-adherent, for example of sulfacrate.
A composition of the present invention may also include additional therapeutic agents useful in the
treatment of peptic ulcers and gastritis, and agents which delay gastric emptying, for example
methylcellulose, guar gum, fats such as triglyceride esters, and triethanolamine myristate.
A composition of the invention may be made up in the form of a swallow tablet, a chewable tablet or
a water dispersible tablet. Alternatively it may be supplied as a water-dispersible powder, either for
dispersion immediately prior to administration or for dispensing in liquid form, as a suspension or as
a liquid emulsion. Suitable water-dispersible formulations include soluble effervescent or noneffervescent powders.
The compositions of the present invention may also contain appropriate additives, for example
preservatives, buffering agents, suspending agents, flavorings, bulking agents, binders, adhesives,
lubricants, disintegrants, colouring agents, sweeteners, adsorbents, thickeners, suspending agents,
and diluents including water, appropriate to their form.
Coatings able to retard the release of pharmaceuticals are well known in the art of pharmaceutical
formulation, and include polymers such as acrylic resins (for example the material sold by Rohm
Pharma under the trade name `Eudragit`) and cellulose esters (for example ethyl cellulose). If
desired, the release of the antimicrobial agent may be altered by changing the particle size of a
coated or encapsulated antimicrobial agent.
An encapsulated or delayed release formulation according to the invention may be any such form
well known in the art. Suitable coating materials include water-based coatings, solvent-based
coatings and colloidal dispersions. Lipids may also be used to form liposome-type formulations.
It has also been found that the activity of bacteriocin antimicrobial agents active against Helicobacter
pylori may be enhanced if these agents are administered in combination with various materials which
are not recognised as antimicrobial agents per se, for example chelating agents, surfactants and
mixtures thereof.
102/1006
Accordingly, bacteriocin antimicrobial agents and compositions containing bacteriocin antimicrobial
agents as hereinbefore described for use in the treatment of Helicobacter pylori, further comprising a
chelating agent, a surfactant or mixtures thereof also form part of the invention.
Suitable chelating agents include alkyldiamine tetraacetates, for example ethylenediaminetetraacetic
acid (EDTA), CaEDTA, and CaNa2 EDTA, EGTA and citrate.
Suitable surfactants include ionic and non-ionic surfactants. Examples of non-ionic surfactants
include glycerides and the materials commercially available under the Trade Names Tweens and
Tritons. Ionic surfactants include fatty acids and quaternary compounds, the anionic surfactant
sodium dodecyl sulphate, and amphoteric surfactants such as cocamidopropyl betainc and
emulsifiers.
A composition of the invention may be administered as often as a physician directs, having regard to
the severity of the H.pylori infection. Normally, it is recommended to take a dose two or three times
daily, advantageously after meals.
Compositions of the invention may be prepared by conventional pharmaceutical techniques. Thus
compositions may, for example, be prepared by mixing together the required ingredients with stirring
or grinding to ensure adequate dispersion. Alternatively, some of the ingredients may be mixed
together before other ingredients are added. Granulation and/or coating techniques may be used at
a convenient stage in the process if required.
The invention will now be illustrated by the following examples.
EXAMPLE 1
Activity of Bacteriocin Antimicrobial Agents against Helicobacter Pylori
The Minimum Inhibitory Concentration (MIC) of bacteriocin antimicrobial agents against a human
strains of H.pylori (H.pylori NCTC 11916 and NCTC 11637) were assessed using a spiral plater and
automatic agar plate pourer, according to the method of Wallace A. S. and Corkill J. E. (J. Microbial.
Methods, 10, 303-310, 1989). MIC values were calculated according to the equation for the
Archimedes spiral.
103/1006
Kill time was calculated as the time taken to reduce microbe levels to <200 viable organisms using a
drug concentration equal to 4 times the average MIC value for the test compound.
______________________________________
MIC Value (mg/l)
Compound NCTC 11916 NCTC 11637 Kill Time
______________________________________
Tyrothricin
1.0-4.0 1.0-5.0 <1
Gramicidin
0.5-3.0 1.0-3.5 >30
Ambicin N
50-300 50-300 >30
______________________________________
EXAMPLE 2
Effect of Chelating Agent on activity of Bacteriocin Antimicrobial Agents against Helicobacter Pylori
A kill time assay was carried out to determine the effect of citrate on antimicrobial activity. Microbe
survival was assessed after treatment with bacteriocin alone and bacteriocin plus citrate. The effect
of citrate alone was also measured. Survival was measured at 1,5 and 30 minutes after inoculation.
Activity is expressed as the mean number of counts of colony forming units (CFU) per ml.
______________________________________
Mean Counts CFU/ml
Compound 1 min 5 min 30 min
______________________________________
Citrate (10 mM)
7.98 .times. 10@4
8.64 .times. 10@4
6.92 .times. 10@4
Ambicin N (300 ppm)
6.31 .times. 10@4
5.71 .times. 10@4
4.72 .times. 10@4
104/1006
Ambicin/Citrate
8.42 .times. 10@4
5.87 .times. 10@4
2.94 .times. 10@4
(Initial Inoculum =
8.92 .times. 10@4)
Citrate (10 mM)
1.59 .times. 10@4
1.42 .times. 10@4
1.95 .times. 10@4
Gramicidin 7.69 .times. 10@3
1.44 .times. 10@4
1.35 .times. 10@4
Gramicidin/Citrate
1.17 .times. 10@3
1.39 .times. 10@3
1.71 .times. 10@3
(Initial Inoculum =
2.39 .times. 10@5)
Citrate (10 mM)
1.59 .times. 10@4
1.42 .times. 10@4
1.95 .times. 10@4
Tyrothricin 2.55 .times. 10@4
1.77 .times. 10@4
1.78 .times. 10@4
Tyrothricin/Citrate
1.08 .times. 10@4
7.94 .times. 10@3
1.74 .times. 10@4
(Initial Inoculum =
2.39 .times. 10@5)
______________________________________
EXAMPLE
105/1006
______________________________________
Formulation Examples
mg.
______________________________________
i) Gramicidin
500
Sucralfate
500
Carboxymethyl sodium starch glycollate, (dried)
30
Magnesium stearate
20
Silica
12
Microcrystalline Cellulose to 1500
ii) Ambicin N
500
Sucralfate
500
Carboxymethyl sodium starch glycollate, (dried)
30
Magnesium stearate
20
Silica
12
Microcrystalline Cellulose to 1500
______________________________________
Method:
All the ingredients except for one third of the magnesium stearate are screened, blended, and
compressed on a rotary tabletting machine to fore slugs. The slugs are milled, blended with the
remaining magnesium stearate, and recompressed to fore the final tablets.
iii) A tablet was prepared according to standard methods including tyrothridn (500 mg) and
encapsulated with POLYCARBOPHIL.
iv) A tablet was prepared according to standard methods including nisin (500 mg) and alginate (1 g).
v) An effervescent powder was prepared containing per 5 g dose:
______________________________________
tyrothricin:
500 mg
citric acid (anhydrous):
2.2 g
sodium bicarbonate: 2.3 g
sodium carbonate: 0.5 g
______________________________________
106/1006
Claims:
We claim:
1. A pharmaceutical composition comprising a bacteriocin antimicrobial agent selected from the
group consisting of nisin, gramicidin, tyrothricin and analogs thereof in combination with a
mucoadherent or bioadhesive release-sustaining agent which is a natural gum, a plant extract,
sucralfate, a cellulose derivative or an acrylic acid or methacrylic acid derivative in a
pharmaceutically acceptable carrier.
2. A method of treating gastric disorders associated with Helicobacter pylori in mammals which
comprises administering to a mammal in need of such treatment a pharmaceutical composition
according to claim 1 comprising an amount of the bacteriocin antimicrobial agent effective to treat
the disorder.
107/1006
11. EP0640291 - 01.03.1995
USE OF BACTERIOCIN PRODUCING MICROORGANISMES TO CURE RAW SAUSAGE
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0640291
Inventor(s):
TICHACZEK PETRA S (DE); POHLE SIMONE B (DE); VOGEL RUDI F DR (DE);
HAMMES WALTER P PROF DR (DE)
Applicant(s):
MUELLER KARL and CO KG (DE)
IP Class 4 Digits: A23B; A23L
IP Class:
A23B4/22; A23L1/314
E Class: A23B4/22; A23L1/314D; A23B4/12; A23L1/317B
Application Number:
EP19940112599 (19940812)
Priority Number: DE19934328368 (19930824)
Family: EP0640291
Equivalent:
DE4328368; DK640291T
Cited Document(s):
CH347421; US3814817; EP0321692
Abstract:
THE INVENTION RELATES TO THE USE OF BACTERIOCIN-PRODUCING MICROORGANISMS FOR
RIPENING RAW SAUSAGE AND A COMPOSITION FOR RIPENING RAW SAUSAGE WHICH
CONTAINS THE BACTERIOSIN-PRODUCING MICROORGANISMS. THE MICROORGANISMS USED
ARE IN PARTICULAR LACTOBACILLI, INCLUDING LACTOBACILLUS CURVATUS DSM 8430 AND
LACTOBACILLUS SAKE DSM 6742.Description:
108/1006
Die Erfindung betrifft die Verwendung von Bacteriocin erzeugende Mikroorganismen zum Reifen von
Rohwurst sowie ein Mittel zum Reifen von Rohwurst, das Bacteriocin erzeugende Mikroorganismen
enthдlt. Als Mikroorganismen werden insbesondere Laktobazillen eingesetzt, darunter Laktobazillus
curvatus DSM 8430 und Laktobazillus sake DSM 6742, aber auch verschiedene Pediokokken,
insbesondere Stдmme von Pediococcus acidilactici. Die neuen Mikroorganismen wurden bei der
deutschen Sammlung fьr Mikroorganismen und Zellkulturen DSM nach den Bestimmungen des
Budapester Vertrags hinterlegt und werden dort unter den Eingangsnummern DSM 8430 bzw. DSM
6742 gefьhrt.
Es ist seit langem bekannt, zur Fermentierung von rohen Pцkelfleischwaren (Absдuerung)
Milchsдurebildner als Starterkulturen einzusetzen. Diese konventionellen Sдurebildner entstammen
bisweilen fremden Biotopen und sind im allgemeinen auf ihre Leistungsfдhigkeit und ihre
Prдparationsmцglichkeit hin selektiert.
DE 37 39 989 C1 beschreibt den Mikroorganismus Laktobazillus curvatus DSM 4265, der zur
Herstellung von Pцkelfleischwaren geeignet ist. Dieser Mikroorganismus wird in einer Menge von 5 X
10 Keimen pro kg Wurstmasse in gefriergetrocknetem Zustand zugesetzt. Die Reifung unter
kontrollierten Klimabedingungen dauert etwa 10 Tage und fьhrt zu einer Absenkung des pH-Werts,
zur Ausbildung eines typischen Aromas sowie zu einer Stabilisierung der Rohwurst durch
Behinderung der spontanen Sдurebildnerflora.
Insgesamt hat sich aber gezeigt, dass der Reifungsprozess mit solch herkцmmlichen Laktobazillen
bei relativ hohen Reifungstemperaturen von bis zu 25 DEG C immer in Konkurrenz zur Spontanflora
stattfindet. Da der Reifungsprozess relativ langwierig ist, nimmt die Keimzahl der Spontanflora nicht
unerheblich zu, was nicht nur unter hygienischen Gesichtspunkten unerwьnscht ist, sondern auch
unter dem Gesichtspunkt der Kontrollierbarkeit des Reifungsprozesses.
Des weiteren wurden zur Unterdrьckung von unerwьnschter Spontanflora besonders
wachstumsstarke Stдmme der Gattung Laktobazillus entwickelt. Es hat sich aber gezeigt, dass
diese wachstumsstarken und konkurrenzfдhigen Stдmme mit der Zeit zur Selektionierung einer
konkurrenzfдhigen Spontanflora fьhren kцnnen, die den Vorteil der Wachstumsstдrke wieder
zunichte machen. Ausserdem ist es erforderlich, auch diese wachtumsstarken Stдmme in relativ
hohen Keimzahlen einzusetzen, damit sie sich gegenьber der Spontanflora durchsetzen kцnnen.
109/1006
Erst der hierdurch vermittelte Wachstumsvorsprung fьhrt zur weitgehenden Unterdrьckung der
Spontanflora.
Es ist schliesslich bekannt, dass gewisse Stдmme, insbesondere auch der Gattung Laktobazillus,
sogenannte Bacteriocine freisetzen, die die Vermehrung anderer Stдmme der gleichen Gattung und
auch anderer Gattungen unterdrьcken. Bacteriocine sind Peptide, Proteine oder Proteinkomplexe
mit bakteriocider Wirkung, die vor allem gegen artverwandte Mikroorganismen wirksam werden und
deren Wachstum unterdrьcken. Beispiele bekannter Bacteriocine sind Curvacin, Sakacine sowie
Pediocine.
Ziel der Erfindung ist die Bereitstellung eines Mikroorganismus, der als Starterkultur zur Reifung von
Fleisch- und Wurstwaren, insbesondere von Rohwurst eingesetzt werden kann und geeignet ist, die
konkurrierende Spontanflora weitestgehend zu unterdrьcken. Gleichzeitig soll eine solche
Starterkultur zu einem gleichmдssigen, qualitativ und geschmacklich einwandfreien Produkt mit
einer durchgehenden Umrцtung und einer stabilen Pцkelfarbe fьhren.
Dieses Ziel wird durch die Verwendung Bacteriocin erzeugender Mikroorganismen, besonders der
Gattung Laktobazillus und insbesondere der Stдmme Laktobazillus curvatus DSM 8430 und
Laktobazillus sake DSM 6742 erreicht. Besonders geeignet sind diese Mikroorganismen fьr die
Reifung von schnittfester Rohwurst, aber auch fьr die Erzeugung streichfдhiger Rohwurst.
Es wurde gefunden, dass die von diesen Mikroorganismen erzeugten Bacteriocine die spontane
Konkurrenzflora praktisch vollstдndig unterdrьcken, ohne dass sich Nachteile fьr die Haltbarkeit
und Vertrдglichkeit der erzeugten Wurstwaren ergeben. Die damit hergestellten Produkte genьgen
geschmacklich und qualitativ allen an sie gestellten Anforderungen und weisen eine optimale
Umrцtung auf.
Die Erfindung betrifft ferner ein Mittel zum Reifen von Rohwurst, das die oben genannten
Mikroorganismen enthдlt. Zweckmдssigerweise enthдlt das Mittel den jeweiligen Stamm in
gefriergetrocknetem Zustand, ggf. neben ьblichen weiteren Bestandteilen. Ьbliche weitere
Bestandteile sind beispielsweise Nдhrstoffe und -salze. Bacteriocinresistente ьbliche Co-Starter, der
Gattungen Micrococcaceae oder Staphyloccaceae kцnnen ebenfalls zugegen sein.
Vorzugsweise wird das Mittel in Form einer Einheitspackung konfektioniert, die eine fьr die jeweils
gewьnschte Rohwurstmenge ausreichende Keimzahl des jeweiligen Mikroorganismus enthдlt.
110/1006
Die Erfindung betrifft ferner die Bacteriocin erzeugende Mikroorganismen Laktobazillus curvatus
DSM 8430 und Laktobazillus sake DSM 6742, sowie unter Verwendung dieser und anderer
Bacteriocine erzeugender Mikroorganismen gereifte Fleischwaren, insbesondere Rohwurst.
Die erfindungsgemдssen Mikroorganismen werden in einer Menge von 10 bis 10 CFU/kg
Wurstmasse eingesetzt, insbesondere in einer Menge von 10 bis 10 CFU/kg.
Die erfindungsgemдssen Mikroorganismen Laktobazillus curvatus DSM 8430 und Laktobazillus sake
DSM 6742 haben u. a. den Vorteil, dass sie ьber eine Plasmiduntersuchung schnell und einfach
nachgewiesen werden kцnnen.
Es wurde ьberraschend befunden, dass die von den Mikroorganismen der Erfindung erzeugten
Bacteriocine, darunter Curvacin A (L. curvatus DSM 8430) und Sakacin P (L. sake DSM 6742) sich
im Laufe des Reifungsverfahrens im Produkt selbst abbauen. Bei der Lagerung bei 4 DEG C nahm
die Bacteriocinaktivitдt innerhalb von 8 Tagen auf einen Wert von 50 % ab; nach 21 Tagen Lagerung
war bei dieser Temperatur keine Bacteriocinaktivitдt mehr nachweisbar. Dies bedeutet, dass unter
den bei der Rohwurstherstellung ьblichen Lager- und Nachreifebedingungen die
Bacteriocinaktivitдt aus dem Produkt verschwindet, bevor es in den Handel gelangt.
Die erfindungsgemдssen Starterkulturen fьhren bei guter Sдuerung der damit behandelten
Rohwurst zu einer kontrollierten und standardisierten Fermentierung. Die Behinderung der spontanen
Mikroorganismenflora vermindert das hygienische Risiko und fьhrt zur Ausbildung eines fьr den
erfindungsgemдssen Mikroorganismus charakteristischen Fermentationsaromas. Die bei der
Fermentierung ausgebildete typische Pцkelfarbe erweist sich ьber die Haltbarkeitsdauer der Wurst
als stabil.
Die Fermentierungseigenschaften des Mikroorganismus Laktobazillus curvatus DSM 8430 wurden,
teilweise in Konkurrenz zu anderen Laktobazillen, nдher untersucht. Die Ergebnisse sind in den
beigefьgten Abbildungen dokumentiert. Von diesen Abbildungen zeigt
Fig. 1 das Wachstum von L. curvatus DSM 8430 in Abhдngigkeit von der Zeit und dem
eingesetzten Inokulum sowie den aus der Fermentierung resultierenden pH-Wert;
Fig. 2A das Wachstum von L. curvatus DSM 8430 und L. curvatus LTH 683 bei Co-Inoculation
sowie
111/1006
Fig. 2B das Wachstum von nicht Bacteriocin erzeugenden Derivaten des Stammes L. curvatus DSM
8430 bei Co-Inoculation mit L. curvatus LTH 683 in Abhдngigkeit von der Zeit.
In Fig. 1 ist der Einfluss der inoculierten Menge L. curvatus DSM 8430 auf die schliesslich erreichten
Endwerte der Zelldichte (offene Symbole) und den schliesslich in der Rohwurst erreichten pH-Wert
(schwarze Symbole) dargestellt. Drei Chargen einer herkцmmlichen Rohwurstmischung wurden mit
10 (Quadrate), 10 (Kreise) und 10 (Dreiecke) CFU/g L. curvatus DSM 8430 inoculiert. Aus der Masse
wurden Wьrste hergestellt und Proben auf die Gesamtzahl der Laktobazillen untersucht. Die
Gegenwart von Bacteriocin produzierenden Mikroorganismen wurde durch Replikationsanalyse auf
BSM Agar (P.S. Tichaczek et al., System. Appl. Microbiol. 15, 460 (1992)) und Plasmidprofilanalyse
von Isolaten auf MRSS Agar (R.F. Vogel et al., System. Appl. Microbiol. 15, 129 (1992)) untersucht.
Das Wachstum von L. curvatus DSM 8430 in den Fermentationsmischungen ist in Fig. 1
wiedergegeben.In Wьrsten, die mit 10 oder 10 CFU/g des Mikroorganismus inoculiert worden waren,
wurden nur Bacteriocin erzeugende Kolonien in allen Fermentationsstadien nachgewiesen, d. h. alle
Kolonien erzeugten einen deutlichen Hemmhof im Indikatorrasen und alle Plasmidprofile waren
identisch mit dem von L. curvatus DSM 8430. In mit 10 CFU/g Mikroorganismus inoculierten Wьrsten
machte der Bacteriocinproduzent > 80 % aller nach 5 Tagen auf MRSS Agar reisolierten
Laktobazillen aus. Da im Ausgangsmaterial fьr die Wurstherstellung 2 X 10 CFU/g Laktobazillen vom
Wildtyp vorhanden waren, folgt, dass der zugesetzte Starter in der Lage ist, die Spontanflora trotz
ihres zweifachen Ьberschusses zu dominieren. In den mit 10 und 10 CFU/g inokulierten
Wurstmischungen wurde keine Spontanflora mehr nachgewiesen.
Ausweislich Fig. 1 wird mit allen zugesetzten Startermengen eine zufriedenstellende Sдuerung der
Rohwurstmasse von etwa pH 4,9 erreicht (schwarze Symbole).
Die relative Konkurrenzfдhigkeit des Bacteriocin produzierenden Stammes L. curvatus DSM 8430
wurde in Rohwьrsten untersucht, die mit verschiedenen Kombinationen von Laktobazillen als Starter
inoculiert wurden. Bei Inoculation mit 5 X 10 CFU/g L. curvatus und DSM 8430 und 5 X 10 CFU/g
eines hoch konkurrenzfдhigen kommerziellen Starters, L. curvatus LTH 683, der kein Bacteriocin
erzeugt, macht der Bacteriocin erzeugende Starter nach 3- bzw. 5-tдgiger Fermentierung 100 % der
in der Rohwurstmasse nachgewiesenen Laktobazillen aus (Fig. 2A). Dies zeigt die absolute
Dominanz des Bacteriocin erzeugenden Starters. In Fig. 2A bezeichnen die Kreise die Keimzahlen
an L.c. LTH 683, die (teilweise ьberlagerten) Rauten die an L.c. DSM 8430 und die schwarzen
Quadrate die Gesamtkennzahl der Laktobazillen.
112/1006
In einer weiteren Untersuchung wurde der Beitrag der Bacteriocinproduktion auf die
Konkurrenzfдhigkeit des Bacteriocinerzeugers in einer Vergleichsstudie mit einer Variante von L.
curvatus DSM 8430 (Bac Ery) untersucht, der die Fдhigkeit zur Bacteriocinerzeugung fehlt. Es
wurden jeweils 5 X 10 CFU/g L. curvatus DSM 8430 (Bac) und L. curvatus LTH 683 inoculiert. Zur
Erleichterung der Identifizierung wies die nicht Bacteriocin erzeugende Variante von DSM 8430 ein
Erythromycin-Resistenzgen auf (Ery). Nach 5-tдgiger Fermentierung in einer StandardRohwurstmasse machte der Stamm L.c. DSM 8430 (Bac Ery) etwa 50 % der in der Rohwurstmassen
vorhandenen Laktobazillen aus, der Stamm LTH 683 ebenfalls etwa 50 %. Eine Plasmidprofilanalyse
zeigte, dass andere Stдmme der Spontanflora nicht vorhanden waren. Das Ergebnis ist in Fig. 2B
dargestellt (L.c. LTH 683: Kreise; L.c.DSM 8430 (Bac Ery): Dreiecke; Laktobazillen gesamt: schwarze
Quadrate). Es zeigt sich, dass der Stamm DSM 8430 (Bac Ery) in etwa die gleiche Konkurrenzstдrke
hat, wie der bekannte Stamm LTH 683. Ein entsprechendes Ergebnis wurde mit L.c. DSM 8430 (Bac),
einer Variante ohne die Fдhigkeit Bacteriocin zu erzeugen und ohne das Resistenzgen sowie L.c.
LTH 683 erzielt. Die Erhцhung der Konkurrenzfдhigkeit gemдss Darstellung in Fig. 2A beruht somit
ausschliesslich auf der Fдhigkeit von DSM 8430, Bacteriocin zu erzeugen.
Zum Vergleich wurden die sensorischen Eigenschaften von mit L.c. DSM 8430, LTH 683 sowie L.s.
LTH 677 und 673 bestimmt. Die bekannten Starter L.c. LTH 683 und L.s. LTH 677 werden zur
Erzeugung von fermentierten Rohwьrsten hoher Qualitдt verwandt. Mit jeweils 10 CFU/g der oben
genannten Stдmme inokulierte Wurstmassen wurden nach Standardverfahren gereift. Das
Wachstum der Starterkulturen und der Abnahme des pH-Werts verliefen gleichfцrmig und fьhrten
jeweils zu Endwerten von 8 X 10 CFU/g der jeweiligen Starterkultur und einem pH-Wert von 5,2 nach
5-tдgiger Fermentierung. Durch Plasmidprofilanalyse konnte in allen Fдllen sichergestellt werden,
dass der Ausgangsstarter im Produkt dominierte. Nach 21-tдgiger Fermentierung und Reifung
wurden die Produkte optisch und sensorisch nach Chow et al., Taiwan Sugar 34, 19 (1987) bestimmt.
Hinsichtlich Geschmack und Aussehen schnitt L.c. DSM 8430 am besten ab. L.s. DSM 6742 erzielte
ebenfalls дusserst zufriedenstellende Werte.
Es ist zu berьcksichtigen, dass mit den erfindungsgemдssen Bacteriocinerzeugern das Inoculum
ohne weiteres auf 10 bis 10 CFU/g abgesenkt werden kann, da aufgrund der Wachstumsstдrke des
Mikroorganismus die Konkurrenzflora auch bei diesem Inoculum noch wirksam unterdrьckt wird.
Der Vorteil der erfindungsgemдss eingesetzten Bacteriocin erzeugenden Mikroorganismen und
insbesondere Laktobazillen liegt vor allem in der Verminderung des hygienischen sowie des
113/1006
sensorischen qualitдtsbeeinflussenden Risikos, das aus dem Wachstum unerwьnschter
Spontanflora herrьhrt. Das hygienische Risiko ist zwar bei Rohwьrsten mit sehr niedrigen pH-Werten
vergleichsweise gering, steigt jedoch mit zunehmendem Wassergehalt und zunehmenden pHWerten des Produktes stark an. Verbunden ist dieses hygienische Risiko in allen Fдllen auch mit
unerwьnschten optischen und geschmacklichen Verдnderungen am Produkt. Wie die vorstehend
geschilderten Experimente zeigen, beruht die Verminderung dieses qualitativen und hygienischen
Risikos vor allem auf der Erzeugung von Bacteriocinen durch den eingesetzten Starter mit dieser
Fдhigkeit.Dabei wurde auch gefunden, dass die Bacteriocine nicht nur zu einer spezifischen
Inhibierung von anderen Laktobazillen fьhren, sondern auch geeignet sind, Mikroorganismen
anderer Gattungen zu unterdrьcken, beispielsweise Listeria monocytogenes. Im ьbrigen weist
insbesondere L. curvatus DSM 8430 eine ausgezeichnete Konkurrenzstдrke auf, die die
Grцssenordnung des bekannt konkurrenzstarken L. curvatus LTH 683 ьbertrifft.
Es wurde schliesslich auch gefunden, dass das von L. curvatus DSM 8430 erzeugte Bacteriocin
Curvacin A keinen Einfluss auf das Wachstum von Micrococcus varians und Staphylococcus
carnosus hat, die in einigen Starterzubereitungen zusammen mit Laktobazillen eingesetzt werden.
L. curvatus DSM 8430 ist ausgesprochen kochsalztolerant bis zu einer Menge von 40 g/l. Dabei hat
die Kochsalzkonzentration keinen Einfluss auf die Fдhigkeit des Mikroorganismus, Curvacin A zu
erzeugen. Die Fermentierung kann ferner bei Temperaturen unterhalb 25 DEG C bis hinunter zu
Temperaturen unterhalb 15 DEG C erfolgen, ohne dass die Fдhigkeit zur Erzeugung von Curvacin A
verlorengeht. Das Optimum der Curvacin A-Produktion liegt bei Temperaturen zwischen 15 und 20
DEG C. Diese Fдhigkeit zur Bacteriocinerzeugung auch bei niedrigen Fermentierungstemperaturen
ist ein nicht zu unterschдtzender Vorteil, da erfahrungsgemдss die Fermentierungstemperatur einen
Einfluss auf den Verlauf der Fermentierung, die die hygienischen Eigenschaften des Produkts und
die Vermehrung der Spontanflora hat.
Die Erfindung wird durch das folgende Ausfьhrungsbeispiel nдher erlдutert.
Beispiel
Laktobazillus curvatus DSM 8430 wurde zur Herstellung von schnittfester Rohwurst (Salami)
verwandt.
114/1006
Materialzusammenstellung
30 kg Rindfleisch, grob entsehnt, sichtbarer Fettanteil 5 %,
30 kg Schweinefleisch, sehnenfrei, sichtbarer Fettanteil 5 %,
20 kg Schweinebauch, sichtbarer Fettanteil 60 %,
20 kg Rьckenspeck, ohne Schwarte
Zusдtze je 1 kg Material
28,0 g Kochsalz
0,3 g Kaliumnitrat
4,0 g Glukose
2,0 g Pfeffer, weiss, gemahlen
5 X 10 CFU Laktobazillus curvatus DSM 8430, gefriergetrocknet
5 X 10 CFU Staphylococcus carnosus, gefriergetrocknet (zur Optimierung der Umrцtung,
handelsьbliche Qualitдt)
Zur Herstellung der Rohwurstmasse wurden die Materialien jeweils in Stьcke geschnitten, das
Rindfleisch, der Schweinebauch und der Speck hartgefroren und das Schweinefleisch gut
durchgekьhlt und unmittelbar vor der Verarbeitung in ьblicher Weise gewolft.
Die gefriergetrockneten Starterkulturen Laktobazillus curvatus DSM 8430 und Staphylococcus
carnosus wurden in 500 ml Wasser mit einer ьblichen Reaktivierungsmischung eingerьhrt.
Das Rindfleisch wurde bei Zugabe der Starterkulturen im Kutter so lange vorzerkleinert, bis die
Masse etwas bindet. Danach wurden das Kaliumnitrat, die Glukose sowie der Pfeffer hinzugefьgt
und die Masse noch eine kurze Zeit im Kutter weiter laufengelassen, um eine ausreichende
Vermischung zu erzielen. Danach wurden der Speck und der Schweinebauch zugesetzt. Es wurde
so lange weitergekuttert, bis der Speck eine Kцrnung von 6 - 8 mm aufwies. Anschliessend wurde
das vorgewolfte Schweinefleisch mit dem Kochsalz eingekuttert und die Gesamtmasse so lange
weitergekuttert, bis das Fett die ьbliche Kцrnung von etwa 2 mm und die Wurstmasse Bindung
aufwies.
115/1006
Die fertige Wurstmasse mit einer Temperatur von -2 DEG C wurde in wasserdurchlдssige
Hautfaserdдrme vom Kaliber 70 mm eingefьllt. Anschliessend wurden die gefьllten Dдrme zur
Reifung in die Klimakammer verbracht.
Zunдchst wurden die Wьrste bei 70 % relativer Luftfeuchtigkeit und etwa 18 DEG C Temperatur 6
Stunden vorkonditioniert, um das дussere Schwitzwasser wegzutrocknen. Danach wurde zur
weiteren Konditionierung die relative Luftfeuchtigkeit 18 Stunden auf 94 % erhцht. Die eigentliche
Reifung erfolgte bei einer Anfangstemperatur von 24 DEG C ьber einen Zeitraum von 36 bis 48
Stunden, wobei die Temperatur und Luftfeuchtigkeit langsam auf ьbliche Bedingungen
zurьckgenommen und die Wьrste fertiggereift wurden.
Nach der Trocknung und der ьblichen Nachreife wurde ein pH-Wert von etwa 5,0 festgestellt. Die
Wьrste wiesen eine hervorragende Umrцtung sowie eine sehr haltbare und ansprechende
Pцkelfarbe im Anschnitt auf. Die sensorische Prьfung ergab ein qualitativ ьberdurchschnittliches
Produkt. Claims:
1. Verwendung von Bacteriocin erzeugenden Mikroorganismen, insbesondere der Gattung
Laktobazillus zum Reifen von Fleischwaren, insbesondere von Rohwurst.
2. Verwendung nach Anspruch 1, dadurch gekennzeichnet, dass die Bacteriocin erzeugenden
Mikroorganismen in einer Menge von 10 bis 10 CFU/kg Rohwurstmasse eingesetzt werden.
3. Verwendung nach Anspruch 2, dadurch gekennzeichnet, dass die Bacteriocin erzeugenden
Mikroorganismen in einer Menge von 10 bis 10 CFU/kg Rohwurstmasse eingesetzt werden.
4. Verwendung nach einem der vorstehenden Ansprьche, dadurch gekennzeichnet, dass als
Bacteriocin erzeugende Mikroorganismen Laktobazillus curvatus DSM 8430 oder Laktobazillus sake
DSM 6742 eingesetzt werden.
5. Mittel zum Reifen von Rohwurst, dadurch gekennzeichnet, dass es Bacteriocin erzeugende
Mikroorganismen, insbesondere der Gattungen Laktobazillus und Pediococcus enthдlt.
116/1006
6. Mittel nach Anspruch 5, dadurch gekennzeichnet, dass es die Mikroorganismen in
gefriergetrocknetem Zustand enthдlt.
7. Mittel nach Anspruch 5 oder 6 in Form einer Einheitspackung mit einer fьr eine gewьnschte
Rohwurstmenge ausreichenden Keimzahl.
8. Mittel nach einem der Ansprьche 5 bis 7, dadurch gekennzeichnet, dass es als Mikroorganismen
Laktobazillus curvatus DSM 8430 oder Laktobazillus DSM 6742, enthдlt.
9. Laktobazillus curvatus DSM 8430 und Laktobazillus sake DSM 6742.
10. Unter Verwendung der Mikroorganismen nach Anspruch 9 oder eines Mittels nach einem der
Ansprьche 5 bis 8 gereifte Rohwurst.
117/1006
12. EP0705843 - 10.04.1996
BACTERIOCIN FROM ENTEROCOCCUS FEACIUM ACTIVE AGAINST LISTERIA MONOCYTOGENES
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0705843
Inventor(s):
HUGAS MAURICI MARTA (ES); GARRIGA TURON MARGARITA (ES); MONFORT
BOLIVAR JOSEP M (ES); YLLA ULLASTRE JOSEP (ES)
Applicant(s):
INST RECERCA I TECNOLOGIA AGRO (ES); CASA TARRADELLAS SA (ES)
IP Class 4 Digits: A23L; C07K
IP Class:
C07K14/315; A23L3/3526
E Class: A23L3/3463A; C07K14/315; A23L3/3526
Application Number:
EP19940924890 (19940829)
Priority Number: WO1994ES00081 (19940829); ES19930001882 (19930831)
Family: EP0705843
Equivalent:
ES2068157; WO9506663
Abstract:
THE PRESENT INVENTION RELATES TO A NEW BACTERIOCINE AGAINST LISTERIA
MONOCYTOGENES, OBTAINED FROM THE CULTURE OF A NEW STRAIN OF ENTEROCOCCUS
FAECIUM NAMED CTC492. THE BACTERIOCINE IS A PEPTIDE HAVING A MOLECULAR WEIGHT
OF 4,282 DALTON AND COMPRISES A SEQUENCE OF 35 AMINOACIDS AND MAY BE APPLIED
WITH SUBSTANTIAL EFFICIENCY FOR PREVENTING AND AVOIDING THE GROWTH AND
PROPAGATION OF LISTERIA MONOCYTOGENES IN FOOD, PARTICULARLY IN MEAT AND MEAT
PRODUCTS AND IN MILK AND MILK PRODUCTS.Description:
118/1006
FIELD OF THE TECHNIQUE
The present invention refers to a new bacteriocin, obtained from a new strain of Enterococcus
faecium, with a bactericide action against Listeria monocytogenes and with an application for
inhibiting growth and propagation of said patogenic microorganism on foods, especially on meat and
meat products and on milk and diary products.
PRIOR ART
Listeria monocytogenes is a patogenic microorganism that produces severe upsets on human
beings and animals and can be easily transmitted by means of contaminated foods, especially by
means of meat and meat products and milk and dairy products. Said patogenic microorganism
shows to be cooling resistant but results sensitive to acid conditions, so that low pH values.
In many cases, it is not possible to store and offer foods with a low pH value. In this way, nowadays,
the consumer refuses those meat products which are too acid and his preferences are directed to
fermented meat products with a low acid taste, then resulting in a sizeable increase of possible
contamination and growth of Listeria monocytogenes on them.
In accordance with Tagg, J.R. et al.; Bacteriol. Review, 40; 722-756 (1976), the term "Bacteriocin"
refers to peptide-like substances, produced by microorganisms, able to specifically inhibit the
growth of other bacteria by means of absortion to their specific receptors.
There have been various active bacteriocins disclosed against Listeria monocytogenes, useful for
combating food contamination by the same. In this way, Vendenbergh et al. (European Patent
Number 0 326 062) describe one method for inhibiting Listeria monocytogenes by using a
bacteriocin obtained from a strain of Pediococcus acidilactici. The same authors disclose in the
European Patent Application Number 0 453 719 that the bactericide activity is related to a 4,629
molecular weight fragment, isolated from the original 16,500 molecular weight bacteriocin.
119/1006
Marugg et al. describe in the European Patent Application Number 0 493 779 a cloned gene
encoding for a bacteriocin in Pediococcus acidilactici, useful for inhibiting the growth of Listeria on
foods. Kawase, Kozo et al. describe in the European Patent Application Number 0 503 939 an
antimicrobian peptide capable of inhibiting Listeria monocytogenes, among other microorganisms,
obtained from bovine Lactoferrine hydrolysis.
On the other hand, it is known that bacteria of Streptococcus faecium species, nowadays called
Enterococcus faecium, are capable of producing low molecular weight substances with
antimicrobian activity, which are not bacteriocins and are useful in preventive and curative kind
therapeutical treatements. In this way, Kawai, Yasuo et al. disclose, in Published Japanese Patent
Applications Numbers: 6153293 and 61109728, the use of a compound, having a molecular formula
of C11H15O5N5, obtained from Streptococcus faecium, in toothcare products. In turn, Lewenstein et
al. disclose, in European Patent Application Number 0 405 569, a product having antimicrobian
activity, obtained from Streptococcus faecium cultures, for the treatement and prevention of the
gastrointestinal disorders caused by, among other microorganisms, bacteria of genus Listeria.Said
product has a molecular weight lower than 1,000 and it is not a bacteriocin.
Bacteriocins against Listeria monocytogenes described heretofore have various advantages and
drawbacks but there still remains the necessity to find alternative high efficiency and availability ways
which would allow the Food Industry to efficiently attack the risks of contamination of its products by
Listeria monocytogenes.
The authors of the present invention have discovered a new bacteriocin produced by a new strain of
Enterococcus faecium, with a surprising specific activity against Listeria monocytogenes, and which
is very accessible and easy to be obtained.
OBJECT OF THE INVENTION
The present invention is directed to provide a new bacteriocin against Listeria monocytogenes,
highly effective combating the growth and propagation of Listeria monocytogenes on foods.
Another object of the present invention consist of providing a new bacterial strain of Enterococcus
faecium capable of producing said bacteriocin.
120/1006
A further object of the present invention comprises the use of said bacteriocin or said bacterial strain
in order to prevent and avoid the growth and propagation of Listeria monocytogenes in foodstuff.
DESCRIPTION OF THE INVENTION
The bacteriocin against Listeria monocytogenes which is object of the present invention is
characterized by being a peptide with a molecular weight larger than 4.000, obtained by means of
culturing the bacterial strain CTC492 of Enterococcus faecium. CTC492 is the designation provided
to said strain by the authors of the present invention and it corresponds to the "Colecciуn de Cultivos
del Centre de Tecnologia de la Carn, Institut de Recerca i Tecnologia Agroalimentбries" (Culture
Collection of The Centre of Meat Technology, Food-Farming Research & Technology Institute),
domiciled in Granja Camps i Armet s/n, 17121 Monells (Gerona), Spain.
A culture of said strain has been deposited in accordance with Budapest Treaty in relation to
international acceptance of microorganism deposits towards the procedure on the subject of the
CECT, Colecciуn Espa ola de Cultivos Tipo, de la Universidad de Valencia (Spanish Culture
Colection, University of Valencia), domiciled in Campus de Burjasot, 46100 Burjasot (Valencia),
Spain, which has assigned the deposit-number CECT 4453 to the same.
The strain CTC492 of Enterococcus faecium comprises Gram-positive, Catalase-negative non
sporulated cocci whose principal fermentation product is Lactic acid and which are capable of
producing alpha -hemolysis in blood-agar (48 hours at 30oC). This strain ferments Melibiose; it is
unable to ferment Melezitose and does not reduce Tripheniltetrazolium (TTZ) in a 0,1% Thallium
acetate medium. It presents a positive growth at 45oC and in 6,5% Sodium Chloride and positively
reacts with D-antigen. Its profile of several sugars fermentation is as follows:
Columns=3
Glucose +Ribose +Melibiose +
Mannose +Celobiose +Ramnose Saccharose +D-Rafinose -Melezitose Lactose +Maltose +Trehalose +
121/1006
All these characteristics agree with those described in "Bergey's Manual of Systematic Bacteriology",
vol 2, Baltimore; William and Willains (1986) for Enterococcus faecium, formerly Streptococcus
faecium, clasification.
Members of Enterococcus faecium group are passed by The International Diary Products Federation
as Food quality bacteria useful as starter cultures for dairy products.
Strain CTC492 can be cultured with an excellent yield on MRS culture broth (lactobacilli culture broth
according to De Man, Rogosa and Sharpe commercialized by companies DIFCO, OXOID, MERCK,
etc.) in aerobic conditions at a temperature of about 30oC.
The bacteriocin against Listeria monocytogenes which is object of the present invention, obtained
from strain CTC492, has a molecular weight of 4,282 Dalton and is a peptide that comprises 35
aminoacids in the following sequence:
Said amino-acid sequence is new and presents certain resemblance with the bacteriocins Pediocin,
Sakacin-A, Leucocin-A and Curvacin-A.
By using a degenerated probe which comprises residues from 6 to 15, the authors of the present
invention have been able to determine, by said probe hybridation with the chromosomic and
pasmidic DNA of strain CTC492, that the producing gene of the bacteriocin object of the present
invention may be located in a chromosomic fragment EcoR1 of 12 to 14 Kb.
The bacteriocin object of the present invention, designated as Entericin CTC492 by its authors,
becomes inactive by means of treatment with proteolytic enzimes such as Tripsine, Nagarse or
Proteinase K, but it is resistant to treatement at temperature of 100oC for 20 minutes.
Whole strain CTC492 as much as the bacteriocin (Entericin CTC492) culture are capable of inhibiting
the growth of Listeria monocytogenes, Listeria innocua and Listeria welshimeri in agar drop assay,
agar difussion assay and direct antagonism assay under conditions in which the inhibition is
suppressed due to Hydrogen Peroxyde and acid. Inhibitory action is of bactericide-kind.
122/1006
Assays of growth inhibition have also been carried out for various strains of Listeria in contaminated
food products, e.g., contaminated crushed-meat doughs, with a drastic decrease in number of
colony forming units (cfu) observed, in periods of about 24 hours, after the addition of Entericin
CTC492 of the invention.
Entericin CTC492 may be obtained by the procedure consisting of cultivating the strain CTC492 of
Enterococcus faecium on MRS broth, culture centrifugation to obtain the supernatant, cold
treatement of the same with Ammonium sulphate and subsequent centrifugation of the same to
recover the sediment which consists of a highly active concentrate of Entericin CTC492, that can be
re-suspended in a suitable amount of Phosphate buffer, pH 7,0.
The activity against Listeria is measured in Activity Units, AU, in accordance with the method
described by Barefoot, S. et al. in Appl. Environ. Microbiol. 45 (6), 1808-1815 (1983), conveniently
adapted. Accordingly, an AU is defined as the reciprocal of the highest dilution which shows
complete inhibition of the strain of Listeria monocytogenes on a culture of approximately 100,00
cfu/ml. The AU are expressed in millilitre (AU/ml) in the case of liquid preparations or in grammes
(AU/g) in the case of solid preparations.
In relation to chemical and biochemical recognition studies, the obtained Entericin CTC492
concentrate can be additionally purified by means of a protocol of four steps comprising its
precipitation in Ammonium acetate solutions, Ion Exchange Chromatography, Hydrophobic
Interaction Chromatography and Reverse Phase Chromatography in the FPLC system (Fast
Performance Liquid Chromatography), eluting in a 30% Isopropanol isocratic gradient in a C2-C18
column.
Purified Entericin CTC492 molecular weight is determined by SDS-PAGE (Sodium DodecylsulphatePolyacrilamide Gel Electrophoresis) and by Mass Spectrometry.
Entericin CTC492, object of the present invention, can be obtained by known genetic-engineering
techniques too; for example, by cloning an auxiliar strain of any suitable microorganism with a vector
plasmid obtained from the chromosomic fragment responsible for production of said Entericin
CTC492.
The Entericin CTC492 concentrate obtained by procedures mentioned herein above may be used in
foods, as a liquid suspension or in a solid form, by drying and mixing it with any kind of appropriate
123/1006
excipient or by undergoing a lyophilization procedure, using sufficiently known techniques in every
case.
Also the culture of the strain of Enterococcus faecium itself in a frozen or not, liquid, dried or
lyophilized form, as well as any preparation resulting from the same having different activity levels
can be used for the application in foods.
Entericin CTC492 as well as the culture of strain CTC492 of Enterococcus faecium or preparations
coming from it can be used with a high effectiveness to, thanks to its addition into foods by any
suitable known way, prevent and avoid the growth and propagation of Listeria monocytogenes on
them, even when their acididity were low. Due to the innocuousness of Entericin CTC492 and strain
CTC492 of Enterococcus faecium, there is no counterindication about the kind of food in which they
can be used in priciple, although the use is especially directed to avoid contamination on meat and
meat products and on milk and diary products.
Entericin CTC492, the culture of strain CTC492 of Enterococcus faecium or derivable preparations
can also be used for the treatement of food containers, thereby forming a protective barrier against
contamination by Listeria monocytogenes. For example, they may be incorporated to a food
protective film by known techniques, such as those described in European Patent Application
Number 0 384 319.
Entericin CTC492 object of the present invention can appear in the form of material compositions
which would contain other kinds of natural or non-natural chemicals or biological products which may
be appropriate.
DESCRIPTION OF THE DRAWINGS
The present description is accompanied by a sheet of drawings, to represent, in an explanatory but
not restrictive way, the object of the present invention, as follows:
Figure 1 shows a diagram of the effect of the addition of Entericin CTC492 to an exponential phase
culture of Listeria monocytogenes. Entericin was added at concentrations of 800 AU/ml and 3,200
AU/ml and the results are shown in a comparative manner with those from a control culture to which
124/1006
no Entericin is added. At the abscissa axis, the lapsed time is measured, whereas at the ordinate
axis, the logarithm of the colony forming units, per millilitre (log [cfu/ml]) is shown.
EXAMPLES:
Following examples are shown also in an explanatory but not restrictive manner for the object of the
present invention.
EXAMPLE 1. OBTENTION OF A ENTERICIN CTC492 CONCENTRATE
500 ml of MRS culture broth of DIFCO Company are inoculated with a 1% of culture volumen in
stationary phase of Enterococcus faecium CTC492 on MRS; and resulting culture is then incubated
at 30oC during 12-18 hours. Subsequently, the culture is centrifuged at 10,000 rpm for 10 minutes,
the supernatant is collected and a 40% in weight of Ammonium sulfate is added thereto. Resulting
mixture is stored between 0oC and 5oC for 30 minutes and then centrifuged at 10,000 rpm for 35
minutes. The obtained precipitate is suspended in 10 ml 3mM Potassium Phosphate Buffer at pH 7,0.
The obtained suspension constitutes the Entericin CTC492 concentrate which can be used as such
or diluted at different concentrations.
EXAMPLE 2. INHIBITION ASSAY BY AGAR DIFUSSION
An Enterococcus faecium CTC492 culture on MRS broth is centrifuged at 10,000 rpm, incubated for
20-24 hours at 30oC. The supernatant fluid is collected, its pH is adjusted to 6.5 and it is filtersterilized through a 0.45 micrometre pore diameter filter.
A 0.2% Glucose MRS soft agar tube is inoculated with 200 microlitres of an overnight culture of
Listeria monocytogenes, poured onto a 0.2% Glucose MRS agar plate and allowed to solidify. Holes
are then made in the agar by a sterile 3 mm diameter pipette and 30 microlitres of the previously
125/1006
obtained supernatant from the culture of Enterococcus faecium are added into each hole. The plate
is incubated in anaerobic conditions at 25oC for 24 hours and a 10 mm inhibition halo is then
observed to be produced arround each hole where the supernatant is deposited.
EXAMPLE 3. INHIBITION OF LISTERIA MONOCYTOGENES GROWTH BY MEANS OF THE
BACTERIOCIN ENTERICIN CTC492.
In the same manner, three exponential phase cultures of Listeria monocytogenes are prepared in a
TSBYE medium (triptic soya broth from DIFCO Co.) to which 0.6% yeast extract is added. One
culture is reserved as a control and 800 and 3,200 AU/ml Entericin CTC492 prepared as in Example
1 are added to the other two cultures respectively. The cultures are incubated at 30oC and plate
tests are carried out in TSBYE agar at different time intervals, diluting as much as necessary, for
counting the Listeria monocytogenes colony former units up.The obtained results, illustrated as
log(cfu/ml), are as follows:
Columns=4
Head Col 1: Time (min)
Head Col 2: Control
Head Col 3: +800AU
Head Col 4: +3200AU
0.05.435.435.43
40.05.681.901.30
70.05.772.001.30
130.06.342.201.90
190.06.482.402.00
In Figure 1, the graphic representation of the obtained values is shown. From its observation, the
high efficiency of Entericin CTC492 to inhibit the growth of Listeria monocytogenes is unquestionably
deduced.
EXAMPLE 4. LISTERIA INNOCUA INHIBITION ASSAYS IN MEAT PRODUCTS
126/1006
Because of security of research workers, these assays were carried out with Listeria innocua,
species from genus Listeria that, unlike Listeria monocytogenes, does not produce damages in case
of being ingested by humans.
Two shares of meat dough for elaboration of salchichуn were prepared according to traditional
recipe in meat industry, and both were contaminated by Listeria innocua with 10,000 cfu per gramme
of meat dough. 800 AU Entericin CTC492, as obtained in Example 1 , per gramme of meat dough
were added to one of the shares. Then, putting into skins was carried out.
After 24 hours, cfu/g count decreased to 100 in the case of sausages made of Entericin CTC492
treated share and count decreased to 50 cfu/g after 8 days.
The pH evolution in control share as well as in Entericin CTC492 treated one was totally similar, for
which it may be deduced that cfu/g recount decrease is due to the action of Entericin CTC492 and
not to variations on meat dough acidity.
INFORMATION ON MICROORGANISM DEPOSITE
A deposite of microorganism has been carried out, in accordance with Budapest Treaty's provisions
on the international recognition of the deposit of microorganism for the purpose of Patent procedure,
with the International Authority for Deposites CECT, Colecciуn Espa ola de Cultivos Tipo, de la
Universidad de Valencia (Spanish Type Culture Colection, University of Valencia), Campus de
Burjasot, 46100 Burjasot (Valencia), Spain.
Columns=3
Head Col 1: Applicant identification
Head Col 2: CECT Number
Head Col 3: Date of Deposite
Enterococcus faecium CTC492CECT 445307-14-93
127/1006
The culture is deposited and is at public disposal without restrictions, under Budapest Treaty
conditions, although said disposal cannot be interpreted as a licence for carrying out the object of
the present invention, infringing present patent applicant rights.
Claims:
1. Bacteriocin against bacteria of the genus Listeria, characterized by being a peptide with a
molecular weight of 4.282 Daltons which has the following amino-acid sequence:
2. Bacteriocin according to Claim 1, characterized by being obtained from a culture of the bacterial
strain CTC492 of Enterococcus faecium, obtainable from CECT 4453.
3. Bacteriocin, according to Claim 1, characterized by being obtained by cloning an auxiliar strain of
any suitable microorganism with a vector plasmid obtained from a suitable chromosomic fragment of
the bacterial strain CTC492 of Enterococcus faecium, obtainable from CECT 4453.
4. Bacteriocin, according to any one of Claims from 1 to 3, characterized by being disposed as a
watery suspension.
5. Bacteriocin, according to any one of Claims from 1 to 3, characterized by being disposed as a
dried or lyophilized form.
6. Compositions of material characterized by containing the bacteriocin of Claim 1.
7. The bacterial strain CTC492 of Enterococcus faecium, obtainable from CECT 4453, capable of
producing the bacteriocin of Claim 1.
8. Use of the bacteriocin of Claim 1 to prevent and avoid the growth and propagation of Listeria
monocytogenes on foods.
128/1006
9. Use of the bacteriocin of Claim 1, according to Claim 8, to prevent and avoid the growth and
propagation of Listeria monocytogenes on meat and meat products.
10. Use of the bacteriocin of Claim 1, according to Claim 8, to prevent and avoid the growth and
propagation of Listeria monocytogenes on milk and dairy products.
129/1006
13. EP0759469 - 10.02.2004
BACTERICIDE COMPOSITIONS PREPARED AND OBTAINED FROM MICROCOCCUS VARIANS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0759469
Inventor(s):
MOLLET BEAT (CH); PEEL JOHN (CH); PRIDMORE DAVID (CH); REKHIF NADJI
(CH); SURI BRUNO (CH)
Applicant(s):
NESTEC SA (CH)
IP Class 4 Digits: C12N; A23L; C07K; C12P; A01N; A61K
IP Class:
C07K14/305
C12N1/20; C12P21/02; A23L3/3526; C12N1/21; A01N37/18; A61K7/00;
E Class: A23B4/22; A23L3/3463A; A61K8/64; A61K8/99; A61Q11/00; A61Q17/00; A61Q19/00;
C07K14/305
Application Number:
US19960693353 (19960806)
Priority Number: EP19950810497 (19950807)
Family: AU716251
Equivalent:
AU6194396; AU716251; BR9603325; CA2182810; CN1150601; DE69529982D;
DE69529982T; DK759469T; ES2193180T; FI963093; JP9121874; NO963282; NZ299124; ZA9606676
Abstract:
A BACTERIOCIN IS OBTAINED BY CULTURING CELLS OF A STRAIN OF MICROCOCCUS VARIANS
WHICH, UPON CULTURING IN A CULTURE MEDIUM, PRODUCES A BACTERIOCIN WHICH HAS
AGAR WELL INCUBATION INHIBITION TEST ACTIVITY AGAINST AT LEAST ONE OF
LACTOBACILLUS, LACTOCOCCUS, STREPTOCOCCUS, ENTEROCOCCUS, LISTERIA, BACILLUS,
CLOSTRIDIA AND STRAPHYLOCOCCUS BACTERIA. THE STRAIN IS CULTURED TO OBTAIN
CULTURED CELLS IN A CONCENTRATION OF FROM 10<7 >TO 10<11 >ORGANISMS PER ML OF
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THE MEDIUM, AND THE CULTURE MEDIUM SUPERNATANT IS SEPARATED FROM THE
CULTURED CELLS TO OBTAIN THE SUPERNATANT WHICH CONTAINS THE BACTERIOCIN,
WHICH IS ALSO IDENTIFIED BY HAVING AN AMINO ACID SEQUENCE FROM SEQ ID NO: 1 OR A
SEQUENCE DIFFERING FROM SEQ ID NO: 1 BY FROM 1 TO 4 AMINO ACIDS. THE SUPERNATANT,
AS IS, OR A CONCENTRATE THEREOF OR A BACTERIOCIN PRODUCT ISOLATED FROM THE
SUPERNATANT BY DEHYDRATION OR OTHERWISE ISOLATED AND PURIFIED THEREFROM IS
ADDED TO A FOOD OR COSMETIC PRODUCT TO INHIBIT THE GROWTH OF BACTERIA AGAINST
WHICH AGAR WELL INCUBATION INHIBITION TEST ACTIVITY IS EXHIBITED BY THE
BACTERIOCIN.Description:
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a bacteriocin, to a strain which produces this bacteriocin, to a
process for preparing this bacteriocin, and to the use of this bacteriocin and/or a strain producing
this bacteriocin in the manufacture of foodstuffs and cosmetics.
STATE OF THE ART
[0003] Bacteriocins have been isolated from numerous Gram-positive and Gram-negative bacteria.
Bacteriocins are molecules which are essentially proteinaceous in nature and which possess a
bactericidal effect and, for this reason, a bacteriocin provokes an antagonistic reaction between the
bacterium which produces it and one or more different bacterial species. Furthermore, the inhibition
spectrum of a bacteriocin is often limited to the species which are closely related to the bacterial
species which produces it.
[0004] Bacteriocins have, in particular, been demonstrated in lactic acid bacteria. For example, EP
0643136 (Sociйtй des produits Nestlй) describes the identification of two bacteriocins from
Streptococcus thermophilus. Similarly, a bacteriocin has been isolated from Lactococcus lactis (App.
and Env. Microbio. 58, 279-284, 1992; J. of Bio. Chem. 268, 16361-16368, 1993).
[0005] However, to date, no bacteriocin is known which is derived from Micrococcus varians,
Micrococcus varians is now much used within the foodstuff sphere, in particular in the fermentation of
meat for the purpose of manufacturing delicatessen products such as salamis and sausages, for
example. It would, therefore, be very useful to have available a bacteriocin-producing strain in order
to combat pathogenic genes.
[0006] The object of the present invention is to respond to this need.
SUMMARY OF THE INVENTION
[0007] To this end, the present invention provides a bacteriocin which is prepared and obtained from
Micrococcus varians, which bacteriocin has agar well incubation inhibition test activity against at
least one of Lactobacillus, Streptococcus, Enterococcus, Listeria, Bacillus, Clostridia and
Straphylococcus. Further to this end, the bacteriocin according to the present invention is a
131/1006
bacteriocin from Micrococcus varians, which bacteriocin exhibits the amino acid sequence SEQ ID
NO:1 or any amino acid sequence differing from the sequence SEQ ID NO:1 by one substitution, one
deletion and/or one insertion of from 1 to 4 amino acids. Furthermore, any nucleotide fragment
encoding this bacteriocin, in particular the nucleotide fragment exhibiting the sequence SEQ ID NO:2,
also comes within the scope of the present invention.
[0008] Similarly, the strain according to the present invention is a Micrococcus varians strain which
produces this bacteriocin, in particular the Micrococcus varians strains CNCM I-1586 and CNCM I1587.
[0009] In the process for preparing the bacteriocin according to the present invention, a
Micrococcus varians strain which produces the bacteriocin, in particular the strain CNCM I-1586 or
the strain CNCM I-1587, is cultured, in a medium and under conditions which are favorable for
growth, so as to obtain a culture medium containing from 10to 10organisms of this strain per ml, after
which the supernatant is isolated from the resulting culture by separating the supernatant from the
cultured cells to obtain a supernatant containing the bacteriocin, and to effect separation, the
resulting culture is centrifuged and a supernatant extract containing the bacteriocin is obtained. The
supernatant may be concentrated to obtain a concentrate comprising the bacteriocin, and the
bacteriocin may be isolated from the supernatant and concentrate by dehydration to obtain a powder,
and an isolated and purified bacteriocin may be obtained from the supernatant and concentrate and
may be dehydrated.
[0010] Finally, the use of the Micrococcus varians bacteriocin according to the invention comprises
using its nucleotide sequence, as well as its signal sequence, and using the supernatant extract
containing the bacteriocin, and a Micrococcus varians strain which produces the bacteriocin, for
preparing foodstuffs and cosmetics.
DETAILED DESCRIPTION OF THE INVENTION
[0011] In that which follows, the bacteriocin according to the present invention will be termed
"variacin".
[0012] Within the meaning of the present invention, an arbitrary unit (au) is defined as the inverse of
the value of the largest dilution at which a sample still exhibits a bactericidal effect in the test which is
known to the skilled person as the "agar well test".
[0013] Within the meaning of the present invention, the term "fragment" or "DNA fragment" is to be
understood to mean a single-stranded or double-stranded DNA fragment which is partially or entirely
coding and which can be synthesized, replicated in vitro by, for example, the known polymerase
chain reaction method, or replicated in vivo in a bacterium of the Escherichia coli type, for example.
[0014] Within the meaning of the present invention, a "homologous fragment" is understood to mean
any fragment which only differs from the fragments according to the invention by the substitution,
132/1006
deletion or insertion of a small number of bases. Within this context, two DNA fragments which
encode one and the same polypeptide, due to the degeneracy of the genetic code, will, in particular,
be regarded as being homologous. That fragment will also be regarded as being an homologous
fragment which exhibits more than 80% homology with the fragment according to the invention. In
this latter case, the homology is determined by the ratio between the number of bases in a
homologous fragment and the number in a fragment according to the invention.
[0015] Finally, within the meaning of the present invention, "homologous fragment" is also understood
to mean any fragment which is able to hybridize with the fragments according to the present
invention by the Southern blot method (Sambrook et al., Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory Press, U.S.A., 1989, chapter 9.31 to 9.58). Preferably, the
hybridization is carried out under rigorous or stringent conditions so as to avoid non-specific
hybridizations or hybridizations which are relatively unstable.
[0016] A proteinaceous factor, in this instance a bacteriocin possessing a powerful bactericidal
effect, has been isolated from the strains CNCM I-1586 and CNCM I-1587. This bacteriocin, which is
derived from Micrococcus varians and which consequently exhibits the amino acid sequence SEQ
ID NO:1, which is described in the sequence list below, has been termed variacin.
[0017] In view of the interest afforded by variacin, the invention also relates to any bacteriocin which
possesses an amino acid sequence which differs from the sequence SEQ ID NO:1 by one
substitution, one deletion and/or one insertion of from 1 to 4 amino acids. Thus, the said bacteriocin,
which exhibits an amino acid sequence differing from the sequence SEQ ID NO:1 by one substitution,
one deletion and/or one insertion of from 1 to 4 amino acids, may have an inhibition spectrum for a
bacterial genus or bacterial species which is wider than that of the said variacin, for example.
[0018] It has also been possible to select a chromosomal nucleotide fragment encoding the variacin
according to the invention from the two strains CNCM I-1586 and CNCM I-1587. The said fragment
exhibits the sequence SEQ ID NO:2 given in the sequence list below.
[0019] In view of the interest afforded by the present invention, the invention also relates to any
nucleotide fragment which encodes the variacin according to the present invention, in particular to
nucleotide fragments which are homologous to or which hybridize with the sequence SEQ ID NO:2.
[0020] In particular, the invention relates to nucleotides 88 to 153 of the sequence SEQ ID NO:2,
encoding the signal peptide of the variacin, to nucleotides 154 to 228 of the sequence SEQ ID NO:2,
encoding the secreted variacin according to the present invention, and/or to nucleotides 88 to 228 of
the sequence SEQ ID NO:2, encoding the bacteriocin fused to its signal peptide.
[0021] The present invention relates also to the bacteriocin fused to its signal peptide which exhibits
the amino acid sequence SEQ ID NO:3, which is described in the sequence list below.
133/1006
[0022] The fragment encoding the secreted variacin may be advantageously used to express the
variacin according to the present invention in a plant or in a microorganism other than Micrococcus
varians. To this end, nucleotides 154 to 228 of the sequence SEQ ID NO:2 can be cloned into an
expression vector downstream of a promoter or of a signal sequence and upstream of a terminator,
while paying due regard to the reading frame, and the said vector can then be introduced into a
plant, a bacterium or a yeast so as to increase their spectrum of inhibition towards certain bacteria,
for example.
[0023] Use can be made of the signal sequence according to the invention by fusing nucleotides 88
to 153 of the sequence SEQ ID NO:2 to a gene of interest, while paying due regard to the reading
frame, and by then cloning the whole into a Micrococcus varians expression vector, so as to enable
the protein encoded by the said gene of interest to be expressed and secreted in Micrococcus
varians, for example.
[0024] Nucleotides 88 to 228 of the sequence SEQ ID NO:2 can be cloned into a Micrococcus
varians expression vector and introduced into another strain of Micrococcus varians so that this latter
strain produces the variacin according the present invention.
[0025] In addition, the Micrococcus varians strain which contains, integrated into its genome or by
means of an expression vector, a DNA fragment encoding the variacin according to the invention is
also part of the subject-matter of the present invention. In particular, the Micrococcus varians strains
which were deposited on Jun. 7, 1995, in accordance with the Budapest Treaty, in the Collection
National de Cultures de Microorganismes [National collection of microorganism cultures], INSTITUT
PASTEUR, 25 Rue du Docteur Roux, F-75724 PARIS CEDEX 15, France, where they were given the
deposition numbers CNCM I-1586 and CNCM I-1587, are part of the subject-matter of the present
invention.
[0026] Micrococcus varians bacteria are Gram-positive, catalase-positive, aerobic bacteria which
are permanently immobile. They are spherical in shape and are found in the form of tetrads which are
arranged irregularly. Micrococcus varians colonies are coloured yellow on BHI medium. The
optimum temperature for growing the said strains is 25-37[deg.] C.
[0027] Strains CNCM I-1586 and CNCM I-1587, which are part of the subject-matter of the present
invention, metabolize both glucose and fructose. The CNCM I-1587 strain additionally metabolizes
sucrose and furanose.
[0028] In addition, strain CNCM I-1586 harbours two plasmids, of 4 and 12 kb, while strain CNCM I1587 only harbours a single plasmid of 7 kb.
[0029] The culture supernatants of strains CNCM I-1586 and CNCN I-1587 exhibit a relatively wide
inhibition spectrum with regard to the growth of other bacteria. The following may be included, by
way of example, among the bacteria which are sensitive to the said supernatants: Lactococcus lactis,
134/1006
Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii
subsp. lactis, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus acidophilus, Lactobacillus
johnsonii, Lactobacillus plantarum, Lactobacillus sake, Lactobacillus curvatus, Leuconostoc
carnosum, Leuconostoc mesenteroides subsp. mesenteroides, Streptococcus thermophilus, Listeria
monocytogenes, Enterococcus faecalis subsp. faecalis, the spores and vegetative cells of Bacillus
subtilis, Bacillus cereus, Bacillus polymyxa, Bacillus circulans, Bacillus pumulus and Bacillus
liqueniformis, and the Clostridia.
[0030] Preferably, in the process for preparing the variacin, a Micrococcus varians strain which
produces the said bacteriocin is cultivated, in a medium and under conditions which are favourable
for growth, so as to obtain a culture medium containing from 10to 10organisms of the said strain per
ml, after which the resulting culture is centrifuged and a supernatant extract containing the said
bacteriocin is obtained.
[0031] In order to produce this extract, the Micrococcus varians strain according to the present
invention which produces the variacin, in particular strain CNCM I-1586 or strain CNCM I-1587, can
be cultured in a medium and under conditions which are favourable for the growth of Micrococcus
varians. To this end, cultivation can take place, in particular, in BHI medium, at 25-37[deg.] C. under
aerobic conditions and with shaking, until a concentration of 10-10organisms per ml of medium is
obtained, for example. The standard culture which is thus obtained is centrifuged at 4000-6000 g
and the supernatant extract containing the said bacteriocin is collected.
[0032] The present invention also relates to the use of variacin, in particular in extract form, or the use
of a Micrococcus varians strain which produces this bacteriocin, for preparing foodstuffs and
cosmetics.
[0033] A culture of one of the said Micrococcus varians strains according to the present invention
may, in particular, be used in the fermentation of meat in order to prepare salami so as to combat
contamination with Clostridia, for example.
[0034] Variacin, in crude extract or purified form, may be used, when added to the leaven which is
obtained employing bacteria which are resistant to the said variacin, in the preparation of cheeses, in
particular cheeses of the mozzarella type, in order to avoid the holes which are produced by Bacillus
polymixa whose spores survive the fermentation, and of the vacherin type in order to combat
contamination with Listeria monocytogenes, for example.
[0035] Variacin, in particular in crude extract or purified form, or one of the two strains, may be used
as an additive or agent which is active against pathogenic bacteria in the preparation of dessert
mousses such as pasteurized custards, so as to combat the growth of spores such as Clostridia and
Bacillus cereus, or of bacterial strains such as Listeria, for example.
135/1006
[0036] In addition, variacin, in crude extract or purified form, or one of the two strains, may be used
as an additive or agent which is active against pathogenic bacteria in the preparation of cosmetics,
such as moisturizing creams or deodorants, so as to combat pathogenic bacteria of the skin, for
example.
ILLUSTRATIVE DESCRIPTION OF VARIACIN CHARACTERISTICS AND ACTIVITY
[0037] The variacin according to the present invention is characterized in more detail below with the
aid of various microbiological, biochemical and genetic findings which illustrate its properties. The
percentages are given by weight.
[0038] Unit of antibacterial activity-"agar well test"
[0039] Within the context of the present exposition, bactericidal activity is defined in terms of arbitrary
units.
[0040] A supernatant of a standard culture of Micrococcus varians according to the present invention,
which supernatant is prepared, for example, under the conditions described in Example 1, typically
exhibits an activity of 640 au/ml. Similarly, a concentrate of the said supernatant, which concentrate
is prepared, for example, under the conditions described in Example 2, typically exhibits an activity
of approximately 24000 au/ml.
[0041] The said agar well test is used to determine whether a sample still displays bactericidal
activity at a given dilution value.
[0042] In order to do this, 15 ml of MRS agar medium containing an indicator strain at a
concentration of 10-10CFU/ml are inoculated into a Petri dish. A strain which is sensitive to variacin,
in this case the strain Lactobacillus bulgaricus (YL 5) or the strain Lactobacillus helveticus (N2), for
example, is used as the indicator strain.
[0043] Holes of 5 mm in diameter are bored in the culture medium. The samples of the supernatant
or of the supernatant concentrate to be tested are poured into the holes at the rate of 50 [mu]l per
hole. The dish is preincubated at 4[deg.] C. for 2 h and then incubated overnight at either 30[deg.] C.
or 37[deg.] C. depending on the indicator strain employed. Following the incubation, the indicator
strain has grown and inhibition holes are visible. The dilution value at which a sample no longer
exhibits bactericidal activity is the dilution value starting from which an inhibition halo is no longer
discerned.
[0044] Inactivation by enzymes
[0045] The said agar well test is used to determine whether the variacin which has been isolated in
accordance with the present invention is inactivated in the presence of a proteolytic enzyme or in the
presence of catalase.
136/1006
[0046] In order to do this, enzyme is added, at the rate of 5 mg/ml, to a concentrate of the culture
supernatant described in Example 2. The enzyme is allowed to act at the incubation temperature for
60 min before the sample is deposited in the agar well test well.
[0047] A control is prepared in parallel using the same concentrate at pH 7 and without the addition
of enzyme. This control sample is incubated at 37[deg.] C. for 60 min before it is deposited in the
agar well test well for the purpose of comparing the inhibition halos obtained in the presence of
enzyme with the inhibition halo of the control. The diameter of the control inhibition halo is 27 mm.
[0048] Table I below gives the results which were obtained with the enzymes which were tested
using the indicator strain Lactobacillus helveticus (N 2). In this table, as in Table II, the enzyme is
designated by its type, the name of the supplier and the supplier's catalogue number. The numeral 0
indicates that there is no longer any halo, in other words that the bactericidal activity of the variacin
has been compromised by incubating the latter with the enzyme. The numeral 27 indicates that there
is still a halo of 27 mm, corresponding to the full bactericidal activity of the variacin.
TABLE I
Incubation temperatureInactivation
Enzymes([deg.] C.)(mm)
Catalase (SIGMA C-10)3027
Pronase E (SIGMA P-8038)370
Proteinase K (MERCK 1000 144)370
Ficin (SIGMA F-3266)370
[0049] Table II below gives the results which were obtained with the enzymes which were tested
using the indicator strain Lactobacillus bulgaricus (YL 5).
TABLE II
Incubation temperatureInactivation
Enzymes([deg.] C.)(mm)
Catalase (SIGMA C-10)3027
Pronase E (SIGMA P-8038)370
Proteinase K (MERCK 1000 144)370
Ficin (SIGMA F-3266)370
[0050] The results given in Tables I and II show that all the proteolytic enzymes suppress the
bactericidal activity of the variacin. These results demonstrate the fact that variacin is proteinaceous
in nature and that this proteinaceous moiety is involved in the bactericidal activity.
[0051] The fact that catalase is not found to exert any influence on the bactericidal activity of the
variacin also demonstrates that inhibition of the growth of the two indicator strains is not due to the
137/1006
antibacterial activity of H2O2, which is known to have a similar activity to that of the bacteriocins,
since H2O2 would have been degraded by the catalase.
[0052] Inhibition spectrum of the culture supernatant containing the variacin
[0053] The agar well test is used to determine whether the bactericidal activity of the culture
supernatant containing the variacin according to the present invention exhibits an activity which is
inhibitory to the growth of the different strains of spores and bacteria. The inhibition spectrum of the
supernatant is thus determined.
[0054] In order to carry out these assays, 15 ml of a standard medium, which is inoculated with 15
[mu]l of a culture of the strain to be tested, which culture was prepared during the preceding night,
are poured with Petri dishes so as to obtain a bacterial concentration of 10-10per ml of standard
medium. The standard medium is the medium which is favourable to the growth of the strain to be
tested.
[0055] Furthermore, when the strain to be tested has to grow from spores, 10-10spores are
inoculated per ml of covering medium.
[0056] A hole of 5 mm in diameter is bored in each Petri dish. A sample of 50 [mu]l of culture
supernatant, as described in Example 1, is deposited therein. The dishes are preincubated at 4[deg.]
C. for 2 h and are then incubated at a temperature which is favourable to the growth of the strain
under test for the time which is required for the strain to cover the plate with a visible bacterial lawn.
[0057] The effect, or the degree of inhibition, herein the "agar well incubation inhibition activity" is
characterized by the diameter of the observed inhibition. The inhibition is regarded as being very
strong (++++) if the halo exhibits a diameter of 18-28 mm, strong (+++) if the diameter is 10-17 mm,
average (++) if the diameter is 5-9 mm, weak (+) if the diameter is 1-4 mm, and zero (-) if no halo is
observed.
[0058] 32 strains of lactic acid bacteria of different species and subspecies are tested in this way
and it is noted that none of them is resistant to the supernatant. The detailed results of these tests are
presented in Table III below. In this Table III, as in the following tables, the strain designation or strain
no, indicated is the no. which is attributed to it in the Nestlй collection (Address: NESTEC S. A.,
Centre de Recherche, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland). The temperature
which is indicated is the incubation temperature during the test. The medium which is indicated is the
standard medium favourable to the growth of the strain to be tested.
TABLE III
SpeciesNo.T ([deg.] C.)MediaInhibition
Lactococcus lactisSL237MRS+++
Lactobacillus helveticusN 237MRS+++
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N 25837MRS+++
N 26237MRS+++
N 27137MRS+++
LBL 437MRS++++
Lactobacillus debrueckiiN 937MRS++
subsp. lactisN 6237MRS++
Lactobacillus delbrueckiiLFi 137MRS+++
subsp. bulgaricusLFi 537MRS+++
YL 537MRS+++
YL 1837MRS+++
Lactobacillus delbrueckiiN 837MRS+++
subsp. delbrueckiiN 18737MRS+++
Lactobacillus acidophilusLa 337MRS+++
La 1037MRS++
La 2737MRS++
La 2837MRS++
Lactobacillus johnsoniiLa 130MRS++
Lactobacillus plantarum6030MRS+
3.4 RP30MRS+
Lactobacillus sakeLSK30MRS+
Lactobacillus curvatus1830MRS++
Leuconostoc carnosumLCA 337M 17++++
Leuconostoc mesenteroidesA 7430MRS+
subsp. mesenteroidesB 5030MRS+
Streptococcus thermophilusSFi 1337Elliber++
SFi 1637Elliber++
SFi 2137Elliber++
SFi 2537Elliber++
ST 1137Elliber++
ST 137Elliber++
[0059] In Table III, it is noted that the inhibition spectrum of the supernatant is broad in the sense that
the degree of inhibition is of about the same level for the different species tested.
[0060] The inhibition spectrum of the supernatant of a culture producing the variacin of the invention
is also regarded as being broad in the sense that it is not limited to species of lactic acid bacteria but
extends to other species of Gram-positive bacteria, in particular to the undesirable or pathogenic
139/1006
bacteria Listeria innocua, Listeria welhia, Listeria monocytogenes, and to the spores of the Bacilli, for
example, as is attested by the results presented in Table IV below.
TABLE IV
SpeciesNo.T ([deg.] C.)MediaInhibition
Enterococcus faecalisJ37Elliber++
subsp. faecalis
Enterococcus faectumD 32537Elliber++
MB 2637Elliber++
Listeria innocua730BHI+++
Listeria monocytogenesFSM30BHI++++
122
Listeria welhia230BHI++
bacillus subtilisA 130BHI+++
(spores and vegetative cellsA 230BHI++
A 330BHI++
A 430BHI++
A 1330BHI+++
Bacillus subtilisA 1530BHI++
(spores and vegetative cells2430BHI++
15230BHIBacillus cereusC 130BHI++
(spores and vegetative cells)C 230BHI++
C 530BHI++
C 630BHI++
7930BHI++++
C 1530BHI+
Bacillus amyloliquefaciens22630BHI++++
Bacillus polymyxa25230BHI++++
Bacillus liqueniformis6430BHI++++
Bacillus stearothermophilus1030BHIBacillus circulans1530BHI++++
Bacillus pumulusB 130BHI++
(spores and vegetative cells)
B 230BHI+++
21330BHI++++
140/1006
Clostridium botulinum10000345DRMC++
(spores and vegetative cells)10000645DRMC++
10001945DRMC++
10002345DRMC++
Clostridium butyricum10200145DRMC++
(spores and vegetative cells)
Clostridium tyrobutyricum10700245DRMC+
(spores and vegetative cells)
Clostridium perfringens10300145DRMC+
(spores)
Clostridium sporogenes10400145DRMC+
(spores and vegetative cells)
Clostridium acetobutilycum10600145DRMC+
(spores and vegetative cells)
Clostridium10500145DRMC+
thermosaccharolyticum
(spores and vegetative cells)
Staphylococcus aureus330BHI+
1530BHI+
4430BHI+
6030BHI++
Staphylococcus xylosus130BHI+
Staphylococcus simulans130BHI+
Staphylococcus carnosus130BHI++
1430BHI++
Staphylococcus saprophyticus130BHI+
1530BHI+
Staphylococcus warneri130BHI+
Staphylococcus cohrii330BHI+
[0061] The results shown in Table IV make it possible, inter alia, to forecast attractive uses for this
supernatant, or for the purified variacin, as an additive in the preparation of foodstuffs, in the role of
an agent which is active against pathogenic bacteria, in particular against Clostridia in meat
products, against Listeria monocytogenes in cheeses, against Bacillus cereus and Listeria in dessert
mousses, or against the Bacilli in fresh pastes or sauces for fresh pastes, from precisely which
bacteria, for example, the above strains originate.
141/1006
[0062] Finally, variacin does not exert any inhibitory effect on the growth of Gram-negative bacteria
as can be noted from the results shown in Table V below.
TABLE V
SpeciesNo.T ([deg.] C.)MediaInhibition
E. coli130BHIEnterobacter chloacae7230BHISalmonella anatumXIV/2030BHISalmonella typhimuriumXIV/27430BHIPseudomonas fluorescens330BHI[0063] Inhibition spectrum of a concentrate of the supernatant containing variacin
[0064] The procedure is as described above except that the inhibitory effect on the growth of
different strains of spores and bacteria is determined which is produced by a supernatant
concentrate obtained as described in Example 2.
[0065] The same species and subspecies are tested as previously. The results of these tests are
presented in Tables VI, VII and VIII below. The strain designation or strain no. indicated is the no.
which is attributed to it in the Nestlй collection (Address: NESTEC S. A., Centre de Recherche, Verschez-les-Blanc, CH-1000 Lausanne 26, Switzerland). The temperature which is indicated is the
incubation temperature during the test. The medium which is indicated is the standard medium
favourable to the growth of the strain to be tested.
TABLE VI
SpeciesNo.T ([deg.] C.)MediaInhibition
Lactococcus lactisSL237MRS+++
Lactobacillus helveticusN 237MRS++++
N 25837MRS++++
N 26237MRS++++
N 27137MRS++++
LBL 437MRS++++
Lactobacillus delbrueckiiLFi 137MRS++++
subsp. bulgaricusLFi 537MRS++++
YL 537MRS++++
YL 1837MRS++++
Lactobacillus delbrueckiiN 937MRS+++
subsp. lactisN 6237MRS+++
Lactobacillus delbrueckiiN 837MRS++++
subsp. delbrueckiiN 18737MRS++++
142/1006
Lactobacillus acidophilusLa 337MRS++++
La 1037MRS++++
La 2737MRS+++
La 2837MRS+++
Lactobacillus johnsoniiLA 130MRS+++
Lactobacillus plantarum6030MRS++
3.4 RP30MRS++
Lactobacillus sakeLSK30MRS+++
Lactobacillus curvatus1830MRS+++
Leuconostoc carnosusLCA 337M 17++++
Leuconostoc mesenteroidesA 7430MRS++
subsp. mesenteroidesB 5030MRS++
Streptococcus thermophilusSFi 1337Elliber+++
SFi 1637Elliber+++
SFi 2137Elliber+++
SFi 2537Elliber+++
ST 137Elliber+++
ST 1137Elliber+++
TABLE VII
SpeciesNo.T ([deg.] C.)MediaInhibition
Enterococcus faecalisJ37Elliber+++
subsp. faecalis
Enterococcus faeciumD 32537Elliber+++
MB 2637Elliber+++
Listeria inaocua730BHI++++
Listeria monocytogenesFSM 12230BHI++++
Listeria welhia230BHI++++
Bacillus subtilisA 130BHI+++
(spores and vegetativeA 230BHI++
cells)A 330BHI++
A 430BHI++
A 1330BHI+++
A 1530BHI++
2430BHI++
15230BHI-
143/1006
Bacillus cereusC 130BHI++
(spores andC 230BHI++
vegetative cellsC 530BHI++
C 630BHI++
7930BHI++++
C 1530BHI+
Bacillus amyloliquefaciens22630BHI++++
Bacillus polymyxa25230BHI++++
Bacillus liqueniformis6430BHI++++
Bacillus stearothermophilus1030BHIBacillus circulans21530BHI++++
Bacillus pumulusB 130BHI++
(spores andB 230BHI+++
vegetative cells)21330BHI++++
Clostridium butyricum10200145DRMC+++
(spores and
vegetative cells)
Clostridium perfringens10300145DRMC++
(spores)
Clostridium tyrobutyricum10700245DRMC++
(spores and
vegetative cells)
Clostridium sporogenes10400145DRMC++
(spores and
vegetative cells)
Clostridium acetobutilycum10600145DRMC++
(spores and
vegetative cells)
Clostridium10500145DRMC++
thermosaccharolyticum
(spores and
vegetative cells)
Clostridia botulinum10000345DRMC+++
(spores and10000645DRMC+++
vegetative cells)10001945DRMC+++
144/1006
10002345DRMC+++
Staphylococcus aureus330BHI++
1530BHI++
4430BHI+
6030BHI+++
Staphylococcus xylosus130BHI+
Staphylococcus simulans130BHI+
Staphylococcus carnosus130BHI+++
1430BHI+++
Staphylococcus saprophyticus130BHI++
1530BHI++
Staphylococcus warneri130BHI++
Staphylococcus cohrii330BHI++
TABLE VIII
SpeciesNo.T ([deg.] C.)MediaInhibition
E. coli130BHIEnterobacter chloacae7230BHISalmonella anatumXIV/2030BHISalmonella typhimuriumXIV/27430BHIPseudomonas fluorescens330BHI[0066] The results shown in Tables VI, VII and VIII demonstrate the increased efficacy of the
supernatant concentrate, as compared with the supernatant, in inhibiting the growth of many of the
strains tested. Inhibition spectra are observed for the same species and subspecies, but at a higher
level of inhibition.
[0067] This suggests the preparation of a variacin supernatant concentrate, as described in Example
2, and its use for combating pathogenic bacteria in the preparation, for example, of foodstuffs and
cosmetics.
[0068] Resistance to pH
[0069] The agar well test is used to determine whether the variacin which has been isolated in
accordance with the present invention is pH-dependent.
[0070] To this end, the agar is inoculated with an indicator strain, as previously described in the
"agar well test". Lactobacillus helveticus (N 2) is used as the indicator strain.
[0071] The pH of an extract of the concentrate of the culture supernatant described in Example 2 is
adjusted to a pH of from 2 to 10 with 2N NaOH and/or 2N HCl, the extract is incubated at 37[deg.] for
145/1006
60 min and the pH is then readjusted to 6-7 before a sample of the extract is deposited in the agar
well test well.
[0072] A control, using the same supernatant concentrate at pH 7, is set up in parallel and incubated
at 37[deg.] C. for 60 min before the control sample is deposited in the agar well test well so as to
compare the inhibition halos of the test samples with the inhibition halo of the control. The diameter of
the inhibition halo of the control is 27 mm.
[0073] In Table IX, as in Tables X and XI, the numeral 27 indicates that there is still a halo of 27 mm,
corresponding to the full bactericidal activity of the variacin.
TABLE IX
pHInhibition halos
227
427
627
827
10 27
[0074] The results shown in the above table demonstrate that the bactericidal activity of the variacin
is not compromised. It is therefore possible to conclude that the bactericidal activity of variacin is not
pH-dependent.
[0075] Resistance to heat
[0076] The agar well test is used to determine whether the variacin which has been isolated in
accordance with the present invention is heat-dependent.
[0077] To this end, the agar is inoculated with an indicator strain, as previously described in the agar
well test. Lactobacillus helveticus (N 2) is used as the indicator strain.
[0078] A concentrate extract of the culture supernatant, described in Example 2 and adjusted to pH
7, is incubated at 100[deg.] C. for 15 to 60 min before a sample of it is deposited in the agar well test
well.
[0079] A control, obtained using the same supernatant concentrate at pH 7, is set up in parallel and
incubated at 37[deg.] C. for 60 min. This enables the inhibition halos of the temperature test samples
to be compared with the inhibition halo of the control. The diameter of the inhibition halo of the control
is 27 mm.
TABLE X
Temperature ([deg.] C.)Incubation time (min)Inhibition halos (mm)
1001527
1003027
146/1006
1006027
[0080] These results demonstrate that variacin is not heat-dependent. Thus, the bactericidal activity
of variacin is not compromised even after a 60 min incubation at 100[deg.] C.
[0081] The resistance of variacin to heat is a biochemical characteristic which is of great importance
in relation to using variacin in the preparation of foodstuffs and cosmetics. Thus, variacin can be
used, in particular in crude extract or purified form, in the preparation of pasteurized foodstuffs, so as
to combat the growth of spores, such as, for example, the Bacilli, which are resistant to heat.
[0082] Resistance to heat and to pH
[0083] In addition, the stability of variacin is tested when combining pH and heat.
[0084] To this end, the agar is inoculated with an indicator strain, as previously described in the
"agar well test". Lactobacillus helveticus (N 2) is used as the indicator stain.
[0085] The culture supernatant concentrate extract described in Example 2 is adjusted to pH 4 or 7
with 2N HCl and/or 2N NaOH and incubated at 115[deg.] C. for 20 min; the pH of the extract is then
readjusted to 6-7 before a sample of it is deposited in the agar well test well.
[0086] A control, obtained at pH 7, is set up in parallel at 37[deg.] C. for 20 min, before depositing
the control sample in the agar well test well so as to compare the inhibition halos of the test samples
with the inhibition halo of the control. The diameter of the inhibition halo of the control is 27 mm.
TABLE XI
Incubation
pHtime (min)Incubation temperature ([deg.] C.)Inhibition halo (mm)
42011527
72011527
[0087] The results given in the above table demonstrate that the bactericidal activity of variacin is not
compromised at pH 4 or 7, combined with an elevated temperature.
[0088] Purification of variacin
[0089] 4 l of BHI medium are inoculated with a culture of Micrococcus varians, which culture
produces the variacin according to the present invention. This standard culture is incubated at
30[deg.] C. overnight under aerobic conditions and while shaking, after which it is centrifuged at
5000 g so as to collect the supernatant in a recipient vessel, to which 72 g of XAD-7 resin (Amerblite
(R)) are added.
[0090] The mixture is stirred at 25[deg.] C. for 30 min in order to facilitate adhesion of the variacin
molecules to the resin, and the whole is then transferred onto a sintered glass where the supernatant
is filtered in vacuo.
147/1006
[0091] The resin is washed successively in 3 buffers containing 20 mM sodium citrate, pH 4, and
isopropanol. The first buffer contains 10% isopropanol, the second buffer contains 15% isopropanol,
and the third buffer contains 20% isopropanol.
[0092] The resin is transferred into a column and the variacin is eluted with 700 ml of buffer
containing 20 mM sodium citrate, pH 4, and 25% isopropanol. The bactericidal activity of the variacin
is monitored using the agar well test as described previously.
[0093] The active fractions are mixed and the isopropanol is evaporated. A 5 ml S-Resource column
for FPLC (Pharmacia) is prepared by equilibrating it with 20 mM sodium citrate buffer. The
evaporated mixture of active fractions is loaded onto this column and the contents of the column are
then eluted with an NaCl buffer having a gradient of from 100 mM to 400 mM.
[0094] Fractions are collected and the bactericidal activity of the variacin, which has thus been
purified, is checked using the agar well test.
[0095] Sequencing the variacin
[0096] The N-terminal part of the variacin purified from strains CNCM I-1586 and CNCM I-1587 is
sequenced using an Applied Biosystems 4774 automatic sequencer.
[0097] This reveals the peptide sequence of 5 amino acids whose sequence is identical to that for
the N-terminal part of the sequence SEQ ID NO:1, described in the sequence list below.
[0098] It was not possible to demonstrate the presence of a peptide of more than 5 amino acids
during the sequencing.
[0099] In addition, variacin which has been purified from strains CNCM I-1586 and CNCM I-1587 is
hydrolysed with 6N HCl for 10 min. 3 peptides are obtained which are isolated in the usual manner
by HPLC. It was only possible to sequence one of the three peptides which were isolated since the
other two most probably contain peptide modifications. The sequence of the said peptide which was
isolated in this way is identical to that comprising amino acids 19 to 22 of the sequence SEQ ID NO:1,
described in the sequence list below.
[0100] Finally, a fraction containing the variacin which has been purified from strains CNCM I-1586
and CNCM I-1587 is subjected to mass spectrometry and the variacin is found to have a molecular
weight of 2659 daltons.
[0101] Homology
[0102] Homology was demonstrated between the sequence of lacticin 481 from Lactococcus lactis
and that of the variacin according to the present invention. This homology relates, in particular, to the
sequences of the N-terminal part of the two bacteriocins. Thus, amino acids 1 to 5 of the N-terminal
sequence of variacin are identical to amino acids 3 to 7 of the sequence SEQ ID NO:8 of lacticin 481,
described in the sequence list below. Nevertheless, it is only a matter of partial homology and not of
identity.
148/1006
[0103] In addition, secreted lacticin 481 is shown by mass spectrometry to have a molecular weight
of 2900 daltons, whereas the molecular weight of secreted variacins is 2659 daltons, as we have
previously seen.
[0104] When the strain of Lactococcus lactis which produces lacticin 481 is inoculated in the
presence of a variacin extract, as previously described in the inhibition spectrum test, the growth of
the said strain is seen to be inhibited. The strain of Lactococcus lactis which produces lacticin 481 is
immune to its own bacteriocin, lacticin 481, but is not immune to the variacin which is produced by
either of the two Micrococcus varians strains according to the present invention. These results, which
were obtained in the previously described inhibition spectrum test, confirm that these two
bacteriocins are different.
[0105] The above genetic findings demonstrate that while lacticin 481 and variacin exhibit sequence
homologies, their sequences are not identical. The biochemical findings, as well as the
microbiological findings, confirm that lacticin 481 and variacin are two different bacteirocins.
[0106] Sequencing the variacin gene
[0107] The degenerate nucleotide sequence SEQ ID NO:4, which is described in the sequence list
below and which corresponds to the C-terminal part of the peptide of the previously sequenced
variacin, is constructed in a conventional manner. The mixture of SEQ ID NO:4 sequences is then
rendered radioactive by the action of T4 polynucleotide kinase.
[0108] A preparation of chromosomal DNA is made from strains CNCM I-1586 and CNCM I-1587 in a
conventional manner. The said DNA preparation is digested with SalI, SacI, SphI and BamHI in
accordance with the recommendations of the enzyme suppliers, 2 [mu]g of the digestion product are
then run on an agarose gel. The DNA on the gel is washed with 250 mM HCl and the migration
product is then transferred, in alkaline medium, from the gel onto a "Zetaprobe" (Biorad) membrane.
The Zetaprobe membrane is then prehybridized at a temperature of 55[deg.] C., which temperature
is lowered by 5[deg.] C. every 2 h. down to a temperature of 40[deg.] C., in a medium comprising 6*
SSC, 1% SDS and 0.25% skimmed milk. This membrane is hybridized with the degenerate
radioactive probe exhibiting the sequence SEQ ID NO:4 in the previous hybridization medium and
under the same temperature conditions. It is then left to incubate at 40[deg.] C. for 4 hours, after
which the membrane is washed at 40[deg.] C. in 6* SSC. Finally, it is exposed on an autoradiography
film at -80[deg.] C. for 16 h.
[0109] The hybridization demonstrates a variety of migration bands: a SalI band of 7 kb, a SacI band
of 1.4 kb, a BamHI band of 1.8 kb and an SphI band of greater than 15 kb.
[0110] 5 [mu]g of genomic DNA from strain CNCM I-1586 is then digested with the restriction
enzyme BamHI and a fragment of 1.6-2 kb is separated by agarose gel chromatography followed by
elution of that part of the gel containing the fragment. The eluted DNA fragment is ligated to the
149/1006
vector pK 19 (Gene, 56 (1987) 309-312), which has previously been digested with BamHI and then
treated with calf intestinal phosphatase (Boehringer Mannheim, part No. 713023).
[0111] The Escherichia coli strain BZ 234 (Biozentrum collection-University of Basle, Switzerland),
which has previously been rendered competent, is then transformed, in a conventional manner, with
the ligation medium. The clones containing the insert are identified on agar medium which is
supplemented with 50 [mu]g/ml kanamycin, 60 ng/ml IPTG (Boehringer Mannheim, part No. 724815)
and 300 ng/ml X-gal (Boehringer Mannheim, part No. 651745) and which is incubated at 37[deg.] C.
for 16 h.
[0112] The white colonies, which normally contain an insert, are picked out into 96-well microtitre
plates. Each white colony is picked out into one of the said wells, with each well containing 150 [mu]l
of LB medium supplemented with 50 [mu]g/ml kanamycin, and incubated at 37[deg.] C. for 20 h in
order to produce mini cultures.
[0113] Two primers of opposite orientation are prepared because the orientation of the gene in
vector pK 19 is not known. To this end, the said primers are constructed by assembling them from a
nucleotide fragment exhibiting the sequence SEQ ID NO:5, partially encoding lacticin 481, which is
linked to one or other of the universal probes of the pUC vectors, which probes exhibit the sequence
SEQ ID NO:6 or the sequence SEQ ID NO:7.
[0114] 1 [mu]l from each well is mixed with 100 pmol of one of the said primers, 6 [mu]l of 2 mM
dTNPs and 2.5 [mu]l of Taq buffer (P. H. Stehelin & Cie AG, cat. no. TP05b), and the whole is
covered with a drop of Dyna-wax (Finnzymes Oy, 02201 Espoo, Finland) and heated at 98[deg.] C.
for 10 min so as to lyse the bacteria; the PCR is then carried out.
[0115] The positive clones then give a band of 800 bp on an electorphoresis gel.
[0116] The positive clones are selected in this way and the plasmid DNA of these clones is extracted;
the DNA fragment which is cloned into the pK 19 vector is then sequenced by the dideoxynucleotide
method using a sequencing kit (Pharmacia Biotech, part No. 27-1682-01) and universal primers,
followed by specific primers which are based on the sequence thus obtained.
[0117] This results in a nucleotide sequence, SEQ ID NO:2, which is described in the sequence list
below, being obtained. The said nucleotide sequence encodes the sequence SEQ ID NO:1, which
corresponds to the amino acid sequence of variacin, prior to maturation.
[0118] Variants of the protein conserving the bactericidal activity
[0119] Variants of the protein having the amino acid sequence SEQ ID NO:1 are created. To this end,
the encoding DNA sequence of the variacin without the peptide signal (recombinant vector pK19
above) is inserted into the polyclonal site of plasmid pKK232-8 (Pharmacia, UK). Chemical
mutagenesis with hydroxylamine are performed on the expression vector according to the process
described by Yoast et al. (Applied and Env. Micro., 60, 1221-1226, 1994). Other methods could also
150/1006
be used, such as the method described by Dunn et al. (Protein Engineering, 2, 283-291, 1988)
dealing with the creation of precise mutations.
[0120] The mutagenized vectors are transferred into competent E. coli BZ 234 and transformants are
selected.
[0121] Transformants are isolated and cultivated. Supernatants are prepared and concentrated
according to Example 2.
[0122] Each supernatant is then screened according to the "agar well test" described above.
[0123] Results show that some supernatants provide an inhibition activity against Lactobacillus,
Lactococcus, Streptococcus, Enterococcus, Listeria, Bacillus, Clostridia and/or Staphylococcus, for
example.
[0124] DNA analysis of vectors expressing the bacteriocin show that some bacteriocins present a
DNA sequence which is different to the encoding sequence SEQ ID NO:2. The amino acid sequence
of these variants are generally different by from 1 to 4 amino acids.
EXAMPLES
[0125] The examples below are presented by way of illustrating the process for producing, and the
uses of, the bacteriocin according to the present invention. The percentages in the examples are
given by weight unless otherwise indicated.
Example 1
[0126] BHI culture medium is inoculated, and a culture containing organisms of the Micrococcus
varians strain is added to it. The whole is incubated overnight at 30[deg.] C. with shaking and under
aerobic conditions, after which the medium contains 10organisms of the strain per ml. The standard
culture thus obtained is centrifuged. The standard supernatant is collected.
Example 2
[0127] The supernatant concentrate is obtained from 750 ml of the said supernatant, which has been
obtained as described in Example 1 and to which is added 15 g of XAD-7 resin (Amberlite (R)). The
mixture is shaken at 4[deg.] C. for 60 min and is then filtered through a No. 604 Schleicher & Schuell
(Germany) filter. The filter is then washed with 1% sodium citrate in order to elute all the nonadsorbed proteins. The resin is isolated and transferred into a recipient vessel containing sodium
citrate and the whole is shaken for 2 min. The resin, together with the sodium citrate, is transferred
into a column and the sodium citrate is eluted with 50% acetonitrile and 0.1% TFA. The eluate is
evaporated and the residue is resuspended in 50 mM phosphate buffer at pH 6.8.
Example 3
[0128] 10 liters of a culture of the Micrococcus varians strain are produced in BHI medium overnight
at 30[deg.] C. with shaking and under aerobic conditions. 200 g of XAD-7 resin (Amberlite) are then
151/1006
added directly to the culture and the whole is shaken gently at 4[deg.] C. for 1 h. The mixture is then
filtered through a No. 604 Schleicher & Schuell (Germany) filter, and the resin which is retained on
the filter is then washed with 10 liters of a 50 mM acetic acid, pH 5.2, solution in order to remove the
bacteria. After that, 450 ml of a solution containing 100% ethanol and 20 mM ammonium acetate are
added to the resin and the whole is filtered in order to remove the resin; the filtrate is then lyophilized
in order to obtain a powder containing the variacin according to the invention, which can be used in
the foodstuff industry.
[0129] The bactericidal activity of this powder, which has been previously diluted in water, is then
determined by means of the previously described agar well test. This powder possesses at least
10au/g powder.
[0130] Finally, the above powder is added, at the rate of 0.5 g/kg, to a meat foam while it is being
prepared in a traditional manner. This results in a meat foam, each g of which contains 50 au of
bacteriocins which are able to completely inhibit the development of pathogenic bacteria, in
particular Clostridia.
Example 4
[0131] This example relates to the preparation of a moisturizing cream for skin care containing the
powder described in Example 3 at the rate of 0.05 g/kg, that is, therefore, variacin, which is capable
of inhibiting the development of undesirable bacteria on the skin, in particular Staphylococcus
aureus and Streptococcus pyogenes, at the rate of 5 au/g.
[0132] In order to prepare this emulsion, the components of lipid phase A are mixed and heated at
75[deg.] C. Aqueous phase B is prepared and also heated at 75[deg.] C.; lipid phase A is then
added, while mixing slowly, and, after that, the whole is cooled, under slow mixing, down to ambient
temperature, that is approximately 25[deg.] C. The C constituents are added slowly, at this
temperature, in the order of the formula.
%
Peg-6 stearate, glycerate and peg-20-cethyl ether (peg:15
polyethylene glycol)
Liquid paraffin5
Wheat germ oil stabilized with 0.1% phenylindane (antioxidant)3
and 1% soybean phospholipid (see EP94109355.1)
Sweet-almond oils2
Cetyl alcohol1
Isostearyl isostearate2
2-Octyldodecyl myristate1
Lanolin wax1
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Aqueous phase B
Methylisothiazoline0.1
Demineralized water59.6
Human placenta protein2
C Additives
Propylene glycol and calendula extract2
50% soluble collagen in demineralized water5.8
Perfume0.3
2.5% bacteriocin powder in accordance with Ex. 3 in0.2
demineralized water
Example 5
[0133] The bacteriocin powder described in Example 3 is added to a mouthwash at the rate of 0.5
g/kg. This mouthwash is consequently capable of inhibiting the development of pathogenic bacteria,
in particular Streptococcus sobrinus, Streptococcus sanguis, Streptococcus mutans and
Actinomyces viscosus, in the buccal cavity.
Example 6
[0134] A solution comprising the bacteriocin powder of Example 3, which is diluted in water at the
rate of 1%, is sprayed onto a foodstuff which is intended to be sterilized in order to prevent postcontamination during packaging.[sequence listing - see original document]Claims:
We claim:
[0135] 1. A process for preparing a composition having bactericidal activity comprising:culturing
cells of a strain of Micrococcus varians, which upon culturing in a culture medium, produces a
bacteriocin which has agar well incubation inhibition test activity against bacterial strains including
Listeria monocytogenes to obtain cultured cells in a concentration of from 10to 10organisms per ml
of the medium and a supernatant comprising the bacteriocin; and separating the supernatant from
the cultured cells to obtain the supernatant for obtaining a supernatant composition comprising the
bacteriocin.
[0136] 2. A process according to claim 1 wherein the bacteriocin comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1 and a sequence differing from SEQ ID
NO:1 by from 1 to 4 amino acids.
[0137] 3. A process according to claim 1 wherein the bacteriocin comprises amino acid sequence
SEQ ID NO:1.
153/1006
[0138] 4. A process according to claim 1 wherein the strain comprises a nucleotide fragment
comprising nucleotide sequence SEQ ID NO:2.
[0139] 5. A process according to claim 1 wherein the strain is selected from the group consisting of
strain CNCM I-1586 and strain CNCM I-1587.
[0140] 6. A process according to claim 1 or 2 further comprising isolating the bacteriocin from the
supernatant composition to obtain a purified bacteriocin product.
[0141] 7. A process according to claim 5 further comprising isolating the bacteriocin from the
supernatant composition to obtain a purified bacteriocin product.
[0142] 8. A process according to claim 6 further comprising lyophilizing the purified bacteriocin
product to obtain a lyophilized product.
[0143] 9. A process according to claim 7 further comprising lyophilized the purified bacteriocin
product to obtain a lypholized product.
[0144] 10. A process according to claim 1 or 2 further comprising concentrating the supernatant
composition to obtain a concentrate comprising the bacteriocin.
[0145] 11. A process according to claim 5 further comprising concentrating the supernatant
composition to obtain a concentrate comprising the bacteriocin.
[0146] 12. A process according to claim 10 further comprising drying the concentrate to obtain a
powder comprising the bacteriocin.
[0147] 13. A process according to claim 11 further comprising drying the concentrate to obtain a
powder comprising the bacteriocin.
[0148] 14. The supernatant composition of the process of claim 1 or 2.
[0149] 15. The supernatant composition of the process of claim 5.
154/1006
[0150] 16. The purified bacteriocin product of the process of claim 6.
[0151] 17. The purified bacteriocin product of the process of claim 7.
[0152] 18. The powder of the process of claim 12.
[0153] 19. The powder of the process of claim 13.
[0154] 20. A cell-free Micrococcus varians culture-medium-supernatant composition comprising a
bacteriocin which has agar well incubation inhibition test activity against bacterial strains including
Listeria monocytogenes.
[0155] 21. A cell-free culture-medium-supernatant composition according to claim 20 wherein the
bacteriocin comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1
and a sequence differing from SEQ ID NO:1 by from 1 to 4 amino acids.
[0156] 22. A cell-free culture medium supernatant according to claim 20 wherein the bacteriocin
comprises amino acid sequence SEQ ID NO:1.
[0157] 23. A bactericide which comprises a bacteriocin isolated from a bacteria and purified and
which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and a
sequence differing from SEQ ID NO:1 by from 1 to 4 amino acids.
[0158] 24. A bactericide according to claim 23 wherein the bacteriocin comprises amino acid
sequence SEQ ID NO: 1.
[0159] 25. A bactericide according to claim 23 or 24 in a dehydrated powder form.
[0160] 26. A process for inhibiting growth of bacterial strains in a composition for human use,
wherein the composition is selected from the group consisting of a food composition and a cosmetic
composition, comprising adding to the composition a bactericide composition selected from the
group consisting of a cell-free Micrococcus varians bacteriocin supernatant composition, including a
concentrate thereof, and a bacteriocin composition isolated from one of the supernatant and
concentrate and purified, wherein the bactericide composition has agar well incubation inhibition test
activity against bacterial strains including Listeria monocytogenes.
155/1006
[0161] 27. A process according to claim 26 wherein the bactericide composition comprises a
bacteriocin which comprises an amino acid sequence selected from the group consisting of SEQ ID
NO: 1 and a sequence differing from SEQ ID NO: 1 by from 1 to 4 amino acids.
[0162] 28. A process according to claim 26 wherein the bactericide composition comprises amino
acid sequence SEQ ID NO: 1.
[0163] 29. A process according to claim 1 wherein the bacteriocin further has agar well incubation
inhibition test activity against Listeria innocus and Listeria welhia and Bacillus polymyxa.
[0164] 30. A process according to claim 2 wherein the bacteriocin further has agar well incubation
inhibition test activity against Listeria innocua and Listeria welhia and Bacillus polymyxa.
[0165] 31. A cell-free Micrococcus varians culture-medium-supernatant composition according to
claim 20 wherein the bacteriocin further has agar well incubation inhibition test activity against
Listeria innocua and Listeria welhia and Bacillus polymyxa.
[0166] 32. A cell-free Micrococcus varians culture-medium-supernatant composition according to
claim 21 wherein the bacteriocin further has agar well incubation inhibition test activity against
Listeria innocua and Listeria welhia and Bacillus polymyxa.
156/1006
14. EP0811014 - 25.04.2000
BACTERIOCIN PISCICOLIN 126
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0811014
Inventor(s):
WETTENHALL RICHARD EDWARD HUGH (AU); DAVIDSON BARRIE ERNEST (AU);
HILLIER ALAN JAMES (AU); HARMARK KIM (AU); JACK RALPH WILSON (DE); HICKEY MALCOLM
WAYNE (AU); COVENTRY JOHN (AU); WAN JASON (AU)
Applicant(s):
LIMITED (AU)
UNIV MELBOURNE (AU); COMMW SCIENT IND RES ORG (AU); DARATECH PTY
IP Class 4 Digits: A61K
IP Class:
A61K38/16
E Class: A23L3/3571; C07K14/195
Application Number:
US19970894483 (19971208)
Priority Number: AU1995PN01310 (19950222); WO1996AU00096 (19960222)
Family: NZ301613
Equivalent:
CA2213561; JP11505407T; NZ301613; WO9626216
Abstract:
PCT NO. PCT/AU96/00096 SEC. 371 DATE JUN. 26, 1998 SEC. 102(E) DATE JUN. 26, 1998 PCT
FILED FEB. 22, 1996 PCT PUB. NO. WO96/26216 PCT PUB. DATE AUG. 29, 1996THE PRESENT
INVENTION PROVIDES A BACTERIOCIN HAVING A MOLECULAR WEIGHT OF ABOUT 4.4 KDA
AND METHODS INVOLVING THE USE OF THIS BACTERIOCIN. THE BACTERIOCIN PREFERABLY
HAS AN AMINO ACID SEQUENCE AS SHOWN IN SEQ ID NO:5 (FIG. 3). THE PRESENT INVENTION
ALSO PROVIDES A DNA MOLECULE ENCODING THIS BACTERIOCIN.Description:
157/1006
This invention relates to novel bacteriocin molecules having antimicrobial activity. In one particular
application, the bacteriocin according to the invention is used as a food preservative.
BACKGROUND OF THE INVENTION
There is a continual need for new food preservatives bearing new and useful properties. Further,
there is growing interest in replacing traditional "chemical" food preservatives with effective "natural"
preservatives, especially those which are specific for pathogenic microorganisms and do not harm
beneficial food-producing strains. In this regard, considerable research has been conducted on
bacterial peptides known as bacteriocins which are often heat stable and have antimicrobial activity.
Two such bacteriocins which are commercially produced for use as food preservatives are nisin and
pediocin PA-1. Nisin has been given the status of Generally Regarded as Safe for human
consumption (GRAS) by the United States FDA. Nisin and pediocin PA-1 however, have broad
spectrum activity, affecting not only pathogenic, but also beneficial (in the food system)
microorganisms.
SUMMARY OF THE INVENTION
Novel bacteriocins having a range of activity that is different to and preferably narrower than those of
nisin and pediocin PA-1 would be desirable. The present inventors have now identified and isolated
a new bacteriocin which has a limited range of activity in comparison to, for example, nisin and
pediocin PA-1.
Thus, in a first aspect, the present invention consists in a substantially pure preparation of a
bacteriocin having a molecular mass of about 4.4 kDa and antimicrobial activity substantially
according to Table 2.
In a preferred embodiment of the present invention the bacteriocin has an amino acid sequence
substantially identical to the sequence shown in FIG. 3.
In a second aspect the present invention consists in a biologically pure culture of bacterial strain
JG126.
158/1006
A sample of bacterial strain JG126 was deposited under the Budapest Treaty with Australian
Government Analytical Laboratories (AGAL), 1 Saukin Street, Pymble, New South Wales, Australia,
on Dec. 21 1994, and was accorded accession No. N94/61478.
In a third aspect the present invention consists in an isolated DNA molecule encoding the
bacteriocin of the first aspect of the present invention.
In a preferred embodiment of this aspect of the present invention the DNA molecule includes a
nucleotide sequence as shown in FIG. 6.
In a fourth aspect the invention consists in a method of preserving foods or beverages, the method
comprising adding to the food or beverage an effective antimicrobial amount of a bacteriocin of the
first aspect of the present invention.
The bacteriocin of the present invention may be isolated from natural sources, or produced by
recombinant DNA techniques well known in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the nature of the present invention may be more clearly understood, preferred forms
thereof will now be described with reference to the following non-limiting examples and the
accompanying figures.
Figure Legends
FIGS. 1A-1C: Purification of piscicolin 126 by reversed-phase HPLC
(A) C18 reversed-phase HPLC separation of piscicolin 126 on a 4.6.times.250 mm Spherisorb ODS
II (80 .ANG. pore size, 5 .mu.m particle size) column developed with the gradient of increasing
acetonitrile concentration indicated by the dashed line. The piscicolin 126 had been previously
partially purified by ion-exchange chromatography (not shown). The peak indicated by the arrow was
collected and shown to have antimicrobial activity. (B) Rechromatography by C18 reversed-phase
HPLC of the peak of piscicolin 126 collected as described in panel A. The column (2.1.times.250 mm
Spherisorb ODS II [80 .ANG. pore size, 5 .mu.m particle size]) was developed with the gradient of
increasing acetonitrile concentration indicated by the dashed line. (C) Electropherogram of purified
159/1006
piscicolin 126 separated by CZE through a 50 .mu.m.times.50 cm uncoated silica capillary in 20 mM
sodium citrate (pH 2.5) at 20 kV.
FIG. 2. MALDI-TOF mass spectrum of purified piscicolin 126.
A sample of purified piscicolin 126 was co-analysed along with the bacteriocin SA-FF22 (m=2794.3)
and porcine insulin (m=5777.6) and the instrument was calibrated accordingly. From 36 mass
spectra recorded, a mass estimate of m=4416.6 .+-.1.9 was calculated for piscicolin 126.
FIG. 3. Amino Acid Sequence of Piscicolin 126.
FIGS. 4A-C: Analysis of the products formed following treatment of piscicolin 126 with the alkylating
agent 4-vinylpyridine in either the presence or absence of a reducing agent.
FIG. 4A: Reversed-phase HPLC separation of piscicolin 126 following treatment with 4-vinylpyridine
in the (A) absence of and (B) presence of the reducing agent 2-mercaptoethanol. The collected
peaks from each separation were analysed by MALDI-TOF mass spectrometry.
FIG. 4B: (C) peak material from A and
FIG. 4C: D peak material from B. No mass increase was observed in A (sample without reducing
agent) suggesting that piscicolin 126 does not contain free sulphydryl groups while the mass
increase in D (210 Da) corresponds to bi-pyridethylation and suggests the piscicolin 126 contains a
disulphide bridge between the two cysteine residues.
FIG. 5. DNA sequence of the 607 bp Hinf I/Sau 3A DNA fragment encoding the structural gene for
Piscicolin 126.
FIG. 6. DNA sequence and translation of gene encoding Piscicolin 126 and its putative signal
peptide.
EXAMPLES
The invention will now be described in more detail with reference to the following examples.
160/1006
Example 1
Isolation and Characterisation of Bacteriocin-Producing Strain JG126
A number of Gram-positive bacteria were collected from a wide range of sources and assessed for
antimicrobial activity. One strain, JG126, was isolated from ham and found to be a catalase negative,
Gram-positive rod.
To establish the identity of this organism, 16S rDNA was amplified by the polymerase chain reaction
(PCR) from chromosomal DNA of the producing organism using the primers
5'GAGTTTGATCCTGGCTCAG and 5'TACAAGGCCCGGGAACG, corresponding to nucleotides 9-27
and 1394-1378, respectively, in the Eschericia coli 16S rRNA numbering system. A partial nucleotide
sequence of the PCR product was determined using primer 9-27 and the sequence compared to all
16S rRNA sequences in the ribosomal database of Australian National Genomic Information Service
(ANGIS) using the program FastA. The nucleotide sequence was 100% identical to Carnobacterium
piscicola, accession no. M58812 in GenBank and it was concluded that JG126 was a strain of C.
piscicola.
A sample of this bacterium was deposited under the Budapest Treaty with Australian Government
Analytical Laboratories (AGAL), 1 Saukin Street, Pymble, New South Wales, Australia, on Dec. 21,
1994, and was accorded accession No. N94/61478.
The activity of the bacteriocin (designated herein as piscicolin 126) was determined as follows by
bioassay with Listeria monocytogenes 4A. Bacteriocin was serially diluted with sterile distilled water
and aliquots of each dilution were applied to an MRS (de Man, Rogasa and Sharpe medium) agar
plate and overlaid with 7 ml TSBYE (Tryptohe Soy broth supplemented with 0.6% (w/v) yeast extract)
soft agar (0.6% (w/v)) seeded with 10@6 cfu ml@-1 of Listeria monocytogenes 4A and incubated at
30 DEG C. for 16 h. The activity of piscicolin 126 was expressed in Arbitrary Units (AU), defined as
the reciprocal of the titre of the highest dilution demonstrating a detectable zone of growth inhibition
in the indicator lawn culture.
Example 2
Purification of Piscicolin 126
161/1006
Piscicolin 126 was purified in the following manner:
Overnight cultures of bacterial strain JG126 in acetate-free MRS grown at 22 DEG C. were clarified
by centrifugation.
The peptide was precipitated from the culture supernatant by the addition of ammonium sulphate to
80% saturation and the precipitate was recovered by centrifugation.
Peptide was purified by chromatography:
1. by ion exchange chromatography on CM-Sepharose Fast Flow; then
2. by semi analytical Reversed-phase HPLC on Spherisorb ODSII 4.6 .times.250 mm, 80 .ANG., 5
mm particle (FIG. 1A); then
3. by analytical reversed-phase HPLC on Spherisorb ODSII 2.1.times.250 mm, 80 .ANG., 5 mm
particle (FIG. 1B).
This resulted in a single peak of peptide (FIG. 1C).
Example 3
Characterisation of the Properties of Piscicolin 126.
The molecular weight (Mr) of piscicolin 126 was determined by MALDI-TOF mass spectrum analysis
(FIG. 2). The peak of the piscicolin 126 corresponds to an Mr of 4416.6.+-.1.9 Da.
Piscicolin 126 was digested with a variety of enzymes, including some with proteolytic activity, and
the digests were assessed for antimicrobial activity. The results are presented in Table 1 and
indicate that the piscicolin 126 contains peptide material.
Table 1. Enzyme Inactivation of Piscicolin 126
20 .mu.l enzyme solution (200 .mu.g/ml in 0.2 M phosphate buffer, pH 7.0) was added to 200 .mu.l
of filter sterilised bacteriocin preparation. and incubated at 37 DEG C. for 2 h. Residual bacteriocin
activity (Arbitrary Units ml@-1) was determined by bioassay with Listeria monocytogenes 4A.
______________________________________
162/1006
Activity
Enzyme Treatment AU ml@-1
%@1
______________________________________
Control
12800 100
Catalase
12800 100
.alpha.-Chymotrypsin
0 0
.beta.-Chymotrypsin
0 0
Lipase
12800 100
Lysozyme
12800 100
Pepsin
12800 100
Protease I 0 0
Protease XIV 400 3
Protease XXIII 0 0
Trypsin
800 6
______________________________________
@1 % refers to residual bacteriocin activity (following treatment) as
a % of original activity, i.e., unaffected = 100%. completely destroyed =
0%.
Example 4
Determination of the Structure of Piscicolin 126
The amino acid sequence of the piscicolin 126 was determined. The sequence is presented in FIG.
3.
The presence or absence of free sulphydryl groups in piscicolin 126 was determined by analysis of
the products formed following treatment of piscicolin 126 with the alkylating agent 4-vinylpyridine in
either the presence or absence of the reducing agent 2-mercaptoethanol (FIG. 4). No mass increase
was observed for the sample that had been reacted with 4-vinylpyridine in the absence of reducing
agent FIG. 4C), indicating that piscicolin 126 does not contain free sulphydryl groups. The mass
increase of 210 Da observed for the sample reacted with 4-vinylpyrridine in the presence of the
163/1006
reducing agent (FIG. 4D) corresponds to bipyridethylation and indicates that piscicolin 126 contains
a disulphide bridge between two cysteine residues.
Example 5
Isolation and characterisation of the Gene Encoding Piscicolin 126
High molecular weight total DNA was isolated from bacterial strain JG126 and partially digested with
restriction endonuclease Sau3AI. The cohesive ends were partially filled using Klenow DNA
polymerase, dGTP and dATP and ligated into the partially filled Xho I sites of the arms of the
LambdaGEM.RTM.-12 vector. The ligated DNA was packaged into phages and used to transfect
E.coli strain LE392.
A piscicolin 126 specific gene probe was prepared by PCR amplification using the degenerate
oligo-nucleotide primers 5'TG(CT)AA(CT)AA(AG)AA(CT)GG(ACGT)TG(CT) and
5'GC(CT)TT(AG)TTCCA(ACGT)CC(ACGT)GC(ACGT)GC(ACGT)CC and C. piscicola JG126 genomic
DNA as template. The 107 bp PCR product was cloned into the pGEM.RTM.-T vector in the E.coli
host PMC112 and the nucleotide sequence of the cloned fragment determined. Translation of the
sequence confirmed that part of the piscicolin 126 gene had been cloned.
The 107 bp PCR fragment was used to screen the genomic lambda library and a positive clone
containing an insert of approximately 20 kb was isolated. From the lambda clone a 607 bp Hinf I/Sau
3A fragment was subcloned into pGEf.RTM.-7 in the E.coli host PMC112 and the nucleotide
sequence of both strands determined (FIG. 5). Translation of the DNA sequence confirmed that the
gene encoding the piscicolin 126 and its putative signal peptide had been cloned (FIG. 6).
Example 6
Spectrum of Antimicrobial Activity of Piscicolin 126
The spectrum of the antimicrobial -activity of piscicolin 126 has been tested against a number of
pathogenic and non pathogenic bacteria and yeast strains.
The spectrum was determined in at least two separate tests against Gram-positive and Gramnegative bacteria and yeasts. Where a variable result occurred in the first two tests, the indicator
164/1006
reaction was tested a third time and the result used to determine an overall positive or negative
reaction. The results are presented in Table 2.
Table 2. Antimicrobial Activity of Piscicolin 126.
Aliquots (5 .mu.l) of a preparation of piscicolin 126 concentrated by ammonium sulphate
precipitation were applied to the surface of agar plates and allowed to dry prior to overlaying with
indicator cultures. After incubation, the inhibition of the indicator strain by piscicolin 126 was scored
by the presence (+) or absence (-) of a zone of growth inhibition in the culture lawn.
______________________________________
AFRICC
Accession
No Indicator
Activity
______________________________________
0110 Aeromonas hydrophila 0303 Bacillus cereus
0312 Bacillus polvmyxa 0315 Bacillus stearothermophilus
0316 Bacillus stearothermophilus var. calidolactis
0320 Bacillus subtilis 0602 Brocothrix thermosphactum
0603 Brocothrix thermosphactum
0604 Brocothrix thermosphactum
0605 Brocothrix thermosphactum
+
0901 Clostridium botulinum type B
0914 Clostridium sporogenes 1001 Corynebacterium sp. -
165/1006
1105 Debaryomyces hansei 1201 Enterobacter aerogenes NCTC 10006
1301 Escherichia coli NCTC 8196
2001 Lactococcus diacetylactis
2002 Lactococcus cremoris 2010 Lactococcus lactis 2011 Lactococcus lactis 2101 Lactobacillus sake +
2102 Lactobacillus plantarum 2103 Lactobacillus curvatus +
2104 Lactobacillus sp. 2201 Leuconostoc mesentroides var. cremoris
+
2206 Leuconostoc cremoris 2208 Leuconostdc dextranicus +
2301 Listeria denitrificans ATCC 14570
2303 Listeria grayi
+
2305 Listeria innocua +
2306 Listeria ivanovii +
2310 Listeria monocvtogenes 4A
+
2311 Listeria monocytogenes 4B
+
2312 Listeria monocytogenes +
2320 Listeria seeligeri +
2321 Listeria seeligeri +
2405 Micrococcus luteus 2406 Micrococcus luteus 2415 Micrococcus varians 2702 Pediococcus acidilactici
-
166/1006
2703 Pediococcus acidilactici
+
2704 Pediococcus pentosaceus +
3020 Proteus vulgaris 3101 Pseudomonas aeruginosa 3105 Pseudomonas fluorescens 3301 Saccharomvces cerevisiae
3410 Salmonella salford 3412 Salmonella typhimurium 3501 Serratia marcescens 3601 Staphylococcus aureus NCTC 6571
3602 Staphylococcus aureus 3603 Staphylococcus aureus 3605 Staphylococcus camosus 3609 Staphylococcus epidermidis NGTC 6513
3715 Streptococcus thermophilus
+
3716 Streptococcus thermophilus
+
3801 Yersinia enterocolitica 3901 Enterococcus faecalis +
3902 Enterococcus faeceum +
4001 Carnobacterium sp. +
______________________________________
The results indicate that the bacteriocin has a limited spectrum of activity, and appears to act
against food-borne pathogens and spoilage microorganisms but should have limited effect against
beneficial bacteria such as cheese starter culture strains used in food systems.
Example 7
Quantitation of antimicrobial activity of piscicolin 126 against cheese starter bacteria.
167/1006
The sensitivity of cheese starter bacteria to piscicolin 126 was tested in both artificial growth media
and whole milk (Tables 3, 4 and 5).
Table 3. Minimum Inhibitory Concentration of piscicolin 126 tested against cheese starter bacteria.
A preparation of piscicolin 126 concentrated by ammonium sulphate precipitation was serially
diluted in sterile distilled water and applied (5 .mu.l) to the surface of MRS agar plates and allowed to
dry prior to overlaying with indicator cultures. The amount of bacteriocin JG 126 activity in the
concentrated preparation was determined as 204,800 AU ml@-1 by bioassay with Listeria
monocytogenes 4A. After incubation. the minimum inhibitory concentration of piscicolin 126 against
each strain of cheese starter bacteria was expressed as the lowest amount of activity of piscicolin
126 which demonstrated a distinct zone of growth inhibition in the culture lawn.
______________________________________
Starter
Culture
Accession
MIC
No. Indicator
(AU ml@-1)
______________________________________
Mauri Laborataries Culture Collection (MLCC)
M124 Lactococcus lactis subsp. cremoris
>204,800
M126 Lactococcus lactis subsp. cremoris
51,200
M145 Lactococcus lactis subsp. cremoris
>204,800
M149 Lactococcus lactis subsp. cremoris
>204,800
M193 Lactococcus lactis subsp. cremoris
>204,800
M379 Lactococcus lactis subsp. cremoris
>204,800
M229 Lactococcus lactis subsp. lactis
>204,800
168/1006
M238 Lactococcus lactis subsp. lactis
>204,800
M262 Lactococpus lactis subsp. lactis
>204,800
M276 Lactococcus lactis subsp. lactis
>204,800
M392 Lactococcus lactis subsp. lactis
>204,800
M474 Lactococcus lactis subsp. lactis
>204,800
DRC Lactococcus lactis subsp. lactis var
>204,800
diacetyladis
FDLD3 Lactococcus lactis subsp. lactis var
>204,800
diacetylactis
HBD1 Lactococcus lactis subsp. lactis var
>204,800
diacetylactis
HDD1 Lactococcus lactis subsp. lactis var
>204,800
diacetylactis
FLIL1 Leuconostoc sp. >204,800
HLD1 Leuconostoc sp. >204,800
ST3 Streptococcus thermophilus
>204,800
ST41 Streptococcus thermophilus
>204,800
ST42 Streptococcus thermophilus
>204,800
AFRI Culture Collection (AFRICC)
2201 Leuconostoc mesentroides var. cremoris
102,400
2208 Leuconostoc dextranicus
102,400
169/1006
3715 Streptococcus thermophilus TS1
2,048
3716 Streptococcus thermophilus TS2
3,200
______________________________________
The results indicate that, in plate
bioassays with Listeria monocytogenes 4A, cheese starter bacteria are generally not sensitive to less
than 2,048 AU ml@-1 of piscicolin 126 and most are not sensitive to 102,400 AU ml@-1 and higher
concentrations.
Table 4. The effect of Piscicolin 126 on cheese starter culture activity in whole milk.
The effect of piscicolin 126 (2,048 AU ml@-1) on starter culture activity (rate of pH decrease) was
determined in sterile whole milk at 30 DEG C. (except for strains 3715 and 3716, where an incubation
temperature of 37 DEG C. was required to support more optimal growth). Cultures were grown in
sterile skim milk (10% w/v) for 16 h at 30 DEG C. or 37 DEG C. (strains 3715 and 3716) and
inoculated (10% v/v) into sterile whole milk to test starter activity in the absence and presence (2,048
AU ml@-1) of piscicolin 126. Uninoculated milk samples with and without added piscicolin 126 were
used as controls. The pH of aliquots (2.5 ml) of samples removed under sterile conditions were
determined at the times indicated.
______________________________________
Starter culture pH
Accession No.
JG126 0 h 2 h 4 h 6 h 8.5 h
______________________________________
MLCC M276 - 5.92 5.70 5.14 4.53 4.27
(Bacteriocin
+ 5.87 5.66 5.12 4.50 4.28
insensitive)
MLCC M126 - 6.00 5.72 5.15 4.54 4.25
(Bacteriocin
+ 6.04 5.76 5.27 4.64 4.30
sensitive)
AFRICC 3715
- 5.89 5.75 5.47 5.22 5.04
170/1006
(Bacteriocin
+ 5.87 5.76 5.44 5.18 5.01
sensitive)
______________________________________
The results indicate that piscicolin 126 at a level of 2,048 AU ml@-1 (as determined by plate
bioassay with Listeria monocytogenes 4A) does not inhibit starter activity.
Table 5. The effect of piscicolin 126 on viable count of cheese starter cultures and Listeria
monocytogenes 4A in whole milk.
The effect of piscicolin 126 (2,048 AU ml@-1) on viable count of the most bacteriocin-sensitive
starter culture from each of the Mauri Laboratories (strain M126) and AFRI (strain 3715) culture
collections (Table 3) and Listeria monocytogenes 4A (at initial levels of 10@4 and 10@6 cfu ml@-1)
was determined in sterile whole milk (at 30 DEG C. for strains M126 and 2310 and at 37 DEG C. for
strain 3715) at the times indicated. Viable counts were determined (as 0.1 ml spread plates of an
appropriate dilution of the cultures) on M17 Agar (strains M126 and 3715) and Listeria Selective Agar
(strain 2310).
______________________________________
Viable count
(log cfu ml@-1)
Culture JG126 0 h 6 h
______________________________________
Starter cultures
MLCC M126 - 6.71 8.51
(on M17 Agar)
+ 6.63 8.52
AFRICC 3715 - 7.15 8.08
+ 7.18 8.00
Listeria inoculum 10@4 cfu ml@-1
- 4.38 5.95
monocytogenes 4A
+ <1.0 <1.0
AFRICC 2310
inoculum 10@6 cfu ml@-1
- 6.24 7.72
171/1006
(on Listeria
+ <1.0 2.94
Selective Agar)
______________________________________
The results indicate that the viable counts of cheese starter cultures were unaffected, whereas the
viable count of Listeria monocytogenes was substantially reduced by the presence of piscicolin 126
in whole milk.
The piscicolin 126 therefore should find application as a preservative for foods and beverages.
Example 8
Antimicrobial Activity of Piscicolin 126 in Devilled Ham Paste and Ricotta Cheese
Ricotta cheese (250 g) and Devilled Ham Paste (260 g) were inoculated with Listeria
monocytogenes (AFRICC No. 2310) at 10@3 -10@6 cfu g@-1 in sterile stomacher bags. An aliquot
(10 ml) of concentrated piscicolin 126 preparation was added and mixed thoroughly (Colworth
stomacher, 60 s) to provide a final concentration of piscicolin 126 of approximately 2,048 AU g@-1
(as determined by plate bioassay). In control samples, sterile water was substituted for the piscicolin
126 preparation. Portions (10 g) of the mixtures were sealed in sterile stomacher bags and incubated
at 10 DEG C. for the length of the trial. At the times indicated. aliquots (90 ml) of peptone solution
(0.1% w/v, pH 7) were added to portions of Ricotta cheese and mixed (Colworth stomacher, 60 s).
Viable counts were determined on aliquots (0.1 ml) of appropriate dilutions of the mixed samples (in
peptone solution) spread onto the surface of Listeria Selective Agar (Oxoid) plates. Viable count
determinations were performed on each of three portions of Ricotta cheese and Devilled Ham Paste
at the times indicated and are expressed as the mean value in Tables 6 and 7.
Table 6. Effect of Piscicolin 126 on Listeria monocytogenes 4A in Challenged Ricotta Cheese.
The effect of piscicolin 126 (2,048 AU g@-1) on the viable count of Listeria monocytogenes 4A (initial
inoculum 10@4 cfu g@-1) in Ricotta cheese spread was determined on Listeria Selective Agar (Oxoid)
plates as the mean value of triplicate samples after incubation at 10 DEG C., at the times indicated.
______________________________________
Viable count on Listeria Selective Agar (Log cfu g@-1)
172/1006
Treatment
Day 0 Day 1 Day 2 Day 5 Day 7 Day 21
______________________________________
Control 4.49 4.85 5.57 7.20 7.67 8.26
Piscicolin 126
2.12 2.00 2.52 4.08 4.95 7.87
______________________________________
Table 7. Effect of Piscicolin 126 on Listeria monocytogenes 4A in Challenged Devilled Ham Paste.
The effect of piscicolin 126 (2,048 AU g@-1) on the viable count of Listeria monocytogenes 4A (initial
inoculum 10@3 cfu g@-1) in devilled ham paste was determined on Listeria Selective Agar (Oxoid)
plates as the mean value of triplicate samples after incubation at 10 DEG C., at the times indicated.
______________________________________
Viable count on Listeria Selective Agar (Log cfu g@-1)
Day Day Day Day Day Day Day
Treatment
0 1 4 6 7 14 21
______________________________________
Control 3.26 3.16 5.10 6.05 6.87 9.49 9.76
Piscicolin 126
<2.0 <2.0 <2.0 <2.0 <2.0 <2.0 5.83
______________________________________
The results indicate that under the experimental conditions growth of Listeria monocytogenes in
Devilled Ham Paste is prevented by piscicolin 126 for between 14 and 21 days, while growth of
Listeria monocytogenes in Ricotta cheese is inhibited for up to 7 days.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may
be made to the invention as shown in the specific embodiments without departing from the spirit or
scope of the invention as broadly described. The present embodiments are, therefore, to be
considered in all respects as illustrative and not restrictive.
__________________________________________________________________________
173/1006
#
SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 8
- (2) INFORMATION FOR SEQ ID NO: 1:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 19 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / - #desc = "Primer"
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 19
CAG
- (2) INFORMATION FOR SEQ ID NO: 2:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 17 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / - #desc = "Primer"
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 17
G
- (2) INFORMATION FOR SEQ ID NO: 3:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 26 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / - #desc = "Primer"
174/1006
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
#3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#
26 GGAC GTTGCT
- (2) INFORMATION FOR SEQ ID NO: 4:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 37 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: / - #desc = "Primer"
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
# 37
TCCA CGTGCACGTG CACGTCC
- (2) INFORMATION FOR SEQ ID NO: 5:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 44 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (v) FRAGMENT TYPE: N-terminal
- (vi) ORIGINAL SOURCE:
(A) ORGANISM: Carnobacteri - #um piscicola
(B) STRAIN: JG126
#5: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Lys Tyr Tyr Gly Asn Gly Val Ser Cys Asn Ly - #s Asn Gly Cys Thr Val
#
15
- Asp Trp Ser Lys Ala Ile Gly Ile Ile Gly As - #n Asn Ala Ala Ala Asn
#
30
- Leu Thr Thr Gly Gly Ala Ala Gly Trp Asn Ly - #s Gly
175/1006
# 40
- (2) INFORMATION FOR SEQ ID NO: 6:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 607 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
(A) ORGANISM: Carnobacteri - #um piscicola
(B) STRAIN: JG126
#6: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- CGATGTTACA ATCAATTAAC TTTATAAGTT CATGAATAAT ATCGTGATAG TT - #CAGGAATA
60
- AAAAATCTAT AAGTAAAAAA GATGTGATAC AGTCAGCATG TTGTAAAAAA TA - #TTTTAAAA
120
- AGGAGCGTGT TTACGCATGA AAACTGTTAA AGAACTTAGC GTTAAAGAAA TG - #CAACTAAC
180
- TACAGGAGGT AAGTATTACG GAAATGGCGT TTCCTGTAAT AAAAATGGTT GT - #ACTGTAGA
240
- TTGGAGCAAA GCTATTGGGA TTATAGGAAA CAATGCAGCA GCAAATTTGA CT - #ACAGGTGG
300
- AGCCGCTGGT TGGAACAAAG GATAATTAAA GTCTCTTATT TTTTATCTTG TA - #AAAAAGAT
360
- GATACGCATC AATGCTGTGA CATAACATAG ATGGGTCTTT ATATTTGTAA GT - #TACATTTA
420
- AAACAAAAAT AAATATATAA AAATATTTTT TTATAGTCTT AGGAATTATG TT - #ATACTAAC
480
- AAAAATAGGC TAGTTTCAAC ATGATGTAAA GAAACTTATA CTATCAACTA AA - #ATCATAAA
540
- TATATAAAAT TAAGGAGTGA TATTTTATGG GTAAGTTAAA ATGGTTTTCT GG - #AGGAAAAG
600
#
607
176/1006
- (2) INFORMATION FOR SEQ ID NO: 7:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 189 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
(A) ORGANISM: Carnobacteri - #um piscicola
(B) STRAIN: JG126
#7: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- ATGAAAACTG TTAAAGAACT TAGCGTTAAA GAAATGCAAC TAACTACAGG AG - #GTAAGTAT
60
- TACGGAAATG GCGTTTCCTG TAATAAAAAT GGTTGTACTG TAGATTGGAG CA - #AAGCTATT
120
- GGGATTATAG GAAACAATGC AGCAGCAAAT TTGACTACAG GTGGAGCCGC TG - #GTTGGAAC
180
# 189
- (2) INFORMATION FOR SEQ ID NO: 8:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 62 amino
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (v) FRAGMENT TYPE: N-terminal
- (vi) ORIGINAL SOURCE:
(A) ORGANISM: Carnobacteri - #um piscicola
(B) STRAIN: JG126
#8: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
- Met Lys Thr Val Lys Glu Leu Ser Val Lys Gl - #u Met Gln Leu Thr Thr
177/1006
# 15
- Gly Gly Lys Tyr Tyr Gly Asn Gly Val Ser Cy - #s Asn Lys Asn Gly Cys
# 30
- Thr Val Asp Trp Ser Lys Ala Ile Gly Ile Il - #e Gly Asn Asn Ala Ala
# 45
- Ala Asn Leu Thr Thr Gly Gly Ala Ala Gly Tr - #p Asn Lys Gly
# 60
__________________________________________________________________________ Claims:
What is claimed is:
1. A substantially pure bacteriocin in which the bacteriocin has an amino acid sequence as shown in
SEQ ID NO:5.
2. A method of preserving foods or beverages, the method comprising adding to the food or
beverage an effective antimicrobial amount of a bacteriocin as claimed in claim 1.
3. A composition of matter comprising a cheese starter culture and a bacteriocin as claimed in claim
1.
178/1006
15. EP0830061 - 09.06.1998
MOIST BACTERIOCIN DISINFECTANT WIPES AND METHODS OF USING THE SAME
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0830061
Inventor(s):
BLACKBURN PETER (US); DE LA HARPE JON (US)
Applicant(s):
AMBI INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K9/70
E Class: A01N37/46+M; A01N63/02+M
Application Number:
US19950479280 (19950607)
Priority Number: US19950479280 (19950607)
Family: IL122103
Equivalent:
AU715384; AU7629696; BR9608603; CA2219924; CN1123293C; CN1186413;
CZ9703847; DE69607958D; DE69607958T; DK830061T; ES2147649T; GR3033983T; HU9901082;
IL122103; JP11507363T; NO975635; NZ310798; PL185880B; PL323717; RU2163145; SK164797;
WO9639842; ZA9604330
Abstract:
DISCLOSED ARE NOVEL, MOIST PAPER OR FABRIC WIPES CONTAINING A BACTERIOCIN
DISINFECTANT FORMULATION, WHICH HAVE A LOW ALCOHOL CONTENT AND WHICH AFFORD
RAPID, ONE-STEP DISINFECTION AND DRYING OF SURFACES. ALSO DISCLOSED IS A METHOD
OF DISINFECTION AND DRYING OF SKIN, INCLUDING COW TEAT SKIN, EITHER PRIOR TO OR
AFTER MILKING, WHICH EMPLOYS THE WIPES.Description:
179/1006
BACKGROUND OF THE INVENTION
Moist disinfectant wipes or towelettes are currently available commercially and are produced in a
variety of forms. The wipes currently available have a number of uses including the disinfection of
hands, skin, food lines and hospital surfaces, as well as applications in the dairy industry.
Moist disinfectant wipes presently available typically contain a germicide such as chlorhexidine,
chlorhexidine digluconate or povidone-iodine and/or an alcohol as the disinfecting agent(s). The
alcohol component, generally in a concentration of 70% or greater, also serves as a drying agent.
While efficient drying of a surface exposed to a moist wipe is important, the high concentration of
alcohol commonly used poses a problem of excess drying and chapping of skin. There is a further
concern with the flashpoint of formulations with such a high alcohol content. Furthermore, the
disinfectants commonly used are not of ideal potency, and the amounts required to obtain
reasonable disinfection properties can present toxicity or sensitization problems.
One particular area wherein the problems have not been completely addressed by the disinfecting
methods currently known is the dairy industry. The dairy industry incurs tremendous losses, upwards
of 2 billion dollars per year in the United States alone as a result of mastitis. The practice of
appropriate udder hygiene prior to, during, and after milking is recommended in order to control
mastitis infection.
Preparation of a dairy cow for milking is viewed as the most labor intensive and time consuming part
of the milking procedure. Commonly, dairy farmers use one of two different methods. The most
prevalent method is udder washing. This practice consists of using an udder wash solution,
individual paper towels and water. The udder wash solution may be injected into the stream in a
water hose and sprayed onto the udder. Alternatively a towel is soaked in the udder wash solution in
a bucket and the damp paper towel is used to wash and massage the udder. In each case the udder
is then dried with a paper towel.
Predipping with a germicidal teat dip has replaced udder washing in approximately 40% of the U.S.
dairy farms. The herdsman will dip or spray the cows' teats (as opposed to the complete udder) with
a teat dip, allowing the dip to stay in contact with the teat for at least 15 seconds. The teat dip
solution is then wiped off with a paper towel.
180/1006
While each of these methods has certain advantages, neither effectively addresses all of the
concerns encountered in this milieu. Among the concerns are effective cleaning and drying,
contamination of milk by udderwash runoff and/or predip residues, and efficient use of labor and time
in preparing the cow.
Much effort has been put into remedying the widespread and costly bacterial contamination of milk.
Various improved methods for pre-milking treatment of udders and methods for the prevention and
treatment of bovine mastitis have been described (see, e.g., U.S. Pat. No. 4,206,529 to Neumann;
U.S. Pat. Nos. 5,124,145 and 5,234,684 to Sordillo, et al.; U.S. Pat. No. 4,253,420 to Hoefelmayr and
U.S. Pat. No. 5,366,732 to Zighelboim). Others have described improved antimicrobial compositions
which have particular application to the problem of contaminating residues in premilking sanitizing
operations (see, e.g., U.S. Pat. No. 5,139,788 to Schmidt) or have described improved systems of
general applicability for delivery of moist wipes (see, e.g., U.S. Pat. No. 4,775,582 to Abba, et al. and
U.S. Pat. No. 4,853,281 to Win, et al.). Berg, et al., J. Dairy Sci. 68, 457-461 (1985); Pankey, et al.
Veterinary Clinics of North America 9, 519-530, 1993; McKinnon, et al., J. Dairy Res. 50, 153-162,
1983; Murdough, et al., J. Dairy Sci. 76, 2033-2038, 1993 and Ansari, et al., Am. J. Infect. Control 19,
243-249, 1991 provide further description of the present state of the art and describe the evolution of
udder hygiene in terms of various aspects of the commonly applied procedures for pre-milking
sanitization of teats.
There is thus a need for moist disinfectant wipes which provide efficient one-step disinfection and
drying of surfaces but which employ disinfecting agents with greater bactericidal potency yet fewer
possible undesirable side effects. The improved wipes should also provide efficient drying without
chapping or other loss of integrity of sensitive surfaces.
SUMMARY OF THEE INVENTION
The present invention concerns novel, moist paper or fabric wipes which afford rapid, one-step
disinfection and drying of surfaces. The wipes contain a liquid disinfectant formulation typically
comprising a bacteriocin as the disinfecting agent, a stabilizer for the bacteriocin, a chelating agent,
a surfactant, a salt, a skin conditioner or humectant, and an agent to promote rapid drying. The
bacteriocin disinfecting agent can also be combined with commonly used germicidal agents, as
appropriate. In the wipes of the present invention, said germicidal agents can be employed in much
lower amounts, thus alleviating toxicity and sensitization concerns. Because the inventive wipes
employ bacteriocins, i.e., far more potent bactericidal agents, the alcohol component is required
181/1006
primarily as a drying agent, and thus the required concentration of alcohol is substantially lowered
relative to that required for bactericidal action. This provides a remedy for the typically encountered
problem of chapping of sensitive surfaces by high-alcohol formulations.
The wipes of the instant invention are suited to disinfection and drying of any surface where
sanitization is required. One particular application of the inventive wipes is the rapid and efficient
disinfection and drying of cow teats. The present invention further concerns a method of reducing the
incidence of mastitis infection in dairy animals which employs the novel wipes.
U.S. Pat. Nos. 5,135,910; 5,217,950; 5,260,271; 5,304,540 and 5,334,582 all disclose broad range
bactericidal compositions comprising a lanthocin (a lanthionine-containing bacteriocin) such as nisin,
and a chelating agent. The patents further disclose a number of uses for the compositions based on
their bactericidal properties. Consept, a nisin-containing formulation within the scope of the
compositions disclosed in the cited patents, has been on the market for a number of years. The
wipes of the instant invention typically contain formulations such as those disclosed in the abovecited patents, which are incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention provides a disposable wipe of a paper or cloth fabric typically with a
bacteriocin-based formulation further comprising a chelating agent, a salt component, a stabilizer, a
drying agent and a surfactant. The wipes of the instant invention provide efficient one-step
disinfection and drying of surfaces and have applicability to any situation requiring sanitization of a
surface. One particular application is in the disinfection and drying of cow teats.
The wipes of the instant invention contain a disinfectant formulation typically comprising as active
ingredient a bacteriocin in combination with a salt component. The preferred active agent is a
lanthocin (a lanthionine-containing bacteriocin) such as nisin, subtilin, epidermin, gallidermin,
cinnamycin, duramycin, ancovenin or Pep 5. Other peptide bacteriocins such as lysostaphin may
also suitably be employed. The bacteriocin agents of the instant invention are much more potent than
commonly used germicides and do not exhibit undesirable side effects. The active agents of the
formulations according to the instant invention are not confined, however, to peptide bacteriocins;
other antibacterial agents such as chlorhexidine may suitably be employed in combination with the
bacteriocins of the invention. The presence of the bacteriocin component allows the employment of
the chlorhexidine or other non-bacteriocin germicidal component in much lower concentrations, thus
182/1006
eliminating concerns regarding unwanted side effects. Furthermore, the active agent may comprise
two or more bacteriocins in combination or a bacteriocin in combination with another antibacterial
agent. The most preferred embodiment of the instant invention known at this time comprises nisin as
active ingredient.
The use of bacteriocins such as nisin in conjunction with a paper or cloth wipe has inherent
difficulties due to the peptide nature of the active ingredient. It would be expected that such
compounds adsorbed onto a wipe would not easily be released from the wipe, in the instant case
onto the site where disinfection is desired. Accordingly, the formulations of the instant invention
further comprise a component to increase the ionic strength and which thus serves to loosen the
links between the bactericidal agent and the surface of the wipe. This component has also been
found to be a factor in enhancing the stability of the active agent while in contact with the wipe.
Suitable agents for increasing the ionic strength are halide salts of carboxylates, hydroxyacid salts,
salicylates, glycolates, phosphates and polyphosphates. A preferred component is NaCl in a
concentration range of 10 to 100 mM. The preferred concentration of NaCl is 50 mM. Another
preferred component is sodium citrate in a concentration range of 1 to 10 mM. The preferred
concentration of citrate is 5 mM.
It has been found (see U.S. Pat. Nos. 5,135,910; 5,217,950; pending U.S. application Ser. No.
08/386,122 incorporated herein by reference) U.S. Pat. Nos. 5,260,271; 5,304,540 and 5,334,582,
incorporated herein by reference) that lanthocins in combination with a chelating agent exhibit
enhanced potency and broader range as bactericides. Accordingly,the formulations of the instant
invention typically contain a chelator component. Suitable chelating agents are, for example, EDTA,
CaEDTA, CaNa2 EDTA and other alkyldiamine tetraacetates, as well as EGTA and citrate. The
preferred chelating agents are EDTA (1-10 mM) and/or citrate (1-30 mM). The most preferred
concentrations of EDTA and citrate are 3 mM and 5 mM, respectively. The chelating agents may be
used alone or in combination.
The preferred peptide bacteriocins of the instant invention are somewhat labile, and degradative
losses can be incurred when the bacteriocin is in contact with the wipe. It has been found (pending
U.S. application Ser. No. 08/386,122, incorporated herein by reference) that methionine and related
thioether compounds act to protect bacteriocins, particularly nisin, against degradation. Accordingly,
methionine is typically a component of the formulations of the instant invention. Methionine is
employed in a concentration range of 1-10 mM, the most preferred concentration being 2 mM. A
183/1006
combination of EDTA and citrate has also been found not only to impart the properties described
above for chelators, but additionally to enhance the stability of the bacteriocin component.
Also along the lines of stabilization of the active agent, catalase may be added to the formulation to
destroy any peroxides which may be present. Catalase may typically be employed at a concentration
range of 6 to 600 units/ml. The most preferred concentration of catalase is 60 units/ml.
Another important component of the formulation is a drying agent. A fine balance must be achieved
between formulations which are too "wet" and ones which are too "dry." As described above, too
much moisture during the disinfecting procedure can interfere with the disinfection process; enhance
the chances of cross contamination; and, particularly in the case of sensitive surfaces such as skin
and teats, cause irritation. On the other hand, a minimal amount of "wetness" must be maintained in
order that the bacteriocin component function optimally; the wipes according to the instant invention
are not "moisture activated," but a minimally moist environment must be maintained to assure the
desired efficacy. Furthermore, formulations which promote too rapid or complete drying can promote
irritation of sensitive surfaces. Accordingly, the formulations of the instant invention typically further
comprise a drying agent such as an alcohol. Because of the bacteriocin component employed in the
inventive wipes, the alcohol component is required only in the capacity of drying and not in that of
disinfection. Therefore, the formulations of the instant invention contain far less alcohol than typically
required in known wipe formulations. The typical concerns of surface sensitivity and flashpoint
associated with high alcohol content are thus circumvented. Among the alcohols suitable for use in
the inventive wipes are methanol, ethanol, 1-propanol, 2-propanol, and benzyl alcohol. The preferred
drying component is 1-propanol in a concentration of 10 to 20% w/v. The most preferred
concentration for 1-propanol is 12% w/v.
The patents cited above as directed to nisin-chelator compositions also disclose that a surfactant
component may further enhance the potency and range of activity of lanthocin-containing
bactericidal compositions. The formulations of the instant invention may further comprise such a
component. Such components include nonionic and amphoteric surfactants and emulsifiers,
quaternary compounds, monoglycerides and fatty acids. More particularly such components may be
selected from among glycerol monolaurate (0.03 to 0.3% w/v); nonionic surfactants such as
polysorbate 20 (0.1 to 3% w/v), Arlasolve 200 (0.1 to 3% w/v), and Triton X-100 (0.1 to 3% w/v);
cationic agents such as lauramine oxide (0.1 to 3% w/v); or zwitterionic agents such as
cocoamidopropyl betaines (0.1 to 3% w/v). The preferred surfactant component presently known is
the nonionic surfactant polysorbate 20 at a concentration of 1% w/v.
184/1006
The wipe formulation of the instant invention may further comprise a conditioner/humectant
component and a thickening agent. The conditioner is particularly useful when the wipe is applied to
easily irritated skin surfaces. Typically this component may be selected from propylene glycol,
glycerol, sorbitol and lactylate each in the concentration range of 1 to 10% w/v. The preferred
conditioner is propylene glycol at a concentration of 10% w/v. The thickening agent may be selected
from hydroxyethyl cellulose, methyl cellulose, polyvinyl pyrrolidone or mixtures of these agents, each
in the concentration range 0.1 to 1% w/v. Hydroxyethyl cellulose at 0.35% w/v is the preferred
component according to the instant invention. The liquid-carrying capacity of the wipe may be
increased when a thickener is a component of the wipe formulation.
The pH of the formulations is adjusted to the range from 3.0 to 5.0. The preferred final pH of the
formulations is 3.5.
The '910, '950, '271, '540 and '582 patents cited above disclose a number of compositions and
formulations which comprise various combinations of ingredients recited above in the description of
the instant formulations. The patents are incorporated by reference for their disclosure of the
production of nisin chelator compositions, compositions which may be used in the wipes of the
instant invention.
The choice of paper wipe to be used in conjunction with the nisin-based formulation is also
important to the successful carrying out of the invention. Some papers, for example, do not allow
advantageous adsorption of the active agent. This could result either in insufficient adsorption of
active ingredient by the wipe or difficulty in releasing the active ingredient from the wipe to the target
area. Other considerations in this regard are cost, texture with respect to impact on skin, liquid
capacity per towelette, liquid residue left on skin and tensile strength of the paper when wet. The
best papers which are currently known to the inventors are hydroentangled cellulose.
According to the instant invention, the wipes may be dispensed by a variety of means, all of which
are designed to preserve the integrity of unused wipes from soiling and to prevent premature drying
of the wipes. The wipes may be dispensed from a center-pull dispenser via a roll, in a layered
manner, an interfold/interleaved manner or a pop-up manner from a canister. Another aspect of the
invention provides for the dispensing of wipes from a recyclable, disposable, sealed plastic bag.
185/1006
The dispenser may take the form of a belt holster for the convenience of the user or mounted close
at hand to each workstation. A "disposable bag"-type dispenser would be suitable for mounting
throughout the area of use. One aspect of the invention deals with reusable dispensers, a concept
which addresses the problem of waste disposal.
The packaged product may suitably take a number of forms, including a package prefilled with both
towelette and liquid formulation, a canister containing dry towelettes with the liquid component to be
added immediately prior to dispensing, a reusable canister to which towelettes and liquid may be
added at the appropriate time, or a package containing dry towelettes impregnated with reagents to
which water or formulated liquid components could be added at the appropriate time prior to use.
One application of the wipes of the present invention provides a method of pre-milking preparation
of cows' teats, combining the advantages of udder washing (removal of excess soilage, mechanical
stimulation of the udder to promote milk "let-down"); of pre-dipping with a germicide (sanitize the teat
skin and teat end, remove excess residues and dry off with a paper towel); speed and economy of
effort in one step. As dairies become larger and the practice of milking cows three times a day
becomes more prevalent, so grows the importance of time-saving practices and means of increasing
milking efficiency. The inventive disposable moist wipes and one-step procedure according to the
instant invention address the primary concerns with regard to pre-milking preparation set forth by the
National Mastitis Council (NMC) in its guidelines. The treatment according to the instant invention
decreases the required time and labor for preparing the animals and increases milking efficiency.
The practice of premilking hygiene promotes the reduction of bacterial contamination of milk which
has been the focus of studies for many years and remains of prime importance.
Pre-milking treatment of the teats with the towelettes of the instant invention removes any debris or
soilage from the milking-unit-contact-surface of the teat, stimulates the release of oxytocin by
massage of the udder, thus enhancing milk "letdown," provides a germicidal action to kill mastitisinducing bacteria on the teat, and leaves a minimum and quick drying residue of liquid on the teat,
thus eliminating the need to dry the teat by a separate step.
The application of the disposable wipes according to the instant invention can likewise provide time
and labor-saving benefits when used to clean and sanitize the skin during gloveless food handling
(while minimizing the risk of contaminating the food with germicidal residues) The application of the
instant invention would provide similar benefits when used to clean and sanitize food contact hard
surfaces or other food contact inanimate surfaces. The application of the disposable wipes
186/1006
according to the instant invention can likewise provide time and labor-saving benefits when used to
cleanse the skin of the head, neck and face, or any skin surface that may benefit from combined
cleansing and sanitization with a moist wipe leaving behind a rapidly drying moist residue. the
cleansing quality of the wipe can provide a fresh or cleansing benefit to the user. Likewise, the
cleansing and germicidal qualities can be used before and after minor surgical procedures or prior
to other procedures which may entail breaking the integrity of the skin, such as puncturing as
encountered during application of a hypodermic needle.
The towelettes may be located in a stationary HDPE plastic container or alternatively in a beltmounted dispenser that is strapped around the waist. The towelettes could be in the form of a centerpull roll in a cylindrical tub or interleaved in a rectangular container and soaked in the germicidal
solution according to the instant invention. A dispenser with a pull-through mechanism would
minimize the likelihood of soiling the stock of towelettes.
Employment of the towelettes of the instant invention has a number of advantages over currently
employed procedures. Among these are that cleaning and sanitizing become a single operation,
saving time and effort, and the operator's hand is simultaneously sanitized each time at the point of
application. For the dairyman current procedures comprise separate steps of removing dirt,
sanitization with teat dip, and drying with a paper towel; now a single product can accomplish all
these steps. Furthermore, a single-use paper wipe eliminates cross-contamination. The disclosed
treatment effectively addresses the NMC recommended practice of avoiding excess liquid from
udderwashing running down the teat; minimal or no liquid residue remains on the teat and gets into
the milk supply.
The wipes provide for optimal measured delivery of adequate germicidal agent and optimal drying
of treated area. In addition the wiping action physically removes bacteria (>90%) as well as
superficial debris and, when used with dairy cows, further provides for simultaneous stimulation of
milk let-down by massaging action. The amount of sanitizer required is reduced due to elimination of
the necessity for frequent discard of product from the teatdip cup, required to overcome/prevent
crosscontamination by soiled germicidal solution in the cup.
The treatment according to the instant invention can also be used beneficially in a post-milking
regimen. This aspect of treatment provides mechanical removal of residual milk from "open" teat
orifice and concomitant reduction of likelihood of infection, as well as a "dry" teat protected from the
187/1006
risk of chapping and "freezing" in cold weather. The postmilking sanitization also affords an additional
measure of prevention of mastitis.
EXAMPLE 1
To determine whether methionine could protect the nisin from degradation when the nisin is exposed
to the paper towels, nisin-based sanitizer formulation (PDF) was prepared with methionine at
concentrations from 0 to 100 mM. Fresh sections of paper towel were incubated in these solutions
and samples of the PDF were analyzed for nisin content at intervals over 6 days. Nisin content was
determined as absorbance at 210 nm after separation by HPLC. Methionine was seen to give
protection against loss of nisin. Chromatograms of the samples showed degradation of the nisin into
multiple components in the control samples without methionine and protection against this
degradation in samples containing methionine.
______________________________________
1-propanol
24 ml
propylene glycol 6 ml
10% polysorbate 20 6 ml
100 mM EDTA
1.2 ml
water
qs 60 ml
pH adjusted to 3.5
______________________________________
______________________________________
Met (200 mM)
nisin (10 mg/ml)
PDF H2 O
______________________________________
1. 0 ml 0.045 ml 8.96 ml
9.0 ml
2. 0.45 ml 0.045 ml 8.96 ml
8.55
3. 0.9
0.045 ml 8.96 ml
8.1
4. 1.8
0.045 ml 8.96 ml
188/1006
7.2
5. 4.5
0.045 ml 8.96 ml
4.5
6. 9.0
0.045 ml 8.96 ml
0
______________________________________
To 50 ml polypropylene tubes were added 100 cm@2 (10 cm.times.10 cm) pieces of paper towel
and 9 ml aliquots of the given test solution. The tubes were then rocked gently at room temperature
and 400 ul aliquots were taken at various times for nisin analysis. The data suggest that the presence
of methionine protects against the loss of nisin by degradation. Studies on the stability of nisin
exposed to the paper wipes also revealed loss of nisin without the corresponding appearance of
degradation products. This loss suggested there was a further source of nisin instability in the moist
wipes product and that methionine did not protect against this second form of loss.
EXAMPLE 2
Nisin in a wipes formulation is stabilized from degradation by methionine and from adsorptive loss
by addition of salt.
The results indicate that the presence of sodium chloride reduces the adsorptive loss of nisin, and
that methionine and sodium chloride together stabilize nisin in the wipes formulation and allow
recovery of >90% of the nisin in the samples after 6 days at room temperature.
EXAMPLE 3
Nisin in a wipes formulation is stabilized from degradation by addition of methionine and from
adsorptive loss by the addition of salt. Paper types from various sources encompassing a range of
compositions, weights, feel and tensile strengths were evaluated for their suitability with the wipes
formulation.
PDF (25 ug/ml nisin) was prepared as outlined in Table I. For each condition in the experiment, a
100 cm@2 section of towel was placed in a 50 ml conical polypropylene tube, 9 ml test solution was
added, and the tubes were placed on a rocker table at room temperature. Samples of liquid (1ml)
were withdrawn for analysis after 3 days (Table II).
189/1006
The data shown in Table II confirm that nisin in the wipes formulation is stabilized by the addition of
salt and methionine to the formulation. The data further illustrate that this permits a wide range of
papers to be used, but that hydroentangled cellulose was the most suitable choice of paper.
TABLE I
______________________________________
PDF Formulations
A B C
regular +Met +Met +NaCl
______________________________________
1-propanol (%)
20
20 20
propylene glycol (%)
10
10 10
polysorbate 20 (%)
1
1 1
EDTA mM 1
1 1
methionine mM -10 10
NaCl mM --- 100
nisin ug/ml 25
25 25
DI water qs
qs qs
______________________________________
TABLE II
______________________________________
Evaluation of towel samples for softness, strength, and residual nisin
concentration in the liquid phase after 3 days at room temperature.
control
weight
softNisin, +Met
g/yd@2
ness* strength**
-- g/ml
+Met +NaCl
190/1006
______________________________________
polyester/
57 4 4 0 4 28
cellulose
cotton 100% 3 1 7 12 23
Rayon/poly45 3 3 10 15 26
propylene
Rayon acrylic
45 4 3 9 17 25
carded Rayon
26 4 2 0 3 20
100%
carded Rayon
32 4 2 0 0 12
100%
Rayon/ 26 3 2 1 3 26
cellulose wetlaid
paper crepe
23#/R 2 2 4 9 29
control
26 27 27
Rayon 70/30
30 4 3 6 10 19
cellulose hydro31 3 3 22 26 28
entangled
______________________________________
*1 = harsh 4 = soft
**1 = tears easily 4 = resists tearing
EXAMPLE 4
The germicidal potency of wipes formulations was evaluated against bacterial suspensions of E. coli
strain ATCC 8739 and S. aureus strain ATCC 6538. The formulation compositions were identical to
that illustrated in Table I, but with 12% 1-propanol and with 10% propylene glycol substituted with 5%
191/1006
glycerol. In addition, a range of concentrations of benzyl alcohol and/or citrate were added to the
formulations. Further, the test formulations were evaluated for germicidal potency in the presence of
50% by volume whole milk to act as an organic load. Formulations were preincubated 2 hours in the
presence of milk, then their germicidal performance was evaluated after 1 minute incubation with
bacteria suspensions at 37 DEG C.
The data in Tables III and IV illustrate that addition of citrate enhances the germicidal potency of
nisin-based formulation, particularly in the presence of the organic load provided by milk. The
combination of the chelators, citrate and EDTA, in the formulation was surprisingly effective at
overcoming divalent cations present in the milk. Also, the germicidal performance of the formulations
was further improved by the incorporation of benzyl alcohol.
TABLE III
______________________________________
E. coli (cfu/ml)*
% benzyl alc
% citrate -- 50% milk
______________________________________
1.5 3.0
<5 <5
1.5 2.5
<5 <5
1.5 2.0
<5 <5
1.5 1.5
<5 <5
1.5 1.0
<5 <5
1.5 0.5
<5 <5
1.5 0.0
<5 <5
3.0
<5 5
2.5
<5 <5
2.0
<5 170
1.5
<5 <5
1.0
<5 10
0.5
<5 3.75 .times. 10@6
0.0
<5 5.70 .times. 10@7
______________________________________
*Initial viable count: 3.7 .times. 10@9 cfu/ml
192/1006
TABLE IV
______________________________________
S. aureus (cfu/ml)*
% benzyl alc
% citrate
50% milk
______________________________________
1.5 3.0
<5 20
1.5 2.5
<5 10
1.5 2.0
<5 70
1.5 1.5
<5 330
1.5 1.0
<5 6.45 .times. 10@3
1.5 0.5
<5 4.00 .times. 10@5
1.5 0.0
<5 9.65 .times. 10@7
3.0
<5 1.30 .times. 10@3
2.5
<5 6.15 .times. 10@5
2.0
<5 5.04 .times. 10@6
1.5
<5 2.68 .times. 10@7
1.0
<5 1.72 .times. 10@8
0.5
<5 4.40 .times. 10@7
0.0
5 2.74 .times. 10@8
______________________________________
*Initial viable count: 2.35 .times. 10@8 cfu/ml.
EXAMPLE 5
Wipes were formulated with PDF as in Table I except that Arlasolve 200 was substituted for
polysorbate 20, nisin concentration was 50 ug/ml, NaCl was 300 mM where present, and citrate was
1% where present. Paper from various sources was used in the various wipes formulations. Nisin
stability was monitored by HPLC after storage of the wipe formulations at room temperature. The
results are presented in Tables V and VI.
The data confirms that several papers are suitable, including hydroentangled cellulose (HEC), when
the formulations contain methionine to prevent degradation of nisin, plus either NaCl or citrate to
prevent adsorptive losses of nisin.
193/1006
TABLE V
______________________________________
Trial #
MA3-16Paper
ProductNaCl
+/-Met
day 1 day 6 day 18 day 28
+/-Nisin Concentration ug/ml
______________________________________
1 Chubbs + - 22.2 35.6 30.6 26.5
2 Chubbs - - 45.4 46.9 43.1 43.2
3 New Chubbs
+ + 49.2 49.0 47.1 42.1
4 New Chubbs
+ - 42.1 37.4 29.7 25.2
5 New Chubbs
- + 52.5 53.6 50.6 44.9
6 New Chubbs
- - 44.5 35.2 25.3 21.1
7 AMBI #546 + + 49.6 52.9 53.0 40.8
8 AMBI #546 + - 52.7 43.7 37.9 36.0
9 AMBI #546 - + 46.7 56.9 60.2 48.2
10 AMBI #546 - - 45.6 42.2 34.5 26.2
11 HEC paper + + 50.6 53.4 56.6 46.8
12 HEC paper + - 41.5 42.5 45.1 33.9
13 HEC paper - + 53.2 55.2 61.8 48.8
14 HEC paper - - 44.2 40.4 38.0 29.2
15 Scott 1480
+ + 49.6 56.1 61.4 51.0
16 Scott 1480
+ - 43.8 44.4 43.5 33.1
17 Scott 1480
- + 53.1 56.9 60.5 49.3
18 Scott 1480
- - 43.5 38.0 27.4 22.8
194/1006
19 Scott 129 + + 49.4 52.1 55.9 31.7
20 Scott 129 + - 46.0 44.9 45.2 35.5
21 Scott 129 - + 44.1 41.7 29.1 26.7
22 Scott 129
Stock - 35.9 29.3 16.5 16.1
Solution
23 MA3-15-1 + + 52.1 49.3 48.7 39.0
24 MA3-15-2 + - 45.6 37.6 30.8 26.0
25 MA3-15-3 - + 55.1 52.3 48.6 39.6
26 MA3-15-4 - - 45.6 36.3 25.6 25.5
______________________________________
TABLE VI
______________________________________
Trial #
Paper Citrate Methionine
Nisin Concentration
MA3-29 Product +/- +/- day 0
day 14
day 28
______________________________________
1 New + + 38.5 31.4 42.0
Chubbs
2 New + - 21.0 17.8 23.6
Chubbs
3 New - + 34.6 26.3 21.8
Chubbs
4 New - - 13.5 28.0 27.3
Chubbs
5 AMBI + + 42.1 39.2 48.2
#546
6 AMBI + - 24.0 20.8 30.2
#546
7 AMBI - + 34.9 7.4 7.6
#546
195/1006
8
AMBI - - 13.5 3.8 6.3
#546
9 HEC* + + 40.0 38.2 46.9
paper
10 HEC + - 22.0 24.9 37.9
paper
11 HEC - + 33.3 17.8 15.9
paper
12 HEC - - 16.3 4.8 11.3
paper
13 MA3-29- + + 40.8 37.1 41.9
1
STOCK
14 MA3-29- + - 22.6 17.2 23.0
2
STOCK
15 MA3-29- - + 39.6 33.5 39.9
3
STOCK
16 MA3-29- - - 19.3 30.2 36.5
4
STOCK
______________________________________
*Hydroentangled cellulose paper
EXAMPLE 6
This study was performed to determine the amount of liquid sufficient to wet a towelette for use as a
Wipe product. Measured areas of each paper sample were weighed, then wet with PDF so as to be
thoroughly moist without being dripping wet, and weighed again to determine the amount of
formulation required for this condition. The results are seen in Table VII.
TABLE VII
______________________________________
Total Weight of
196/1006
Weight of
Liquid
ml liquid
Paper Area Paper Wet Paper
Added /cm@2 paper
______________________________________
New Chubbs
344.0 cm@2
1.92 g 8.20 g 6.4 ml
.019
#546 310.6 cm@2
1.17 g 3.60 g 2.3 ml
.007
#565 288.5 cm@2
1.13 g 4.50 g 3.3 ml
.011
hydroentangled
cellulose
______________________________________
EXAMPLE 7
Formulated wipes prepared as shown in Table I but with 300 mM NaCl were evaluated for
germicidal performance on live cow teat skin according to the following protocol A.
Protocol A
Ten cows were prepped and cleaned for protocol A. This involved removing the hair from the bottom
side of the udders and a series of washings. The cows' teats were washed with Theratec (0.5%
iodophor), thoroughly rinsed with water, and then wiped down with alcohol swabs. Collection cups
were labelled and placed beside each cow, four per cow. Each teat dipped to 15 mm with a
suspension of bacteria at approximately 10@8 cells/ml. After ten minutes, the teat was dipped with
the appropriate test solution to a depth of 30 mm or wiped with one towelette. The wiping action
consisted of grabbing the teat at its base, pulling down and off, turning the hand 90- and grabbing
the teat at its base once again, pulling down and off. After one minute, surviving cells were harvested
197/1006
using a syringe,filled with 10 ml of quenching solution to neutralize the action of nisin and to collect
bacteria from the surface of the teat into a collection cup. Collection cups were immediately capped
and placed on ice in a cooler. Approximately one hour later, the 80 samples were diluted and plated
in duplicate on blood agar plates. Plates were incubated at 37 DEG C. for 24-48 hours. Colony
forming units (cfu) were scored and reductions in cfu were calculated relative to cfu recovered from
the control teats. All four teats were tested on each cow, and a total of 20 cows were tested. The two
hind teats were the control teats, that is they were dipped in water. The two front teal-s were tested
with the one of the germicidal products.
Results, expressed as log reduction in cfu, for S. aureus, are presented in Table VIII. The log
reduction value per cow was calculated by subtracting the mean log cfu of the two test (front) teats
from the mean log cfu of the two control (hind) teats. The Total Mean Log Reduction for the five cows
in each test condition is also reported in Table VIII.
The PDF wipes performed comparably to the PDF dip. The action of wiping alone, with a water wipe,
yielded a 1.7-log reduction.
TABLE VIII
__________________________________________________________________________
Theratec PDF
(0.5% (25 ug/ml
PDF Water
COW lodophor)
COW nisin)
COW Wipes
COW Wipes
__________________________________________________________________________
#1 0.5 #6 3.3 #11 3.6 #16 1.7
#2 0.7 #7 3.2 #12 2.7 #17 1.9
#3 0.4 #8 2.7 #13 1.9 #18 1.8
#4 0.5 #9 4.2 #14 2.4 #19 1.6
#5 0.8 #10 1.6 #15 2.2 #20 1.4
TOTAL
0.6 TOTAL
3.0 TOTAL
198/1006
2.6 TOTAL
1.7
MEAN MEAN MEAN MEAN
LOG LOG LOG LOG
RED RED RED RED
__________________________________________________________________________
EXAMPLE 8
Wipes formulation (PDF) was prepared as described in Table I except that 1% Arlasolve 200 was
used in place of polysorbate 20 and 1% citrate was used in place of NaCl, and nisin was at 50 ug/ml.
Where present, 1-propanol was at 20%. The test formulations, #1 with and #2 without 1-propanol,
were evaluated for germicidal performance toward S. aureus on live cow teat skin according to
protocol A (above), and compared with the performance of Theratec (0.5% iodophor) and PDF teat
dip.
The results are presented in Table IX and demonstrate that both wipes formulations performed
comparably against S. aureus, and were equivalent to the performance of the 0.5% iodophor.
TABLE IX
__________________________________________________________________________
WIPES WIPES
#1 #2 Theratec PDF
50 ug/ml 50 ug/ml 0.5% 25 ug/ml
COW nisin
COW nisin
COW Iodophor
COW nisin
__________________________________________________________________________
#1 1.9 #6 2.4 #11 2.4 #16 4.3
#2 3.4 #7 2.4 #12 3.1 #17 2.3
#3 3.4 #8 3.9 #13 1.7 #18 2.5
#4 2.3 #9 2.6 #14 2.2 #19 4.7
#5 2.9 #10 3.1 #15 2.7 #20 4.7
TOTAL
199/1006
2.8 TOTAL
2.9 TOTAL
2.4 TOTAL
3.7
MEAN MEAN MEAN MEAN
LOG LOG LOG LOG
RED RED RED RED
__________________________________________________________________________
EXAMPLE 9
Wipes formulation (PDF) was prepared as described in example 8 and used to prepare moist paper
wipes with liquid content at 3 g, 5 g, and 7 g per wipe (6 in.times.6.75 in). These wipes were
evaluated for germicidal activity against E. coli on live cow teat skin according to protocol A (above),
and compared with the performance of Theratec (0.5% iodophor).
The results are presented in Table X and demonstrate that 7 g liquid / wipe provide germicidal
activity superior to that of the wipes with lower liquid loading.
TABLE X
______________________________________
log
reduction
positive
date formulation target treatment
control
______________________________________
2/2/95 PDF wipes, E. coli 2.2 3.2
3 g/wipe
PDF wipes, 5
3.4
g/wipe
PDF wipes, 7
3.7
g/wipe
______________________________________
EXAMPLE 10
200/1006
Wipes formulation (PDF) was prepared as described in example 8 and used to prepare moist paper
wipes. These wipes were evaluated for germicidal activity against a range of organisms on live cow
teat skin according to protocol A (above), and compared with the performance of several teat dips
as positive controls.
The results are presented in Table XI and demonstrate that the PDF formulated wipes have effective
germicidal activity on cows' teats against the major pathogenic organisms implicated in mastitis
infections.
TABLE XI
______________________________________
log reduction
cow teats (n=)
target treatment positive control
______________________________________
10
S. aureus 2.5
0.2*
10
S. agalactiae
4.3
0.7*
10
S. uberis 3.0
3.3*
10
Klebsiella
2.6
3.2*
10
E. coli 2.3
10
S. aureus 3.4
10
S. uberis 2.8
2.5$
10
K. 3.1
4.1$
pneumoniae
10
S. aureus 3.4
3.0$
10
E. coli 2.0
4.0$
______________________________________
*Consept Pre + Post Teat Dip
$Teat Guard 1% iodophor Teat Dip
EXAMPLE 11
201/1006
Formulated wipes prepared as shown in Table I but with 12% 1-propanol, 3 mM EDTA, and 5 mM
citrate, were used in an Experimental Challenge trial following the standard protocol of the NMC. A
dairy herd of 160 cows at Cornell University Veterinary College was used to test the efficacy of the
PDF wipes in the field. The four treatment conditions are summarized in Table XII, there were 40
cows in each treatment group. To create extreme infectious conditions promoting the likelihood of
mastitis infections each cow's teats were dipped in a suspension of S. aureus and S. agalactiae
(10@8 cfu/ml for each organism) immediately after the afternoon milking Monday to Friday. The
bacterial challenge was followed immediately by the postmilking sanitization appropriate for that
group.
The data presented in Table XII show that the PDF wipes used as a post-milking treatment, or as a
premilking treatment in conjunction with post dipping with Consept teat dip, reduce the incidence of
mastitis infections comparable to the effect of Theratec (0.5% iodophor) teat dip.
TABLE XII
______________________________________
mastitis
postmilking
infections
group premilking treatment
treatment at 2 weeks
______________________________________
A - ve
wash with water-soaked paper
none 13
control
towel
dry with separate paper towel
B + ve
wash with water -soaked paper
dip with
control
towel
Theratec 0.5%
dip with Theratec 0.5% iodophor
iodophor
202/1006
dry with separate paper towel
C clean and sanitize with PDF Wipes
dip with nisin6
based Consept
teat dip
D wash with water-soaked paper
sanitize with
7
towel PDF Wipe
dry with separate paper towel
______________________________________ Claims:
We claim:
1. A moist paper or fabric wipe containing a liquid formulation comprising a suitable amount of a
bacteriocin, a chelating agent, a stabilizer, a surfactant, a salt and an alcohol drying agent.
2. A wipe according to claim 1 wherein the bacteriocin is a lanthocin; the chelating agent is selected
from one or more of an alkydiamine tetraacetate, EGTA and citrate; the stabilizer is a thioether
compound; the surfactant is a nonionic surfactant, a cationic surfactant, a monoglyceride or a fatty
acid; and the salt is NaCl.
3. A wipe according to claim 2 wherein the lanthocin is nisin, the chelating agent is EDTA, the
stabilizer is methionine, the surfactant is polysorbate 20 and the drying agent is 1-propanol.
4. A wipe according to claim 3, wherein the chelating agent comprises EDTA and citrate in
combination.
5. A wipe according to claim 3 wherein the concentration of nisin is in the range of 25 to 500 ug/ml
and the concentration of NaCl is in the range of 10 to 300 mM.
6. A wipe according to claim 3 wherein the concentration of EDTA is in the range of 0.1 to 10 mM.
203/1006
7. A wipe according to claim 4 wherein the concentration of EDTA is in the range of 0.1 to 10 mM
and the concentration of citrate is in the range of 1 to 30 mM.
8. A wipe according to claim 3 wherein the concentration of methionine is in the range of 1 to 10 mM.
9. A wipe according to claim 3 wherein the concentration of polysorbate 20 is in the range of 0.1 to
3%.
10. A wipe according to claim 3 wherein the concentration of 1-propanol is in the range of 10 to 20%.
11. A wipe according to claim 1 wherein the formulation further comprises a conditioner and a
thickener.
12. A wipe according to any one of claims 1-4, wherein the formulation further comprises
chlorhexidine.
13. A wipe according to any one of claims 1-4 wherein the paper wipe is of a non-woven material.
14. A one-step method for disinfecting and drying a cow teat prior to milking which comprises
wiping the teat with a wipe according to any one of claims 1-4.
15. A one-step method for disinfecting and drying a cow teat after milking which comprises wiping
the teat with a wipe according to any one of claims 1-4.
16. A one-step method for disinfecting and drying a skin surface which comprises wiping the skin
surface with a wipe according to any one of claims 1-4.
17. A one-step method for cleansing a skin surface which comprises wiping the skin surface with a
wipe according to any one of claims 1-4.
18. A one-step method for disinfecting and drying a food contact surface which comprises wiping
the food contact surface with a wipe according to any one of claims 1-4.
19. A method for reducing the incidence of mastitis infection in dairy animals, which comprises
wiping the animals' teats after milking with a wipe according to any one of claims 1-4.
204/1006
20. A method for reducing the incidence of mastitis infection in dairy animals, which comprises premilking wiping of the animals' teats with a wipe according to any one of claims 1-4 in conjunction with
post-dipping with Consept teat dip.
205/1006
16. EP0851751 - 27.02.1997
A CHEWING GUM COMPOSITION CONTAINING A BACTERIOCIN ANTIBACTERIAL AGENT
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP0851751
Inventor(s):
MCCONVILLE PETER SCOTT (GB)
Applicant(s):
SMITHKLINE BEECHAM PLC (GB); MCCONVILLE PETER SCOTT (GB)
IP Class 4 Digits: A61K
IP Class:
A61K7/16
E Class: A61K7/16D13
Application Number:
WO1996EP03653 (19960819)
Priority Number: GB19950017113 (19950821)
Family: EP0851751
Equivalent:
JP2000512124T
Cited Document(s):
WO8912399
DE4400408; WO9412150; WO9405251; WO9311738; EP0545911;
Abstract:
A GUM COMPOSITION WHICH COMPRISES A GUM BASE WHICH HAS A WATER CONTENT OF
LESS THAN 2 % BY WEIGHT OF THE FINAL COMPOSITION CHARACTERISED IN THAT THE
COMPOSITION CONTAINS AN ANTIBACTERIALLY EFFECT AMOUNT OF A BACTERIOCIN
ANTIBACTERIAL AGENT. THE COMPOSITIONS ARE USEFUL IN ANTIPLAQUE AND BREATH
FRESHNESS THERAPY.Description:
206/1006
A CHEWING GUM COMPOSITION CONTAINING A BACTERIOCIN ANTIBACTERIAL AGENT
This invention relates to oral hygiene compositions and in particular to oral hygiene gums
comprising particular antibacterial agents which compositions are useful in antiplaque and breath
freshness therapy.
A particular class of antibacterial agents are the bacteriocins. These have been defined as
proteinaceous substances produced by bacteria and which have antibacterial activity only against
species closely related to the species of origin. More recently it has been found that,
at least in certain instances, the spectrum of antibacterial activity may in fact be broader.
An example of a bacteriocin which has already found commercial application is nisin. This is
a lanthocin, comprising the atypical amino acid lanthionine. Nisin is a polypeptide with
antibacterial properties which is produced naturally by various strains of the bacterium
Streptococcus lactis. It is also a naturally occurring preservative found in low concentration in milk
and cheese. Nisin has recently been recognised by the FDA as a direct food ingredient. A summary
of nisin's properties is to be found in Advances in Applied
Microbiology 27 (1981), 85-123.
Recently, a purified form of nisin has been made available by Applied Microbiology Inc under the
trade name AMBICIN Nut). It has been suggested for use in a variety of applications including oral
care, as disclosed in PCT Application WO 89/12399 to
Blackburn et al.. and now in issued US Patent No. 5,135,910 for use in the oral cavity,
however the only specific disclosure of an oral hygiene product is as an oral rinse, for use as a
broad spectrum disinfectant. UK Application 9510719.9 discloses a nisin oral care
compositions for controlling Candida as well as previously indicated antibacterial activity against
plaque and gingivitis. There is no reference in any of the abovementioned patents or
applications to gum formulations containing nisin or AMBICIN N8.
The formulation of compositions comprising highly purified proteins, such as the compound
nisin, are particularly challenging as a protein in this state is rendered sensitive to many of
the processes that are commonly required in the preparation of successful formulations.
These problems are highlighted in 'Stability of Protein pharmaceuticals' part B, In vivo
207/1006
Pathways of Degradation and Strategies for Protein Stabilization, Edited by Tim J. Alien and Mark C.
Manning.
Chewing gum compositions are, in general, composed of a water-insoluble or base component and
a water-soluble chewing gum component, and as a result it is known that moisture loss and gain can
effect the life of gum products.
Chewing gum compositions which have low moisture content and/or low moisture pickup during
storage are known. EP-A-0472 428 (WM Wrigley JR.Company) describes a low moisture gum which
includes about 30 to 90% of a gum base with a softening point from about 70"C to about 100"C;
from about 0. l to about 20% of a water-insoluble plasticizer; from about 0 to 65% solid bulk
sweetener; less than about 0.5% water; and is substantially free of humectant. EP-A-0328 849
(Warner-Lambert Company) claims a sugarless, anhydrous chewing gum composition having a lowmoisture pick-up which includes from about 10 to 75% of a gum base, a low-moisture pick-up
bulking agent which both inhibits moisture pick-up and provides enhanced firmness, and a high
intensity sweetener.
Preferably the low-moisture pick-up bulking agent is an isomalt.
It has thus been found that when a gum, in particular an oral healthcare gum which contains
bacteriocin derivatives, particularly nisin, is prepared by applying usual, conventional formulations
and known processes, an unacceptable, unstable product results. In such cases the antibacterial
agent will break down during processing and further on storage.
It has also been found that use of a water soluble purified protein, such as nisin in a gum
composition can be released in a controlled and sustained manner to provide extended antibacterial
activity. Nisin also benefits as a protein moiety, from being a safe and ingestible active material.
It has therefore become desirable to prepare a gum composition which does not possess the
instability problems associated with the use of nisin and that is stable and acceptable to the
consumer, thus increasing its shelf life. By careful selection, control and order of addition of the
ingredients that make up the composition it has been possible to prepare a gum containing the
active bacteriocin derivatives.
208/1006
Accordingly, the present invention provides a gum composition which comprises a gum base and
has a water content of less than 2% by weight of the final composition characterised in that the
composition contains an antibacterially effect amount of a bacteriocin antibacterial agent.
In a further aspect of the invention there is provided a gum composition which comprises a gum
base and an antibacterially effect amount of a bacteriocin antibacterial agent and which has a water
content of from 1.0 to 1.5% by weight of the final composition.
The gum compositions of the present invention are considered anhydrous or substantially anhydrous
compositions as they have moisture contents of below 2%, preferably 1.5%, and most preferred
1.0% by weight of the final gum composition. It is only by use of this very low moisture content in the
composition that it possible to achieve a gum containing
AMBICIN N# that is acceptably stable. Therefore in accordance with the present invention, moisture
(in the form of water) is all but entirely removed from the compositions, and in as many cases as
possible, materials in their powder or anhydrous form are incorporated in the compositions.
Suitable bacteriocin antibacterial agents include nisin, gramicidin and tyrothricin and purified forms
of bacteriocins such as AMBICIN N(!3). Nisin and in particular the purified nisin preparation, in the
form of AMBICIN Ng)are especially preferred. AMBICIN N8 may be used in the encapsulated form
to protect against degradation, for example by embedding in a carbohydrate glass such as the
sugar trehalose.
In compositions ofthe present invention, the gum comprises from 0.001 to 5.0%, preferably from
0.005 to 2.0%, advantageously from 0.02 to 1.0 % of bacteriocin antibacterial agent by weight of the
final composition. In an alternative manner the level of bacteriocin agent needed is one which
reaches a sufficient level in the oral cavity to inhibit the desired microrganisms. An effective level of
a bacteriocin agent in the oral cavity, and more specifically nisin, to inhibit the desired organisms is a
level of about 0.99 ppm.
With regard to the gum composition the amount of gum base employed will vary depending on
various factors such as the type of base used, consistency required in the final product and the
other components used in the composition. A typical gum base composition comprises elastomers,
resins, fillers and softeners. The gum base will be selected from those commercially available. The
elastomer component of the gum base can be selected from, but not limited to, synthetic elastomers
209/1006
for example styrene-butadiene copolymer, isobutylene-isoprene copolymer, polyisobutylene, and
natural elastomers like chicle, natural rubber and jelutong and mixtures thereof.
The gum base usually includes a resin which acts as an elastomer solvent and can be selected from
terpene resins, glycerol esters of hydrogenated, partially hydrogenated, polymerised and partially
dimerised rosin and mixtures thereof. The resins may be used in an amount of from 10 to 50% by
weight of the gum base. Fillers such as talc, calcium carbonate and magnesium silicate in quantities
of from 20 to 50% by weight of the gum base may also be present in the gum base. In this particular
invention, talc is the preferred filler. The gum base may also include if desired, softeners, texture
modifiers such as waxes, partially hydrogenated vegetable oils and emulsifiers, particularly useful
being lecithin, fatty acid mono, di and triglygerides, glycerol monostearate and triacetin in amounts
of about 5% by weight of the gum base.Other optional ingredients include antioxidants, preservatives
and colorants.
The gum base may be either a soft or a hard gum base with no subsequent restriction on the
softening point temperature. Typically a soft gum base would be for example a bubble gum and a
hard gum would be a medicated gum. Suitably a hard gum base is used in the present invention
Suitably the gum base will be present in the amount of from 10 to 70%, preferably from 15 to 45%
and most preferably from 30 to 40% by weight of the final composition.
Compositions of the present invention will include at least one sugar alcohol (polyols) ingredient,
used as non-sugar bulk sweetener, as particularly in the instance of sugar-free gum compositions.
The sugar alcohols include sorbitol, xylitol, mannitol, lactitol and maltitol. Hydrogenated glucose
syrup, which is known by the Trade name Lycasin or polyhydric alcohols such as glycerine or
mixtures thereof may also be used as bulk sweeteners. The exact selection of the bulk sweetener will
be determined by the required texture of the final gum to meet processing requirements, product
stability and consumer acceptance criteria. Particle size is important in controlling elasticity and
firmness, especially in low moisture compositions.
Suitably the sugar alcohol will be present in the range of from 15 to 70%. preferably from 25 to 65%
and more preferably from 55 to 65% by weight of the final composition.
When sorbitol is used as the sugar alcohol either alone or in combination thereof, it may be
incorporated in either the liquid or the anhydrous powder form. Liquid sorbitol is sold as a 70%
aqueous solution, therefore if the liquid form is to be used in the composition, it will be necessary to
210/1006
keep the level as low as possible, thus excluding as much water as possible from the final
composition, but in anycase keeping the moisture content below the critical value of 2.0% by weight
of the final composition.
When hydrogenated glucose syrup is used as the bulk sweetener either alone or in combination
thereof, suitably it will be present in the range of from 0.1 to 5.0%, preferably from 2.0 to 3.5%, most
preferably 3.0% by weight ofthe final composition.
The gum composition according to the present invention may contain a variety of flavours alone or in
admixture. Particularly suitable flavours include essential oils, such as cinnamon, spearmint,
peppermint and the like and synthetic flavours. Natural flavours, for example those derived from the
essence of fruits may also be used. The flavour can be a liquid and/or powder form to give a long
lasting effect. Sweetening agents, for example sodium saccharin, Aspartame, Acesulpham K,
Neohesperidine Dichalconate and Talin and solubilisers such as lecithin and Cremophor
(polyethoxyhydrogenated castor oil) which is a
Tradename of BASF, may be added to the composition to help release and modify flavour.
Suitably any flavour that is used in the present invention will be added in the range of from 0.1% to
3.0% by weight of the final composition.
Selection of flavour and solubiliser may also modify the texture of the gum composition.
Additions of vegetable oils may also be used to keep the gum soft. These oils may be incorporated
at levels of 0.5 to 3% by weight of the final composition.
In a preferred aspect, compositions according to the present invention comprise a gum base; one or
more sugar alcohols such as, for instance, sorbitol and xylitol; flavour and AMBICIN Nut). Glycerine
is to be avoided or minimised in such compositions.
Compositions according to the present invention may usefully comprise a fluoride ion source, to
provide an anti-caries activity. A fluoride ion source is found to be compatible with the bacteriocin
peptide antibacterial agent. Suitable fluoride ion sources include metal fluoride salts, for instance
alkali metal fluoride salts such as sodium fluoride, amine fluoride salts, alkali metal
monofluorophosphate salts such as sodium monofluorophosphate and amine monofluorophosphate
211/1006
salts. Suitably the fluoride ion source would, if present, be included to provide from 50 to 3500 ppm,
preferably 100 to 2500 ppm of fluoride ions.
The gum composition may also comprise further optional ingredients may be added to the
composition such as buffering agents, for example urea and bicarbonate, dyes, pyrophosphates,
whitening agents, for example titanium dioxide and sodium tripolyphosphate (STP), preservatives,
chelators to broaden the spectrum of activity, for example ethylenediaminetetraacetic acid (EDTA)
and citrate, antisensitivity agents such as strontium and potassium salts, polishing agents and
anticalculus agents such as tetraalkali and dialkaliimetal pyrophosphate salts. It will be appreciated
that in each instance, an optional ingredient, if included, will be compatible with the bacteriocin.
The gums according to the present invention may be either sugar-free or sugar containing.
Sweeteners are well known in the art and include sugars such as sucrose, glucose, dextrose and
mixtures thereof. In instances where a sugar-free containing gum is required, auxiliary sweeteners
are incorporated into the compositions. The gums may be presented in any of the presentations as
conventionally used in the art, for instance as sticks, strips and dragees.
Compositions according to the invention will have a pH which is orally acceptable and within which
the antibacterial activity of the bacteriocin is not substantially compromised.
Also citrates, maleates or oxalates may be added as enzyme inhibitors to reduce the fall in plaque
pH. Gum compositions according to the invention may be prepared by conventional mixing/kneading
procedures, comprising admixing the ingredients together in the appropriate relative amounts in any
order that is convenient with the proviso that the nisin must be added during the final step of the
procedure and preferably not in aqueous solution.
Suitably, all excipients are dried, such that, for example the gum base, sugar alcohol, flavour and
nisin are in the anhydrous form and not in aqueous solution. An example of this process would
involve firstly melting the gum base at room temperature, thereafter increasing the temperature to
approximately 50 to 60"C. The sugar alcohol, flavour and additives are added thereafter with
continuous mixing. Nisin is then added at this final stage of the process, preferably in a premix with
any sugar alcohol and at this point the temperature should preferably be between 50 and 70"C. The
mixing time after adding nisin should be minimised but sufficient to ensure adequate dispersion.
212/1006
Control of temperature, mixing, sheer and time is important. The gum may then be formed into its
desired shape, such as sticks, strips or dragees.
To optimise the shelf life of the gum composition moisture pick up should be avoided or controlled.
Hygroscopic humectants may usefully be used in this respect.
The gum composition can be coated inside a candy coating of sucrose or preferably sugar alcohol
such as sorbitol, mannitol or xylitol. An intermediate coating can also be used to minimise moisture
migration between for example dragee centre and coating. These intermediate coatings include gum
arabic, gelatin, and other film forming ingredients like ethyl cellulose and copolymers of methacrylic
acid and ethyl acrylate which is sold under the
Tradename of Eudragit from Rohn Pharma.
Coatings for gums are well known in the art, but typically when a gum coating is required for a
dragee of the present invention this will comprise of approximately 99.63% of sorbitol solution, 0.3%
liquid flavour and 0.07% carnuba wax. The weight ratio of gum centre to coating in this instance will
be 0.9-1 .0g: 0.25-0.35g.
Gum compositions of the present invention are effective against oral bacteria and as such will be of
use in antiplaque and breath freshness therapy.
Accordingly, the present invention provides a method of reducing or preventing the formation of
dental plaque, which method comprises applying an antiplaque effective amount of a composition
according to the present invention to a patient in need thereof.
Accordingly, in a further aspect, the present invention also provides a method of freshening the
breath, which method comprises applying a breath freshness effective amount of a composition
according to the present invention to a patient in need thereof (E) indicates a registered trademark.
The selection of a composition comprising the water insoluble gum base the water soluble bulk
sweetener and other excipients is made to optimise the release of the antibacterial agent, nisin, over
an extended time to provide an exposure time for the active ingredient.
See Figs 1 and 2 for this release data.
213/1006
The invention will also be illustrated by reference to the following examples:
EXAMPLE 1 2 3 4 5 6 7 8
Ingredient % w/w % w/w % w/w % % % % w/w % w/w
w/w w/w w/w
Gum Base 35.00 35.00 35.00 35.00 35.00 35.00 36.00 36.00
Xylitol 20.00 20.00 20.00 - 57.00 20.00 20.00 20.00
Sorbitol powder 37.05 36.85 38.30 57.05 - 29.85 37.25 34.85
Sorbitol liquid, 3.00 - - 3.00 3.00 - - 70%
Liquid flavour 1.75 1.75 2.50 1.75 1.75 1.75 2.50 1.75
Menthol 1.50 1.50 1.00 1.50 1.50 1.50 0.75 1.50
Powder flavour 1.50 1.50 - 1.50 1.50 1.50 - 1.50
Ambicin N 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
Lycasin - 3.00 3.00 - - - 3.00 3.00
Mannitol - - - - - 10.00 EDTA - 0.20 - - - 0.20 0.20 0.20
Lecithin - - - - - - 0.10
Vegetable fat - - - - - - - 1.00
Claims:
1. A gum composition which comprises a gum base and has a water content of less than 2% by
weight of the final composition characterised in that the composition contains an antibacterially effect
amount of a bacteriocin antibacterial agent.
2. A gum composition which comprises a gum base and an antibacterially effect amount of a
bacteriocin antibacterial agent and which has a water content of from 1.0 to 1.5% by weight ofthe
final composition.
3. A composition according to claim 1 or 2, wherein the bacteriocin antibacterial agent include nisin,
gramicidin and tyrothricin and purified forms of bacteriocins such as
214/1006
AMBICIN N8.
4. A composition according to claim 3, wherein the bacteriocin antibacterial agents are present in an
amount of from 0.001 to 5.0% by weight ofthe final composition.
5. A composition according to claim 4 wherein the bacteriocin antibacterial agent is
AMBICIN N8.
6. A composition according to any one of the preceding claims wherein the gum base is present in
an amount of from 10 to 70% by weight of the final composition.
7. A composition according to any one of the preceding claims which additionally comprises a nonsugar bulk sweetener.
S. A composition according to claim 7 wherein the non-sugar bulk sweetener is present in the range
of from 15 to 70% by weight of the final composition.
9. A process for the preparation of a composition according to any one of the preceding claims
which comprises admixing the ingredients together in the appropriate relative amounts in any order
that is convenient with the proviso that the nisin must be added during the final step of the procedure
and preferably not in aqueous solution.
10. A method of reducing or preventing the formation of dental plaque, which method comprises
applying an antiplaque effective amount of a composition according to any one of the preceding
claims to a patient in need thereof.
215/1006
17. EP1090035 - 21.12.2004
SPRAY -DRIED BACTERIOCIN POWDER WITH ANTI-MICROBIAL ACTIVITY
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP1090035
Inventor(s):
ROSS REYNOLDS PAUL (IE); HILL COLIN (IE)
Applicant(s):
TEAGASC AGRIC FOOD DEV AUTHORI (IE); NAT UNIVERSITY OF IRELAND (IE)
IP Class 4 Digits: A23L; C12P
IP Class:
A23L3/3526; C12P21/04
E Class: A23L3/3463; A23C9/123B; A23C9/13; A23C19/11; A23L3/3463A; C07K14/315
Application Number:
US20010720382 (20010307)
Priority Number: IE19980000500 (19980622); WO1999IE00058 (19990622)
Family: AU767838
Equivalent:
AU4529799; AU767838; NZ509267; WO9967287
Abstract:
THE PRODUCTION OF A SPRAY-DRIED BACTERIOCIN LACTICIN 3147 POWDER IS DESCRIBED.
THE POWDER IS SHOWN TO HAVE EFFECTIVE ANTI-MICROBIAL ACTIVITY IN A RANGE OF
FOODSTUFFS, NAMELY INFANT MILK FORMULATIONS, POWDERED SOUP, YOGHURT AND
COTTAGE CHEESE. INCREASED ANTI-MICROBIAL ACTIVITY WAS DEMONSTRATED WHEN THE
LACTICIN 3147 POWDER WAS USED IN CONJUNCTION WITH INCREASED HYDROSTATIC
PRESSURE. THE PROCESS COMPRISES: INOCULATING A MEDIUM WITH A LACTICIN 3147PRODUCING STRAIN OF BACTERIA, FERMENTING THE INOCULATED MEDIUM, ADJUSTING THE
PH OF THE FERMENTATION TO 6.3-6.7, INACTIVATING THE BACTERIAL FERMENTATE AND
EVAPORATING THE FERMENTATE.Description:
[0002] This application is a 371 of PCT/IE99/00058, filed Jun. 22, 1999.
216/1006
[0003] The present invention relates to a spray-dried bacteriocin powder with anti-microbial activity,
and to a method of producing the powder. In particular, the invention relates to a lacticin 3147 spraydried powder.
[0004] 1. Prior Art
[0005] The elimination of food spoilage and pathogenic organisms has become the focus of much
research since, in terms of individuals affected and the cost of treatment, food-borne illnesses have
an enormous impact. It has been estimated that microbial pathogens in food cause 6.5-33 million
cases of human illness annually in the U.S., at a cost of between $2.9-$6.7 billion dollars (2), with
Gram-positive food-borne pathogens accounting for between 25-55% of the costs. In recent years,
consumer demand for fresh minimally processed safe food, in addition to concern over the use of
chemical preservatives in foods, has prompted substantial interest in the application of
biopreservatives. Bacteriocins produced by lactic acid bacteria are seen as alternatives to traditional
preservatives for ensuring food safety and potential applications in foods have been readily identified
(21).
[0006] Nisin, a bacteriocin produced by certain strains of Lactococcus lactis, has been used
successfully to control food spoilage, in a number of different foods, including cheeses, canned
goods and dairy desserts (10). However, its use is subject to certain restrictions. It is most effective
in foods with acidic pH (below pH 6.0) and low protein and fat content. It is poorly soluble above pH
6.0 and as such has limited effectiveness in many foods. A powdered form of nisin, Nisaplin (Aplin
and Barrett, Towbridge, Wiltshire, U.K.) has been developed and is used for the preservation of
foods.
[0007] In addition to the development of Nisaplin, other powdered bacteriocin-containing agents
have been developed for the preservation of foods. Propionibacterium freundenreichii subsp.
shermanni is used to produce Microgard (Wesman Foods, Inc., Beaverton, Oreg.) by pasteurisation
and drying of propionibacteria-fermented skim milk. It is estimated to be used in approximately one
third of all cottage cheese made in the US and is said to be inhibitory to most Gram-negative
bacteria and some fungi (4). The active agents in Microgard include propionic acid, acetic acid,
diacetyl, lactic acid and a heat-stable peptide of approximately 700 daltons which is considered to
be the most active component.
[0008] Lacticin 3147 is a bacteriocin produced by L. lactis DPC3147 which has a similar host
range to that of nisin, in that it is inhibitory to a wide range of Gram-positive organisms, including
Listeria, Clostridium spp., Enterococcus, Staphylococcus and Streptococcus (17). Given that many
of these organisms have been identified as agents of food spoilage and pathogenesis, the
development of a lacticin 3147-based system for control of these organisms has obvious attractions.
This may be achieved in two ways. The first involves the use of starter cultures (including
217/1006
transconjugants) which produce lacticin 3147, and can be used in food fermentations where these
strains can be substituted for the original starter cultures. The genetic determinants for lacticin 3147
are encoded on a 60.2 kb plasmid, pMRC01 which has been fully sequenced (6) and which has
been mobilised to a number of cheese starter cultures (3). Lacticin 3147 is the subject of PCT
Application No. PCT/IE96/00022, published as WO 96/32482.
[0009] Recently, it has been shown that a lacticin 3147 producing transconjugant can inhibit
Listeria monocytogenes in Cottage cheese (13). This starter has also been used to control the
proliferation of non-starter lactic acid bacteria in Cheddar cheese. The second approach to
improving food safety through the use of lacticin 3147 involves the development of a spray-dried
form of the bacteriocin. The advantage of such a bio-active powder is that it could be applied as a
food ingredient in a variety of foods. However, it is not at all apparent that the bacteriocin is robust
enough to withstand spray-drying and there was the possibility that spray-drying would result in a
significant loss in bacteriocin activity.
OBJECT OF THE INVENTION
[0010] The object of the invention is to provide a lacticin 3147-enriched food ingredient for
incorporation into foodstuffs. In particular, it is an object to provide a spray-dried lacticin 3147
powder. It could not be predicted that such a spray-dried powder could be produced since spraydrying could have caused heat denaturation of the bacteriocin, bearing in mind that lacticin 3147 is
composed of two peptides, both of which are required for activity. Furthermore, dehydration could
irreversibly inactivate the bacteriocin.
[0011] Described herein is a whey based bio-active powder, with effectiveness in controlling two
representative pathogens, L. monocytogenes and Staphylococcus aureus, in buffer at both neutral
and acidic pH. Also described is its effectiveness in controlling L. monocytogenes in an infant milk
formulation and other foodstuffs. However, it will be apparent to those skilled in the art that the
bacteriocin-powder of the invention need not be dairy based and that it would also be possible to
produce a spray-dried bacteriocin based, for example, on other powders, synthetic materials or the
like.
SUMMARY OF THE INVENTION
[0012] According to the present invention there is provided a process for the production of spraydried lacticin 3147 powder comprising:
(a) inoculating a medium with a lacticin 3147-producing strain of bacteria;
(b) fermenting the inoculated medium;
218/1006
(c) adjusting the pH of the fermentation to pH 6.3 to 6.7;
(d) inactivating the bacterial fermentate;
(e) evaporating the fermentate of step (d).
[0018] The medium which may be inoculated with the bacteria can be selected from milk or dairybased powders including demineralised whey powder, reconstituted skimmed milk powder, whey
protein concentrate powder, pasteurised milk, Cheddar cheese whey, or synthetic laboratory media
such as LM17 or TY broth or the like.
[0019] Preferably the inoculated medium is fermented at about 30[deg.] C. for about 6 to 24 hours.
[0020] Preferably the pH of the fermentation is adjusted to about 6.5.
[0021] Suitably, the bacterial fermentate is inactivated by pasteurisation or treating at ultra-high
temperature.
[0022] Suitably, if the fermentate is pasteurised, it is pasteurised at about 72[deg.] C. for about 15
seconds.
[0023] Preferably the inactivated fermentate is evaporated at about 60[deg.] C. to about 40% total
solids.
[0024] The concentrate of step (e) may then be cooled to about 32[deg.] C., seeded with lactose at
about 0.1% w/w and allowed to crystallise at a cooling rate of about 1[deg.] C. per hour.
[0025] The crystallised concentrate is then spray-dried by methods known in the art.
[0026] The invention also provides a spray-dried lacticin 3147 powder which has the ability to
inhibit organisms which are not resistant to lacticin 3147 and which may suitably have an activity of
about 40,240 au (arbitary units)/per ml.
[0027] The invention also provides a food product comprising a spray-dried lacticin 3147 powder
as defined above. The food product may be an infant milk formulation, a sauce, mayonnaise, a
dessert, a yoghurt, a custard, a tinned food product such as a tinned vegetable or tinned meat
product, a soup, a bakery product or similar products.
[0028] The food product may further have been subjected to increased hydrostatic pressure during
processing, suitably at a pressure of about 150 to 800 MPa.
FIGURE LEGENDS
[0029] The present invention will now be described in greater detail with reference to the
accompanying drawings in which:
[0030] FIG. 1. (A) Growth of L. lactis DPC3147 and lacticin 3147 production in 10% reconstituted
demineralized whey powder at 30[deg.] C., in pH controlled and uncontrolled conditions. (-) cfu/ml
with no pH control imposed, (-) cfu/ml at constant pH of 6.0, (-) cfu/ml at constant pH of 6.5 and (O)
cfu/ml at constant pH of 7.0. () AU/ml with no pH control, (-) AU/ml at constant pH of 6.0, (-) AU/ml at
219/1006
constant pH of 6.5 and () AU/ml at constant pH of 7.0. (B) Inhibitory activity of lacticin 3147 against L.
lactis HP when (a) grown with no pH control and when (b) grown at a constant pH of 6.5.
[0031] FIG. 2. Schematic diagram of temperature profile and lacticin 3147 activity during the
manufacturing of lacticin 3147 powder.
[0032] FIG. 3. Effect of lacticin 3147 powder on the viability of Listeria monocytogenes Scott A in
buffer at 30[deg.] C. (A) at pH 5 and (B) at pH 7. (-) no addition, (-) addition of 10% lacticin 3147
powder.
[0033] FIG. 4. Effect of lacticin 3147 powder on the viability of Staphylococcus aureus 10 in buffer
at 30[deg.] C. (A) at pH 5 and (B) at pH 7. (-) no addition, (-) addition of 15% lacticin 3147 powder.
[0034] FIG. 5. Effect of lacticin 3147 powder on the viability of L. monocytogenes Scott A when
used as a component of infant milk formula. (-) 15% lacticin powder, (-) 10% lacticin powder, 5%
infant milk powder, (-) 5% lacticin powder, 10% infant milk powder, (-) 15% infant milk powder.
[0035] FIG. 6. Effect of lacticin 3147 powder (10%) on the viability of Listeria monocytogenes Scott
A in yoghurt. (-) no lacticin 3147 added, (-) 10% lacticin 3147 added. The 10% here refers to 10 g
lacticin 3147 powder added to 90 g yoghurt.
[0036] FIG. 7. Effect of lacticin 3147 powder (10%) on the viability of Listeria monocytogenes Scott
A in cottage cheese. (-) no lacticin 3147 added, (-) 10% lacticin 3147 added. The 10% here refers to
10 g lacticin 3147 powder added to 90 g cottage cheese.
[0037] FIG. 8. Effect of lacticin 3147 powder (10%) on the viability of Bacillus cereus in (packet)
soup.
(-) no lacticin 3147 added,
(-) 1% lacticin 3147 added,
(-) 5% lacticin 3147 added,
(-) 10% lacticin 3147 added.
The 1, 5, 10% here refers to 1, 5 or 10 g lacticin 3147 powder added to 99, 95 or 90 g packet soup
powder, then reconstituted to the manufacturers instructions.
[0043] FIG. 9. Effect of lacticin 3147 powder (10%) on the viability of Listeria monocytogenes Scott
A in (packet) soup.
(-) no lacticin 3147 added,
(-) 1% lacticin 3147 added,
(-) 5% lacticin 3147 added,
(-) 10% lacticin 3147 added.
220/1006
The 1, 5, 10% here refers to 1, 5 or 10 g lacticin 3147 powder added to 99, 95 or 90 g packet soup
powder, then reconstituted to the manufacturers instructions.
[0049] FIG. 10. The effect of increasing pressures on the activity of lacticin 3147, (a) atmospheric
pressure, (b) 200 MPa. (c) 400 MPa, (d) 600 MPa and (e) 800 MPa.
[0050] FIG. 11. The effect of high pressure and lacticin 3147 on L. innocua DPC1770 viability.
EXAMPLE
Material and Methods
Bacterial Strains and Culture Conditions
[0052] The bacteriocin producer L. lactis subsp lactis DPC3147 and the sensitive indicator strain L.
lactis subsp lactis HP were routinely grown at 30[deg.] C. in M17 (20; Oxoid Ltd., Basingstoke,
Hampshire, England) supplemented with 0.5% (w/v) lactose. Other indicator strains used included L.
monocytogenes Scott A grown in Trypicase Soy Broth (TSB, Becton Dickinson and Co., Cockeysville,
Md. 21030, USA) supplemented with 0.6% (w/v) yeast extract (Oxoid), and Staphylococcus aureus
10 (DPC culture collection, Moorepark, Fermoy, Co. Cork, Ireland) grown in Brain Heart Infusion
broth (BHI, Oxoid), both at 37[deg.] C. Solid media was prepared by the addition of 1%
bacteriological agar (Oxoid).
[0053] A number of different media were investigated for the production of lacticin 3147. These
were made as 10% (w/v) solutions, apart from pasteurised whole milk and Cheddar cheese whey.
The 10% solutions were prepared from demineralized whey powder (95% demineralized),
reconstituted skimmed milk powder (Dairygold, Mitchelstown, Co. Cork, Ireland) and whey protein
concentrate powder (WPC35, 35% protein in dry matter, Moorepark Technology Ltd., Moorepark,
Fermoy, Co. Cork, Ireland). The whey based solutions were sterilised by heating to 95[deg.] C. for 30
minutes. The skimmed milk powder solution was sterilised by autoclaving for 5 min at 121[deg.] C.
Bacteriocin Assay and Activity Determination
[0055] Bacteriocin activity was determined by the agar well diffusion assay as described by
Parente and Hill (15). Molten agar was seeded with an indicator strain and dispensed into petri
dishes. Wells of approximately 4.6 mm in diameter were bored in the agar and a 50 [mu]l volume of a
two fold serial dilution of a bacteriocin preparation was dispensed into each well. Bacteriocin solution
was prepared by centrifuging the culture and heat treating the supernatant at 70[deg.] C. for 10
minutes prior to carrying out the dilution series. The plates were then incubated at either 30[deg.] C.
or 37[deg.] C., depending on the indicator strain used. Bacteriocin activity was calculated as the
inverse of the last dilution that gave a definite zone of clearance after overnight incubation. Activity
units (AU) were expressed per milliliter (1/dilution, *20).
221/1006
Controlled pH Fermentations.
[0057] Controlled pH fermentations were carried out over a 24 hour period, with slow agitation
(approximately 20 rpm) at 30[deg.] C. A 1% inoculum of DPC3147 was used to inoculate 100 ml of
growth media. The pH of the growth media was kept at a constant value by the addition of 1.0 M
NaOH on demand via a 718 STAT Titrino (Metrohm, Ireland). Cell counts and bacteriocin activity
determinations were carried out at hourly if intervals for the first 10 hours, and a final sample was
taken after 24 hours.
Production of a Spray Dried Lacticin 3147 Powder.
[0059] A 170 L volume of demineralized whey powder (10% total solids) was inoculated with 1%
DPC3147 and the pH of the 24 hour fermentation was controlled by the addition of 2.5M NaOH on
demand (pH 6.5). The fermentate was then pasteurised at 72[deg.] C. for 15 sec using an APV SSP
pasteurizer (APV, Silkborg, Denmark). The pasteurised fermentate was then evaporated at 60[deg.]
C. to 40% total solids using a single effect falling film evaporator (Anhydro model F1 Lab). The
resulting concentrate was cooled to 32[deg.] C., seeded with lactose (0.1% w/w) and allowed to precrystallize overnight at a cooling rate of 1[deg.] C. per hour. The pre-crystallized concentrate was
then spray-dried using nozzle atomization in an Anhydro spray drier (Anhydro model Lab 3) at an air
inlet temperature of 190[deg.] C. and a 90[deg.] C. outlet temperature. The powder was aliquoted,
sachet packed in foil-lined sample bags and stored at 4[deg.] C. Bacteriocin activity was assessed
at each step during the process.
Effect of Lacticin 3147 Powder Against Pathogens in Buffer
[0061] Sensitive cells were grown to mid-exponential phase, washed and resuspended at
approximately 10 -10 cfu/ml in 2.5 mM sodium phosphate buffer, pH 7.0 or pH 5.0, and 2.5 mM
sodium phosphate buffer, pH 7.0 or pH 5.0 supplemented with 10 mM glucose. Lacticin 3147
powder was added (at different concentrations depending on the sensitive strain under investigation)
and samples were taken at appropriate time intervals over a 3 hour period to determine the viable
cell count.
Effect of Lacticin 3147 Powder Against L. monocytogenes in an Infant Milk Formulation
[0063] Lacticin 3147 powder was added to a commercially available infant milk formula,
[ingredients listed as follows: demineralized whey powder, vegetable oils, lactose, skimmed milk,
calcium carbonate, potassium citrate, calcium chloride, sodium citrate, magnesium chloride, vitamin
C, emulsifier (soya lecithin), taurine, potassium hydroxide, iron sulphate, zinc sulphate, vitamin E,
nicotinamide, pantothenic acid, vitamin A, copper sulphate, citric acid, thiamin, vitamin B6, (carotene,
manganese sulphate, potassium iodide, folic acid, vitamin K, sodium selenite, vitamin D, biotin).
Manufacturers instructions indicate that the final liquid for infant consumption is a 15% solution (w/v).
In experiments the 15% (w/v) infant milk powder was replaced with either 5% (w/v) lacticin powder
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and 10% (w/v) infant milk powder, or with 10% (w/v) lacticin powder and 5% (w/v) infant milk powder.
L. monocytogenes cells were grown to mid-exponential phase, washed and resuspended at
approximately 10 cfu/ml in the various infant milk formulations at 30[deg.] C. and samples were taken
at appropriate time intervals over a 3 hour period to determine the viable cell count.
Preparation of Lacticin 3147 for Use in High Pressure Inactivation Studies
[0065] For the inactivation of Staph. aureus ATCC6538 a liquid preparation of lacticin 3147 was
prepared using hydrophobic adsorption chromatography. For studies on inactivation of L. innocua
DPC1770 a food grade powdered preparation of lacticin 3147 was manufactured as described
above with the following modification; a 1% demineralised whey powder solution was fermented with
L. lactis subsp. lactis DPC3147 under pH controlled conditions of pH 6.0 for 18 hours.
[0066] Activity of both lacticin 3147 preparations was determined by the agar well diffusion assay
as described by Parente and Hill (15). Molten agar was seeded with the indicator strain L. lactis
subsp. lactis HP and dispensed into petri dishes. Wells of approximately 6.0 mm in diameter were
bored in the agar and a 50 [mu]l volume of a two fold serial dilution of a bacteriocin preparation was
dispensed into each well. The plates were then incubated at 30[deg.] C. Bacteriocin activity was
calculated as the inverse of the last dilution that gave a definite zone of clearance after overnight
incubation. Activity units (AU) were expressed per milliliter (1/dilution, *20). Activity may also be
expressed as zone diameter (mm), where the diameter of the first zone (neat, undiluted sample) of
the dilution series is recorded.
Effect of High Pressure on Staph. aureus ATCC6538 and L. innocua DPC1770 Viability
[0068] Staph. aureus ATCC6538 cells were resuspended in 10% RSM and aliquoted into sterile
700 [mu]l PCR eppendorfs prior to placing in sterile stomacher bags (Seward Ltd., London, UK). Ten
millilitre volumes of L. innocua DPC1770 cells were resuspended in 20% reconstituted demineralised
whey powder aliquoted into sterile stomacher bags. Samples were individually vacuum sealed prior
to placing in the pressure vessel (Stansted Fluid Power Ltd., Stansted, England). The vessel
consisted of a stainless-steel cylinder (37 mm diameter*300 mm height) filled with a 15% (v/v) caster
oil in ethanol solution which acts as the hydrostatic pressurisation medium. Samples were treated for
30 min at 25[deg.] C. in the pressure range 150 to 600 MPa, in addition to a control sample being
held at atmospheric pressure (0.1 MPa). All experiments were carried out in duplicate. The chamber
temperature was determined by means of a thermoregulating system which circulated to maintain
the chamber temperature.
Effect of High Pressure on Lacticin 3147 Activity
[0070] To determine the effect of high pressure on lacticin 3147 activity, reconstituted lacticin 3147
powder and aliquots of liquid lacticin 3147 were vacuum sealed and exposed to pressures ranging
from 100 to 800 Mpa as described above. Pressurised and non-pressurised solutions of lacticin 3147
223/1006
were heat treated at 80[deg.] C. for 10 minutes prior to carrying out activity determination by the well
diffusion assay using L. lactis HP as an indicator strain.
Results
[0072] The objective of this research was to develop a powdered form of lacticin 3147 suitable for
use as an ingredient which could help in the control of undesirable micro-organisms in foods.
Following the optimization of lacticin 3147 production a scale-up fermentation was carried out and
the fermentate was spray-dried to form a bacteriocin-rich powder. This powder was assessed in both
a buffer and an infant milk food system for it, ability to inhibit pathogens.
Lacticin 3147 Production in Various Media
[0074] Following inoculation of DPC3147 (1%) and overnight incubation at 30[deg.] C., lacticin
3147 activity was assessed in a number of different growth media. Most of the media were dairy
based, but two synthetic media were also included (LM17 and TY). Results of production of lacticin
3147 (see Table 1) demonstrated that activity was high in almost all the dairy based media (1,280 to
2,560 AU/ml) apart from WPC35 (320 AU/ml). Highest levels of lacticin 3147 activity were found in
Cheddar cheese whey, whole milk and LM17 (2,560 AU/ml). Both 10% reconstituted demineralized
whey powder and 10% reconstituted skimmed milk powder gave activity of 1,280 AU/ml. Lower
levels of lacticin 3147 activity were observed in TY broth (640 AU/ml).
[0075] Since demineralized whey powder is a commercially and readily available, and good
lacticin 3147 activity was observed in this media, further investigations into the optimization of lacticin
3147 production in demineralized whey powder was carried out.
Optimization of Lacticin 3147 Production in 10% Reconstituted Demineralized Whey Powder
[0077] Bacteriocin production and viable cell counts in pH-controlled and pH-uncontrolled
fermentations revealed that increased levels of lacticin 3147 could be produced by maintaining the
pH of the growth media constant, at pH 6.5 (FIG. 1). Levels of bacteriocin activity reached 10,240
AU/ml in 10% reconstituted demineralized whey powder when the pH of the growth media was held
constant at pH 6.5 (FIG. 1B(a)) compared to 640 AU/ml when no pH control was imposed (FIG.
1B(b)). At both pH 6.0 and pH 7.0 lacticin activity reached 5120 AU/ml. Results of viable cell counts
over a 24 hour period indicated that increased bacteriocin activity corresponded to higher cell
densities. Without pH control viable cell counts reached 1*10 cfu/ml, whereas when the pH of the
growth media was maintained at a constant pH of 6.5 viable cell counts reached 3.8*10 cfu/ml (FIG.
1A). With pH control at 6.0 and 7.0 viable cell counts reached 2.5*10 cfu/ml.
Production of Lacticin 3147 Powder
[0079] A spray-dried lacticin 3147 preparation was manufactured as described in materials and
methods. During the manufacturing process bacteriocin activity was assessed at each step, using L.
lactis HP as the indicator strain (FIG. 2). Following the pH controlled fermentation (in 10%
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reconstituted demineralized whey powder) bacteriocin activity was 10,240 AU/ml. The fermentate
was subjected to pasteurisation to inactivate the bacteriocin producing culture DPC3147.
Pasteurisation had no effect on bacteriocin activity (FIG. 2). Evaporation (from 10% total solids to
40% total solids) led to a concentration of the fermentate and resulted in an increase in bacteriocin
activity to 40,960 AU/ml. Following overnight crystallisation, the activity of the concentrate remained
stable. Spray drying of the concentrate resulted in the production of an active powder. When
resuspended at a concentration of 50 mg/ml (5% solids) the spray dried powder contained 5,120 AU
indicating that the activity of the lacticin powder was 102,400 AU/g (100% solids). Lacticin 3147
activity expressed as AU/g of dry matter remained constant throughout manufacture at 102,400 AU/g,
indicating that no loss in bacteriocin activity occurred during processing.
[0080] The inhibitory activity of the bacteriocin-enriched powder was attributed to the action of
lacticin 3147 rather than other fermentation metabolites such as lactic acid, since it inhibited a
sensitive L. lactis MG1614, but did not show any inhibitory effect against a transconjugant containing
the pMRC01 plasmid.
Effect of Lacticin 3147 Powder on Pathogens
[0082] The lacticin 3147 enriched demineralized whey powder (lacticin 3147 powder) was
investigated for its ability to inhibit two food-borne pathogens. The inhibitory effect of the powder was
investigated at pH 5 and at pH 7, in the presence and absence of 10 mM glucose. The effectiveness
of a 10% (w/v) solution of lacticin 3147 powder against mid-exponential growth phase cells of L.
monocytogenes Scott A demonstrated that approximately a 3.3 log kill (99.95% kill) could be
achieved at pH 5 within 3 hours at 30[deg.] C. (FIG. 3A). Killing of L. monocytogenes Scott A with a
10% (w/v) solution of lacticin powder was slightly more effective at pH 7 (FIG. 3B). A 3.8 log kill
(99.98% kill) was observed within 3 hours at 30[deg.] C.
[0083] S. aureus 10 was found to be more resistant than L. monocytogenes Scott A to the action of
the lacticin enriched powder, for this reason a 15% solution of the powder was used. The
effectiveness of a 15% (w/v) solution of lacticin 3147 powder against mid-exponential phase cells of
S. aureus 10 resulted in approximately a 1.1 log kill (90.4% kill) at pH 5 within 3 hours at 30[deg.] C.
(FIG. 4A). The killing effect of a 15% solution (w/v) of lacticin powder increased dramatically at pH 7,
where almost a 4 log kill (99.98% kill) of S. aureus 10 was observed within 3 hours at 30[deg.] C. (FIG.
4B). The inclusion of 10 mM glucose resulted only slight increases in the level of cell deaths for either
L. monocytogenes Scott A or S. aureus 10 (results not shown).
Effect of Lacticin 3147 Powder Against L. monocytogenes Scott A in an Infant Milk Formulation
[0085] To evaluate the effectiveness of the lacticin 3147 powder in a food system experiments were
carried out in an infant milk formula, since this is an example of a food destined for a high-risk
consumer which contains demineralized whey powder as a major constituent. Results indicated
225/1006
greater that a 99% kill of L. monocytogenes Scott A resulted when part of the infant milk formulation
was substituted with either two thirds (10% lacticin powder and 5% infant milk powder) or one third
lacticin 3147 powder (5% lacticin powder and 10% infant milk powder) (FIG. 5). Counts here were
reduced from approximately 7*10 cfu/ml to 3*10 cfu/ml within 3 hours at 30[deg.] C. In the control
culture with no lacticin 3147 powder present counts increased from approximately 10 cfu/ml to
approximately 10 cfu/ml within the same time period.
Application of Lacticin 3147 Powder in a Range of Foods
[0087] Powdered lacticin 3147 has been assessed for the inhibition of food spoilage and
pathogenic micro-organisms in a number of food systems including infant food formula, powdered
soup, cottage cheese and natural yoghurt. The following are specific examples of the use of lacticin
3147 to inhibit pathogens in food systems.
[0088] The ability of the lacticin 3147 powder to inhibit Listeria monocytogenes Scott A was initially
investigated in an infant milk formulation as described above. To further investigate the inhibitory
effect of the lacticin 3147 powder, inactivation trials were carried out against a number of different
micro-organisms in natural yoghurt, cottage cheese and reconstituted powdered soup, with pHs of
4.5, 4.4 and 6.6 respectively.
[0089] The effect of 10% lacticin 3147 powder on the inhibition of Listeria monocytogenes Scott A
(10 cfu/ml) in natural yoghurt demonstrated that greater than 98.3% of the culture was killed within 5
minutes at 30[deg.] C. Within 60 minutes no viable cells remained, (FIG. 6).
[0090] In the case of cottage cheese inoculated with 10 cfu/ml Listeria monocytogenes 40% of the
population was killed within 5 minutes at 30[deg.] C. in the presence of a 10% lacticin 3147 powder.
After 160 minutes only 14% of the population remained viable, (FIG. 7).
[0091] The effect of 1, 5 and 10% concentrations of lacticin 3147 in powdered soup against
Bacillus cereus at 30[deg.] C., demonstrated that following 24 hours incubation greater than a 99.9%
kill was observed in the presence of the 5 and 10% lacticin 3147 powder concentrations. In the case
of the 1% lacticin 3147 concentration 17% of the population survived, (FIG. 8).
[0092] A similar study was carried out to determine the effect of 1, 5 and 10% concentrations of
lacticin 3147 powder on the survival of Listeria monocytogenes Scott A in powdered soup. A 1%
concentration of lacticin was ineffective at inhibiting Scott A within 24 hours, whereas at a 5%
concentration greater than 10% of the population were inhibited. At a concentration of 10% greater
than 40% of the culture was inhibited, (FIG. 9).
[0093] From these results it can be seen that a powdered form of lacticin 3147 has indeed many
applications in food safety for the control of food pathogens and spoilage organisms.
Effect of Hydrostatic Pressure
226/1006
[0095] The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and
whey with a view to combining both treatments for improving the quality of minimally processed dairy
foods. The system was evaluated using two foodborne pathogens, Staphylococcus aureus
ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed
using concentrated lacticin 3147 prepared from culture supernatant. Results demonstrated greater
than an additive effect when both treatments were used in combination, for example, the combination
of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill
(FIG. 10). Similar results were obtained when a foodgrade powdered form of lacticin 3147
(developed from a spray dried fermentation of reconstituted demineralised whey powder) was
evaluated for the inactivation of L. innocua DPC1770 (FIG. 11). Furthermore, it was observed that
treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in
bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of
high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed
foods at lower hydrostatic pressure levels.
Discussion
[0096] The development of a whey based bio-active food ingredient was achieved following
investigations into lacticin 3147 production in different media. Lacticin 3147 activity was high in all of
the dairy based media investigated, apart from whey protein concentrate (WPC35). A possible
explanation for the low level of activity in the whey protein concentrate could be that bacteriocin
activity fractionated into the pellet upon centrifugation, prior to assaying for activity. Two synthetic
media were investigated for lacticin 3147 production, LM17 broth (20) and TY broth (15). Levels of
lacticin 3147 activity in LM17 were comparable to dairy based media, but this is not unexpected,
since this media was developed for the cultivation of lactococci. However, TY broth, in which low
levels of lacticin 3147 activity was observed, was developed to yield optimal bacteriocin (enterocin
1146) production while minimising peptide levels in the medium (to eliminate peptides that may
interfere with purification). For the development of a powder the use of the most cost effective growth
media is obviously advantageous. Demineralized whey powder, a readily available and cost effective
medium ($20 per 25 Kg) was investigated for the optimization of lacticin 3147 production. However,
other suitable growth media could be used, as described above.
[0097] The effect of pH on bacteriocin production has been well documented, and for a number of
bacteriocin-producing strains control of pH during growth results in higher bacteriocin titres (11, 14,
18). Lacticin 3147 activity increased dramatically when the pH of the growth media was held
constant at pH 6.5, 5 highest bacteriocin titres and highest cell numbers were observed at this pH.
227/1006
Lowest bacteriocin titres and lowest cell numbers were observed when no pH control was imposed.
Increased bacteriocin activity corresponded with increased cell numbers.
[0098] Once lacticin 3147 production had been optimized in 10% reconstituted demineralized
whey powder a large-scale fermentation was set up to generate enough fermentate for spray drying.
The production of an active spray dried powder demonstrated the resilience of the bacteriocin, to the
extremes of the processing conditions. Activity was detected throughout the process and the final
powder had an activity of 102,400 AU/g dry matter, equivalent to the activity present at the beginning
of the process. This unexpected result is significant in that it suggests that no loss in activity
occurred during production.
[0099] Assessment of the inhibitory activity of the bio-active powder demonstrated that it is capable
of inhibiting both L. monocytogenes and S. aureus at pH 5 and at pH 7. In both cases the bio-active
powder exhibited enhanced killing ability at neutral pH. This is a significant finding, since Nisaplin, a
fermented food ingredient for extension of product shelf life and prevention of spoilage is known to
be most effective at acidic pH (below pH 6.0). The development of a food ingredient capable of
killing Gram-positive bacteria at neutral pH indicates that the lacticin 3147 powder may be suitable
for incorporation into a wide range of foods, that hitherto had no opportunity for the prevention of
food spoilage/pathogenesis apart from the inclusion of chemical preservatives.
[0100] The mechanism of action of lacticin 3147 has been elucidated (12). It induces cell death by
permeabilising the membranes of sensitive cells through pore formation, allowing the efflux of K ions
and phosphate. This action results in the dissipation of the proton motive force, hydrolysis of
intracellular ATP and ultimately leads to cell death. Energised cells are more susceptible to the action
of lacticin 3147. Cells incubated in the presence of lacticin powder combined with 10 mM glucose
demonstrated slight increases in killing efficiency (apart from S. aureus 10 at pH 7, results not
shown). This is in keeping with results reported by McAuliffe et al., (12), where energised cells were
observed to be more sensitive to lacticin 3147. Energised cells have a proton motive force which
may favour the insertion of lacticin 3147 molecules into the membrane, as is the case with nisin, a
lantibiotic pore former (7, 8).
[0101] The development of a powdered form of lacticin 3147 would allow it to be applied to a
number of food systems. Since the existing lacticin 3147 powder has been developed from a
demineralized whey powder, this powder has applications in all foods where demineralized whey
powder is an existing ingredient. For example demineralized whey powder is incorporated into a
number of foods including infant milk formulations. Results presented in this paper demonstrate the
ability of this powder to effectively inactivate 99% of L. monocytogenes Scott A spiked into infant
formula, where part of the infant milk powder had been substituted with the lacticin 3147 powder.
Infant milk formulations are manufactured to the highest of standards and incidents of food-borne
228/1006
illness associated with such foods are rare. However more than many other foods infant milk formulas
are susceptible to contamination through domestic contamination, putting the health of infants at risk.
For this reason the inclusion of a lacticin 3147 enriched powder in such formulations may offer
increased protection in the event of contamination, which would be beneficial to both producers and
consumers.
[0102] For manufacturers already using demineralized whey powder as a food ingredient it should
prove possible to substitute this powder (either partially or fully) with a bio-active demineralized whey
powder to further safe guard food products from spoilage and pathogenic Gram-positive organisms.
And indeed, for manufacturers who do not use demineralized whey powder as a food ingredient the
inclusion of low levels of the bio-active powder could be sufficient to confer enhanced protection
without affecting the sensory or functional characteristics of these foods. It is, however, also apparent
that a spray-dried lacticin 3147 powder based on a medium other than whey powder, would be
obtainable by this invention. Such a powder has potential for application as a substitute in areas
where whey powder is not utilised, with the same beneficial effects.
SUMMARY
[0103] The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is
inhibitory to a wide range of Gram-positive food spoilage and pathogenic organisms. A 10% solution
of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate
was spray dried and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was
assessed in buffer at both acidic (pH 5) and neutral pH (pH 7). In addition, the ability of the powder
to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of 8.3
log mid-exponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147enriched powder resulted in a 1000 fold reduction in viable cells at pH 5 and pH 7, after 3 hours at
30[deg.] C. In the case of S. aureus 10, resuspension of 2.5*10 mid-exponential phase cells in a 15%
solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10 fold reduction in viable cell
counts, compared to a 1000 fold reduction at pH 7, following incubation for 3 hours at 30[deg.] C. In
an infant milk formulation the use of the lacticin 3147 powder resulted in greater than a 99% kill of L.
monocytogenes within 3 hours at 30[deg.] C. Similarily, the lacticin 3147 powder was shown to be
effective in inhibiting food spoilage in powdered soup, yoghurt and cottage cheese. Furthermore, the
combination of hydrostatic pressure and lacticin 3147 causes increased killing making this an
attractive method of preventing spoilage in minimally processed foodstuffs. Thus this bio-active
lacticin 3147 food ingredient will find applications in many different foods, including those with pH
close to neutrality.
229/1006
References
2. Buzby, J. C., T. Roberts, C. T. J. Lin, and J. M. MacDonald. 1996. Bacterial Food-borne Disease:
Medical Costs and Productivity Losses. Food and consumer economics division, economic research
service, U.S. Department of Agriculture. Agricultural Economic Report No. 741.
3. Coakley, M., G. F. Fitzgerald and R. P. Ross. 1997. Application and evaluation of the phage
resistance and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures.
Appl. Environ. Microbiol. 63:1434-1440
4. Daeschel, M. A. 1989. Antimicrobial substances from lactic acid bacteria for use as food
preservatives. Food Technol. 43:164-167
6. Dougherty, B., C. Hill, J. F. Weidman, D. R. Richardson, J. C. Venter and R. P. Ross. Sequence
and analysis of the 60 kb conjugative, bacteriocin producing plasmid pMRC01 from Lactococcus
lactis DPC3147. Mol. Microbiol. (in press).
7. Driessen, A. J. M., H. W. van der Hooven, W. Kuiper, M. van de Kamp, H.-G. Sahl, R. N. H.
Konings, and W. N. Konings. 1995. Mechanistic studies of lantibiotic-induced permeabilisation of
phospholipid vesicles. Biochem. 34:1606-1614.
8. Garcia-Garcera, M. J., M. G. M. Elferink, A. J. M. Driessen, and W. N. Konings. 1993. In vitro poreforming activity of the lantibiotic nisin: role of protonmotive-force and lipid composition. Eur. J.
Biochem. 212:417-422.
10. Hurst, A. 1983. Nisin and other inhibitory substances from lactic acid bacteria, p. 327-351. In P.
M. Davidson and A. L. Branen, (ed.), Antimicrobials in food, Marcel Dekker, New York.
11. Joerger, M. C., and T. R Klaenhammer. 1986. Characterization and purification of helveticin J and
evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J.
Bacteriol. 167:439-446
12. McAuliffe, O., M. P. Ryan, R. P. Ross, C. Hill, P. Breeuwer and T. Abee. 1998. Lacticin 3147: a
broad spectrum bacteriocin which selectively dissipates the membrane potential. Appl. Environ.
Microbiol. 64:439-445.
13. McAuliffe, O., C. Hill and R. P Ross. 1998. Inhibition of Listeria monocytogenes in Cottage cheese
manufactured with a lacticin 3147 producing starter culture. Submitted for publication: J. Appl.
Microbiol.
14. Muriana, P. M., and T. R. Klaenhammer. 1987. Conjugal transfer of plasmid encoded
determinants for bacteriocin production and immunity in Lactobacillus acidophilus 88. Appl. Environ.
Microbiol. 53:553-560.
230/1006
15. Parente, E., and C. Hill. 1992. A comparison of factors affecting the production of two
bacteriocins from lactic acid bacteria. J. Appl. Bacteriol. 73:290-298.
17. Ryan, M. P., M. C. Rea, C. Hill and R. P. Ross. 1996. An application in Cheddar cheese
manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin,
lacticin 3147. Appl. Environ. Microbiol. 62:612-619.
18. Schillinger, U., M. E. Stiles, and W. H. Holzapfel. 1993. Bacteriocin production by
Carnobacterium piscicola LV61. Int. J. Food Microbiol. 20:131-147.
20. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their
bacteriophages. Appl. Environ. Microbiol. 29:807-813.
21. Stiles, M. E. 1996. Biopreservation of lactic acid bacteria. In lactic acid bacteria: genetics,
metabolism and applications (Venema, G., huis in't Veld, J. H. J. and Hugenholz, J. eds.)
Proceedings of the Fifth Symposium, veldhoven, the Netherlands, 235-249, Kluwer Academic
Publishers.
[0120] The words "comprises/comprising" and the words "having/including" when used herein with
reference to the present invention are used to specify the presence of stated features, integers, steps
or components but does not preclude the presence or addition of one or more other features,
integers, steps, components or groups thereof.
TABLE 1
Lacticin 3147 activity in various media
after overnight incubation at 30[deg.] C.
Lacticin 3147 activity
Growth Media(AU/ml)
Cheddar cheese whey2560
Whole milk2560
Reconstituted skimmed milk powder1280
Reconstituted demineralised whey powder1280
Whey protein concentrate (WPC3S)320
LM172560
TY broth640Claims:
1. A process for the production of a spray-dried concentrate comprising lacticin 3147, for use as a
food ingredient, comprising:
(a) inoculating a milk or dairy based medium with a lacticin 3147-producing strain of bacteria;
(b) fermenting the inoculated medium;
(c) adjusting the pH of the fermentation to a pH ranging from 6.3 to 6.7;
(d) inactivating the bacteria within the fermentate; and
231/1006
(e) evaporating the fermentate of step (d) thereby producing the lacticin 3147 concentrate for use as
a food ingredient.
2. A process as claimed in claim 1, wherein the medium of step (a) is selected from the group
consisting of milk, reconstituted dairy-based powders, reconstituted demineralized whey powder,
reconstituted skimmed milk powder, reconstituted whey protein concentrate powder, pasteurized
whole milk, Cheddar cheese whey, reconstituted yeast powders, and synthetic laboratory-type media.
3. A process as claimed in claim 1 or 2, wherein the evaporation step of step (e) comprises cooling
the fermentate of step (d), seeding it with lactose at about 0.1% w/w and crystallizing at a cooling
rate of about 1[deg.] C. per hour.
4. A process as in claim 1, wherein the inoculated medium of step (b) is fermented at about 30[deg.]
C. for about 6 to 24 hours.
5. A process as in claim 1, wherein the pH of the fermentation is adjusted in step (c) to about pH 6.5.
6. A process as in claim 1, wherein the fermentate of step (d) is inactivated by pasteurization or ultrahigh temperature treatment.
7. A process as claimed in claim 6, wherein said pasteurization step comprises heating at about
72[deg.] C. for about 15 minutes.
8. A process as in claim 1, wherein step (e) comprises evaporating said bacteria fermentate at about
60[deg.] C. to about 40% total solids.
9. A process as in claim 1, further comprising the step of spray-drying the concentrate.
10. A concentrate comprising a food-grade spray-dried lacticin 3147 produced by the process of
any one of claims 1 to 9.
11. A spray-dried food-grade powder containing lacticin 3147 having the ability to inhibit organisms
which are not resistant to lacticin 3147, and having an activity of greater than about 20,000 AU/ml.
232/1006
12. A food product comprising a lacticin 3147 enriched spray-dried food-grade fermentate produced
by the process of any one of claims 1 to 9 and a foodstuff.
13. The food product as claimed in claim 12, wherein said product is selected from the group
consisting of an infant milk formulation, a sauce, a mayonnaise, a dessert including a custard, a
tinned food, a yogurt, a soup and a bakery product.
14. A food product as claimed in claim 12 or 13, which has been subjected to hydrostatic pressure in
the range from about 150 MPa to about 800 MPa.
233/1006
18. EP1183365 - 06.03.2002
BACTERIOCIN, PREPARATION AND USE THEREOF
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP1183365
Inventor(s):
THONART PHILIPPE (BE); JABRANE ABDELHAMID (BE); DESTAIN JACQUELINE
(BE); PIERRARD ANNICK (BE); DRION RAPHAEL (BE); JACQUES PHILIPPE (BE); LEPOIVRE
PHILIPPE (BE); JIJAKLI HAISSAM M (BE); VALEPYN EMMANUEL (BE); CHEGGOUR ABDELHAMID
(BE); VERHEYDEN CLEM (BE); DECKERS TOM (BE); VERMEIREN DIRK (BE); BEAUDRY THIERRY
(BE)
Applicant(s):
AGROSTAR (BE); FACULTE UNIVERSITAIRE DES SCIE (BE)
IP Class 4 Digits: C12N; C07K; A01N
IP Class:
A01N63/02; C12N15/31; C07K14/24
Application Number:
EP20000934825 (20000609)
Priority Number: EP20000934825 (20000609); WO2000BE00062 (20000609); EP19990870124
(19990611)
Family: EP1183365
234/1006
19. GB2274247 - 29.11.1994
ORAL COMPOSITION
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=GB2274247
Inventor(s):
GAFFAR ABDUL (US); AFFLITTO JOHN (US); SUBRAMANIAN MALATHY (US)
Applicant(s):
COLGATE PALMOLIVE CO (US)
IP Class 4 Digits: A61K
IP Class:
A61K7/16; A61K7/22
E Class: A61K8/64; A61Q11/00; A61K8/55
Application Number:
US19930001480 (19930107)
Priority Number: US19930001480 (19930107)
Family: DE4400408
Equivalent:
DE4400408; FR2700118; IT1271825
Abstract:
AN ANTIPLAQUE COMPOSITION COMPRISING A BACTERIOCIN SUCH AS NISIN AND A
POLYPHOSPHONATE.Description:
This invention relates to oral antiplaque compositions such as dentifrice's, mouthwashes, lozenges,
chewing gums and the like. The use of nisin, or of related anthionine-containing bacteriocins, in
mouthwash and toothpaste is suggested in Blackburn et al, PCT International patent application WO
89/12399, published Dec. 28, 1989.
In accordance with one aspect of this invention, the oral composition contains the nisin together with
an azacycloalkane-2,2-diphosphonate ion, such as azacycloheptane-2,2-diphosphonate ("AHP").
235/1006
In broader aspects of the invention, other polyphosphonates may be used in place of the AHP. These
include the known polyphosphonates which inhibit crystallization of hydroxyapatite and are inhibitors
of the formation of dental claculus. Examples of such agents include pharmaceutically acceptable
ions of azacycloalkane-2,2-phosphonic acids, such as those in which the alkane moiety has five, six
or seven carbon atones, in which the nitrogen atom is unsubstituted (as in AHP) or carries a lower
alkyl substitutent (e.g. methyl), such as those disclosed in Ploger et al U.S. Pat. No. 3,941,772. Other
examples are ions of germinal diphosphonic acids such as ethanehydroxy-1,1,-diphosphonate
(EHDP), ethane-1-amino-1,1-diphosphonate or dichloromethane-diphosphonate, as well as
polymeric phosphonates such as water-soluble polymers and copolymers of vinylphosphonic acid
(e.g. of molecular weight about 5000 to 30,000), including phosphonic polymers disclosed in Gaffar
et al U.S. Pat. No. 5,032,386.
In place of the nisin, other lanthionine-containing peptide bacteriocins (e.g. subtilin, epidermin,
cinnamycin, duramycin, anconvenin and Pep 5) may be employed, alone or in combinations of two
or more such bacteriocins.
The proportion of the bacteriocin such as nisin is preferably such as to exert an antiplaque effect and
the proportion of the phosphonate such as AHP is preferably such as to increase the antiplaque
effectiveness of the bacteriocin. For a mouthrinse the proportion of the phosphonate may, for
instance, be in the range of about 0.01% to 3%, preferably less than about 1%, e.g. within the range
of about 0.05% to 0.5% such as about 0.2% or 0.3% and the proportion of the bacteriocin may, for
instance, be in the range of about 0.01% to 1%, preferably about 0.03% to 0.5%. For a toothpaste (or
other oral composition which, unlike a mouthrinse, becomes considerably diluted by saliva during
use) the proportion of the phosphonate may, for instance, be in the range of about 0.03% to 15%,
preferably within the range of about 0.15% to 6%, such as about 0.5% or 1%, and the proportion of
the bacteriocin may, for instance, be in the range of about 0.01% to 5%, preferably about 0.5% to 2%.
Some aspects of the invention are illustrated in the following Examples.
EXAMPLES 1-3
A mouth rinse is prepared by mixing the following ingredients in the following proportions:
__________________________________________________________________________
1 2 3
236/1006
__________________________________________________________________________
Water
70.68% 77.925% 82.925%
Sodium acetate 0.245% 0.200% 0.15%
Aqueous 10% 5.00% 5.00% 5.00%
solution of acetic
acid
99.5% glycerol 3.00% 0 0
Tween 80 0.40% 0.40% 0.40%
Pluronic (F87)
0.20%
(F127)
1.00%
(F127)
1.00%
Aqueous 1% 10.00% 10.00% 10.00%
solution of nisin
95% ethanol 10.00% 5.00% 5.00%
Mint flavor 0.20% 0.20% 0.20%
Sodium
0.075% 0.075% 0.075%
saccharin
Disodium salt of
0.20% 0.20% 0.25%
azacycloheptane-2,
2,-diphosphonic
acid
__________________________________________________________________________
Tween 80, Pluronic F87 (Ex. 1) and Pluronic F127 (Ex. 2 and 3) are nonionic surfactants. Tween 80 is
polyoxyethylene (20) sorbitan mono-oleate. The Pluronics are polyoxyethylene-polyoxypropylenepolyoxyethylene block copolymers; F87 has an average molecular weight of 7700, an HLB of more
than 24 and a cloud point in aqueous 1% or 10% solution above 100 DEG C.; F 127 has an average
molecular weight of 12600, an HLB of 18-23 and a cloud point in aqueous 1% or 10% solution of
above 100 DEG C.
237/1006
The pHs of the mixtures are 4.42 (Ex. 1), 4.16 (Ex. 2) and 4.06 (Ex. 3). Example 1 gives an initially
clear mixture which becomes cloudy on standing for a few hours. Examples 2 and 3 give mixtures
which remain clear. Each formulation shows superior activity against plaque formation when tested in
an "artificial mouth" apparatus. In that test, diluted whole human saliva is pumped continuously, at a
rate of 1 ml/minute, through a chamber containing two germanium plates. This forms a pellicle on the
plates. After 20 minutes the saliva flow is stopped and the mouthrinse is pumped into the chamber
for 30 seconds at a flowrate of 10 ml/min., after which the saliva flow, at 1 ml/min., is resumed for 30
minutes to wash out residual mouthrinse. Then the diluted saliva, together with 10% of its volume of
trypticase soy broth, is continuously pumped through the chamber at the rate of 1 ml/min. After 24
and 48 hours the same procedure (involving a 30-second mouthrinse treatment) is repeated. After
72 hours the saliva treatment is discontinued and distilled water is circulated through the chamber at
a rate of 5 ml/min. for 10 minutes. The resulting washed plates are removed and allowed to air dry,
and the amount of plaque formed thereon is measured by infrared spectroscopy.
In the foregoing formulations the sodium acetate and acetic acid are present to provide a pH buffer,
the Tween and Pluronic components are nonionic surfactants which aid in dispersing the ingredients,
the flavor is added in solution in the ethanol (which helps to solubilize it in the composition), the
glycerol (a conventional humectant ingredient in mouthwashes) gives increased viscosity and a
moist feel in the mouth and the saccharin is, of course, a sweetening agent.
Generally, a mouthrinse according to this invention may contain, for instance, up to about 20% of
ethyl alcohol, about 0% to about 50% of humectant, about 0.1 to 5% of an emulsifying agent
(surfactant), about 0% to 0.5% of a sweetening agent, about 0.03% to 0.3% of a flavoring agent.
The pH values of the oral compositions of this invention are preferably in the range of about 4 to 8,
such as about 4, 4.5, 5, 5.5, 6, 6.5 or 7. The compositions are preferably acidic; e.g. the pit is below
about 6 or below about 5.
The oral compositions of this invention preferably contain surfactants, such as nonionic, cationic,
zwitterionic and amphoteric surfactants. Many of the suitable surfactants (surface-active agents) are
disclosed in Gaffar et al U.S. Pat. No. 4,889,712; the disclosures of such agents, and their
proportions (e.g. up to about 10%), in that patent are incorporated herein by reference.
Zwitterionic surfactants include quaternary ammonium, phosphonium and sulfonium compounds
having an 8 to 18 carbon atom aliphatic substituent and an aliphatic substituent having an anionic
238/1006
water-solubilizing group (e.g. carboxy, sulfonate, sulfate, phosphate or phosphonate); one example
is 4-(N,N-di(2-hydroxyethyl)-N-octadecylammonio)-butane-1-carboxylate.
Cationic surfactants include quaternary ammonium compounds having an 8 to 18 carbon atom alkyl
substituent, e.g. cetyl pyridinium chloride.
Amphoteric surfactants include secondary and tertiary amines having an 8 to 18 carbon atom
aliphatic substituent and an aliphatic substituent having an artionic water-solubilizing group (e.g.
carboxy, sulfonate, sulfate, phosphate or phosphonate); examples are N-alkyltaurines (e.g. reaction
product of dodecylamine and sodium isethionate), Miranol, etc.
The compositions of this invention may contain fluoride ion, e.g. in the form of sodium fluoride,
sodium fluorophosphate, or other fluoride ion source, such as those listed in Gaffar et al U.S. Pat. No.
4,889,712; the disclosures of such fluoride ion sources, and their proportions, in that patent are
incorporated herein by reference.
EXAMPLE 4
A toothpaste is prepared according to the following formula:
______________________________________
Glycerol 20.0%
Precipitated silica
20.0%
Sodium saccharin
0.75%
Sorbitol (70%) 20.0%
Sodium fluoride 0.243%
Tween 80 1.0%
Pluronic F127 1.0%
Mint flavor 1.0%
Nisin
1.0%
AHP
1.0%
Hydroxyethylcellulose
Thickening amount
Water
Balance
239/1006
______________________________________
In this toothpaste formulation, the glycerol and 70% sorbitol are conventional humectants, the
hydroxyethylcellulose is a conventional binder or gelling agent, and the precipitated silica is a
conventional amorphous silica dental abrasive. Other humectants, binders (thickeners or gelling
agents), abrasives (polishing agents), surfactants and flavoring agents may be used, such as those
listed in Gaffar et al U.S. Pat. No. 4,889,712, in the proportions described in that patent, whose
disclosures thereof are incorporated herein by reference.
EXAMPLE 5
A clear mouthrinse having a pH of 4.25 is prepared from the following ingredients: water 84.625%;
sodium acetate 0.6%; aqueous 10% solution of acetic acid 2.6%; Tego Betain L5351 1%; Pluronic
F127 1%; sodium sacharin 0.075%; nisin 0.1%; aqueous 5% solution of polyvinyl phosphonic acid (of
molecular weight about 300) 10%. The Tego Betain L5351 is an aqueous solution, containing about
33% of cocamidopropyl betain, a zwitterionic surfactant commercially available from Goldschmidt
Chemical Corp.
The composition of this invention are preferably substantially free of strong chelating agents such as
EDTA or citrate ions.
As indicated in the Examples the compositions of this invention contain water, e.g. 2% to 95% water.
For mouthrinses the proportion of water may be, for instance, in the range of about 10 to 95%
preferably about 40% to 85%. For toothpastes the water content may be, for instance, in the range of
about 5 to 50% preferably about 10 to 30%.
This invention has been disclosed with respect to preferred embodiment and it will be understood
that modifications and variations thereof are to be included within the spirit and purview of this
application.
Claims:
We claim:
1. An oral composition comprising antiplaque amounts of a lanthionine-containing bacteriocin and a
polyphosphonate which is an inhibitor of the crystallization of hydroxyapatite.
240/1006
2. An oral composition as in claim 1 comprising nisin.
3. An oral composition as in claim 1 comprising an azacycloalkane-2,2-diphosphonate ion.
4. An oral composition as in claim 2 comprising azacycloheptane-2,2-diphosphonate ion.
5. An oral composition as in claim 1 having an acidic pH.
6. A composition as in claim 1 which is an aqueous mouthrinse containing about 0.01% to 3% of said
polyphosphonate and about 0.01% to 1% of said bacteriocin.
7. Process for reducing the incidence of plaque on teeth in the mouth comprising contacting said
teeth with a composition as in claim 1.
8. An oral composition according to claim 1 in the form of a toothpaste containing about 0.03% to
15% of said polyphosphonate and about 0.01% to 5% of said bacteriocin.
9. A toothpaste composition according to claim 8 comprising AHP and nisin.
10. A toothpaste composition according to claim 9 substantially free of strong chelating agents such
as EDTA and citrate ions.
11. A mouthrinse composition according to claim 6 comprising AHP and nisin.
12. A mouthrinse composition according to claim 11 substantially free of strong chelating agents
such as EDTA and citrate ions.
13. Process for reducing the incidence of plaque on teeth in the oral cavity comprising contacting the
teeth with a composition according to claim 9.
14. Process for reducing the incidence of plaque on teeth in the oral cavity comprising contacting the
teeth with a composition according to claim 11.
15. An oral composition as in claim 1 in the form of an aqueous mouth rinse containing about 0.05%
to 0.5% of AHP and about 0.03% to 0.5% of nisin and having a pH of about 4 to 6.
241/1006
16. An oral composition as in claim 1 in the form of an aqueous mouthrinse containing about 0.2% to
0.3% of AHP and about 0.01% to 1% of nisin and having a pH of about 4 to 6.
17. An oral composition as in claim 1 in the form of a toothpaste containing about 0.5% to 1% of AHP
and about 0.5% to 2% of nisin.
242/1006
20. HU9500561 - 30.10.1995
PHARMACEUTICAL BACTERIOCIN COMPOSITIONS AND METHODS FOR USING THE SAME
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=HU9500561
Inventor(s):
BLACKBURN PETER (US); PROJAN STEVEN J (US); GOLDBERG EDWARD B (US)
Applicant(s):
APPLIED MICROBIOLOGY INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K37/02
Application Number:
HU19950000561P (19950629)
Priority Number: HU19950000561P (19950629)
Family: HU9500561
243/1006
21. HU9500563 - 30.10.1995
PHARMACEUTICAL BACTERIOCIN COMPOSITIONS AND METHODS FOR USING THE SAME
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=HU9500563
Inventor(s):
BLACKBURN PETER (US); PROJAN STEVEN J (US); GOLDBERG EDWARD B (US)
Applicant(s):
APPLIED MICROBIOLOGY INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K37/02; A61K31/14
Application Number:
HU19950000563P (19950629)
Priority Number: HU19950000563P (19950629)
Family: HU9500563
244/1006
22. IE940624L - 22.12.1989
LANTHIONINE-CONTAINING BACTERIOCIN COMPOSITIONS FOR USE ASBACTERICIDES
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=IE940624L
Applicant(s):
CRAIG MED PROD LTD (--)
IP Class 4 Digits: A23C
IP Class:
A23C3/08; A23C9/152
Application Number:
IE19940000624 (19940810)
Priority Number: US19880209861 (19880622); US19890317626 (19890301)
Family: IE940624L
245/1006
23. IL107887 - 23.06.1994
STABILIZED LANTHIONINE BACTERIOCIN COMPOSITIONS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=IL107887
Inventor(s):
BLACKBURN PETER (--); DE LA HARPE JON (--)
Applicant(s):
APPLIED MICROBIOLOGY INC (US)
IP Class 4 Digits: A01N
IP Class:
A01N63/02; A01N25/22; A01N43/78
E Class: A01N37/46+M; A01N63/02+M; A61K7/16D13; A61K7/22; A61K47/18B
Application Number:
WO1993US11884 (19931208)
Priority Number: US19920986671 (19921208)
Family: IL107887
Equivalent:
AU5743094; DE69324159D; DK673199T; EP0673199WO9413143; ES2130401T;
GR3029943T; JP8510716T; ZA9309170
Cited Document(s):
WO8912399; WO9009739; EP0431663
Abstract:
THE INVENTION CONCERNS COMPOSITIONS CONTAINING A LANTHIONINE CONTAINING
BACTERIOCIN SUCH AS NISIN WHICH ARE STABILIZED BY THE PRESENCE OF A THIOETHER
STABILIZING AGENT AGAINST DEGRADATION. THE THIOESTER COMPOUND IS PREFERABLY A
COMPOUND OF FORMULA (I): R<1>-S-R<1>. IN A PREFERRED EMBODIMENT, THE COMPOUND
OF FORMULA (I) IS THE AMINO ACID METHIONINE OR AN ANALOG THEREOF.Description:
246/1006
STABILIZED LANTHIONINE BACTERIOCIN COMPOSITIONS
Backaround of the Invention
It is particularly difficult to maintain proteins and peptides stable for extended periods when stored
at ambient temperatures, particularly in dilute solution, and this remains a major challenge for
formulation chemists. Proteins and peptides can undergo degradation by various pathways,
including but not limited to the following: peptide bond hydrolysis particularly at extremes of pH,
deamidation under acidic pH, dehydration and desulfurization at alkaline pH, halogenation of
aromatic side chains, oxidation of sulfur-containing and indole side chains, thiol-disulfide
rearrangements, modification of amine groups by reactive carbonyl compounds and amadori
rearrangements with beta-hydroxy carbonyl compounds, polymerization, precipitation, and
denaturation.
The rate of degradation of a protein or peptide can be influenced by the sequence of adjacent
amino acid residues in the molecule; for example Asn-Gly sequences are particularly susceptible to
deamidation and betarearrangement of the intervening peptide bond. The amino acid sequence,
subject to environmental constraints, determines the three-dimensional structure of the molecule
which can further influence the rate of degradation of a protein or peptide. The components of a
formulation and their interactions can create environmental conditions in the formulation which can
influence the structure of a protein or peptide molecule, or they might participate directly in
degradative pathways to positively or negatively affect the stability of a protein or peptide in that
formulation.
Nisin is a bacteriocin, and in particular, is a member of a family of peptides characterized by the
presence of lanthionine-containing ring structures believed to be essential for the integrity and
functionality of the molecule. Other members of this class of peptide include, but are not limited to,
subtilin, duramycin, cinnamycin, ancovenin, Pep 5, epidermin and gallidermin.
Nisin and its related peptides are antimicrobial agents that, among other things, inhibit the
germination and arrest the outgrowth of certain bacterial spores. In this context, a commercial nisin
preparation, Nisaplinw is marketed (Aplin & Barrett, Beaminster, U.K.) as a direct additive in foods
to inhibit the growth of certain pathogens and spoilage organisms, in particular thermostable, sporeforming clostridial species that are responsible for botulism. In addition, nisin and related peptides
are active against vegetative forms of certain bacteria responsible for certain diseases in animals
and humans.
247/1006
It has been found that when nisin and related peptide bacteriocins are combined with chelating
agents and/or various surfactants,the bactericidal activity of the antimicrobial peptide in such
formulations is significantly improved, and is broadened to include a much wider range of bacteria
now including species of both gram negative and gram positive bacteria (see U.S.
Patent No. 5,135,910, the disclosure of which is herein incorporated by reference). In addition, the
performance of the peptide formulation can be further affected by the presence of various excipients
and other carriers useful to facilitate delivery of the formulation to its intended site of action, for
example, under physiological conditions for pharmaceutical formulations.
Adequate performance of formulations of nisin and related peptides requires that the peptide remain
physically stable and biologically active in the various formulations under conditions of use and
storage.
Furthermore, the requirements for stability and integrity of active agents, including biologically active
peptides, are a subject for regulatory scrutiny.
It has been widely accepted that the activity of lanthionine-containing peptides is relatively stable
and can even tolerate extreme temperatures. The nisin preparation, Nisaplin", has been used under
extreme temperatures, for example during pasteurization and even at the retort temperatures used in
canning of certain foods. Despite this apparent stability it has been found that upon storage these
bacteriocin molecules undergo degradative changes some of which, but not all, result in a loss of
bioactivity. It has been shown by Chan et al.
"Isolation and characterization of two degradation products derived from the peptide antibiotic nisin."
FEBS Letters, Vol. 252 No. 1,2, 29-36 (July 1989) that upon storage of the spray-dried preparation
Nisaplinm, nisin in the preparation undergoes degradation with the accumulation of breakdown
products separable by reversed phase high performance liquid chromatography (RPHPLC) on silicabased resins eluted with gradients of organic modifiers.
Compounds as widely diverse as proteins (e.g., albumin), amino acids, surfactants, alcohols,
carbohydrates and various oxygen and radical scavengers have been cited as candidates for the
stabilization of peptides and proteins in solution. While nisin alone in dilute acid or a buffered solution
in the pH range 2 to 5 shows good stability characteristics, it has been found that some substances
248/1006
such as certain emulsifiers and surfactants which enhance the bactericidal activity of lanthioninecontaining bacteriocins in formulations (see
U.S. Patent No. 5,135,910) may also accelerate the degradation of the bacteriocins over the course
of time.
Many commonly used stabilizers and antioxidants are virtually ineffective in overcoming the
degradation of lanthionine-containing peptides. Consequently, new agents were sought which would
counteract the degradation of the bacteriocins in the formulation and which would thus yield
compositions of enhanced and stable shelf life.
Summarv of the Invention
The present invention concerns compositions comprising lanthionine-containing peptide
bacteriocins such as nisin stabilized by the presence of a suitable thioether compound as a stabilizer.
The thioether compound is preferably a compound of the formula I.
R1-S-R2 (I) wherein Rl is an alkyl group containing 1-6 carbon atoms
and R2 is
wherein n is 0 to 5; R3 is hydrogen an amino group, or an hydroxyl group; and R4 is a hydrogen, a
carboxyl group, an ester group or an amido group wherein the amino function is contributed by an
amino acid residue or wherein Rl and R2 together are joined to form, with the sulfur, a thiazolidine
ring.
In a preferred embodiment, the compound of formula I is the amino acid methionine or an analog
thereof.
The present invention further concerns methods of stabilizing nisin and other lanthionine-containing
peptides in solution and in dry mixtures. According to the invention, a compound of formula I is
added to a composition comprising a lanthionine-containing peptide bacteriocin in an amount
sufficient to protect the lanthionine-containing bacteriocin from degradation. The stabilizer compound
may be added to the lanthioninecontaining bacteriocin upon formulation or alternatively it may be
pre-formulated with one or more of the nonbacteriocin components prior to formulation with the
bacteriocin.
249/1006
The addition of a compound of formula I, which is preferably methionine or an analog thereof,
stabilizes the active bacteriocin ingredient over a broad pH range and does not compromise in any
way the potency or utility of the compositions.
Brief Description of the Fissures
Figure 1 shows the effect of different concentrations of methionine on the stabilization of nisin in the
presence of the polysorbate surfactant T-MAZ 20 at pH 6.
Figure 2 shows the stability of nisin formulated with polysorbate (T-MAZ 20) preincubated with
methionine.
Detailed Description of the Invention:
Lanthionine-containing bacteriocins such as nisin can be formulated into a variety of compositions
which exhibit bactericidal activity against gram negative and gram positive organisms. These
bacteriocin compositions which, in addition to the bacteriocin also may contain surfactants,
emulsifiers, chelating agents, humectants and other excipients such as thickening agents, flavors,
fragrances, abrasives and lubricants, are used in a wide variety of applications such as oral rinses,
topical disinfectants, pharmaceutical compositions, dentifrices, disinfectant paper wipes, food
preservatives, germicides, intramammary infusions, etc. Such composition and methods for
preparing them are described in U.S. Patent
No. 5,135,910, whose disclosure is herein incorporated by reference.
The lanthionine-containing bacteriocins used in these bactericidal compositions can be selected
from the group consisting of nisin, subtilin, duramycin, cinnamycin, ancovenin, Pep 5, epidermin, and
gallidermin.
While the lanthionine bacteriocin is not limited to the selection of the above group, the preferred
bacteriocin is nisin.
Suitable surfactants used in combination with lanthionine-containing bacteriocins in such
bactericidal compositions are: polyethoxylated sorbitol esters, e.g.,
Peg(40) sorbitan diisostearate, Tweensm; polycondensates of ethylene oxide and propylene oxide,
e.g., Poloxamers,
250/1006
Pluronic, F127, F68; polyethoxylated hydrogenated castor oil, e.g., Cremophor, El, RH40; sorbitan
fatty esters; long chain imidazoline derivatives, e.g., Miranol C2M; long chain alkyl betaines, e.g.,
Empigon BB; long chain alkyl amidoalkyl betaines, e.g., cocamidopropylbetaine;
D, L-2-pyrrolidone-5-carboxylic acid salt of ethyl-Ncocyl-L-arginate, e.g., CAE; cocamidopropyl PG
diammonium chloride, EGM Monoquat PTC; lauramidopropyl, e.g.,
Monaquat BTL; Tagat, R60, L2, 02, S2; Cetiol HE; Pyroter;
Ryoto sugar; Tensopol; Tegobetaine; Incromine; Solutol
HS15 and laurainine oxide.
The instant invention provides compositions, and methods for producing the same, which are
improved over previously disclosed compositions comprising lanthioninecontaining bacteriocins. The
inventive compositions not only act as enhanced broad range bactericides but in addition, have an
extended shelf life greater than that of the prior art compositions. The inventive bacteriocin
compositions containing a suitable thioether stabilizing agent may be formulated into solutions or dry
compositions such as freeze-dried preparations.
Representative Formulations Comprising Lanthionine Containina Bacteriocins
Bactericidal formulations for use in the present invention may be formulated as disclosed in U.S.
Patent
No. 5,135,910. In addition, these formulations may be stabilized by the addition of suitable thioether
compounds as disclosed herein, and for specific applications excipients may be added to the
formulation suited to the purposes of the commercial application.
Representative formulations and ingredients are set forth below. The concentration and inclusion of
the excipients may be varied by those of ordinary skill in the art so as to obtain the preferred
properties desired for each formulation.
(i) A nisin-containing formulation useful as an oral rinse or dentifrice comprising: ethanol or other
alchohols poloxamer, polysorbate or other emulsifier/surfactants
EDTA, citrate or other chelators coolmint or other flavors glycerol, propylene glycol or other
humectants blue dye or other colors saccharin or other sweeteners nisin or other lanthioninecontaining bacteriocins
May also contain thickeners such as hydroxyethyl cellulose and abrasives such as silica or
diatomaceous earth for use as a dentifrice. May contain xanthan gums or stearate salts useful for
formulating as a tablet.
251/1006
(ii) A nisin-containing formulation useful as a topical germicide comprising: 1-propanol, ethanol or
other alchohols polysorbate or other emulsifier/surfactants propylene glycol, glycerol or other
humectants
EDTA, citrate or other chelators nisin or other lanthionine-containing bacteriocins water qs
May also contain thickeners such as polyvinylpyrrolidone, hydroxyethyl cellulose, alginates or
silicones. In addition may contain fragrances.
(iii) A nisin-containing formulation useful as a deodorant comprising: 1-propanol, ethanol or other
alchohols polysorbate or other emulsifier/surfactants propylene glycol, glycerol or other humectants
EDTA, citrate or other chelators fragrances nisin or other lanthionine-containing bacteriocins water qs
(iv) A nisin-containing formulation useful as an intramammary infusion for treating mastitis comprising:
polysorbate or other emulsifier/surfactants
EDTA, citrate or other chelators glycerol, sorbitol, propylene glycol or other humectants nisin or other
lanthionine-containing bacteriocins water qs
Thioether Stabilizing ComPounds
The inventive thioether stabilizing agents are effective in increasing the stability of bacteriocins such
as nisin when formulated with a wide range of surfactants, chelators, emulsifiers and humectants.
While different components exhibit different degrees of associated degradation, the addition of the
inventive thioether stabilizing agents is expected to have a beneficial effect in all situations in which a
lanthionine-containing bacteriocin is formulated with such components. The thioether stabilizing
agents also increase the stability of nisin in a wide variety of formulations with chelating agents in
combination with the humectants glycerol or sorbitol.
According to the invention, the lanthioninecontaining peptide compositions typically would have a
peptide concentration in the range of 1 yg/ml to 1000 yg/ml, preferably in the range of 30 yg/ml to
300 yg/ml, a surfactant concentration in the range of 0.1% to 10.0% and a concentration of a
thioether stabilizer compound in the range of 1 mM to 50 mM. In most instances, a stabilizer
concentration in the range of 1 to 10 mM will be sufficient to maintain the initial potency of the active
ingredient.
While any suitable thioether stabilizer compound may be used as a stabilizer for these bacteriocin
formulations it is preferred that the stabilizing compounds of the formula I below be used:
Rl-S-R2 (I) wherein Rl is an alkyl group containing 1-6 carbon atoms
252/1006
and R2 is
wherein n is 0 to 5; R3 is hydrogen, an amino group, or an hydroxyl group; and R4 is a hydrogen, a
carboxyl group, an ester group or an amido group wherein the amino function is contributed by an
amino acid residue or wherein Rl and R2 together are joined to form, with the sulfur, a thiazolidine
ring.
Preferably the compound of formula I is methionine, an analog thereof, or a related thioether
compound.
Suitable compounds for use in the invention are methionine, methionine hydroxy analog, methionine
methyl ester, methionine ethyl ester, thiazolidine, and lanthionine. In certain embodiments of the
invention the thioether stabilizing compound may be a peptide or a polymer rich in methionine, or
methionine analog residues.
According to further embodiments of the invention, the thioether stabilizing agent may be defined by
the formula II described below:
R'-S-R2
II wherein Rl is an alkyl group containing 1-6 carbon atoms or
n is 0-5 wherein R3 is hydrogen or
x is 1-3; and R4 may be -H, -CH3, -CH(CH3)2, -CH2CH(CH3)21
-CH(CH3) CH2CH3, -CH2SH, -CH2CH2SCH3, -CH2OH, -CH (OH) CH3, -CH2COOH, - (CH2)
2COOH, -CH2CONH2, - (CH2) 2CONH2,-(CH2) 4NH2,
or -(CH2)3- joined with the amino group of R3 to form a pyrrolidine ring; and
R2 is
wherein R5 is hydrogen, an amino group or a hydroxyl group and R6 is hydrogen or
wherein R7 is hydroxy, alkoxy containing 1-6 carbon atoms or
and n, x and R4 are as defined above.
253/1006
The thioether stabilizing compound must also be suitable for the intended use of the formulation.
Some thioether stabilizing compounds may protect against nisin degradation; however, the
usefulness of such compounds may be limited because of their odor, toxicity or carcinogenicity.
Thus, the thioether stabilizer compounds must also be selected so that they are suitable for the
specific commercial application and so that they do not possess adverse characteristics which
cannot be remedied.
The examples set forth below demonstrate that the thioether stabilizing compounds of the invention
and preferably the compounds of formula I, e.g., methionine and related compounds, are effective,
while commonly used antioxidants are not, in stabilizing lanthioninecontaining peptides against
degradation associated with surfactant, chelating agent or humectant components of the
compositions. The data indicate that the stabilizing agents not only preserve the physical integrity but
also do not interfere with the biological properties of the .active-ingredient peptides.
Methods of Determining Nisin Stability
The stability of nisin may be determined in a number of ways. Those used in the examples in this
application were: (i) analytical reverse phase high pressure liquid chromatography and (ii) the
Minimum Inhibitory
Concentration Assay described below: i) Analytical reverse phase high pressure liquid
chromatography (RPHPLC). The analyte (nisin) in
solution is passed through a column of hydrophic
beads to which the nisin tends to bind. The solution
flowing through the column is then made
progressively more hydrophobic by increasing
acetonitrile concentration until it causes the nisin
to be released from the beads and eluted from the
column. The emergence of the nisin from the column
is detected by measuring the absorbance of light at
210 nm by the effluent.
A second detection system may also be used in
conjunction with RPHPLC, by reacting
amine-containing components emerging from the column
with fluorescamine. The products of this reaction
254/1006
are fluorescent and may be detected by an
appropriate monitor. The advantage of the
fluorescence-based system is that it is sensitive
only to amine-containing analytes (nisin has four
amine groups). This allows the analysis of nisin in
complex formulations where other components
interfere with the detection of nisin by absorption
at 210 nm.
ii) Estimation of the minimum inhibitory concentration
(MIC) of the nisin solution. This measures the
functional activity of the nisin in the solution by
testing its ability to kill or prevent the growth of
a target bacterial population.
A series of two-fold dilutions of the solution of
nisin to be tested is prepared. Aliquots of 5 y1 of
these solutions are pipetted onto a lawn of target
bacteria (Staphylococcus aureus) growing on a gel of
nutrient agar in a petri dish. The dish is covered
and incubated at 37 C overnight (-16 hr). Where the
nisin concentration is sufficiently high to prevent
growth of the bacteria there is a zone of clearance
in the bacterial lawn. The activity of a nisin
solution being tested is given as the lowest nisin
concentration inhibiting the growth of the bacteria
(the minimum inhibitory concentration).
Example 1
Oral rinse formulations containing nisin were formulated with a variety of alternative components to
determine which component(s) was associated with the degradation of the nisin. A series of
formulations was prepared in which each component was omitted in turn.
These formulations and their components are set forth in
Table 1. Residual nisin concentration is shown in yg/ml.
255/1006
The initial nisin concentration was 300 yg/ml.
A reference solution of nisin in 10 mM HC1 was prepared as well as a full formulation with no
omissions.
After incubation at room temperature for 3 days the formulations were analyzed by RPHPLC to
determine the extent of degradation of the nisin.
Table 1
Residual Nisin Concentration In An Oral Rinse With
Sequential Component Omission
Component omitted Nisin
(g/ml) Theoret
ical
conc.
nisin 300 100
standard
full
formulation 206 69
color FD & Blue No. 1 189 63
glycerol 223 74
ethanol 154 51
saccharin 163 54
EDTA 149 50
polysorbate 274 91
Poloxamer 407 146 49
flavor - coolmint 154 51
(Noville)
256/1006
Examination of the RPHPLC chromatograms for the formulations of Table 1 revealed that all the
formulations showed accelerated nisin degradation relative to the reference in 10 mM HCl but that
degradation was minimized in the formulations in which the polysorbate surfactant/emulsifier or the
glycerol humectant were omitted.
Formulations of nisin useful as oral rinses comprising EDTA and the humectant glycerol were also
prepared and analyzed by RPHPLC. These studies revealed a source of nisin degradation which
was dependent on the simultaneous presence of both EDTA and glycerol in the formulation. Neither
compound caused a problem in the absence of the other.
Glycerol and sorbitol from a number of sources, and a number of alternative chelators, were
screened (Tables 2 and 3). No source of glycerol nor of sorbitol was found which was without effect
on nisin stability in these formulations, and none of the chelating agents tested eliminated the
problem, although citrate was superior toEDTA.
Table 2
Glycerol And Sorbitol Batches Screened For
Effects On Nisin Stability
Dow Glycerol
P & glycerol batches # 925-371, #925-647,
# 925-602
Henkel glycerol batches # ODG14, # OGG06,
; OGG07
Witco glycerol batches ; OU4314, ; OR2245,
# 9X5951
Pfizer sorbitol batches # G06150, # GO6200,
# GO6270
Roquette sorbitol batches ; 4878, ;4710, ;4929
Table 3
Chelating Agents Screened For Effects On Nisin Stability
257/1006
EDTA (ethylenediaminetetraacetic acid)
EGTA (ethyleneglycol-bis-(P-aminoethyl ether)
N,N,N',N'- tetraacetic acid)
CDTA (1,2-diaminocyclohexane
N,N,N',N'-tetraacetic acid)
DTPA (diethylaminetriaminepentaacetic acid)
HEEDTA (N-hydroxyethylethylenediaminetriacetic
acid)
EDITEMPA (N,N,N' ,N'-ethylenediaminetetra (methylene
phosphonic acid))
citrate
Example 2
In order to further analyze the nisin degradation associated with one particular surfactant/emulsifier,
polysorbate, material was obtained from as many manufacturers as possible. Where possible,
multiple production batches were obtained from each manufacturer.
Other emulsifiers or surfactants believed to closely resemble polysorbate in their properties as well
as dissimilar surfactants were also tested. An oral rinse formulation containing nisin was prepared
using each of these compounds and, after incubation, samples were analyzed to evaluate nisin
stability. The manufacturers and multiple batches of components tested are listed in
Table 4.
Table 4
Manufacturers And Batches Of Polysorbate Or Similar
Surfactants Tested For Use In Nisin Formulations.
Lonza Polysorbate
Heterene Hetsorb 120 P lots ;18142, ;20716, ; 18410, ;18546
258/1006
Mazer T-MAZ 20 lots ;79779, ;109104, ;105655, ;83371, ;98372,
;96864, ;95338, ;95523, ;82240, ;80777
Croda Crillet 1 lots ;;WB 1181, #WB1651DU, #WB1333DU
ICI Tween 20, 40, 60, 65, 80, and 85
ICI Arlatone B
ICI Arlatone G
ICI Arlatone T
ICI Arlacel 165
ICI Arlasolve 200
Great variation was observed in nisin stability in the presence of the emulsifier/surfactants obtained
from different manufacturers; nisin stability was even markedly different using different batches of the
same product.
However, it was significant that all of the surfactants on the extensive list tested accelerated nisin
degradation to some extent, relative to control formulations in which the emulsifier/surfactant was
omitted.
One possible explanation for this degradation may be the introduction of substances during the
manufacturing process of formulation excipients. For example, to enhance the appearance of
polysorbate the product is bleached by addition of peroxide, a well known oxidizing agent. It seemed
quite possible that the presence of residual peroxide in polysorbate contributed to nisin instability.
Nisin was found to rapidly degrade on exposure to peroxide (Table 5).
Nisin was partially protected by the presence of EDTA. An assay for peroxide was used to screen
the various batches of polysorbate used in nisin formulations described herein, but no relationship
could be seen between residual peroxide levels and nisin stability. Furthermore, samples taken from
a production batch at Mazer Chemicals immediately prior to and immediately after the bleaching
step were found to be equivalent in their effects on nisin stability. Thus peroxide added during
manufacture may likely not be the agent of degradation.
Table 5
Residual nisin concentration ( g/ml) after 8 days at room
temp. Initial nisin concentration was 300 yg/ml. The
concentration of NaOAc was 4mM and the concentration of
259/1006
Fe was 10 yM.
peroxide (pM) glyceraldehyde (pom)
100 10 1 0 1000 100 10 0
NaOAc, NaOAc, Fe, peroxide 86 261 280 294
EDTA, DTT, Fe, peroxide 170 273 297 303
NaOAc, NaOAc, peroxide 154 263 289 283
EDTAI peroxide 175 284 295
NaOAc, NaOAc, 257 291 290
glyceraldehyde
EDTA, EDTA, 273 289 285
glyceraldehyde
Example 3
A number of commonly used antioxidants were tested for their ability to protect nisin in formulation
using the methods described in Example 2. The compounds and combinations tested are listed in
Table 6. None of the compounds tested gave satisfactory protection of nisin.
In fact some compounds in the list aggravated the stability problem, e.g., dithiothreitol (DTT),
ascorbate, sodium sulfite.
Table 6
Commonly Used Antioxidants Tested For Protection
Of Nisin In Formulation.
butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA) propyl gal late alpha-tocopherol
phenylene-diamine ethoxyquin ascorbic acid citric acid hydroquinone dithiothreitol (DTT) sodium
sulfite
BHA + BHT + propyl gallol + citric acid imidazole sodium thiosulfate sodium benzoate
Example 4
260/1006
The stabilization of nisin at 25 yg/ml by a range of sulfur-containing compounds was evaluated in
formulations useful as topical germicides comprising polysorbate (T
MAZ 20, Mazer Chemicals) with propyleneglycol, 1propanol, and either EDTA or citrate. In order to
expedite the execution of these experiments the formulations were subjected to "stressed" conditions,
i.e., pH6 and 400C, both intended to accelerate any degradation effects taking place. The stability of
nisin in the formulations was evaluated by RPHPLC.The data in
Tables 7, 8 and 9 illustrate that the thioether compounds, L- and DL-methionine, DL-methionine
methyl- or ethyl esters, and DL-methionine hydroxy analog are all able to stabilize nisin from
degradation. To a lesser degree, nisin was also stabilized by the thioether compounds thiazolidine
and lanthionine. The disulfide compounds cystine and oxidized glutathione, cysteic acid, methionine
sulfoxide and methionine sulfone, and the sulfhydryl compound cysteine did not stabilize nisin from
degradation. In addition, the dibasic amino acid lysine did not stabilize nisin.
The stabilization of nisin by methionine in formulations useful as topical germicides where other
emulsifier/surfactants were substituted for polysorbate was tested. Formulations were prepared with
nisin at 25 yg/ml and titrated to pH6. They were incubated at 400C for 3 days and then analyzed by
RPHPLC. The data in
Table 10 illustrates that methionine also enhances nisin stability in formulations containing Brij,
Tergitol,
Tyloxapol, and Triton.
Table 7
Residual nisin concentration as a percentage of the
theoretical concentration after 5 days at 400C, pH 6
jd6-35- T-MAZ EDTA Citrate con- Met Lye CySH
H H 20 (mM) % trol (lem) (lmM) (lmM)
Batch
standar 100
d
33 - 36 357 1 87 84 80 85
37 - 40 357 0.1 73 75 71 29
41 - 44 357 = 0.3 71 76 71 36
261/1006
45
49
53
57
61
-
48
52
56
60
64
357
348
348
348
348
1.0 67 73 71 31
1 55 80 56 51
0.1 49 76 45 36
0.3 49 73 47 29
1.0 51 73 49 33
Met = methionine; Lys = lysine; CySH = cysteine
Table 8
Residual nisin concentration after incubation for 2 weeks
at 400C.Nisin Concentration (yg/ml)**
T-MAZ 20 T-MAZ 20
BATCH 357 BATCH 357 348
water control 19.5 9.5
methionine 23.2 23.3
MHA 22.5 23.4
cystine* 11.1 9.0
cysteic acid 19.5 9.6
glutathione (oxidized) 14.1 7.6
lanthionine 18.2 10.7
thiazolidine 17.3 14.9
* Cystine at 5mM came out of solution as the pH was raised to 3.5.
** Nisin was formulated at 25 yg/ml.
Table 9
Residual nisin concentration ( g/ml) after 6 days at
400C.
262/1006
Stabilizer nisin T-MAZ 20
g/ml)
no methionine 10.1 1
DL-methionine 19.7 1
L-methionine 19.9 1
DL-methionine methyl ester 21.9 1
DL-methionine methyl ester 20.8 1
DL-methionine sulfoxide 8.6 1
DL-methionine sulfone 8.6 1
methionine hydroxy analog 18.1 1
(Sigma)
methionine hydroxy analog 19.8 1
(MHA Novus)
no stabilizer, no T-MAZ-20 21.8 0
* The initial concentration of nisin was 25 yg/ml.
Table 10
Stabilizing effect of methionine on nisin at 25 yg/ml in a dermatological formulation under stressed
conditions. Formulations were prepared at pH 6.0, substituting other surfactant agents for
polysorbate (T
MAZ 20). Formulations were incubated for 3 days at 400C to accelerate the degradation of nisin.
Ir no 5mM
methionine methionine
T-MAZ 20 acid 16.9 20.5
Brij 7.2 12.7
eoxycholic acid 20.0 20.3
ergitol 3.8 9.7
yloxapol 10.0 19.4
riton X-305 17.1 20.2
Triton X-100 8.9 12.1
263/1006
Example 5
A series of tests was performed to determine the methionine concentration required for stabilization
of nisin in a topical germicide formulation containing the polysorbate surfactant, T-MAZ 20, with
methionine at concentrations in the range 0 to 5 mM. The stability of nisin in these formulations was
compared to that of nisin in a formulation in which the polysorbate was omitted (Fig. 1).
A topical germicide formulation containing nisin was also prepared using the polysorbate T-MAZ 20
preincubated for the indicated times as a 10% solution containing methionine at 10 mM or 50 mM.
Various preincubation mixtures were prepared and subjected to different preincubation time periods.
Following preincubation, the treated mixture was diluted ten-fold when combined with nisin, resulting
in a composition 1% in T-MAZ 20 and either 1 mM or 5 mM in methionine. The samples were then
incubated at 40 C for 12 days and subsequently analyzed for nisin by RPHPLC. The results are
shown in Fig. 2.
While there appears to be no advantage imparted to stabilization by preincubation, such a
component preincubation manufacturing process may offer other advantages in terms of
manufacturing efficiency, etc.
Example 6
A stabilized nisin formulation useful as an oral rinse was prepared as in Example 1 comprising a)
polysorbate, b) glycerol or sorbitol, c) EDTA or citrate, and d) methionine in the concentrations as
indicated in Table 11 below.
The formulations were adjusted to pH 4.0 and stored at 40"C. After 1 month samples were analyzed
by RPHPLC.
The estimated nisin concentration is shown in Table 11.
Initial nisin concentration in the formulations was 100
Pg/ml.
Table 11
Residual nisin concentrations in oral rinse formulations.
264/1006
10% EDTA citrate methionine Nisin
Humectant (mM) (%) (mM) (g/mi)
sorbitol 1 0 88.9
sorbitol 1 1 94.1
sorbitol 1 2 97.7
glycerol 1 2 94.8
glycerol 0.3 2 2 ~ 84.9
Example 7
A stabilized nisin formulation useful as a deodorant was prepared using polysorbate T-MAZ 20 at pH
3.5, incubated for three (3) months at 40 C, and analyzed by
RPHPLC after 3 months. The results are presented in
Table 12.
Table 12
Residual nisin concentration in deodorant formulations at
pH 3.5.
Sample Ethanol Tween Met Nisin*
No.
1 % lmM (Zg/ml)
Batch #
1 35 357 4.2
2 35 357 + 22.4
3 35 348 8.8
265/1006
4 35 348 + 21.7
5 35 - 9.3
6 35 - + 24.4
* Initial Nisin concentration was 25 yg/ml. Nisin concentrations were determined by RPHPLC.
Deodorant formulations were also prepared with polysorbate T-MAZ 20 and methionine at 0, 1, 3, or
5 mM.
Formulations were titrated to pH 3.5, 4.5, or 6.0 and set to incubate at 40 C. Samples were analyzed
by RPHPLC after 5 days, 18 days, and 60 days. The estimated residual nisin concentration is shown
in Table 13 below.
Table 13
Residual Nisin Concentration in Deodorant Formulations
at a Range of pH 3.5 to 6.0.
pH 3.5 pH 4.5 pH 6.0
days: 5 18 60 5 18 60 5 22 60
Met* Nisin Concentration Hg/ml**
(mM)
0 21.3 16.6 10.7 21.2 14.1 3.9 20.1 10.3 3.3
1 23.7 21.7 16.2 22.7 21.6 13.4 22.1 18.6 11.9
3 24.6 22.9 17.0 24.8 22.0 13.9 23.3 20.0 13.1
5 25.2 23.2 17.5 25.3 22.9 13.8 23.2 19.9 13.3
* Met = Methionine
** Initial nisin concentration was 25 yg/ml.
Example 8
Stable nisin formulations useful as topical germicides were prepared at pH 3.5 and set to incubate
at 40 C. After 2, 4, and 6 months samples were analyzed by
266/1006
RPHPLC. At 6 months the samples were also analyzed for activity in the MIC assay. The RPHPLC
and MIC assay data are presented in Table 14.
Table 14
Nisin concentration and MIC (yg/ml)
after 6 months at pH 3.5, 400C.
nisin
EDTA EDTA citrate T-MAZ 20 Met MIC conc.*
1 niM 1% 1 mM g/ml jug/ml
+ 12.5 17.8
+ + 12.5 22.2
+ + > 50 11.5
+ + + 12.5 19.8
0.1 12.5 13.1
0.1 + 12.5 18.1
0.1 + > 50 3.0
0.1 + + 6.25 15.8
0.3 12.5 13.6
0.3 + 12.5 15.3
0.3 + > 50 4.1
0.3 + + 3.125 14.8
1.0 25 7.7
1.0 + 12.5 10.0
1.0 + 12.5 2.5
1.0 + + 6.25 9.0
* Initial nisin concentration was 25 yg/ml. Claims:
267/1006
WE CLAIM:
1. A lanthionine-containing bacteriocin composition stabilized against degradation comprising a
lanthioninecontaining bacteriocin and a thioether stabilizing agent.
2. The composition of claim 1 wherein the bacteriocin is nisin.
3. A lanthionine-containing bacteriocin composition stabilized against degradation comprising a
lanthioninecontaining bacteriocin and a compound of the formula I
Rl-S-R2 (I) wherein Rl is an alkyl group containing 1-6 carbon atoms
and R2 is
wherein n is 0 to 5; R3 is hydrogen, an amino group or hydroxyl group; and R4 is a hydrogen, a
carboxyl group, an ester group or an amido group wherein the amino function is contributed by an
amino acid residue or wherein Rl and R2 together are joined to form, with the sulfur, a thiazolidine
ring.
4. The composition of claim 3 wherein the composition also comprises a surfactant.
5. The composition of claim 3 wherein the composition also comprises a chelating agent.
6. The composition of claim 3 wherein the bacteriocin is nisin.
7. The composition of claim 4 wherein the surfactant is a polysorbate.
8. The composition of claim 5 wherein the chelating agent is EDTA or citrate.
9. The composition of claim 3 wherein the compound of formula I is methionine or methionine
hydroxy analog.
10. The composition of claim 9 wherein the concentration of methionine is in the range of 1 to 50 mM.
11. The composition of claim 10 wherein the concentration of methionine is in the range of 1 to 10
mm.
268/1006
12. A lanthionine-containing-bacteriocin composition stabilized against degradation comprising the
bacteriocin in a concentration range of .1 to 1000 yg/ml, a surfactant in a concentration range of 0.1
- 10%, a chelating agent in a concentration range of 0.1 - 20 mM and a thioether stabilizing agent in
a concentration range of 1 mM to 50 mM.
13. The composition of claim 12 wherein the bacteriocin is nisin and the stabilizing agent is
methionine.
14. Use of a compound according to Formula I of claim 3 for stabilizing against degradation a
lanthionine bacteriocin component of a composition.
269/1006
24. JP2000102388 - 11.04.2000
BACTERIOCIN GENE FROM SOFT ROT-CAUSING BACTERIUM
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP2000102388
Inventor(s):
KAMIO YOSHIKORE (--)
Applicant(s):
CENTRAL GLASS CO LTD (--)
IP Class 4 Digits: C12N; C12P
IP Class:
C12N15/09; C12P21/02
Application Number:
JP19980278786 (19980930)
Family: JP2000102388
Abstract:
PROBLEM TO BE SOLVED: TO OBTAIN A NEW BACTERIOCIN GENE WHICH CONSISTS OF A DNA
FRAGMENT CODING FOR BACTERIOCIN WHICH IS AN ANTIBIOTIC PROTEIN FROM A SOFT ROTCAUSING BACTERIUM, IS USEFUL FOR CONTROLLING AND DETECTING A SOFT ROT-CAUSING
BACTERIUM WHICH ROTS VEGETABLES, AND CAN BE USED FOR DIAGNOSING THE DISEASE
AND CONTROLLING THE DISEASE INJURY IN AGRICULTURE AND SO ON.
SOLUTION: A NEW DNA FRAGMENT CODING FOR BACTERIOCIN FROM A SOFT ROT- CAUSING
BACTERIUM IS PROVIDED. THE SOFT ROT-CAUSING BACTERIUM IS A PATHOGEN AGAINST
PLANTS, AND ROTS VEGETABLES. THE BACTERIOCIN OF THE SOFT ROT-CAUSING BACTERIUM
IS AN ANTIBIOTIC PROTEIN HAVING AN ACTIVITY AGAINST RELATED BACTERIA. A DNA
FRAGMENT CODING FOR THE GENE IS USEFUL FOR DIAGNOSING AND DETECTING THE SOFT
ROT-CAUSING BACTERIUM AND SO ON. THE DNA FRAGMENT IS OBTAINED BY ISOLATING
GENOMIC DNA FROM SOFT ROT-CAUSING BACTERIA SUCH AS ERWINIA CAROTOVORA SUBSP.
CAROTOVORA CGE234M403, CARRYING OUT PCR USING PRIMERS CONSISTING OF ITS
PARTIAL SEQUENCES, FOLLOWED BY SCREENING A GENOMIC LIBRARY OF E. CAROTOVORA
USING AN OBTAINED DNA FRAGMENT AS A PROBE.
270/1006
25. JP2001335597 - 04.12.2001
NEW BACTERIOCIN AND METHOD FOR PRODUCING THE SAME
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP2001335597
Inventor(s):
KAWAMOTO SHINICHI (--); SHIMA JUN (--); OMOMO SADAHIRO (--); MORI
KATSUMI (--); OGATA SEIYA (--)
Applicant(s):
NATIONAL FOOD RESEARCH INSTITUTE (--)
IP Class 4 Digits: C12N; C07K; C12P
IP Class:
C07K14/315; C12N1/20; C12P21/02
Application Number:
JP20000152586 (20000524)
Family: JP2001335597
Equivalent:
JP3472804B2
Abstract:
PROBLEM TO BE SOLVED: TO OBTAIN AN ANTIBACTERIAL PRODUCT DERIVED FROM LACTIC
ACID BACTERIA, HAVING NO PROBLEM ON SAFETY AND THEN APPLICABLE TO A FOOD
INDUSTRY, AND TO PROVIDE A METHOD FOR EFFICIENTLY PRODUCING THE ANTIBACTERIAL
PRODUCT.
SOLUTION: THIS METHOD FOR PRODUCING ENTEROCIN SE-K4 IS CHARACTERIZED BY
CULTURING THE ENTEROCIN SE-K4 AND HAS THE FOLLOWING PROPERTIES AND
ENTEROCOCCUS FAECALIS K-4 STRAIN (FERM BP-7162) AND COLLECTING THE NEW
BACTERIOCIN FROM THE CULTURE. THE ENTEROCIN SE-K4 (A) HAS ABOUT 5 KDA
MOLECULAR WEIGHT [MEASURED BY A SODIUM DODECYL SULFATE (SDS)-POLYCRYLAMIDE
GEL ELECTRODPHORESIS], (B) HAS ANTIBACTERIAL ACTIVITY TO ENTEROCOCCUS FAECIUM,
(C) HAS HEAT RESISTANCE, (D) HAS ANTIBACTERIAL ACTIONS ON FOOD POISONING
BACTERIA AND (E) HAS A SEQUENCE REPRESENTED BY SEQUENCE NUMBER 1 IN THE
271/1006
SEQUENCE TABLE (REFER TO THE SPECIFICATION) IN AN AMINO ACID SEQUENCE HAVING AN
N TERMINAL.
272/1006
26. JP2002262780 - 28.11.2002
BACTERIOCIN-CONTAINING SORBIC ACID PRODUCT AS ADDITION TO FEEDSTUFFS IN
AGRICULTURAL LIVESTOCK REARING
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP2002262780
Inventor(s):
RACZEK NICO N (DE)
IP Class 4 Digits: A23L
IP Class:
A23L1/28
E Class: A23K1/00C2B; A23K1/16G; A23K1/16D; A23K1/18; A23K1/18K; A23K1/18L2; A23K1/18T;
A23K1/18V
Application Number:
US20020080198 (20020219)
Priority Number: DE20011010431 (20010305)
Family: JP2002262780
Equivalent:
AU2029702; DE10110431; EP1238591; US6780447
Abstract:
THE PRESENT INVENTION RELATES TO A PRODUCT FOR USE IN ANIMAL FEEDSTUFFS. THE
PRODUCT COMPRISES SORBIC ACID AND LIVE OR DEAD MICROORGANISMS WHICH SECRETE
BACTERIOCINS, OR THE BACTERIOCINS THEMSELVES OR COMBINATIONS THEREOF AND,
WHERE APPROPRIATE, A CARRIER. THE INVENTION FURTHER RELATES TO THE USE OF THE
PRODUCT ON ITS OWN IN FEEDSTUFFS OR IN A MIXTURE WITH OTHER FEED ADDITIVES FOR
IMPROVING THE HYGIENIC STATUS OF THE FEED AND FOR IMPROVING PERFORMANCE IN
AGRICULTURAL LIVESTOCK REARING.Description:
BACKGROUND OF THE INVENTION
273/1006
[0001] The invention relates to a product which comprises sorbic acid and at least one bacteriocin
and can be used on its own in feedstuffs or mixed with other feedstuff additives in agricultural
livestock rearing.
[0002] Antibiotics are frequently used to improve performance in the animal feed sector. In some
cases, very similar or identical substances are used in human medicine. The use of antibiotics in the
animal nutrition sector is suspected in principle of being responsible for the dangers derived from
resistant bacteria, which may also endanger human health in the long term. It is therefore necessary
to look for products about which there are fewer health doubts for this purpose of use. Thus, in other
sectors too there is increasing replacement of substances about which there are physiological and
epidemiological health doubts or else which are harmful for the environment, such as, for example,
antibiotics, formaldehyde-emitting materials, halogenated substances, and many others, by materials
about which there are fewer doubts, for example in human foods, feedstuffs, pet food, silages,
pomace or other waste materials from the food industry. The purpose of these materials is, on the
one hand, aimed at maintaining the value of the actual product. However, on the other hand, it is also
intended to improve the hygienic condition thereof and achieve a longer shelf life.
[0003] It is known that sorbic acid can be employed for preserving feedstuffs. Sorbic acid
(trans,trans-2,4-hexadienoic acid) is a colorless solid compound which dissolves only slightly in cold
water and is used around the world as a preservative. The principle of action is determined by sorbic
acid in undissociated form. Sorbic acid therefore displays its best effect in the acidic pH range.
Sorbic acid and its salts have a very good microbiostatic, antimycotic action. At the same time, as
unsaturated fatty acid, sorbic acid is virtually nontoxic, which has been proven by very extensive
data and by the decades of use of this acid in the human food sector, in animal feeds, inter alia.
[0004] Besides sorbic acid, other organic acids have also been employed for some years for
preserving feedstuffs and for improving feed hygiene. The hygienic quality in particular of feed for
young animals must meet special requirements. This is why some organic acids are approved
without a limitation on the maximum amount, on the basis of the national legal provisions concerning
feedstuffs.
[0005] Bacteriocins are specific inhibitors which are secreted by microorganisms and are lethal for
other microorganisms-principally bacteria. Bacteriocins are peptides, polypeptides, proteins or
substances which have at least proteinogenic structures and are composed of amino acids. It is
moreover possible for these bacteriocins which are composed of amino acids also to contain
unusual amino acids such as, for example, lanthionine or [beta]-methyllanthionine. For example,
pediocin L50 contains other modified amino acids (L. M. Cintas et al., "Isolation and Characterization
of Pediocin L50, a New Bacteriocin from Pediococcus acidilactici with a Broad Inhibitory Spectrum",
Applied and Environmental Microbiology, July 1995, pages 2643-2648).
274/1006
[0006] Microorganisms which produce bacteriocin frequently occur naturally, for example in milk and
dairy products (cf. for example, E. Rodriguez et al., "Diversity of bacteriocins produced by lactic acid
bacteria isolated from raw milk", International Dairy Journal 10 (2000) 7-15). Such microorganisms
are moreover continually being isolated from other foodstuffs such as meat and meat products (cf.
for example, Food Science and Technology International (1998) 4, 141-158).
[0007] The microorganisms which secrete bacteriocins have often already been used for several
centuries-often unknowingly-for producing foodstuffs in that the bacteria which are intentionally
added as so-called protective cultures inhibit, by their secretion products, other bacteria which
cause spoilage, are toxic, unwanted or hazardous in other ways. A well-known bacteriocin is nisin.
This is produced commercially and has also been employed for some years as foodstuff additive
against certain microorganisms which cause so-called "late blowing" in cheese.
[0008] The fundamental disadvantage of using bacteriocins is that they are active only against
certain groups of microorganisms, in particular against close relatives. In addition, bacteriocins are
unstable in the foodstuff and decompose after a certain time, so that no activity is available any
longer.
[0009] The other organic acids known as addition to feedstuffs have the disadvantage that some of
them are volatile, have unpleasant odors and, in addition, corrosive effects. The performanceimproving effects which can be achieved with them are associated with considerable disadvantages
in handling.
[0010] The object accordingly was to provide a stable addition which is easy to handle, has a
preservative effect and improves performance but does not have these disadvantages.
BRIEF DESCRIPTION OF THE INVENTION
[0011] This object is achieved by a product (composition) which comprises sorbic acid and at least
one bacteriocin. The bacteriocin(s) may be employed as such but it is also perfectly possible to
employ live or dead microorganisms which produce or contain these bacteriocins. It is preferred to
use bacteriocin-producing microorganisms which occur naturally, for example in dairy or meat
products. Microorganisms to be employed according to the invention are only those which produce
bacteriocins. The table detailed below contains species of microorganisms which may or may not
produce bacteriocins (for example Bacillus cereus); these can accordingly be employed only if they
produce bacteriocins. The bacteriocin-producing or -containing microorganisms or the bacteriocins
themselves can also be employed in encapsulated form or bound to carriers. It is moreover possible
to use products which contain bacteriocins in effective concentrations or detectable amounts. This
also includes mixtures of such products, for example with whey proteins or common salt. Available
275/1006
products of this type are, for example ALTA2341 (Quest Biotechnology, Inc., Sarasota, U.S.A.)
Microgard (Rhфne Poulenc, Courbevois, France).
DETAILED DESCRIPTION OF THE INVENTION
[0012] The bacteriocins/microorganisms mentioned in the following table are preferably employed.
MicroorganismsBacteriocins
Aeromonas hydrophilasakacin A or P
Lactobacillus sakei
Bacillus cereuslactocin-S, lactostrepcin-5,
pediocin-A, pediocin-AcH, sakacin-A
Bacillus coagulansnisin
Bacillus licheniformis
Bacillus stearothermophilus
Clostridium bifermentans
Lactococcus lactis
Bacillus pumilisthermophillin
Bacillus subtilis, 168, JH642subtilin, lacticin-481, nisin,
thermophillin, subtilosin
Bronchothrix thermospactacurvacin-A, pediocin-AcH,
sakacin-A, sakacin-P
Carnobacterium divergens
Carnobacterium piscicola UI 49,carnocin UI 49, carnobacteriocin A,
LV 17 or LV 61B1 and B2; piscicolin 61
Clostridium botulinumnisin, pediocin-A, reuterin, sakacin-A
Clostridium butyricumnisin, reuterin
Clostridium perfringensnisin, pediocin-A, pediocin-AcH,
pediocin-VTT, reuterin, thermophillin
Clostridium sporogensnisin, pediocin-A
Clostridium tyrobutricumlacticin-481, lactocin-S, pediocinAcH
Enterococcus faecalis
Enterococcus faecalis 226, INIA 4enterocin 226NWC, AS-48
Enterococcus faecalis S-48bacteriocin Bc-48
Enterococcus faecium, BFE 900,enterocin 1146, B, A, Cal, ON- 157,
CTC492, cal 1, NIAI157, A, B, P,P, L50A, L50B
276/1006
L 50, G 16, AA13, T136
Enterococcus spp.enterococcins (I-V)
Escherichia colireuterin, thermophillin
Fusobacterium mortiferum,
(e.g.: "FM1025")
Lactobacillus acidophiluslactocicin
Lactobacillus acidophilus 11088,lactacin F, lacidin, acidolin, acidoOSU 133, 2181, DDS1, LAPT,phillin, acidophilucin A, bacteriocin
1060, M46, N2, TK8912,M46, lactacin B, acidocin 8912,
lactacin B
Lactobacillus amylovorusamylovorin L471
DCE 471
Lactobacillus bavaricus MI401bavaricin A
Lactobacillus bulgaricusbulgarican
Lactobacillus brevislactobacillin
Lactobacillus brevisbrevicin
Lactobacillus casei B80caseicin 80, caseicin LHS
Lactobacillus casei LHS
Lactobacillus curvatus LTH 1174,curvacin A, 13
SB 13
Lactobacillus delbrьckii ssp.bulgarican
bulgaricus
Lactobacillus delbrueckii subsp.lacticin B
lactis JCM 1106, JCM 1107, JCM
1248
Lactobacillus fermentum 466bacteriocin 446, proteid
Lactobacillus gasserigassericin A
Lactobacillus helveticuslactocin 27
Lactobacillus helveticus 1829,helveticin V-1829, helveticin J,
481, LP27lactocin 27
Lactobacillus plantarum, A2, BN,plantaricin A and D, lactolin, plantarC-11, LPCO-10, LPCO-10,icin BN, A, S, 406, -B, SIK-83, 35 d
MI406, NCDO 1193, SIK-83, 35
d, CTC 305,
Lactobacillus reuteri LA6reutericin 6
277/1006
Lactobacillus sakei, Lb 706, L45,sakacin-A, lactocin S, sakacin P,
LTH 673, CTC 494, CTC 372,sakacin K and T
148
Lactococcus lactis subsp.diplococcin, lactostrepcin 5,
cremoris, -202, -9B4, -346,lactococcin A, B and M, Bac I, II, III
-9B4, 4G6, LMG 2130, LMG2081,and IV, lactococcin A, G, lacticin
JW 3
Lactococcus lactis subsp. lactislactostrepcin 1, 2, 3, 4 and DR, lact10, 300, 71, ADRIA 85LO30,icin 481, dricin, bac V, VI and VII
CNRZ 481, DRC1, 6F3
Lactococcus lactis subsp. lactisnisin A
ATCC 11454
Lactococcus lactis subsp. lactisnisin Z
NIZO 22186
Lactococcus lactis subsp. lactisbac VIII
var. diacetyictis 6F7
Lactococcus lactis subsp. lactislactocin D, bacteriocin S50, bac
var. diacetyictis DPC938, S50WM4
and WM4
Leuconostoc carnosum e.g.: Lm1leucococin Lcm1
Leuconostoc dextranicum
Leuconostoc geldium e.g.:leucocin A-UAL 187
UAL 187
Leuconostoc gelidium
Leuconostoc mesenteroides
Leuconostoc mesenteroidesmesenterocin 52, 5, Y105
subsp. mesenteroides FR52,
UL5, Y105
Leuconostoc paramesenteroidesleuconocin S
OX
Listeria innocualacticin-481, lactosin-S, pediocin-A,
pediocin-AcH
Listeria ivanoviipediocin-A, pediocin-AcH, pediocinPAC10
Listeria monocytogenes spp.carnobacteriocin A & B, curvacin-A,
278/1006
enterocin-1146, lactacin-B, lacticin481, leucocin-A, nisin, pediocin-A,
pediocin AcH, pediocin-JD, pediocinPA-1, pediocin-PAC10, pediocinVVT, piscicolin-61, reuterin, sakacinA, sakacin-P
Listeria seeligeripediocin-A
Listeria welchiilacticin-481, pediocin-A
Mycobacterium tuberculosisnisin
Pediococcus acidilactic e.a. H, E,pediocin AcH
F, M
Pediococcus acidilactic JD1-23,pediocin JD, PA-1, SJ-1
PAC 1.0, SJ-1,
Pediococcus pentosaceuspediocin A, N5p
FBB-61, L-7230, N5p
Proteus mirabillisnisin
Pseudomonas aeruginosathermophillin
Pseudomonas fluorescens
Salmonella enteritidisreuterin, thermophillin
Salmonella infantispseudiocin-VVT, reuterin
Salmonella typhimuriumreuterin, thermophillin
Shigella sp.reuterin, thermophillin
Staphylococcus aureusnisin, lacticin-481, pediocin-A,
pediocin-AcH, plantarcin-SIK83,
sakacin-A, thermophillin
Staphylococcus carnosuscurvacin, lacticin-481, lactocin-S
pediocin-AcH
Staphylococcus epidermidisnisin
Staphylococcus simulansnisin
Streptococcus thermophilusthermophillin 13, bacteriocin St10,
Sfi13, St10, STB40, STB78bacteriocin STB40, bacteriocin
STB78
Yersinia enterocoliticathermophlillin
[0013] The bacteriocins are obtained by known processes, for example by simple precipitation using
ammonium sulfate, gel filtration (Sephadex G-50), cation exchange chromatography (CM-cellulose),
279/1006
RP-HPLC, adsorption/desorption centrifugation, vortex flow filtration or other technically suitable
methods (see Parente E. and Ricciardi A., Appl. Microbiol. Biotechnol. 1999, 52, 628-638).
[0014] The product of the invention contains from 90.00 to 99.90% by weight, preferably 95.00 to
99.99% by weight, sorbic acid. Percentages by weight are based in this case on the total weight of
the product.
[0015] The bacteriocin(s) are expediently present in the product of the invention in amounts such that
from 2.5 to 50 mg/kg, preferably 5 to 40 mg/kg, in particular 10 to 20 mg/kg, are present in the
animal feed. Preparations which contain bacteriocins are added in appropriately higher dosage (if,
for example, the preparation contains 2.5% bacteriocin as active substance, then preferably from
400 to 800 mg/kg thereof are employed). If bacteriocin-producing microorganisms or combinations
thereof are employed in the products of the invention, these are preferably present in amounts which
correspond to about 10to 10microorganisms per g of feedstuff. It is also possible to use spray-dried
products for this purpose. The bacteriocin content in the animal feed should in this case likewise be
from 2.5 to 50 mg/kg, preferably 5 to 40 mg/kg, in particular 10 to 20 mg/kg.
[0016] Carriers which can be used both for the sorbic acid and for the bacteriocin or the
microorganisms are organic or inorganic materials. These include, for example, starch and other
polysaccharides such as cellulose. To improve dispersion in mixtures with sorbic acid, it is also
possible for the bacteriocins to be present in the mixtures in salts such as common salt or mineral
salts or else whey powder or other products of milk processing.
[0017] A further possibility is for the bacteriocins or the microorganisms to be provided with
microcapsules/microspheres in order thus to resist unwanted effects of digestive juices. It is possible
in this case for the sorbic acid to be put, separate from the bacteriocins, into the microspheres or
else into one of the outer layers of a microcapsule in such a way that sorbic acid is released earlier
and leads, for example in the stomach, to a marked reduction in pH, but the bacteriocins are not
released until later in the gastrointestinal tract. A mixture of encapsulated bacteriocins and sorbic
acid is also possible. Examples suitable for the encapsulation are gelatin, lecithins, steairates,
alginates, tragacanth, xanthan, carrageenan, cassia gum, gum arabic, maltodextrins, modified
starches, celluloses, mono- and diglycerides of edible fatty acids esterified with organic acids or
unesterified, solid triglycerides with, preferably, saturated fatty acids such as tripalmitin, solid fatty
acids such as palmitic acid or mixtures thereof.
[0018] Employed as carrier and for stabilizing the products are >0 to 10% by weight, preferably 2.5
to 7.5% by weight (based on the product), of carrier materials, alone or in combination.
[0019] The product of the invention is produced by, for example, mechanical mixing of the sorbic
acid and bacteriocins, bacteriocin mixtures, preparations which contain bacteriocins, or live or dead
microorganisms which have produced bacteriocins. If the product of the invention comprises a
280/1006
carrier, it is expedient for the microorganism extracts, which are liquid where appropriate, initially to
be applied to the carrier, expediently in a commercially available tumbler mixer or other conventional
mixer, and then for the sorbic acid and the other solid ingredients to be added.
[0020] Examples of suitable animal feedstuffs are green fodder, silages, dried green fodder, roots,
tubers, fleshy fruits, grains and seeds, brewer's grains, pomace, brewer's yeast, distillation residues,
milling byproducts, byproducts of the production of sugar and starch and oil production and various
food wastes. Feedstuffs of these types may be mixed with certain feedstuff additives (e.g.
antioxidants) or mixtures of various substances (e.g. mineral mixes, vitamin mixes) for improvement.
Specific feedstuffs are also adapted for particular species and their stage of development. This is the
case, for example, in piglet rearing. Prestarter and starter feed are used here. The product of the
invention can be added to the animal feedstuff directly or else mixed with other feedstuff additives or
else be added via premixes to the actual feedstuff. The product can be admixed dry with the feed,
be added before further processing (e.g. extrusion) or be metered in and dispersed in the mixture.
An additional possibility is to add the individual ingredients of the product separately to individual
ingredients of the feedstuff. It is expedient to use for these purposes product concentrations between
0.25 and 7.5% by weight (based on the feed), preferably 0.75 to 4.0% by weight.
[0021] The product can be added as sole additive to the animal feedstuffs, for example for cattle,
poultry, rabbit or sheep rearing, particularly preferably to prestarter and starter feeds for piglets, or
be used mixed with other feed additives for these stock. Feedstuffs having the product of the
invention are moreover suitable as milk replacers for the early weaning of lambs or calves.
[0022] Surprisingly, the products of the invention do not show the disadvantages described above.
On the contrary, the products show good handling properties. In addition, effective acidification of
the feed is achieved. It is moreover possible, surprisingly, for there to be a beneficial effect on the
growth performance of young stock even with relatively small amounts of product.
[0023] The products of the invention are in a solid state of aggregation. The present invention avoids
the problems which otherwise arise with the handling of the liquid acids previously used. The product
of the invention is also able to improve the hygienic status in that unwanted organisms and spoilage
microbes, which may otherwise consume nutrients present, are suppressed.
[0024] It has been found, surprisingly, that a marked improvement in performance in relation to
growth rate and feed conversion can be achieved by adding even small amounts of products of the
invention in piglet rearing. To ensure a significant nutritional activity, it is expedient to add products
of the invention in amounts of from 0.25 to 7.5% by weight, based on the feed, preferably from 0.75
to 4.0% by weight.
[0025] The invention is illustrated below by means of examples.
281/1006
EXAMPLE 1
[0026] 0.0075 to 0.015 kg (corresponding to a concentration of at least 20 mg/kg bacteriocin in the
feed) of a product from Lactococcus lactis subsp. cremoris and Lactobacillus plantarum, which has
been sprayed with whey powder, dried and enriched with bacteriocins, is mixed with 1.0 kg of sorbic
acid in a double cone blender with tumbling movements over a period of about 15 min. The
homogeneous mixture is mixed with 100 kg of piglet feed of the following composition (the following
data in % by weight).
Fish meal4.00
Extracted soybean meal18.50
Barley40.00
Wheat33.00
Vegetable oil1.90
L-Lysine HCl0.2
DL-Methionine0.1
L-Threonine0.1
Mineral feed2.2
EXAMPLE 2
[0027] 0.08 kg of a mixture of nisin (Nisaplin Aplin & Barrett, Dorset, U.K.) with whey proteins and
common salt, which contains 2.5 percent of pure substance (equivalent to about 1*10IU/g or
1*10Reading units/g), is mixed with 0.92 kg of sorbic acid in a double cone mixer with tumbling
movements over a period of about15 min to achieve a uniform mixture. This mixture is mixed with 100
kg of piglet feed of the following composition (the following data are in % by weight).
Extracted soybean meal22.00
Barley40.00
Wheat31.00
Vegetable oil2.90
L-Lysine HCl0.40
DL-Methionine0.10
L-Threonine0.10
Mineral feed3.50
[0028] It was found that a marked improvement in performance in relation to growth rate and feed
conversion is achieved even by addition of these amounts of products of the invention in piglet
rearing.Claims:
282/1006
1. Product comprising sorbic acid and at least one bacteriocin.
2. A product as claimed in claim 1, wherein the product contains from 90.00 to 99.90% by weight
sorbic acid.
3. A product as claimed in claim 1, wherein the concentration of the bacteriocin or bacteriocins is
such that from 2.5 to 50 mg/kg of at least one bacteriocin is present in the animal feed in which the
product is employed.
4. A product as claimed in claim 1, wherein the bacteriocin or a bacteriocin-producing
microorganism is selected from one or more of the following materials:
MicroorganismsBacteriocins
Aeromonas hydrophilasakacin A or P
Lactobacillus sakeii
Bacillus cereuslactocin-S, lactostrepcin-5, pediocinA pediocin-AcH, sakacin-A
Bacillus coagulansnisin
Bacillus licheniformis
Bacillus stearothermophilus
Clostridium bifermentans
Lactococcus lactis
Bacillus pumilisthermophillin
Bacillus subtilis, 168, JH642subtilin, lacticin-481, nisin, thermophillin, subtilosin
Bronchothrix thermospactacurvacin-A, pediocin-AcH,
sakacin-A, sakacin-P
Carnobacterium divergens
Carnobacterium piscicola UI 49,carnocin UI 49, carnobacteriocin A,
LV 17 or LV 61B1 and B2; piscicolin 61
Clostridium botulinumnisin, pediocin-A, reuterin, sakacin-A
Clostridium butyricumnisin, reuterin
Clostridium perfringensnisin, pediocin-A, pediocin-AcH,
pediocin-VTT, reuterin, thermophillin
Clostridium sporogensnisin, pediocin-A
283/1006
Clostridium tyrobutricumlacticin-481, lactocin-S, pediocinAcH
Enterococcus faecalis
Enterococcus faecalis 226, INIA 4enterocin 226NWC, AS-48
Enterococcus faecalis S-48bacteriocin Bc-48
Enterococcus faecium, BFE 900,enterocin 1146, B, A, Cal, ON- 157,
CTC492, cal 1, NIAI157, A, B, P,P, L50A, L50B
L 50, G 16, AA13, T136
Enterococcus spp.enterococcins (I-V)
Escherichia colireuterin, thermophillin
Fusobacterium mortiferum,
(e.g.: "FM 1025")
Lactobacillus acidophiluslactocicin
Lactobacillus acidophilus 11088,lactacin F, lacidin, acidolin,
OSU 133, 2181, DDS1, LAPT,acidophillin, acidophilucin A,
1060, M46, N2, TK8912bacteriocin M46, lactacin B,
acidocin 8912, lactacin B
Lactobacillus amylovorus DCEamylovorin L471
471
Lactobacillus bavaricus MI401bavaricin A
Lactobacillus bulgaricusbulgarican
Lactobacillus brevislactobacillin
Lactobacillus brevisbrevicin
Lactobacillus casei B80caseicin 80, caseicin LHS
Lactobacillus casei LHS
Lactobacillus curvatus LTH 1174,curvacin A, 13
SB 13
Lactobacillus delbrьckii ssp.bulgarican
bulgaricus
Lactobacillus delbrueckii subsp.lacticin B
lactis JCM 1106, JCM 1107, JCM
1248
Lactobacillus fermentum 466bacteriocin 446, proteid
Lactobacillus gasserigassericin A
Lactobacillus helveticuslactocin 27
284/1006
Lactobacillus helveticus 1829,helveticin V-1829, helveticin J,
481, LP27lactocin 27
Lactobacillus plantarum, A2, BN,plantaricin A and D, lactolin,
C-11, LPCO-10, LPCO-10,plantaricin BN, A, S, 406,
MI406, NCDO 1193, SIK-83, 35-B, SIK-83, 35 d
d, CTC 305,
Lactobacillus reuteri LA6reutericin 6
Lactobacillus sakei Lb 706, L45,sakacin-A, lactocin S, sakacin P,
LTH 673, CTC 494, CTC 372,sakacin K and T
148
Lactococcus lactis subsp.diplococcin, lactostrepcin 5,
cremoris, -202, -9B4, -346,lactococcin A, B and M, Bac I, II, III
-9B4, 4G6, LMG 2130, LMG2081,and IV, lactococcin A, G, lacticin
JW 3
Lactococcus lactis subsp. lactislactostrepcin 1, 2, 3, 4 and DR,
10, 300, 71, ADRIA 85LO30,lacticin 481, dricin, Bac V, VI and
CNRZ 481, DRC1, 6F3VII
Lactococcus lactis subsp. lactisnisin A
ATCC 11454
Lactococcus lactis subsp. lactisnisin Z
NIZO 22186
Lactococcus lactis subsp. lactisbac VIII
var. diacetylctis 6F7
Lactococcus lactis subsp. lactislactocin D, bacteriocin S50, bac
var. diacetylctis DPC938, S50WM4
and WM4
Leuconostoc carnosum e.g.: Lm1leucococin Lcm1
Leuconostoc dextranicum
Leuconostoc geldium e.g.: UALleucocin A-UAL 187
187
Leuconostoc gelidium
Leuconostoc mesenteroides
Leuconostoc mesenteroidesmesenterocin 52, 5, Y105
subsp. mesenteroides FR52,
UL5, Y105
285/1006
Leuconostoc paramesenteroidesleuconocin S
OX
Listeria innocualacticin-481, lactosin-S, pediocin-A,
pediocin-AcH
Listeria ivanoviipediocin-A, pediocin-AcH,
pediocin-PAC10
Listeria monocytogenes spp.carnobacteriocin A & B, curvacin-A,
enterocin-1146, lactacin-B,
lacticin-481, leucocin-A, nisin,
pediocin-A, pediocin AcH, pediocinJD, pediocin-PA-1, pediocin-PAC10,
pediocin-VVT, piscicolin-61,
reuterin, sakacin-A, sakacin-P
Listeria seeligeripediocin-A
Listeria welchiilacticin-481, pediocin-A
Mycobacterium tuberculosisnisin
Pediococcus acidilactic e.a. H, E,pediocin AcH
F, M
Pediococcus acidilactic JD1-23,pediocin JD, PA-1, SJ-1
PAC 1.0, SJ-1,
Pediococcus pentosaceuspediocin A, N5p
FBB-61, L-7230, N5p
Proteus mirabillisnisin
Pseudomonas aeruginosathermophillin
Pseudomonas fluorescens
Salmonella enteritidisreuterin, thermophillin
Salmonella infantispseudiocin-VVT, reuterin
Salmonella typhimuriumreuterin thermophillin
Shigella sp.reuterin, thermophillin
Staphylococcus aureusnisin, lacticin-481, pediocin-A,
pediocin-AcH, plantarcin-SIK83,
sakacin-A, thermophillin
Staphylococcus carnosuscurvacin, lacticin-481, lactocin-S,
pediocin-AcH
Staphylococcus epidermidisnisin
286/1006
Staphylococcus simulansnisin
Streptococcus thermophilusthermophillin 13, bacteriocin St10,
Sfi13, St10, STB40, STB78bacteriocin STB40, bacteriocin
STB78
Yersinia enterocoliticathermophillin
5. A feedstuff comprising a product as claimed in claim 1.
6. An addition to feedstuffs comprising a product as claimed in claim 1.
7. A feedstuff as claimed in claim 5, comprising from 0.25 to 7.5% by weight, based on the weight of
the feedstuff, of the product.
8. The method of making animal feeds or feedstuffs, comprising incorporating a product as claimed
in claim 1 into animal feeds or feedstuffs.
9. The method as claimed in claim 8 in pig rearing.
10. The method as claimed in claim 8 in cattle rearing.
11. The method as claimed in claim 8 in lamb rearing.
12. The method as claimed in claim 8 in poultry rearing.
13. The method as claimed in claim 8 in rabbit rearing.
287/1006
27. JP2003339377 - 02.12.2003
NEW BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP2003339377
Inventor(s):
KAWAMOTO SHINICHI (--); SHIMA JUN (--); SUZUKI CHISE (--); OMOMO
SADAHIRO (--); HORIKOSHI NAOKO (--); SHITO JUNKO (--); TAKESHITA KAZUKO (--); SAMEJIMA
TAKASHI (--)
Applicant(s):
PRIMA MEAT PACKERS LTD (--)
IP Class 4 Digits: C12N; A23B; A23L; C07K; C12P
IP Class:
C12N15/09; A23B4/22; A23L3/3463; C07K14/315; C12N1/20; C12P21/02
Application Number:
JP20020149621 (20020523)
Family: JP2003339377
Abstract:
PROBLEM TO BE SOLVED: TO PROVIDE A NEW BACTERIOCIN DERIVED FROM LACTOBACILLUS,
BEING AN ANTIBACTERIAL SUBSTANCE READILY DECOMPOSED IN THE BODY, HAVING A HIGH
INHIBITORY SELECTIVITY TO MICROORGANISMS AND PROVIDING HIGH SAFETY, A BACTERIAL
STRAIN PRODUCING THE SAME, A GENE THEREOF, A RELATING GENE THEREOF AND USE OF
THE BACTERIOCIN AND THE BACTERIAL STRAIN TO FOODS.
SOLUTION: A NEW LACTOBACILLUS, ENTEROCOCCUS MUNDTII 7393 STRAIN IS SEPARATED
USING AN ANTIBACTERIAL ACTIVITY TO ENTEROCOCCUS FAECIUM AS AN INDEX AND A NEW
BACTERIOCIN, MUNDTICIN KS IS SEPARATED FROM THE BACTERIAL STRAIN. THE GENE OF
THE NEW BACTERIOCIN, THE RESISTANCE GENE THEREOF AND THE EXTRACELLULAR
EXPORTER GENE THEREOF ARE CLONED FROM THE GENOME GENE OF THE BACTERIAL
STRAIN. THE NEW BACTERIOCIN, THE MUNDTICIN KS AND THE ENTEROCOCCUS MUNDTII 7393
STRAIN ARE USED FOR PRODUCTION OF CONSERVABLE FOODS.
288/1006
28. JP2004313171 - 21.10.2004
NOVEL LACTIC ACID BACTERIA AND BACTERIOCINS PRODUCED THEREFROM, AND METHOD
FOR PROCESSING FISH AND LEGUME FOODSTUFFS USING THE SAME AND THE PRODUCTS
OBTAINED THEREBY
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP2004313171
Inventor(s):
JIANG SHANN-TZONG (TW); YIN LI-JUNG (TW)
IP Class 4 Digits: A23L
IP Class:
A23L1/325
E Class: A23B4/22; A23B7/155; A23L1/211M; A23L1/314D; A23L1/325D; A23L3/3463
Application Number:
US20030652387 (20030829)
Priority Number: TW20030109120 (20030418)
Family: JP2004313171
Abstract:
THE PRESENT INVENTION RELATES TO NOVEL STRAINS PEDIOCOCCUS PENTOSACEUS YJL
WITH THE ACCESSION NUMBERS FERM-BP 8450 (BCRC 910210) AND PEDIOCOCCUS
PENTOSACEUS YJS WITH THE ACCESSION NUMBERS FERM-BP 8449 (BCRC 910211). THE
PRESENT INVENTION ALSO PROVIDES A METHOD FOR PROCESSING FISH FOODSTUFFS
COMPRISING USING LACTIC ACID BACTERIA AND THE FISH FOODSTUFFS OBTAINED THEREBY.
THE PRESENT INVENTION ALSO PROVIDES A METHOD FOR PROCESSING LEGUME
FOODSTUFFS COMPRISING USING LACTIC ACID BACTERIA AND THE LEGUME FOODSTUFFS
OBTAINED THEREBY. THE PRESENT INVENTION FURTHER PROVIDES BACTERIOCINS
PRODUCED FROM THE STRAINS. THE PRESENT METHODS ARE CAPABLE OF PRODUCING
PROCESSED PRODUCTS WITH REDUCED GROWTH OF UNWANTED MICROORGANISMS,
IMPROVED FLAVOR AND ENHANCED ECONOMIC VALUE. THE PENTOCINS ARE EFFECTIVE ON
289/1006
THE INHIBITION OF UNWANTED MICROORGANISMS THEREBY INSURING THE FOODSTUFF A
GOOD QUALITY DURING STORAGE.Description:
[0001] Novel lactic acid bacteria and bacteriocins produced therefrom, and method for
processing fish and legume foodstuffs using the same and the products obtained thereby
FIELD OF THE INVENTION
[0002] The present invention provides two novel strains of lactic acid bacteria (LAB) and
bacteriocins produced therefrom, a method for processing fish foodstuffs comprising using LAB and
the products obtained thereby, and a method for processing legume foodstuffs comprising using
LAB and the products obtained thereby. Specifically, the present invention provides novel strains of
LAB isolated from meat, which find their use in processing fermented foodstuffs wherein fish or
legume is used as raw material. Also, the present invention provides processed products with
reduced growth of unwanted microorganisms, improved flavor and enhanced economic value. The
present invention further provides bacteriocins produced from the novel LAB. The bacteriocins are
effective on the inhibition of unwanted microorganisms thereby insuring the foodstuffs a good quality
during storage.
BACKGROUND OF THE INVENTION
[0003] Traditional foodstuffs gradually cannot meet the needs of consumers. There are lots of
health foods and delicate foods in the markets; it is accordingly known that the development in food
industry focuses on the manufacture of diverse, safe and functional foodstuffs.
[0004] LAB are a group of microorganisms capable of producing lactic acid from the fermentation
of fermentable saccharides. In fermented products such as fermented vegetables, cereals, milks,
meats etc., LAB are used as fermentation starter for enhancing nutrition of foodstuffs (Acton et al.,
1977), for inhibiting the growth of pathogens in the intestines (Bacus and Brown, 1981), for
increasing the availability of lactose (Siddons and Coates, 1985), for preventing cancer (Oda et al.,
1983) and for lowing the cholesterols level. In fermentation process, through the action of LAB
saccharides undergo glycolysis reaction to give products a unique flavor and on the other side,
produce organic acids such as lactic acid, acetic acid etc. to in turn lower the pH value of products
thereby inhibiting the growth of microorganisms so as to prolong the shelf life of the products. Further,
some LAB can produce a variety of antimicrobial substances such as hydrogen peroxide, diacetyl
compounds, bacteriocins etc. to inhibit the growth of saprogenic bacteria or pathogenic bacteria
(Gibbs, 1987; Klaenhammer, 1988; Daeschel, 1989; Schillinger and Lucke, 1989).
[0005] LAB can produce substances which are able to inhibit the growth of pathogenic bacteria
and which are mainly bacteriocins, diacetyl compounds, and hydrogen peroxide and secondary
290/1006
metabolic products. Bacteriocins are macromolecules containing proteins and having the activity on
inhibiting the growth of microorganisms (Scbillinger and Holzapfel, 1996; Roller and Lusengo, 1997).
LAB capable of producing bacteriocins include Lactobacillus fermentum (Deklerk and Smit, 1967)Lactobacillus plantarum (Sedewitz et al., 1983)- Lactobacills helveticus (Joerger and Klaenhammer,
1986)- Lactobacillu acidophilus (Muriana and Klaenhammer, 1987)- Lactobacillu plantarum (West
and Warner, 1988) and Pediococcus pentosaceus (Daeschel and Klaenhammer, 1985).
[0006] Bacteriocins produced by Pediococcus acidilactici are effective on inhibiting the growth of
Listeria monocytogenes in fresh meats, fermented sausages, fermented cabbages, minced beef and
cheeses (Nielsen et al., 1990; Choi and Beuchat, 1994; Motlagh et al., 1992; Parente et al., 1996;
Vignolo et al., 1996; Cutter and Siragusa, 1996) so as to prolong the shelf life thereof in refrigeration.
Bacteriocins produced by Lactobacillus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200
and Pediococcus pentosaceus ATCC 43201, when added into refrigerated processed foodstuffs
containing 3-4% sodium chloride, can inhibit the germination and growth of Clostridium botulinum
spores (Okereke and Montville, 1991). Bacteriocins produced by Lactococcus lactis and
Pediococcus pentosaceous are effective on inhibiting the growth of Gram-positive pathogenic
bacteria such as Bacillus cereus, Clostridium perfringenes, Staphylococcus aureus and Listeria
monocytogenes as well as Gram-negative pathogenic bacteria such as Aeromonas hydrophila AH2,
Escherichia coli O157:H7, Vibrio cholerae 851 and V. parahaemolyticus A865957 (Spelhaug and
Harlander, 1989; Helander et al., 1997). Nisin produced by Lactococcus lactis subsp. lactis has
been categorized as GRAS (generally recognized as safe) by FDA in 1992 and can be used in
refrigerated cheeses for the inhibition of the germination and growth of Clostridium botulinum spores.
[0007] LAB can also produce 2,3-butanedione and hydrogen peroxide (H2O2), besides
bacteriocins. 2,3-Butanedione is a final product produced by LAB from the metabolic intermediate
pyruvate (Kandler, 1983; Monnet et al., 1994). Jay (1982) reports that 2,3-butanedione can inhibit the
growth of Gram-positive bacteria at 200 [mu]g/mL. Though 2,3-butanedione is also on the FDA's
GRAS list, it has a strong flavor and high volatility and thus should be used in a limited amount.
Hydrogen peroxide is produced during the growth of LAB through the action of pyruvate oxidase, Llactase and NADH oxidase respectively on pyruvate, lactose and NADH with oxygen (O2) (Kandler,
1983; Sedewitz et al., 1983) so as to inhibit the growth of harmful microorganisms. Hydrogen
peroxide can form well bacteriostatic substances with other materials, for example, an intermediate
oxidized product formed from the action of lactoperoxidase on thiocyanate is capable of inhibiting
the growth of microorganisms, thereby prolonging the shelf life of foodstuffs (Harnulv et al., 1982).
The aforesaid pathway is called "lactoperoxidase antibacterial system".
[0008] Bacteriocins produced by the genus Pediococcus include Pediocin AcH produced by Ped.
acidilactici H, Pediocin PA-1 produced by Ped. acidilacthci PA1.0, and Pediocin A produced by Ped.
291/1006
pentosaceus FBB61. Further, bacteriocins produced by Ped. cerevesiae FBB63 and Ped. acidilactici
PC are still not named.
[0009] Bhunia et al. (1988) isolated Ped. acidilactici strain H from fermented sausages. The strain
is capable of producing Pediocin AcH with a molecular weight of about 2,700 Da (SDS-PAGE). As
proved by experiments, the bacteriocin is effective on inhibiting the growth of the microorganisms
including Lactobacilli, Leuconostocs, Staphylococcus aureus, Clostridium perfringens,
Pseudomonas putida, Listeria monocytogenes etc. The bacteriocin is sensitive to proteolytic
enzymes and is stable to heat. Since Pediocin AcH can act on cell membrane to cause the loss of
potassium ion etc. from the cell thereby causing the degradation of the cell (Bhunia et al., 1991).
SUMMARY OF THE INVENTION
[0010] In view of the teachings mentioned above, the inventors of the present invention has
conducted elaborated research and found that novel strains Pediococcus pentosaceus YJL and
Pediococcus pentosaceus YJS are effective on improving the processibility of legumes and fishes
such as mackerel. Further, during fermentation the effect of the products produced by LAB on the
quality (texture, flavor, appearance etc.) of fish meat and legumes and the effect of the proteinases
produced by LAB on the texture of fish meat and legumes are studied so as to broaden the
processing field of fishes and legumes and to enhance the applicability thereof. Further, the
fermentation of fish meat or legumes by use of LAB under various conditions lead to products with
various textures, colors, tastes and flavors because of the difference in the type of the substances
produced and in the extent to which the proteinases produced act. As a result, the present invention
provides novel technique in food processing through controlling fermenting conditions such that
various novel products can be produced.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the differentiation of pediococci from other Gram-positive bacteria.
[0012] FIG. 2 shows the SDS-PAGE (8-17.5% gradient polyacrylamide) of purified Pentocin YJL
and Pentocin YJS.
[0013] FIG. 3 shows the photo of fermented fish cheeses prepared in Example 3.
[0014] FIG. 4 shows the photo of fish yogurt prepared in Example 3.
[0015] FIG. 5 shows the photo of soybean pudding prepared in Example 4.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Pediococcus pentosaceus YJL and Pediococcus pentosaceus YJS respectively were
deposited in the International Patent Organism Depositary (IPOD), Japan according to the Budapest
292/1006
Treaty on Aug. 8, 2003 with the accession numbers FERM-BP 8450 and FERM-BP 8449. Also, the
two strains were deposited in Bioresources Collection and Research Center (BCRC), Food Industry
Research and Development Institute, Hsinchu, Taiwan on Jan. 9, 2003 with the accession numbers
BCRC 910210 and BCRC 910211.
[0017] As described above, the present invention therefore provides the following aspects.
[0018] 1. Pediococcus pentosaceus YJL with the accession numbers FERM-BP 8450 (BCRC
910210).
[0019] 2. Pediococcus pentosaceus YJS with the accession numbers FERM-BP 8449 (BCRC
910211).
[0020] 3. A method for processing fish meat wherein LAB of 1. or 2. or other LAB or mixed strains
thereof is (are) used in fermenting the fish meat, comprising the steps of
homogenizing the fish meat with the addition of sodium chloride in an amount of 0.3-2.0 wt % (based
on the weight of the fish meat) and water in an amount 0.5-3 times the weight of the fish meat to
obtain a homogenous substrate;
sterilizing the homogenous substrate at a temperature of from 100 to 115[deg.] C. for 15 to 30
minutes and then cooling the same to a temperature of from 25 to 40[deg.] C.;
adjusting the water content of the substrate by diluting it with water at a dilution ratio of 0-5 times the
weight of the substrate;
adding 1.0-6.0 wt % of a saccharide to the substrate and then inoculating the same with the LAB;
fermenting the substrate inoculated with the LAB at a temperature of from 25-40[deg.] C. for 6-30
hours;
optionally adding a seasoning and a spice; and
optionally packaging the resulting product.
[0028] 4. The method of 3. wherein the other LAB is selected from the group consisting of
Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, and
Lactobacillus helveticus CCRC 14092.
[0029] 5. The method of 3. wherein the fish meat is at least one selected from the group consisting
of red fish meat, white fish meat, frozen surimi and mixture thereof.
[0030] 6. The method of 3. wherein the saccharide is at least one selected from the group
consisting of sucrose, glucose and beet sugar.
[0031] 7. The method of 3. wherein the substrate is a non-diluted or 5-fold-diluted surimi.
[0032] 8. The method of 3. wherein the seasoning is at least one selected from the group
consisting of fresh fruits, processed fruits, sesame and peanuts.
293/1006
[0033] 9. The method of 3. wherein the spice is at least one selected from the group consisting of
ginger, garlic, mirin, wine, and prickly ash.
[0034] 10. The method of 3. wherein the substrate after being fermented has a pH value of from
3.8 to 5.5.
[0035] 11. A processed fish foodstuff obtained from fish meat fermented with LAB of 1. or 2. or
other LAB or mixed strains thereof.
[0036] 12. The processed fish foodstuff of 11. wherein the other LAB is selected from the group
consisting of Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315,
and Lactobacillus helveticus CCRC 14092.
[0037] 13. A processed fish foodstuff obtained by the method of 3.
[0038] 14. A processed fish foodstuff obtained by the method of 3. and then sterilized at a
temperature of from 95-115[deg.] C., shaped, and partly dried to a cheese-like product.
[0039] 15. The processed fish foodstuff of 11., 12., 13., or 14. wherein the fish meat is at least one
selected from the group consisting of red fish meat, white fish meat, frozen surimi and mixture thereof.
[0040] 16. A method for processing legumes wherein LAB of 1. or 2. or other LAB or mixed strains
thereof is (are) used in fermenting the legumes, comprising the steps of
homogenizing the legumes with water and filtering the resulting homogenate;
sterilizing the filtrate thus obtained at a temperature of from 100 to 115[deg.] C. for 15 to 30 minutes
to obtain a substrate and then cooling the substrate to a temperature of from 25 to 40[deg.] C.;
adjusting the water content of the substrate by diluting it with water;
adding 1.0-6.0 wt % of a saccharide to the substrate and then inoculating the substrate with the LAB;
fermenting the substrate at a temperature of from 25-40[deg.] C. for 6-30 hours;
optionally adding a seasoning; and
optionally packaging the resulting product.
[0048] 17. The method of 16. wherein the other LAB is selected from the group consisting of
Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, and
Lactobacillus helveticus CCRC 14092.
[0049] 18. The method of 16. wherein the legumes are at least one selected from the group
consisting of soybean, black soybean and mixture thereof.
[0050] 19. The method of 16. wherein the saccharide is at least one selected from the group
consisting of sucrose, glucose and beet sugar.
[0051] 20. The method of 16. wherein the water content of the substrate is in a range of from 50%
to 98%.
294/1006
[0052] 21. The method of 16. wherein the seasoning is at least one selected from the group
consisting of fresh fruits, processed fruits, sesame and peanuts.
[0053] 22. The method of 16. wherein the substrate after fermenting has a pH value of from 4.5 to
6.0.
[0054] 23. A processed legume foodstuff obtained from legumes fermented with LAB of 1. or 2. or
other LAB or mixed strains thereof.
[0055] 24. The processed legumes foodstuff of 23. wherein the other LAB is selected from the
group consisting of Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC
12315, and Lactobacillus helveticus CCRC 14092.
[0056] 25. A processed legumes foodstuff obtained by the method of 16.
[0057] 26. The processed legumes foodstuff of 23., 24., or 25. wherein the legumes are at least
one selected from the group consisting of soybean, black soybean and mixture thereof.
[0058] 27. A processed legumes foodstuff obtained by the method of 16. and then sterilized at a
temperature of from 95-115[deg.] C., shaped, and partly dried to a cheese-like product.
[0059] 28. A bacteriocin produced by the LAB of 1.
[0060] 29. The bacteriocin of 28. which is an antibacterial substance having a molecular weigh of
from 20 to 30 kDa.
[0061] 30. A bacteriocin produced by the LAB of 2.
[0062] 31. The bacteriocin of 30. which is an antibacterial substance having a molecular weigh of
from 20 to 30 kDa.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention is detailedly described as follows. The present invention provides two
novel strains of lactic acid bacteria and bacteriocins obtained therefrom, a method for processing
fish foodstuffs comprising using LAB and the products obtained thereby, and a method for
processing legume foodstuffs comprising using LAB and the products obtained thereby.
[0064] Among the LAB used in the present invention, two novel strains Pediococcus pentosaceus
YJL and YJS are isolated from meat. The genus Pediococcus is a group of Gram-negative bacteria
having no motility and no productivity of spores and being catalase negative.
[0065] The present invention provides a method for processing fish meat wherein Pediococcus
pentosaceus YJL or YJS or other LAB such as Lactobacillus plantarum CCRC10069, Lactococcus
lactis subsp. lactis CCRC 12315, and Lactobacillus helveticus CCRC 14092, or mixed strains thereof
is (are) used in fermenting the fish meat such as red fish meat, white fish meat, frozen surimi and
mixture thereof, the method comprising the steps of
295/1006
homogenizing the fish meat with the addition of sodium chloride in an amount of 0.3-2.0 wt % (based
on the weight of the fish meat) and water in an amount 0.5-3 times the weight of the fish meat to
obtain a homogenous substrate;
sterilizing the resulting substrate at a temperature of from 100 to 115[deg.] C. for 15 to 30 minutes
and then cooling the same to a temperature of from 25 to 40[deg.] C.;
adjusting the water content of the substrate by diluting with water in a dilution ratio of 0-5 times;
adding 1.0-6.0 wt % of a saccharide such as sucrose, glucose and beet sugar to the substrate and
then inoculating the substrate with the LAB;
fermenting the substrate at a temperature of from 25-40[deg.] C. for 6-30 hours to a final pH value of
from 3.8-5.0;
optionally adding a seasoning, such as fresh fruits, processed fruits, sesame and peanuts, and a
spice, such as ginger, garlic, mirin, wine, and prickly ash; and
optionally packaging the resulting product.
[0073] The present invention further provides a processed fish foodstuff obtained from fish meat
such as red fish meat, white fish meat, frozen surimi and mixture thereof, fermented with
Pediococcus pentosaceus YJL or YJS or other LAB such as Lactobacillus plantarum CCRC10069,
Lactococcus lactis subsp. lactis CCRC 12315, and Lactobacillus helveticus CCRC 14092, or mixed
strains thereof.
[0074] The present invention further provides a processed fish foodstuff obtained from fish meat
such as red fish meat, white fish meat, frozen surimi and mixture thereof, fermented with
Pediococcus pentosaceus YJL or YJS or other LAB such as Lactobacillus plantarum CCRC10069,
Lactococcus lactis subsp. lactis CCRC 12315, and Lactobacillus helveticus CCRC 14092, or mixed
strains thereof and then sterilized, shaped, and partly dried to a cheese-like product.
[0075] The present invention further provides a method for processing legumes wherein
Pediococcus pentosaceus YJL or YJS or other LAB such as Lactobacillus plantarum CCRC10069,
Lactococcus lactis subsp. lactis CCRC 12315, and Lactobacillus helveticus CCRC 14092, or mixed
strains thereof is (are) used in fermenting the legumes, comprising the steps of
homogenizing the legumes such as soybean and black soybean, previously soaked in water, with
the addition of water and filtering the resulting homogenate;
sterilizing the filtrate thus obtained at a temperature of from 100 to 115[deg.] C. for 15 to 30 minutes
to obtain a substrate and then cooling the substrate to a temperature of from 25 to 40[deg.] C.;
adjusting the water content of the substrate by diluting it with water to, for example, 50% to 98%;
adding 1.0-6.0 wt % of a saccharide such as sucrose, glucose and beet sugar to the substrate and
then inoculating the substrate with the LAB;
296/1006
fermenting at a temperature of from 25-40[deg.] C. for 6-30 hours;
optionally adding a seasoning, such as fresh fruits, processed fruits, sesame and peanuts; and
optionally packaging the resulting product.
[0082] The present invention further provides a processed legume foodstuff obtained from
legumes such as soybean and black soybean, and mixture thereof, fermented with Pediococcus
pentosaceus YJL or YJS or other LAB such as Lactobacillus plantarum CCRC10069, Lactococcus
lactis subsp. lactis CCRC 12315, and Lactobacillus helveticus CCRC 14092, or mixed strains thereof.
[0083] The present invention further provides a processed legume foodstuff obtained from
legumes such as soybean and black soybean, and mixture thereof, fermented with Pediococcus
pentosaceus YJL or YJS or other LAB such as Lactobacillus plantarum CCRC10069, Lactococcus
lactis subsp. lactis CCRC 12315, and Lactobacillus helveticus CCRC 14092, or mixed strains thereof
and then sterilized, shaped, and partly dried to a cheese-like product.
[0084] The present invention further provides a bacteriocin produced from Pediococcus
pentosaceus YJL, which is an antibacterial substance having a molecular weigh of from 20 to 30 kDa.
[0085] The present invention further provides a bacteriocin produced from Pediococcus
pentosaceus YJS, which is an antibacterial substance having a molecular weigh of from 20 to 30 kDa.
Preferred Embodiments
[0086] The present invention is further illustrated according to the preferred embodiments
described below, however, the present invention is not limited to the details thereof.
[0087] Apparatus
Cryogenic shaking incubator: Orbital shaking incubator (HOTECH 718, Hotech Instruments Co.,
Taiwan)
High speed cryogenic centrifuge: Automatic high speed cryogenic centrifuge (SCR 20B, Hitachi,
Japan)
Color measurement system: Model TC-1800MK-II, Tokyo Denshoku Co., Japan
pH meter: pH Meter (HM-30S, TOA Electronic Co., Japan)
Constant-temperature-humidity incubator: TC-120HD, Tungtec instruments C., LTD.
Grinding and emulsifying machine: UM-12, Stephan, Germany
Lyophilizer: Model FD-20-84, Fts ststems, INC., U.S.A.
Amino acid analyzer: Amino Acid Analyzer (Hitachi L-8500, Japan)
Rotavapor: Rotavapor (Bьtchi RE111, Bьchi, Switzerland)
Mini electrophoresis: Electrophoresis Cell (Mini-PROTEAN II, Bio-Rad, U. S. A.)
Power supply: Power Supply (Model 200/2.0, Bio-Rad, U.S. A.)
297/1006
Homogenizer: Waring Blender (subjoined with a baffle, Japan)
Refrigerator: -30[deg.] C. and -80[deg.] C., Bio-Freezer (Model 8442, Form a Scientific, U.S.A.).
Colorimeter: Hitachi U-2001, Hitachi, Japan.
EXAMPLE 1
Isolation and Identification of Pediococcus pentosaceus YJL and Pediococcus pentosaceus YJS
Isolation
[0104] Ten different cuts of raw pork meat were purchased from a local supermarket. Samples, 50
g, were weighed aseptically into sterile stomacher bags, sealed, and placed at 5[deg.] C. for up to 3
weeks. At weekly intervals, a sample was added to sterile 0.1% peptone to obtain a 1:10 dilution and
placed in stomacher for 2 min. Serial dilutions were made in 0.1% peptone, spread plated onto MRS
agar in quadruplicate, and incubated anaerobically at 37[deg.] C. for 48 to 72 h until growth was
evident. Anaerobic incubation (GasPak; BBL) was used to rule out any inhibition due to hydrogen
peroxide production. Three plates from the two dilutions having 30 to 300 CFU were overlaid with
approximately 8 mL of brain heart infusion (BHI; Difco) which contained 1% agar. The overlay agar
was seeded with L. monocytogenes CCRC 14845 at a level of 10 to 10 organisms per milliliter. A
fourth plate from these dilutions was saved as a master control plate (no indicator overlay) for use in
future replicate plating. The plates with the overlay were incubated anaerobically overnight at
37[deg.] C. Replica plates of those with inhibition zones were made from the master control plate
onto Trypticase soy agar (without glucose) with a 2.0% yeast extract supplement (TSAYE). TSAYE
plates were used to eliminate acid production due to glucose present in MRS. The indicator overlay
was repeated. Colonies revealed inhibition zones were picked from master control plate with no
indicator overlay into MRS broth incubated at 37[deg.] C.
Identification
1. Differentiation of Pediococci From Other Gram-Positive Bacteria:
[0107] Bacteriocin-producing meat isolates (isolates I and II) were incubated in MRSA broths for
examining their physiological properties according to the protocol for differentiating Lactobacilli
suggested by Schillinger, U.; Lucke, F. K. Identification of Lactobacillili from meat and meat products.
Food Microbiol. 1987, 4, 199-208. The results were shown in Table 1.
TABLE 1
Physiological properties of P. pentosaceus YJL and
P. pentosaceus YJS
P. pentosaceus YJLP. pentosaceus YJS
Determinations(isolate I)(isolate II)
Gram stain++
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Catalase test - Motility-Cellular morphologycoccicocci
Growth
45[deg.] C. (2 days) + +
40[deg.] C. (2 days)++
35[deg.] C. (2 days)++
30[deg.] C. (2 days)++
25[deg.] C. (3 days)++
20[deg.] C. (3 days)++
15[deg.] C. (7 days)++
4[deg.] C. (7 days)+Growth at initial pH
pH 4.0 (3 days)++
pH 5.0 (3 days)++
pH 6.0 (3 days)++
pH 7.0 (3 days)++
Growth at NaCl
concentration %
0.0 (3 days)++
2.5 (3 days)++
4.0 (3 days)++
5.0 (3 days)++
10.0 (3 days)-+
20.0 (3 days)-Final pH (3 days)3.993.91
Both strains were incubated in MRS broth.
"-" not grow or negative response
"+" grow or positive response
incubation time
[0108] Based on the results in Table 1, the isolates I and II were identified as Pediococcus
according to FIG. 1.
[0109] Further, from the results in Table 1, in particular, growth temperature, growth pH and
resistance to NaCl, it was known that both isolates I and II are almost similar, except that the isolate I
can grow at lower temperature (down to 4[deg.] C. for 7 days) and the isolate II show resistance to
299/1006
NaCl (10% NaCl for 3 days). Based on these results and the schemes for identifying species
developed by Schillinger and Lьcke (Food Microbiol. 1987, 4, 199-208), the isolates I and II are
identified as Pediococcus pentosaceus.
[0110] 2. API 50CHL System:
[0111] According to the identification API 50CHL system, the results were obtained as in Table 2.
These two isolates are highly similar to P. pentosaceus CCRC 14024 (purchased from Food Industry
Research and Development Institute, Hsinchu, Taiwan) except in the utilization of D-xylose, salicine
and [beta]-gentiobiose.
TABLE 2
Identification of P. pentosaceus YJL and P. pentosaceus YJS
with API 50CHL system
P. pentosaceusP. pentosaceus YJLP. pentosaceus
CarbohydrateCCRC 14024(isolate I)YJS (isolate II)
2-ceto-gluconate--5-ceto-gluconate--Adonitol--Amidon--Amygdaline+++
Arbutine+++
Cellobiose+++
D-Arabinose--D-Arabitol--D-Fructose+++
D-Fucose--D-Glucose+++
D-Lyxose--D-Mannose+++
D-Raffubose+++
D-Tagatose+++
D-Turanose--Dulcitol--D-Xylose--+
Erythritol--Esculin+++
Galactose+++
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Gluconate--Glycerol--Glycogen--Inuline--L-Arabinose+++
Lactose+++
L-Arabitol--L-Fucose--L-Sorbose--L-Xylose--Maltose+++
Mannitol--Melezitose--Melibiose+++
N-Acetyl+++
glucosamine
Rhamnose+++
Ribose+++
Saccharose+++
Salicine-++
Sorbitol--Trehalose+++
Xylitol--[alpha]-Methyl---D-glycoside
[alpha]-Methyl---D-mannoside
[beta]-Gentiobiose-++
[beta]-Methyl---xyloside
P. pentosaceus99.899.699.3
ID (%)
all results were obtained from anaerobically incubation at 37[deg.] C. for 48 h
"+": positive response
"-": negative response
301/1006
[0112] The results of P. pentosaceus CCRC 14024 showed an identification rate of 99.8% since for
[beta]-gentiobiose the negative result is opposite to the systemic value thereby caused an error.
[0113] The results of the Isolate I (Pediococcus pentosaceus YJL) showed an identification rate of
99.6% since for salicin and [beta]-gentiobiose the negative results are opposite to the systemic
values thereby causing an error.
[0114] The results of the Isolate II (Pediococcus pentosaceus YJS) showed an identification rate of
99.3% since for salicin and [beta]-gentiobiose the negative results are opposite to the systemic
values thereby causing an error.
[0115] Based on the conventional identification (item 1.) and API 50CHL system (item 2.), the
isolates I and II are identified as Pediococcus pentosaceus and named, respectively, Pediococcus
pentosaceus YJL and Pediococcus pentosaceus YJS.
[0116] 3. Biochemical Test:
[0117] Pediococcus pentosaceus YJL and Pediococcus pentosaceus YJS both have hydrolysis
ability on arginine and no utilization ability on urea, tetrazolium red and pyruvic acid. The morphology
of the two strains was determined under Transmission Electron Microscopy (Hitachi, H-7000, Hitachi
Co. Japan) to be tetrad without flagella.
[0118] 4. Sensitivity to Antibiotics:
[0119] After adjusting the cell density in MRS broth to about 1.5*10 CFU/mL, the cultures of
isolates were streaked onto MRS agar plates and then the paper susceptibility discs with a diameter
of 6 mm was stuck on. The resulting samples were then incubated at 37[deg.] C. for 12 h and then
recorded the size of the inhibition zone. The antibiotics used in this study were all from BBL. The
results were shown in Table 3.
TABLE 3
Antibiotic sensitivity of P. pentosaceus YJL and P. pentosaceus YJS
AntibioticConc.(mcg)R I MS S P. pentosaceus YJLP. pentosaceus YJS
Ampicillin10 21 22-2930MSMS
Cefotaxime301415-2223MSMS
Ceftazidime301415-2223RMS
Cefuroxime301415-2223MSS
Clindamycin2.01415-1617SS
Erythromycin151314-1718SS
Gentamicin101213-1415IR
Imipenem1016SS
Moxalactam301415-2223RR
Nalidixic acid301314-1819RR
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Netilmicin301213-1415SS
Penicillin101920-2728MSS
Tetracyclin301415-1819SI
Ticarcillin751415-1920SS
Vancomycin30 910-1112SR
R, resistant; I, intermediate; MS, moderately susceptible; S, susceptible
diameter of inhibition zone (mm)
[0120] As shown in Table 3, Pediococcus pentosaceus YJL is resistant to ceftazidime (30 mcg),
moxalactam (30 mcg), and nalidixic acid (30 mcg); intermediate resistant to gentamicin (10 mcg);
moderately susceptible to ampicillin (10 mcg), cefotaxime (30 mcg), cefuroxime (30 mcg), and
penicillin (10 mcg); and susceptible to clindamycin (2 mcg), erythromycin (15 mcg), imipenem (10
mcg), moxalactam (30 mcg), netilmicin (30 mcg), tetracyclin (30 mcg), ticarcillin (75 mcg), and
vancomycin (30 mcg).
[0121] Pediococcus pentosaceus YJS is resistant to gentamicin (10 mcg), moxalactam (30 mcg),
nalidixic acid (30 mcg), and vancomycin (30 mcg); intermediate resistant to tetracyclin (30 mcg);
moderately susceptible to cefotaxime (30 mcg) and ceftazidime (30 mcg); and susceptible to
ampicillin (10 mcg), cefuroxime (30 mcg), clindamycin (2 mcg), erythromycin (15 mcg), imipenem
(10 mcg), netilmicin (30 mcg), penicillin (10 mcg), and ticarcillin (75 mcg).
[0122] Based on the testing results shown above, the two strains were different in respect of
physiological and biochemical properties. They were, therefore, denominated as Pediococcus
pentosaceus YJL and Pediococcus pentosaceus YJS, respectively.
EXAMPLE 2
Processed Fish Utilizing Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis
CCRC 12315, Lactobacillus helveticus CCRC 14092, Pediococcus pentosaceus YJL and P.
pentosaceus YJS
[0124] Frozen mackerel (Scomber australasicus) was thawed in running tap water (about room
temperature) and then gutted, eviscerated, deboned, and finally passed through a strainer (Meat
Strainer, Model 5000, mesh: 0.3 cm, Chu-Hwa Co., Keelung, Taiwan) to remove the scale, pin bones,
debris and connective tissues. The resulting samples were homogenized with equal volume of 2%
NaCl solution and then diluted with water (2*), followed by sterilizing at 100[deg.] C. for 20 min and
then cooled to 40[deg.] C. Finally, they were mixed with 4% sucrose, 1% glucose and LAB listed in
Table 4 (inoculated to a final level of 10 CFU/g) with stirring by a sterilized glass rod to obtain
homogenous mixtures. The mixtures were fermented at 37[deg.] C. for 48 h. The changes in pH,
viable counts of LAB, aerobic plate counts (APC) and volatile basic nitrogen (VBN) were measured.
303/1006
The Pseudomonas, Staphylococcus and Enterobacteriaceae, which were frequently detected on
fresh or processed seafood and considered as main microflora, were also measured to evaluate the
antimicrobial ability of these LAB. Also, sensory evaluation was carried out after the addition of
mulberries and sugar to the fermented samples. The results were described below and shown in
Tables 4 and 5.
[0125] At the end of fermentation, the pH of the five fermented samples with the addition of LAB
was reduced from 6.2-6.3 to 4.5-4.7 while the pH of that without the addition of LAB was increased to
7.4-7.6. The VBN of samples without LAB increased rapidly from 8.2-8.6 to 50.2-51.4 mg/100 g after
24 h fermentation and further increased to 70.1-71.3 mg/100 g after 48 h fermentation, while those
with LAB increased slowly from 8.1-8.6 to 21.0-24.8 mg/100 g. These data suggested that
fermentation with LAB used in this study could substantially inhibit the accumulation of VBN. Also, the
produced bacteriocins could inhibit the growth of saprogenic bacteria or pathogens such as
Pseudomonas, Staphylococcus and Enterobacteriaceae. The Hunter L (indicator of transparency), b
(indicator of yellow/blue) and whiteness of the five samples with LAB were significantly higher than
that without LAB. Specifically, L increased from 47.61-49.98 to 59.03-65.06; b increased from 7.148.64 to 9.35-11.68; and whiteness increased from 46.8-49.3% to 57.9-63.5%. Further, as shown in
Table 5, high acceptability on taste, flavor, texture and overall acceptance was obtained from the five
samples with LAB. The sensory quality of the 24 h LAB-fermented samples seemed to be higher than
that of 48 h LAB-fermented samples, though there was no significant difference between 24 h and 48
h fermented samples. It was also found that there was also no significant difference in sensory
quality among samples fermented with different LAB.
TABLE 4
Changes in viable counts of aerobic bacteria, lactic acid bacteria, Enterobacteriaceae,
Staphylococcus and Pseudomonas of LAB
fermented mackerel minces fermented with 1 volume
of water (v/w) during 48 h fermentation at 37[deg.] C.
Ferment TimeBacteria (log CFU/mL)
Starters*(h)APCLABEntero.Staph.Pseudo.
NS04.20 +- 0.31 3.34 +- 0.15 2.78 +- 0.13 2.97 +- 0.13 4.10 +- 0.15
247.78 +- 0.36 6.35 +- 0.32 9.04 +- 0.27 6.63 +- 0.23 8.04 +- 0.26
488.17 +- 0.33 7.24 +- 0.17 9.08 +- 0.18 7.54 +- 0.15 9.04 +- 0.21
A06.23 +- 0.18 6.40 +- 0.16 3.15 +- 0.15 2.77 +- 0.21 3.22 +- 0.11
249.38 +- 0.33 9.46 +- 0.19 3.66 +- 0.21 3.38 +- 0.22 4.00 +- 0.26
489.27 +- 0.24 9.49 +- 0.17 4.10 +- 0.18 4.13 +- 0.22 4.20 +- 0.30
B06.28 +- 0.21 6.34 +- 0.21 3.08 +- 0.19 3.10 +- 0.17 3.31 +- 0.22
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248.20 +- 0.32 8.65 +- 0.17 3.38 +- 0.13 3.20 +- 0.15 3.52 +- 0.19
489.32 +- 0.32 8.85 +- 0.21 4.40 +- 0.23 4.43 +- 0.18 4.53 +- 0.20
C06.28 +- 0.20 6.34 +- 0.21 3.18 +- 0.19 3.10 +- 0.17 3.31 +- 0.21
248.20 +- 0.31 8.65 +- 0.17 3.38 +- 0.13 3.20 +- 0.15 3.32 +- 0.19
489.32 +- 0.35 8.85 +- 0.22 4.50 +- 0.24 4.43 +- 0.19 4.33 +- 0.20
D06.43 +- 0.19 6.45 +- 0.12 3.19 +- 0.11 2.80 +- 0.25 3.3 +- 0.14
249.36 +- 0.20 9.26 +- 0.12 3.95 +- 0.21 3.00 +- 0.16 3.49 +- 0.20
489.20 +- 0.27 9.18 +- 0.20 4.30 +- 0.15 4.21 +- 0.20 4.33 +- 0.17
E06.43 +- 0.19 6.45 +- 0.12 3.19 +- 0.11 2.80 +- 0.25 3.3 +- 0.14
249.36 +- 0.21 9.26 +- 0.14 3.95 +- 0.20 3.00 +- 0.16 3.09 +- 0.21
489.20 +- 0.31 9.18 +- 0.21 4.30 +- 0.14 4.21 +- 0.20 4.33 +- 0.19
*NS: No starter added; A: Lactobacillus plantarum CCRC10069; B: Lactococcus lactis subsp. lactis
CCRC12315; C: Lactobacillus helveticus CCRC14092; D: Pediococcus pentosaceus YJL; E:
Pediococcus pentosaceus YJS.
Values in this Table are mean +- SD from 3 replicates; Values with unlike superscripts in the same
colunm within each treatment differ significantly against fermentation time (p < 0.05).
[0126]
TABLE 5
Sensory quality of the LAB fermented mackerel minces ground with 1
volume of water (v/w) during 48 h fermentation at 37[deg.] C.
Incubation time (h)
Starter*Evaluation items02448
NSTaste4.1 +- 1.1 -***Flavor4.2 +- 1.2 -Texture3.3 +- 0.5 -Overall acceptance4.0 +- 0.5 -ATaste4.2 +- 1.1 7.6 +- 1.2 6.9 +- 1.0
Flavor3.8 +- 1.2 7.6 +- 1.1 6.8 +- 0.8
Texture3.3 +- 1.1 8.2 +- 1.1 7.1 +- 1.1
Overall acceptance3.9 +- 1.0 7.9 +- 0.8 6.8 +- 0.7
BTaste4.2 +- 1.2 7.7 +- 1.2 7.1 +- 1.1
Flavor4.0 +- 1.1 7.9 +- 1.0 6.9 +- 0.9
Texture3.4 +- 0.9 8.3 +- 1.2 7.2 +- 0.7
Overall acceptance4.0 +- 1.2 7.7 +- 1.2 7.0 +- 1.0
CTaste3.6 +- 0.7 7.9 +- 0.9 6.8 +- 1.2
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Flavor3.9 +- 1.0 7.9 +- 1.1 7.1 +- 1.1
Texture3.7 +- 0.6 8.3 +- 1.1 7.3 +- 0.8
Overall acceptance3.9 +- 1.0 8.2 +- 1.1 7.0 +- 1.3
DTaste4.0 +- 1.0 7.7 +- 0.7 7.0 +- 1.3
Flavor4.0 +- 0.8 7.9 +- 1.1 7.1 +- 1.2
Texture3.7 +- 0.6 8.4 +- 1.2 7.4 +- 1.1
Overall acceptance4.0 +- 1.1 8.2 +- 1.1 7.0 +- 1.1
ETaste3.7 +- 1.0 7.9 +- 0.9 7.0 +- 1.1
Flavor4.0 +- 0.8 8.0 +- 1.1 7.1 +- 1.2
Texture3.9 +- 0.6 8.2 +- 1.0 7.2 +- 1.0
Overall acceptance4.1 +- 1.1 8.0 +- 1.1 7.1 +- 1.2
*Refer to the footnote of Table 4.
**Values in this Table are mean +- SD from 3 replicates; Values with unlike superscripts in the same
column differ significantly (p < 0.05).
***spoiled.
EXAMPLE 3
Processed Fish Cheese and Yogurt Utilizing Lactobacillus plantarum CCRC10069, Lactococcus
lactis subsp. lactis CCRC 12315, Lactobacillus helveticus CCRC 14092, Pediococcus pentosaceus
YTL and P. pentosaceus YTS
[0128] Frozen nemipterid surimi was thawed at 5[deg.] C. overnight. The surimi was homogenized
with equal volume of 1.0% NaCl solution and then sterilized at 100[deg.] C. for 15 min and cooled to
30[deg.] C. Finally, the homogenate was mixed with 4% sucrose and LAB (inoculated to a final level
of 10 CFU/g) with stirring by a sterilized glass rod to obtain homogenous mixtures. The mixtures were
fermented at 37[deg.] C. for 24 h. The sensory evaluation was carried out after the addition of
sesame or peanut powder to the fermented samples, sterilization at 100[deg.] C. for 15 min and
shaping to a rectangle form. The resulting fish cheese was shown in FIG. 3. In the similar way, one
sample was processed to a yogurt-like product as shown in FIG. 4.
[0129] The results were described below. The pH of five fermented samples with the addition of
LAB was reduced to 4.6-4.8. High acceptability on taste, flavor, texture and overall acceptance was
obtained from the five samples with LAB.
EXAMPLE 4
Processed Legume Utilizing Pediococcus pentosaceus YJL and P. pentosaceus YJS
306/1006
[0131] Soaked soybean was homogenized with water, filtered and sterilized at 100[deg.] C. for 20
min and then cooled to 30[deg.] C. The water content was adjusted to 60%. Finally, the homogenate
was mixed with 4% sucrose, 1% glucose and LAB (inoculated to a final level of 10 CFU/g) with
stirring by a sterilized glass rod to obtain homogenous mixtures. The mixtures were fermented at
37[deg.] C. for 24 h. The changes in pH, viable counts of LAB, aerobic plate counts (APC) were
measured. The Pseudomonas, Staphylococcus and Enterobacteriacea were also measured. Also,
sensory evaluation was carried out after the addition of strawberries and sugar to the fermented
samples. The results were described below and shown in Tables 6 and 7. Further, one sample was
processed to a pudding-like product as shown in FIG. 5.
[0132] At the end of fermentation, the pH of the two fermented samples with the addition of LAB
was reduced from 6.0-6.2 to 4.7-4.9 while the pH of that without the addition of LAB was increased to
7.5-7.7. The growth of Pseudomonas, Staphylococcus and Enterobacteriaceae is all inhibited
effectively (Table 6). The Hunter L, b and whiteness of the two samples with LAB were significantly
higher than that without LAB (p<0.05). Further, as shown in Table 7, high acceptability on taste,
flavor, texture and overall acceptance was obtained from the two samples with LAB.
TABLE 6
Changes in viable counts of aerobic bacteria, lactic acid bacteria,
Enterobacteriaceae, Staphylococcus and Pseudomonas of LAB
fermented soybean after 24 h fermentation at 37[deg.] C.
Ferment TimeBacteria (log CFU/mL)
Starters*(h)APCLABEntero.Staph.Pseudo.
NS04.20 +- 0.31 3.34 +- 0.15 2.78 +- 0.13 2.97 +- 0.13 4.10 +- 0.15
247.79 +- 0.37 6.33 +- 0.33 9.14 +- 0.27 6.73 +- 0.23 8.10 +- 0.26
A06.43 +- 0.19 6.45 +- 0.12 3.19 +- 0.11 2.80 +- 0.25 3.3 +- 0.14
249.37 +- 0.20 9.29 +- 0.12 3.55 +- 0.21 3.10 +- 0.16 3.29 +- 0.20
B06.23 +- 0.19 6.35 +- 0.12 3.09 +- 0.11 2.90 +- 0.25 3.20 +- 0.14
249.32 +- 0.21 9.28 +- 0.14 3.45 +- 0.21 3.05 +- 0.11 3.09 +- 0.20
*NS: No starter added; A: Pediococcus pentosaceus YJL; B: Pediococcus pentosaceus YJS.
Values in this Table are mean +- SD from 3 replicates; Values with unlike superscripts in the same
column within each treatment differ significantly against fermentation time (p < 0.05).
[0133]
TABLE 7
Sensory quality of the LAB fermented soybean after
24 h fermentation at 37[deg.] C.
StarterEvaluation Items024
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NSTaste4.1 +- 1.1 **4.0 +- 1.3 **
Flavor4.2 +- 1.2 4.1 +- 1.5
Overall acceptance4.0 +- 0.5 4.0 +- 0.8
ATaste4.3 +- 1.1 **7.6 +- 1.2
Flavor4.0 +- 1.2 7.6 +- 1.1
Overall acceptance4.0 +- 0.7 7.9 +- 0.8
BTaste4.2 +- 1.1 **7.7 +- 1.2
Flavor4.2 +- 1.4 7.9 +- 1.0
Overall acceptance4.1 +- 0.6 7.7 +- 1.2
*Refer to the footnote of Table 6.
**Values in this Table are mean +- SD from 3 replicates; Values with unlike superscripts in the same
column differ significantly (p < 0.05).
EXAMPLE 5
Isolation and Identification of Bacteriocins from Pediococcus pentosaceus YJL and P. pentosaceus
YJS
1. Isolation of Bacteriocins:
[0136] After 48 h incubation at 37[deg.] C., the MRS broth was centrifuged at 5,000*g, 25[deg.] C.
for 30 min and then filtered through a membrane (0.45 [mu]m, No. 4654, Gelman) to remove the cells.
The filtrates were further washed with 2 volumes of sterile distilled water and concentrated to about
30 mL using Amicon ultrafiltration (cutoff: 1,000, 180 mL, Model 8400). The L. monocytogenes CCRC
14845 was employed to detect the inhibition ability of the purified bacteriocins. The concentrated
fractions were adjusted to pH 6.0 and used as crude bacteriocins for the further purification. The
filtrates after Amicon ultrafiltration had no inhibition ability against L. monocytogenes CCRC 14845.
2. Chloroform Extraction:
[0138] 400 mL of MRS broth was inoculated with 0.1% of an overnight culture of Pediococcus
pentosaceus YJL and Pediococcus pentosaceus YJS and incubated for 18 h at 37[deg.] C. The
culture was centrifuged at 9,500 g for 15 min (4[deg.] C.) and the bacteriocin-containing supernatant
was filtrated through a 0.45 [mu]m filter. The filtrate was stirred vigorously using a magnetic stirrer for
20 min with 400 mL of chloroform. The mixture was then centrifuged at 10,400 g, 4[deg.] C. for 20
min. Four phases were observed in chloroform-containing mixture. The solvent-aqueous interface
layer which had high antibacterial activity was collected and dissolved in 5-10 mL buffer (0.1 M TrisHCl, pH 7.0), since there was no activity in aqueous phase, solvent phase and precipitates. The
bacteriocin-containing buffer was concentrated with vacuum evaporator (40[deg.] C.) (Rotavapor
R114, BЬCHI) to remove the residual chloroform. The final volume of the concentrated bacteriocin
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was 2-3 mL (Burianek and Yousef, 2000). The bacteriocins were named Pediocin L and Pediocin S,
respectively.
[0139] 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE):
[0140] To confirm the purity of purified bacteriocin and to determine its molecular weight, purified
bacteriocins in a dissociating buffer (62.5 mM Tris-HCl buffer, pH 6.8, containing 3% SDS and
0.002% bromophenol blue) was heated in a water-bath at 100[deg.] C. for 5 min. The purity of the
purified bacteriocins was determined using 8-15% gradient polyacrylamide SDS-PAGE, while the
MW was determined by 15% SDS-PAGE according to Laemmli (1970). After electrophoresis, the gel
plate was immobilized with 15% TCA, stained with Coomassie brilliant blue G-250, and destained
with 25% methanol. Finally, the gel plate was dried in cellophane paper.
4. Protein Concentration:
[0142] Protein concentrations of purified bacteriocins during purification were determined by a
dye-binding method of Bradford (1976). Bovine serum albumin was used as a standard protein.
5. Inhibition Assay
[0144] The agar diffusion method was used to assay the inhibition ability of bacteriocins according
to Piddock (1990). The broth was inoculated with indicator organisms (Table 8) and incubated at
37[deg.] C. for 24 h. After adjusting the level of cells in broth to about 1.5*10 CFU/mL, approximate
0.1 mL of indicator broth was mixed uniformly with 15 mL of warm agar. The agar was poured into
petridish and stood at 5[deg.] C. for 1 h. After punching the agar using a stainless ring with a
diameter of 6 mm, approximate 25 [mu]L of samples were added into the hole and incubated at
5[deg.] C. for 24 h to allow the bacteriocins diffusion. The resulting samples were then incubated at
37[deg.] C. for 6 h and then the size of inhibition zone was recorded to qualifying the inhibition ability.
6. Biochemical Properties
6.1 Sensitivity of Bacteriocin to the Proteolytic Enzymes:
[0147] After adjusting the pH of the purified bacteriocins to 4.0, 5.0, 6.0, 7.0 and 8.0 using 1.0 N
HCl or 1.0 N NaOH, proteases were added and incubated at 37[deg.] C. for 2 h. The reaction was
stopped by heating at 80[deg.] C. water bath for 15 min. After cooling to room temperature, the
inhibition ability of resulted bacteriocins was determined according to Piddock (1990). Proteases
used in this study included pepsin (from Porcine Stomach Mucosa, Sigma), [alpha]-chymotrypsin
(from Bovine Pancreas, Sigma), pronase (from Streptomyces griseus, Sigma) and bromelain (from
Pineapple stem, Sigma).
6.2 Thermostability of Purified Bacteriocins
[0149] After adjusting the pH of purified bacteriocins to 4.0, 5.0, 6.0, 7.0 and 8.0 using 1.0 N HCl
or 1.0 N NaOH, the resulted samples were incubated at 80[deg.] C. or 100[deg.] C. for 15, 30, 45
309/1006
and 60 min or 121[deg.] C. for 15 min. They were cooled down to room temperature and the
inhibition ability was determined according to Piddock (ibid).
6.3 Bacteriocin Spectrum of Activity
[0151] Pentocin YJL and Pentocin YJS were screened for activity against a pathogen panel and
spoilage bacteria as shown in Table 8.
TABLE 8
The cultivation conditions of tested strains for inhibition spectrum
experiment
Culture medium/
OrganismTemp. Source
Alcaligenes faecalis ATCC 8750NA/37[deg.] C.CCRC
Aeromonas faecalisNA/37[deg.] C.Prof. Tsai
Bacillus circulans ATCC 11059NA/37[deg.] C.CCRC
Bacillus subtilis ATCC 10225NA/37[deg.] C.CCRC
Bacillus subtilis ATCC 10254NA/37[deg.] C.CCRC
Bacillus cereus ATCC 11778NA/37[deg.] C.CCRC
Clostridium sporogenous ATCC 11259TSB/37[deg.] C.CCRC
Enterobacter aerogenes ATCC 13048NA/37[deg.] C.CCRC
Escherichia coli ATCC 11229NA/37[deg.] C.CCRC
Escherichia coli ATCC 11303NA + 0.5% NaCl/CCRC
37[deg.] C.
Klebsiella oxytoca ATCC 13182NA/37[deg.] C.CCRC
Listeria monocytogenes RIITSBYE/37[deg.] C.NLFD
Listeria monocytogenes LMTSBYE/37[deg.] C.NLFD
Listeria monocytogenes CCRC 14845TSBYE/37[deg.] C.CCRC
Pseudomonas fluorescens ATCC 13523NA/26[deg.] C.CCRC
Proteus vulgaris ATCC 13315NA/37[deg.] C.CCRC
Staphylococcus aureus ATCC 25923TSA/37[deg.] C.CCRC
Staphylococcus epidermidis ATCC 14990NA/37[deg.] C.CCRC
Streptococcus faecalis DS-5MRS/37[deg.] C.CCRC
Shigella dysenteriae ATCC 13983NA/37[deg.] C.CCRC
Vibrio choleraeNA + 0.5% NaCl/CCRC
26[deg.] C.
Incubation time: 24 h.
310/1006
CCRC: Culture Collection and Research Center, Food Industry Research and Development Institute,
Hsinchu, Taiwan.
Lab of Prof. Tsai, Dept. Food Sci., National Taiwan Ocean University, Taiwan.
NLFD: National Laboratories of Foods and Drugs, Development of Health, Executive Yuan, Taiwan.
7. Result 7.1 Purification of Bacteriocins From Pediococcus pentosaceus YJL and P. pentosaceus
YJS
[0154] According to the results from ultra-filtration and SDS-PAGE electrophoresis (FIG. 2) and the
indication of no activity in proteolytic enzymes test, it was known that pentocins YJL and YJS are
proteins and have a molecular weight of 27 and 25 kDa, respectively. They were very stable at
temperatures below 80[deg.] C., pH 4.0-8.0. There was more than 80% activity left even after 30 min
heating at 80[deg.] C., pH 4.0-8.0 for pentocin YJL, and pH 4.0-6.0 for pentocin YJS. About 41% and
37% activity of pentocin YJS were left even after 30 min heating at 100[deg.] C., pH 4.0 and 5.0,
respectively, and 34% activity of pentocin YJL was left after 30 min heating at 100[deg.] C., pH 4.0.
This phenomenon suggested the potential of these bacteriocins for using in preservation of foods.
7.2 Bacteriocin Spectrum of Activity
[0156] The spectrum of antimicrobial activity of the pentocins YJL and YJS is shown in Table 9. As
shown in the Table, pentocin YJL inhibited the growth of gram-negative strains including Shigella, E.
aerogenes, P. vulgaris, S. dysenteriae, and V. cholerae, and gram-positive strains including B.
subtilis, B. cereus, B. circulans, L. monocytogenes, and S. epidermidis. Pentocin YJS inhibited the
growth of gram-negative strains including Shigella, K. oxytoca, and V. cholerae, and gram-positive
strains including B. subtilis, B. cereus, B. circulans, and L. monocytogenes. This result indicated that
the two pentocins are broad-spectrum bacteriocins.
TABLE 9
Antibacterial spectrum of Pentocins YJL and YJS
OrganismPentocin YJLPentocin YJS
G (-)
Alcaligenes faecalis ATCC 8750-*Aeromonas faecalis-Enterobacter aerogenes ATCC 13048+Escherichia coli ATCC 11229-Escherichia coli ATCC 11303+Klebsiella oxytoca ATCC 13182++
Pseudomonas fluorescens ATCC 13523-Proteus vulgaris ATCC 13315++
Shigella dysenteriae ATCC 13983++
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Vibrio cholerae++
G (+)
Listeria monocytogenes Ram II++
Listeria monocytogenes LM++
Listeria monocytogenes CCRC 14845++
Staphylococcus aureus ATCC 25923-Staphylococcus epidermidis+ATCC 14990
Streptococcus faecalis DS-5-Spore-forming bacteria
Bacillus circulans ATCC 11059++
Bacillus subtilis ATCC 10225++
Bacillus subtilis ATCC 10254++
Bacillus cereus ATCC 11778++
Clostridium sporogenous ATCC 11259++
*a: "-" inhibition zone 6 mm. 7.3 Inhibition of the Germination and Growth of Spore-Forming Bacteria
[0158] As shown in Table 9, pentocins YJL and YJS inhibited the growth of Bacillu and Clostridium.
According, a further study was carried out on Bacillus subtilis ATCC 10225, B. subtilis ATCC 10254
and B. cereus ATCC 11778 and the result was shown in Table 10. It is found that the two pentocins
effectively inhibited the germination of spores. For pentocin YJL, the inhibition zones in the sporeforming bacteria Bacillus subtilis ATCC 10225, B. subtilis ATCC 10254 and B. cereus ATCC 11778
were 120.3, 174.3 and 236.3 mm , respectively, and in the spores were 64.0, 96.3 and 189.0 mm .
For pentocin YJS, the inhibition zones in the spore-forming bacteria were 189.0, 146.3 and 236.3
mm , respectively, and in the spores were 85.0, 108.0 and 189.0 mm . It is therefore concluded that
both pentocins YJL and YJS had maximum inhibition zone in B. cereus and its spore, namely, up to
236.3 and 189.0 mm , respectively. Further, both pentocins had more inhibition activity on the sporeforming bacteria than on their spores.
TABLE 10
Effect of Pentocins YJL and YJS on the growth of bacterial
vegetative cell and spores
Inhibition zone (mm )
BacteriumPentocin YJLPentocin YJS
Bacillus subtilis ATCC 10225
vegetative cell120.3189.0
spore64.085.0
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B. subtilis ATCC 10254
vegetative cell174.3146.3
spore96.3108.0
B. cereus ATCC 11778
vegetative cell236.3236.3
spore189.0189.0
[0159] The present invention is not limited to the preferred embodiments described above, any
modification and variation to the invention without departing from the spirit of the invention is well
known to people skilled in this art.
INDUSTRIAL APPLICABILITY
[0160] The present invention provides two novel strains of lactic acid bacteria (LAB) and
bacteriocins thereof, a method for processing fish foodstuffs comprising using LAB and the products
obtained, and a method for processing legume foodstuffs comprising using LAB and the products
obtained. Specifically, the present invention provides novel LAB isolated from meat useful in the
methods for processing fermented foodstuffs wherein fish or legume is used as raw material. Also,
the present invention provides processed products with reduced growth of unwanted
microorganisms, improved flavor and enhanced economic value. The present invention further
provides bacteriocins obtained from the novel LAB. The bacteriocins are effective on the inhibition of
unwanted microorganisms thereby insure the foodstuff a good quality during storage.
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1193. FEMS Microbiol. Lett. 49: 163-169.Claims:
1. Pediococcus pentosaceus YJL with the accession numbers FERM-BP 8450 (BCRC 910210).
2. Pediococcus pentosaceus YJS with the accession numbers FERM-BP 8449 (BCRC 910211).
315/1006
3. A method for processing fish meat wherein Pediococcus pentosaceus according to claim 1 or 2 or
other LAB or mixed strains thereof is (are) used in fermenting the fish meat, comprising the steps of
homogenizing the fish meat with the addition of sodium chloride in an amount of 0.3 to 2.0 wt %
(based on the weight of the fish meat) and water in an amount of 0.5 to 3 times of the weight of the
fish meat to obtain a homogenous substrate;
sterilizing the resulting homogenous substrate at a temperature of from 100 to 115[deg.] C. for 15 to
30 minutes and then cooling the same to a temperature of from 25 to 40[deg.] C.;
adjusting the water content of the substrate by diluting it with water in a dilution ratio of 0 to 5 times;
adding 1.0 to 6.0 wt % of a saccharide to the substrate and then inoculating the substrate with the
LAB;
fermenting the substrate inoculated with the LAB at a temperature of from 25 to 40[deg.] C. for 6 to
30 hours;
optionally adding a seasoning and a spice; and
optionally packaging the resulting product.
4. The method according to claim 3 wherein the other LAB is selected from the group consisting of
Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, and
Lactobacillus helveticus CCRC 14092.
5. The method according to claim 3 wherein the fish meat is at least one selected from the group
consisting of red fish meat, white fish meat, frozen surimi and mixture thereof.
6. The method according to claim 3 wherein the saccharide is at least one selected from the group
consisting of sucrose, glucose and beet sugar.
7. The method according to claim 3 wherein the substrate is a non-diluted or 5-fold-diluted surimi.
8. The method according to claim 3 wherein the seasoning is at least one selected from the group
consisting of fresh fruits, processed fruits, sesame and peanuts.
9. The method according to claim 3 wherein the spice is at least one selected from the group
consisting of ginger, garlic, mirin, wine, and prickly ash.
10. The method according to claim 3 wherein the substrate after fermenting has a pH value of from
3.8 to 5.5.
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11. A processed fish foodstuff obtained from fish meat fermented with Pediococcus pentosaceus
according to claim 1 or 2 or other LAB or mixed strains thereof.
12. The processed fish foodstuff according to claim 11 wherein the other LAB is selected from the
group consisting of Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC
12315, and Lactobacillus helveticus CCRC 14092.
13. A processed fish foodstuff obtained by the method according to claim 3.
14. A processed fish foodstuff obtained by the method according to claim 3 and then sterilized at a
temperature of from 95 to 115[deg.] C., shaped, and partly dried to a cheese-like product.
15. The processed fish foodstuff according to claim 11 wherein the fish meat is at least one selected
from the group consisting of red fish meat, white fish meat, frozen surimi and mixture thereof.
16. The processed fish foodstuff according to claim 13 wherein the fish meat is at least one selected
from the group consisting of red fish meat, white fish meat, frozen surimi and mixture thereof.
17. A method for processing legumes wherein Pediococcus pentosaceus according to claim 1 or 2
or other LAB or mixed strains thereof is (are) used in fermenting the legumes, comprising the steps of
homogenizing the legumes with water and filtering the resulting homogenate;
sterilizing the filtrate thus obtained homogenate at a temperature of from 100 to 115[deg.] C. for 15 to
30 minutes to obtain a substrate and then cooling the substrate to a temperature of from 25 to
40[deg.] C.;
adjusting the water content of the substrate by diluting it with water;
adding 1.0 to 6.0 wt % of a saccharide to the substrate and then inoculating the substrate with the
LAB;
fermenting the substrate inoculated with the LAB at a temperature of from 25 to 40[deg.] C. for 6 to
30 hours;
optionally adding a seasoning; and
optionally packaging the resulting product.
317/1006
18. The method according to claim 17 wherein the other LAB is selected from the group consisting of
Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, and
Lactobacillus helveticus CCRC 14092.
19. The method according to claim 17 wherein the legumes are at least one selected from the group
consisting of soybean, black soybean and mixture thereof.
20. The method according to claim 17 wherein the saccharide is at least one selected from the group
consisting of sucrose, glucose and beet sugar.
21. The method according to claim 17 wherein the water content of the substrate is in a range of from
50% to 98%.
22. The method according to claim 17 wherein the seasoning is at least one selected from the group
consisting of fresh fruits, processed fruits, sesame and peanuts.
23. The method according to claim 17 wherein the substrate after fermenting has a pH value of from
4.5 to 6.0.
24. A processed legume foodstuff obtained from legumes fermented with Pediococcus pentosaceus
according to claim 1 or 2 or other LAB or mixed strains thereof.
25. The processed legumes foodstuff according to claim 24 wherein the other LAB is selected from
the group consisting of Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC
12315, and Lactobacillus helveticus CCRC 14092.
26. A processed legumes foodstuff obtained by the method according to claim 17.
27. The processed legumes foodstuff according to claim 23 herein the legumes are at least one
selected from the group consisting of soybean, black soybean and mixture thereof.
28. The processed legumes foodstuff according to claim 25 wherein the legumes are at least one
selected from the group consisting of soybean, black soybean and mixture thereof.
318/1006
29. A processed legumes foodstuff obtained by the method according to claim 17 and then sterilized
at a temperature of from 95 to 115[deg.] C., shaped, and partly dried to a cheese-like product.
30. A bacteriocin produced by Pediococcus pentosaceus YJL according to claim 1.
31. The bacteriocin according to claim 30, which is an antibacterial substance having a molecular
weigh of from 20 to 30 kDa.
32. A bacteriocin produced by Pediococcus pentosaceus YJS according to claim 2.
33. The bacteriocin according to claim 32, which is an antibacterial substance having a molecular
weigh of from 20 to 30 kDa.
319/1006
29. JP3039090 - 09.01.1991
CLONED GENE ENCODING FOR BACTERIOCIN FROM PEDIOCOCCUS ACIDILACTICI
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP3039090
Inventor(s):
(US)
VANDENBERGH PETER A (US); PUCCI MICHAEL J (US); GONZALEZ CARLOS F
Applicant(s):
MICROLIFE TECHNICS (US)
IP Class 4 Digits: C12N; A23L; A01N
IP Class:
C12N15/31; A01N63/00; A23L3/34
E Class: A23L3/3571; C07K14/195; C12N15/70
Application Number:
EP19900109060 (19900514)
Priority Number: US19890375344 (19890703)
Family: EP0406545
Equivalent:
AU5056290; AU618661; CA2009336; DE406545T; ES2051657T; GR91300137T;
JP2635198B2; NZ232403
Cited Document(s):
EP0293547; WO8706264
320/1006
Abstract:
ISOLATION OF A GENE ENCODING FOR A BACTERIOCIN IN PEDIOCOCCUS ACIDILACTICI
CLONING OF THE GENE IN A VECTOR PLASMID AND TRANSFORMATION TO ESCHERICHIA COLI
IS DESCRIBED. THE BACTERIOCIN IS PARTICULARLY USEFUL FOR INHIBITING LISTERIA AND
SALMONELLA IN FOOD PRODUCTS.Description:
CLONED GENE ENCODING FOR BACTERIOCIN FROM PEDIOCOCCUS ACIDILACTICI
Cross Reference to Related Application
This application is a continuation-in-part of co-pending application Serial No. 012,619, filed February
9, 1987.
BACKGROUND OF THE INVENTION
321/1006
(1) SUMMARY OF THE INVENTION
The present invention relates to a method for isolating a gene encoding for a bacteriocin from
Pediococcus acidilactici and cloning the gene into a vector which is transformed into Escherichia coli.
In particular, the present invention relates to a gene encoding for a bacteriocin derived from a
plasmid in Pediococcus acidilactici.
(2) Prior Art
The pediococci are a diverse group of Gram-positive homofermentative lactic acid bacteria often
found as saphrophytes on vegetable material (Gonzalez, C. F., and B. S. Kunka, Appl. Environ.
Microbiol. 53:2534-2538 (1987); and Mundt, J. O., W. G. Beattie, and F. R. Wieland, J. Bacteriol.
98:938-942 (1969)). Commercially, pediococci are used in the fermentation of vegetables (Pederson,
Bacteriol. Rev. 13:225-232 (1949) and meats (Smith, J. L., and S. A. Palumbo, J. Food Prot. 46:9971006 (1983).
Some strains of P. pentosaceus and P. acidilactici have been found to contain resident plasmids
although the roles of most of these remain unknown (Gonzalez, C. F., and B. S. Kunka, Appl. Environ.
Microbiol. 46:81-89 (1983); Graham, D. C., and L. L. McKay, Appl. Environ. Microbiol. 50:532-534
(1985); and Raccach, M., CRC Crit. Rev. Microbiol. 14:291-309 (1987). Recently, the association of
raffinose fermentation and plasmid DNA has been reported (Gonzalez, C. F., and B. S. Kunka, Appl.
Environ. Microbiol. 51:105-109 (1986). There have also been several reports which associate the
production of bacteriocins with host plasmid DNA (Daeschel, M. A., and T. R. Klaenhammer, Appl.
Environ. Microbiol. 51:1538-1541 (1985); Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol.
53:2534-2538 (1987); Graham, D. C., and L. McKay, Appl. Environ.Microbiol. 50:532-534 (1985); and
Bhunia et al, J. Applied Bact. 65:261-268 (1988)). The cloning of genes for the production of the
bacteriocin has not been described and this would be useful for producing bacteriocin in significant
quantities in genera unrelated to Pediococcus.
322/1006
Cloned Gram-positive genes for different unrelated proteins have been shown to express in
Escherichia coli (Gilmore, M. S., Curr. Top. Microbiol. Immunol. 118:219-234 (1985); Rogeson, J. P.,
R. G. Barletta, and R. Curtiss III, J. Bacteriol. 153:211-221 (1983); and Smorawinska, M., J. C. Hsu, J.
B. Hansen, E. K. Jagusztyn-Krynicka, YH. Abiko, and R. Curtiss III, J. Bacteriol. 153:1095-1097
(1983)).
OBJECTS
It is therefore an object of the present invention to provide an isolated and purified gene segment
which is cloned into a vector plasmid and expressed in a bacterium, preferably unrelated to the
genus Pediococcus, so that the bacteriocin can be produced in a broad spectrum of genera of
bacteria. Further, it is an object to produce the bacteriocin for use in foods and the like to inhibit
spoilage bacteria, Listeria, Salmonella and Pediococcus. These and other objects will become
increasingly apparent by reference to the following description and the drawings.
IN THE DRAWINGS
Figure 1 shows a restriction endonuclease site map of pSRQ11. P. acidilactici PAC1.0 plasmid
pSRQ11 is 9.4 kbp and contains the gene for PA-1 bacteriocin.The approximate position of the PA-1
gene containing restriction endonuclease sites HindIII, XbaI, ClaI, and PvuII is shown.
Figures 2A and 2B show restriction endonuclease site maps of pSRQ11.1 and pSRQ11.2. Both
plasmids are 14.6 kbp and contain erythromycin resistance (Em) genes at the locations indicated.
The Escherichia coli origin of replication (ori) and the PA-1 bacteriocin gene position on each
plasmid are shown. Numbered triangles ( 1 DELTA and DELTA 2) indicate areas of each plasmid
which had been subsequently deleted. Heavy shaded lines indicate Escherichia coli plasmid
pVA891 portions while the remainder of the plasmids are PAC1.0 pSRQ11 DNA.
Figure 3 shows a restriction endonuclease site map of PSRQ220. Plasmid PSRQ220 is 8.7 kbp and
is a chimera of Escherichia coli plasmid pBR322 and PAC1.0 plasmid pSRQ11 digested with EcoRI
and SalI and ligated together.The heavy shaded line indicates the pBR322 portion of plasmid. The
Escherichia coli origin of replication (ori), ampicillin resistance (Ap) gene, and the approximate
location of the PA-1 bacteriocin gene are indicated. Regions of the plasmid which were subsequently
deleted are denoted by numbered triangles ( DELTA 1, DELTA 2 and DELTA 3)
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Figure 4 shows SDS-PAGE profiles of PAC1.0 and PAC1.14 extracellular proteins. Lane 1 indicates
seven molecular mass standards. These are, from top to bottom, 200 kDa, 97 kDa, 68 kDa, 43 kDa,
29 kDa, and 18.4 kDa, and 14.3 kDa. Lane 2 indicates PAC1.0 extracellular proteins while lane 3
displays PAC1.14 extracellular proteins. Arrow indicates the PA-1 polypeptide.
GENERAL DESCRIPTION
The present invention relates to a gene segment of isolated and purified DNA from a Pediococcus
encoding for a polypeptide which is a bacteriocin having a molecular mass of between about 19,000
to 20,000 daltons by SDS-PAGE analysis.
The present invention further relates to a chimeric plasmid for transformation to Escherichia coli,
which is Gram-negative and unrelated to Pediococcus, including a gene segment of DNA from
Pediococcus encoding for a polypeptide which is a bacteriocin having a molecular mass of between
about 19,000 to 20,000 daltons by SDS-PAGE analysis and a plasmid vector for the Escherichia coli
linked to the segment so that the bacteriocin is expressed in the Escherichia coli transformed with
the chimeric plasmid.
Finally the present invention relates to a transformed Escherichia coli containing a chimeric plasmid
including a gene segment of DNA Pediococcus encoding for a polypeptide which is a bacteriocin
and having a molecular mass of between about 19,000 and 20,000 daltons by SDS-PAGE analysis
and a plasmid vector for the Escherichia coli linked to the segment so that the bacteriocin is
expressed by the transformed Escherichia coli.
The gene segment is preferably derived from Pediococcus acidilactici NRRL-B-18050 also known
herein as PAC1.0, which is deposited with the Northern Regional Research Laboratory in Peoria,
Illinois under the Budapest Treaty. The gene is carried in a 9.4 kbp plasmid designated herein as
pSRQ11. A gene segment pSRQ220 (SalI to EcoRI; 6kbp) is ligated in purified form in a vector
plasmid V871 (pSRQ220) in Escherichia coli NRRL-B-18429 and deposited at the same depository
under the Budapest Treaty. Other gene segments of Pediococcus encoding for a bacteriocin can be
isolated and cloned into other genera or species of bacteria using procedures well known to those
skilled in the art.
324/1006
U.S. Serial No. 12,619, filed February 9, 1987 and assigned to a common assignee describes the
isolation of a bacteriocin from Pediococcus acidilactici NRRL-B-18050. A plasmid in this strain was
disclosed to encode for the bacteriocin which was described to be useful in foods to inhibit bacterial
spoilage.
U.S. application Serial No. 148,044 assigned to a common assignee describes a method of using the
bacteriocin to inhibit Listeria monocytogenes which produces a severe illness in humans. The
plasmid pSRQ11 was described as the source of the bacteriocin.
SPECIFIC DESCRIPTION
The following Examples show the cloning of the gene for bacteriocin PA-1 into a vector,
transformation into Escherichia coli and characterization of the bacteriocin produced. The
bacteriocin PA-1 produced by a cloned gene from pSRQ11 was found to express in Escherichia coli
in both liquid and solid media. By analysis of deletion derivatives, the coding region of the gene for
PA-1 was localized to an area encompassing closely clustered HindIII, XbaI, ClaI, and PvuII
restriction sites. This was further verified by insertional inactivation via insertion of a DNA fragment
into the XbaI site. Removal of the XbaI insert led to restoration of bacteriocin activity. The
approximate molecular mass of a PA-1 bacteriocin polypeptide produced by the gene was found to
be 19,000 to 20,000 daltons by comparison with the protein profile from a plasmid-cured derivative
on SDS-PAGE slab gels.Partial purification of the bacteriocin was obtained using cation exchange
HPLC.
Bacterial strains and media. The bacterial strains used are listed in Table 1. Pediococcus spp.
were routinely maintained on MRS agar (Difco Laboratories, Detroit, MI). Escherichia coli strains were
routinely carried on Lennox L agar (Gibco/BRL, Gaithersburg, Md.). Escherichia coli strains were
also grown on modified MRS agar (no citrate or acetate) or in M9 medium (Maniatis, T., E. F. Fritsch,
and J. Sambrook, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY (1982)) supplemented with 1% yeast extract (Oxoid, Ltd., Basingstoke,
Hampshire, U.K.) and 1% Hy Case TM (Sheffield Products, Norwich, NY) for bacteriocin assays.
Purification studies used FMC (Terleckyj, B., N. P. Willett, and G. D. Shockman, Infect. Immun.
48:303-311 (1975)) medium supplemented with 2% yeast extract.Selective antibiotic concentrations
325/1006
were as follows: ampicillin, 25 ug/ml; tetracycline, 10 ug/ml; erythromycin, 50 ug/ml; and
chloramphenicol, 25 ug/ml. All antibiotics were purchased from Sigma Chemical Co., St. Louis, MO.
Bacteriocin assays. Production of bacteriocin was assayed as previously described (Gonzalez, C. F.,
and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)). Strains were patched on MRS agar
or modified MRS agar for Escherichia coli and incubated at 35 DEG C for 18 hours. The plates were
then overlaid with soft agar (0.8%) seeded with indicator cells. Isolates which produced a clear,
defined zone of inhibition were considered as bacteriocin producers.
Isolation and analysis of plasmid DNA. Covalently closed circular plasmid DNA was isolated from
Escherichia coli by the method of Clewell and Helinski (Clewell, D. B., and D. R. Helinski,
Biochemistry 59:4428-4440 (1970)). Escherichia coli strains were screened for plasmid content as
previously described (Macrina, F. L., J. A. Tobian, K. R. Jones, R. P. Evans, and D. B. Clewell, Gene
19:345-353 (1982)). Pediococcus plasmid DNA was obtained by a scaled up modification of the
LeBlanc and Lee procedure (LeBlanc, D. J., and L. N. Lee, J. Bacteriol. 140:1112-1115 (1979)) as
described by Gonzalez and Kunka (Gonzalez,C. F., and B. S. Kunka, Appl. Environ. Microbiol. 46:8189 (1983)). Plasmid DNA and restriction endonuclease digests were analyzed by agarose gel
electrophoresis on 0.8% agarose (Bethesda Research Laboratories, Inc., Gaithersburg, MD) slab
gels.Size standards were Escherichia coli V517 (Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J.
Ayers, and S. McCowen, Plasmid 1:417-420 (1978)) for undigested plasmid DNA and HindIII digested bacteriophage lambda DNA (Bethesda Research Laboratories) for restriction endonuclease
- cleaved plasmid DNA.
DNA enzymology. Restriction endonuclease digestions were performed in low-, medium-, or high-salt
buffers, as recommended by Maniatis et al. (Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)) .
Restriction enzymes were obtained from Bethesda Research Laboratories. DNA ligation reactions
were carried out with T4 DNA ligase (Bethesda Research Laboratories) at 4 DEG C for 18 hours
according to conditions recommended by the manufacturer.
Bacterial transformations. Escherichia coli was transferred by the CaCl2 heat shock method
Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cob Spring Harbor, NY
(1982)) with cells harvested at an optical density at 660 nm of 0.2 to 0.3.
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Sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Polypeptides were separated by
sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (Laemmli, U. K., and M. Favre., J.
Mol. Biol. 80:575-599 (1973)) on a 12% polyacrylamide slab gel with a 4% stacking gel and
visualized by Coomassie blue staining. Proteins were boiled for 5 minutes in sodium dodecyl sulfate
sample buffer before electrophoresis. Molecular mass standards were purchased from Bethesda
Research Laboratories.
Example 1
Restriction endonuclease map of pSRQ11. The gene encoding the bacteriocin PA-1 was previously
shown to be associated with the presence of a 9.4 kilobase plasmid, designated pSRQ11 (Gonzalez,
C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)). Plasmid pSRQ11 was
digested with a number of restriction endonucleases to generate the restriction site map shown in
Figure 1. The plasmid contained several unique sites including EcoRI, NdeI, XbaI, SalI, and SstI.
Other restriction enzymes which cleaved the plasmid were ClaI, HindIII, PvuII, and EcoRV. The
following restriction sites were not found on pSRQ11: AvaI, BamHI, SphI, NruI, PstI, and BglII.
Example 2
Expression of PA-1 bacteriocin in E. coli. Plasmid pSRQ11 was digested with EcoRI and cloned into
the EcoRI site on plasmid pVA891 (Macrina, F. L., et al., Gene 25:145-150 (1983)), which contains an
erythromycin resistance marker expressed in both Escherichia coli and streptococci. Recombinant
plasmids were obtained with pSRQ11 inserted in both orientations and were designated pSRQ11.1
and pSRQ11.2 as shown in Figure 2. These Escherichia coli strains were assayed for expression of
the PA-1 bacteriocin as previously described (Gonzalez, C. F., and B. S. Kunka, Appl. Environ.
Microbiol. 53:2534-2538 (1987)). The strains were grown on modified MRS medium and overlaid with
Pediococcus pentosaceus FBB63 indicator strain.Escherichia coli strains containing pSRQ11.1 and
pSRQ11.2 both produced zones of inhibition in the indicator lawn while the control Escherichia coli
V850 strains showed no zone of inhibition. PA-1 activity by Escherichia coli strains was also observed
in liquid medium. Escherichia coli strain V850 carrying pSRQ161 (Table 1) was grown in M9 medium
supplemented with 1% yeast extract and 1% Hy Case. After overnight growth, the culture
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supernatant yielded 400 AU/ml PA-1 bacteriocin activity. This strain grown in the above medium
gave the best results of those assayed. Example 2 shows that the cloned PA-1 bacteriocin from
Pediococcus acidilactici PAC 1.0 can be expressed and is functional in an Escherichia coli host
strain.
Example 3
Deletion derivative analysis of pSRQ11 subclones. In order to localize the region encoding the PA-1
gene, SalI and PvuII deletion derivatives of pSRQ11.1 and pSRQ11.2 were obtained (Figure 1). The
SalI deletion of pSRQ11.1 retained activity while the PvuII deletion derivatives displayed no zones of
inhibition against the indicator strain (Table 1). Both the PvuII and SalI deletion derivatives of
pSRQ11.1 expressed no PA-1 activity (Table 1). These data suggested that the bacteriocin gene was
located on the approximately 6.0 kbp EcoRI-SalI fragment of pSRQ11.1 as shown in Figure 2A.
The 6.0 kbp EcoRI-SalI fragment then was subcloned into the EcoRI and SalI restriction sites on the
Escherichia coli plasmid, pBR322 (Bolivar, F., R. L. Rodriguez, P. J. Greene, M.C. Betlach, H. L.
Heyneker, H.W. Boyer, J. H. Crosa, and S. Falkow, Gene 2:95-113 (1977)), and the resulting chimeric
plasmid was designated pSRQ220 (Figure 3). The Escherichia coli strain containing pSRQ220 was
assayed and found to express bacteriocin activity while ClaI, HindIII, and PvuII deletion derivatives
(Figure 3) were assayed and found to be negative for PA-1 bacteriocin activity (data not shown). Two
additional subclones were obtained: pSRQ210, which consisted of the pSRQ11 XbaI-SalI fragment
cloned into Escherichia coli vector pACYC184 (Chang, A.C.Y., and S. N. Cohen., J.Bacteriol.
134:1141-1156 (1978)), and pSRQ211, which consisted of pSRQ11 HindIII fragment c (from map
coordinates 2.3 to 4.6, Figure 1) also cloned into pACYC184. Neither of these two strains expressed
PA-1 bacteriocin activity. Together, all of these data suggest that the gene encoding the PA-1
bacteriocin lies in the region of the plasmid containing the closely clustered HindIII, XbaI, ClaI, and
PvuII restriction sites (see Figure I) spanning approximately 500 bp of pSRQ11 DNA.
Example 4
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Insertional inactivation of the PA-1 bacteriocin gene. Since the XbaI restriction site is unique on both
pSRQ11 and pSRQ220 and lies within the PA-1 coding region, it was chosen as a site to insert a
foreign DNA fragment and interrupt transcription of the bacteriocin gene. Plasmid pACYC184,
approximately 4 kbp in size and also containing a single XbaI site, was cloned into the XbaI site on
pSRQ220. The strain containing the resulting recombinant plasmid, pSRQ221, was assayed for PA-1
activity and proved negative (data not shown). When the pACYC184 insert was removed by XbaI
digestion followed by religation, activity was once again restored (data not shown). Another construct
where the XbaI-EcoRI fragment of pSRQ220 was replaced by the XbaI-EcoRI fragment of pACYC184
also was negative for bacteriocin activity (data not shown).These data indicate that the XbaI site lies
within or very close to the coding region of the PA-1 bacteriocin gene.
Example 5
Molecular weight determination of PA-1 bacteriocin. The molecular mass of PA-1 bacteriocin was
estimated by SDS-PAGE. Pediococcus acidilactici PAC1.0 and the cured derivative PAC1.14 were
grown in the semi-defined FMC-yeast extract medium and Escherichia coli UW-11 with pSRQ161
grown in M9 minimal medium-supplement with 1% by weight lactose, yeast extract and Hycase TM
(casein hydrolysate). The two culture supernatants were concentrated by ammonium sulfate
precipitation. The buffer resuspended precipitates were dialyzed and run side-by-side on a 12%
polyacrylamide slab gel. As shown in Figure 4, a polypeptide of approximately 19,000 to 20,000
daltons molecular mass is present in the UW-11 and PAC1.0 lanes but absent in the lane containing
extracellular proteins from the cured derivative, PAC1.14.
Example 6
Cloning of the EcoR1 - HindIII fragment 4.0 kbp fragment from pSRQ220 into the expression vector
pKK223-3.
The E. coli expression vector (pKK223-3) contains the tac promoter which is repressed but may be
derepressed by the addition of isopropyl B-D-thiogalactoside (IPTG). This cloning vector also
contains a multiple cloning site which facilitates the insertion of genes behind the promoter and
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ribosomal binding site. This cloning vector, pKK223-3 (20 ug) was cleaved to completion with the
restriction enzymes EcoR1 and then HindIII.
The linear EcoR1-HindIII fragment was ligated with the plasmid pSRQ220. Prior to ligation the
pSRQ220 (75 ug) was first cut to completion with EcoR1 and then partially digested with HindIII. The
partial HindIII digestion of pSRQ220 was achieved by increasing the NaCl concentration in the
HindIII buffer to 300 mM.
The DNA mixture was allowed to ligate for 18 hours at 16 DEG C then transformed into E. coli JM105.
The transformants were selected on media containing carbenicillin (50 ug/ml) and then checked for
their ability to produce PA-1. Various transformants were lysed and DNA was purified on a CsClEthidium bromide gradient. The 4.0 kbp EcoR1-HindIII-HindIII fragment from pSRQ220 was cloned
into the EcoR1-HindIII site of pKK223-3. This isolate was designated as JM105 (pSRQ225) which
produced PA-1. The initial EcoR1-HindIII (1.75 kbp) fragment from pSRQ220 was also cloned into
pKK223-3 and transformed into E. coli JM105. This isolate designated as JM105 (pSRQ226) did not
produce the bacteriocin PA-1.
The molecular mass of the PA-1 protein was previously reported to be approximately 16,500 daltons
(Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)). The estimate was
obtained by ascending gel filtration chromatography, which is less accurate. The semi-defined
supplemented FMC medium used herein had fewer contaminating peptides than the previously used
MRS medium and polypeptide profiles were examined on SDS-PAGE which is more accurate.
As can be seen from the foregoing Examples, Pediococcus acidilactici PAC1.0 produces a
bacteriocin designated PA-1, which is plasmid-encoded. The results presented in the Examples map
the position of the PA-1 bacteriocin gene on the 9.4 kilobase plasmid, pSRQ11. Deletion derivative
analysis of the Escherichia coli -Pediococcus chimerics localized the gene to an area of clustered
restriction endonuclease sites within an approximately 500 bp span between HindIII and PvuII (about
2.3 kbp to 2.8 kbp on Figure 1). Included in this cluster of restriction sites was a unique XbaI site.
As a further verification of the proposed location of the PA-1 gene, plasmid pACYC184 was digested
with XbaI and inserted into the unique XbaI site of pSRQ220. The resulting chimeric plasmid,
pSRQ221, when transformed back into Escherichia coli, was found to be bacteriocin negative. When
the XbaI fragment was removed by XbaI digestion followed by religation, the strain containing this
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recombinant plasmid was once again producing PA-1 bacteriocin. These data indicate that the XbaI
site lies within or very near the PA-1 coding region.
Escherichia coli strains containing chimeric plasmids pSRQ161, pSRQ220, or others (see Table 1)
were shown to be capable of PA-1 expression on plate assays. Activity was experienced in
Escherichia coli culture supernatant although reduced from that seen in PAC1.0 culture supernatants.
The examples show that PA-1 bacteriocin is produced and expressed when cloned into Escherichia
coli.
The bacteriocin PA-1 was found to inhibit food sspoilage bacteria, Listeria monocytogenes and
Salmonella newport. It is as useful in foods as is the bacteriocin from the uncloned gene. Claims:
1. A gene segment of isolated and purified DNA from a Pediococcus encoding for a polypeptide
which is a bacteriocin having a molecular mass of between about 19,000 to 20,000 daltons by SDSPAGE analysis.
2. The gene segment of Claim 1 wherein the Pediococcus is Pediococcus acidilactici.
3. The gene segment of Claim 1 derived from Pediococcus acidilactici NRRL-B-18050 as the
Pediococcus.
4. The gene segment of Claim 1 derived from plasmid pSRQ11 as carried in Pediococcus acidilactici
NRRL-B-18050.
5. The gene segment of Claim 4 wherein the pSRQ11 is digested with EcoR1 as a restriction enzyme
to provide the gene segment.
6. The gene segment of Claim 1 as carried in Escherichia coli NRRL-B-18429.
7.A chimeric plasmid for transformation to Escherichia coli including a gene segment of DNA from
Pediococcus encoding for a polypeptide which is a bacteriocin having a molecular mass of between
about 19,000 to 20,000 daltons by SDS-PAGE analysis and a plasmid vector for the Escherichia coli
331/1006
linked to the segment so that the bacteriocin is expressed in the Escherichia coli transformed with
the vector plasmid.
8. The chimeric plasmid of Claim 7 wherein the Pediococcus is Pediococcus acidilactici.
9. The chimeric plasmid of Claim 7 wherein the gene is derived from Pediococcus acidilactici NRRLB-18050 as the Pediococcus.
10. The chimeric plasmid of Claim 7 wherein the gene segment is derived from plasmid pSRQ11 as
carried in Pediococcus acidilactici NRRL-B-18050.
11.The chimeric plasmid of Claim 10 wherein the plasmid pSRQ11 is digested with EcoR1 as a
restriction enzyme to provide the gene segment.
12. The chimeric plasmid of Claim 7 as carried in Escherichia coli NRRL-B-18429.
13. A transformed Escherichia coli containing a chimeric plasmid including a gene segment of DNA
Pediococcus encoding for a polypeptide which is a bacteriocin having a molecular mass of between
about 19,000 to 20,000 daltons by SDS-PAGE analysis and a plasmid vector for the Escherichia coli
linked to the segment so that the bacteriocin is expressed by the transformed Escherichia coli.
14. The transformed Escherichia coli of Claim 13 wherein the gene is derived from Pediococcus
acidilactici NRRL-B-18050 as the Pediococcus.
15.The transformed Escherichia coli of Claim 13 wherein the gene segment is derived from plasmid
pSRQ11 as carried in Pediococcus acidilactici NRRL-B-18050.
16. The transformed Escherichia coli of Claim 15 wherein the plasmid pSRQ11 is digested with
EcoR1 as a restriction enzyme to provide the gene segment.
17. Transformed Escherichia coli NRRL-B-18429.
18. The purified bacteriocin produced by the gene of Claim 1 which inhibits Listeria monocytogenes,
Salmonella newport, and Lactobacillus bifermentans.
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19. The purified bacteriocin produced by the bacterium of Claim 13 which inhibits Listeria
monocytogenes, Salmonella newport and Lactobacillus bifermentans.
20. A gene segment of isolated and purified DNA from a Pediococcus encoding for a polypeptide
produced from a plasmid designated as pSRQ11 and carried in Pediococcus acidilactici NRRL-B18050.
21. A chimeric plasmid for transformation to E. coli including a gene segment of isolated and purified
DNA from a Pediococcus encoding for a polypeptide produced from a plasmid designated as
pSRQ11 and carried in Pediococcus acidilactici NRRL-B-18050 and a plasmid vector for the E. coli
linked to the segment so that the bacteriocin is expressed in the E. coli transformed with the chimeric
plasmid.
22. A transformed E. coli containing a chimeric plasmid including a gene segment of isolated and
purified DNA from a Pediococcus encoding for a polypeptide produced from a plasmid designated
as pSRQ11 and carried in Pediococcus acidilactici NRRL-B-18050 and a plasmid vector for the E.
coli linked to the segment so that the bacteriocin is expressed in the Escherichia coli transformed
with the chimeric plasmid.
333/1006
30. JP4217999 - 18.09.1991
METHOD FOR INHIBITING BACTERIA USING A NOVEL LACTOCOCCAL BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP4217999
Inventor(s):
KUNKA BLAIR S (US); VANDENBERGH PETER S (US); WALKER SHIRLEY A (US)
Applicant(s):
MICROLIFE TECHNICS (US)
IP Class 4 Digits: A23L; C07K; C12P; A61L
IP Class:
A23L3/3472; A23L3/3526; C12P21/00; C07K15/00; A61L15/32
E Class: A23L3/3463A; C07K14/315; A23L3/3571; A01N63/02
Application Number:
EP19910101784 (19910208)
Priority Number: US19900492969 (19900313)
Family: EP0446619
Equivalent:
AU631486; AU7199691; CA2034425; DE446619T; ES2033627T; GR92300024T;
JP2514118B2; NZ236795
Cited Document(s):
EP0293547
Abstract:
A NOVEL BACTERIOCIN PRODUCED BY LACTOCOCCUS LACTIS NRRL-B-18535 IS DESCRIBED.
THE BACTERIOCIN IS USEFUL IN FOODS AND OTHER MATERIALS AND HAS A WIDE SPECTRUM
OF ACTIVITY AGAINST GRAM-POSITIVE BACTERIA IN A PH RANGE BETWEEN 2 AND
8.Description:
334/1006
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a novel bacteriocin derived from a Lactococcus and method of use
to inhibit bacteria, particularly in foods and other materials in need of protection from the bacteria.
The present invention particularly relates to a bacteriocin produced by Lactococcus lactis LL-1
deposited as NRRL-B-18535 (previously known as Streptococcus lactis).
(2) Prior Art
The lactic streptococci have been previously described to produce a variety of polypeptide
antibiotics, diplococcin, lactostrepcins and bacteriocins (Klaenhammer, T. R., Biochemie 70: 337349 (1988)). The term nisin describes a family of polypeptide antibiotics produced by Lactococcus
lactis that prevents the outgrowth of Clostridium and Bacillus spores (Eapen, K. C., et al., J. Fd. Sci.
Technol. 20: 231-240 (1983)). Bacteriocins are also produced by pediococci.
Diplococcin is an antimicrobial agent produced by Lactococcus cremoris. This inhibitor does not
inhibit sporeformers and is only active against other dairy lactococci (Davey, G. P. and B. C.
Richardson., Appl. Environ. Microbiol. 41:94-89 (1981)).
Lactostrepcins are inhibitory proteins produced by the lactococci that inhibit other streptococci.
These molecules are active at relatively low pH and activity is completely lost when the pH is raised
to 7.0 (Kozak, W., et al., J. Dairy Res. 45: 247-257 (1978)).
Bacteriocins produced by lactic lactococci have been observed in many commercial strains (Geis,
A., et al., Appl. Environ. Microbiol. 45:205-211 (1983)). Eight bacteriocin types (I-VIII) have been
identified on the basis of their activity spectrum, proteolytic enzyme susceptibility, heat stability and
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cross-reaction with other bacteriocin producers (Geis, A., et al. Appl. Environ. Microbiol. 45:205-211
(1983)).
The problem is that the bacteriocins are not active over a wide pH range. It would be very desirable
to provide a bacteriocin which is useful in a wide variety of foods regardless of whether they are
acidic or basic.
OBJECTS
It is therefore an object of the present invention to provide a novel bacteriocin which is effective at a
pH between pH 2 and 8. It is further an object of the present invention to provide a bacteriocin which
can be relatively easily isolated from a particular strain of Lactococcus lactis. These and other
objects will become increasingly apparent by reference to the following description and the drawings.
IN THE DRAWINGS
Figure 1 is a high pressure liquid chromatographic (HPLC) amino acid profile of the bacteriocin of
the present invention.
GENERAL DESCRIPTION
The present invention relates to a bacteriocin produced by a Lactococcus which comprises: a
protein having a molecular weight of about 6000 daltons, which is inactivated by protease and not
inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of
bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus
cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus
fermentum, Lactobacillus bifermentans and Lactobacillus plantarum and has an optimal pH fob
inhibition between about pH 2 and 8.
336/1006
Further the present invention relates to a method for inhibiting Gram-positive bacteria which can
occur with a material which comprises: providing a bacteriocin with the material in an effective
amount which inhibits the Gram-positive bacteria, wherein the bacteriocin is derived from a
Lactococcus lactis and wherein the bacteriocin is a protein having a molecular weight of about 6000
daltons, is inactivated by protease and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin
and lysozyme, inhibits the growth of bacteria selected from the group consisting of Staphylococcus
aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus,
Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides,
Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillus
plantarum and has an optimal pH for inhibition between about pH 2 and 8.
Further the present invention relates to a composition which comprises: an unspoiled food system
which is spoiled by Gram-positive bacteria and a bacteriocin derived from cells of a Lactococcus
lactis, wherein the composition contains an amount of the bacteriocin to provide between about 10
and 100,000 AU of the bacteriocin per gram of the food system sufficient for the bacteriocin to inhibit
the Gram-positive bacteria and wherein the bacteriocin is a protein having a molecular weight of
about 6000 daltons, is inactivated by protease and not inactivated by alpha-chymotrypsin, trypsin,
lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the group consisting of
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus
pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc
mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and
Lactobacillus plantarum and has an optimal pH for inhibition between about pk 2 and 8.
Further the present invention relates to a device which comprises: a material on the device which can
become infected with Gram-positive bacteria, and a bacteriocin provided with the material in an
amount sufficient to inhibit the Gram-positive bacteria, wherein the bacteriocin is from cells of a
Lactococcus lactis and is a protein having a molecular weight of about 6000 daltons, is inactivated
by protease and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits
the growth of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus,
Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis,
Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus
bifermentans and Lactobacillus plantarum and has an optimal pH for inhibition between about pH 2
and 8.
337/1006
Further the present invention relates to a method for producing a bacteriocin which comprises:
incubating live cells of a Lactococcus lactis in a growth medium for the cells so as to produce the
bacteriocin in the growth medium, and wherein the bacteriocin is a protein having a molecular weight
of about 6000 daltons, is inactivated by protease and not inactivated by alpha-chymotrypsin, trypsin,
lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the group consisting of
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus
pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc
mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and
Lactobacillus plantarum and has an optimal pH for inhibition between about pH 2 and 8.
The strain Lactococcus lactis LLA 1.0 has been deposited under the Budapest Treaty with the
Northern Regional Research Laboratory in Peoria, Illinois as NRRL-B-18535. It is available only upon
request by name and deposit number. The strain has the following fermentation characteristics: it is
able to ferment dextrose, mannitol, sucrose, maltose, salicin, rhamnose, trehalose, cellobiose,
mannose, fructose and N-acetyl-glucosamine.
The strain had three resident plasmids measuring about 33.42, 28.57 and 5.82 Mdal in size.
SPECIFIC DESCRIPTION
The following Examples show the production of the bacteriocin from Lactococcus lactis NRRL-B18535 and its use in foods and other materials. It also shows the amino acid profile of a hydrolyzate
of the bacteriocin. The determination of the approximate molecular weight is also shown.
Example 1
Production of the Bacteriocin
Bacterial Strains and Media. The bacterial strains used in this study were routinely grown on MRS
lactobacillus broth (Difco, Detroit, MI).
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Bacteriocin assay. Production of bacteriocin was assayed by spotting cells on MRS agar (Difco
Laboratories, Detroit, MI) that contained 2.0% MES ([N-morpholino] ethanesulfonic acid buffer;
Sigma, St. Louis, MO). These plates were incubated at 35 DEG C for 18 hours. Assay plates were
exposed to chloroform vapor for 30 minutes and overlaid with soft agar (.075%) seeded with
indicator cells.Plates were incubated at 32 DEG C for 18 hours. Isolates producing a clear zone were
considered as producing bacteriocin.
Inhibitory spectrum of LL-1. The plate assay system was used to evaluate the spectrum of
bacteriocin activity. The strain NRRL-B-18535 showed activity against strains of Staphylococcus
aureus, S. epidermidis, S. carnosus, Pediococcus pentosaceus, P. acidilactici, Lactococcus
cremoris, Lactococcus lactis, Lactobacillus fermentum, Lactobacillus bifermentans, Leuconostoc
mesenteroides, Lactobacillus bulgaricus and L. plantarum. Strains of Streptococcus mutans, S.
sanguis, S. faecalis and Listeria monocytogenes were not sensitive to the bacteriocin LL-1. The strain
was resistant to nisin.
Example 2
Purification and Characterization of the Bacteriocin
One liter of MRS broth (Difco) was inoculated at 1% with an 8 hour old culture of LLA 1.0 grown in the
medium of Example 1 and was incubated statically at 32 DEG C for 24 hours. After 24 hours the cells
were removed by centrifugation at 16,000 x g for 20 minutes at 4 DEG C. The supernatant was
filtered through a 0.22 micron (pore size) filter (Millipore Corp., Bedford, MA). The supernatant was
assayed for bacteriocin activity by spotting 5 microliters of a serial two-fold dilution series onto MRS
plates overlaid with soft agar seeded with indicator cells. Assay plates were incubated at 35 DEG C.
The indicator strain was Pediococcus pentosaceus FBB63C. One arbitrary unit (AU) of bacteriocin
was defined as 5 microliters of the highest dilution of culture supernatant yielding a definite zone of
growth inhibition on the indicator lawn.The titer was expressed as the reciprocal of the highest
dilution showing inhibition.
Ammonium sulfate (Sigma Chemical Co., St. Louis, MO) was added to the filtered supernatant to
50% (wt/vol) saturation at 4 DEG C. After precipitation for 18 hours at 4 DEG C, the mixture was
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centrifuged at 16,000 x g for 15 minutes at 4 DEG C. The precipitate was reconstituted in 25 ml of
0.05 M sodium citrate buffer, pH 6.0. The reconstituted precipitate was dialyzed against the 0.05 M
sodium citrate buffer at 4 DEG C by using Spectra/Por no. 6 membrane tubing (Spectrum Medical
Industries, Inc., Los Angeles, CA) and the titer of its activity was determined. The reconstituted
dialyzed precipitate was then subjected to further purification with gel filtration chromatographs using
Spectra/Gel AcA202 (Spectrum Medical Industries, Inc., Los Angeles, CA).
The bacteriocin preparation (6 ml) was applied to an ascending Spectra/Gel AcA 202 column (2.6 by
30 cm) in 0.05 M sodium citrate buffer (pH 6.0). Fractions were collected (4 ml) and assayed for
bacteriocin activity. The active factors were then collected and concentrated 10-fold in the dialysis
tubing by the removal of water with Carbowax 20 (Fisher Scientific Co., Pittsburgh, PA). This active
concentrated fraction was then applied to an ascending Spectra/Gel AcA 202 column (1.6 by 60 cm)
in 0.05M sodium citrate buffer (pH 6.0). The titer of the partially purified bacteriocin was determined
and was used for partial characterization of the bacteriocin.
Effects of heat treatment and enzymes. A partially purified sample of bacteriocin LL-1 (6,400 AU/ml)
was assayed for thermostability and enzymatic effects on activity.The bacteriocin was incubated with
each enzyme at a final concentration of 50 micrograms/ml for 60 minutes. Incubation in the presence
of alpha-chymotrypsin and trypsin was at 25 DEG C, and all other enzyme-bacteriocin mixtures were
incubated at 37 DEG C. Inactivation of the enzymes was achieved by boiling them for 3 minutes. It is
considered to be a type VI bacteriocin because the strain that produces it is resistant to nisin and
does not inhibit S. sanguis. Temperature stability of the bacteriocin was assessed by heating a
solution of bacteriocin to 80 DEG C for 60 minutes, 100 DEG C for 10 minutes, and 121 DEG C for 15
minutes. After each treatment, bacteriocin samples were assayed for activity.
Enzymes. All enzymes were obtained from Sigma. Alpha-chymotrypsin (type II; 47 U/mg) and lipase
(type 1; 8.6 U/mg) were dissolved in 0.05 M Tris hydrochloride (pH 8.0) containing 0.01 M CaCl2;
protease (type V; 1 U/mg), lysozyme (grade I, 41, 400 U/mg) and trypsin (type IX; 15,000 U/mg) were
dissolved in 0.05 M Tris hydrochloride (pH 8.0); and pepsin (3,200 U/mg) was dissolved in 0.2 M
citrate buffer (pH 6.0).
pH stability of activity. Partially purified bacteriocin (1 ml) was dialyzed against buffers of various
pH's. The bacteriocin solution (12,800 AU/ml) was dialyzed for 18 hours with 2 changes against 0.05
M glycine hydrochloride buffer (pH 2.0), 0.05 M citrate buffer (pH 3 to 6), 0.05 M Tris hydrochloride
(pH 7 to 9), and 0.05 M carbonate-bicarbonate buffer (pH 10 to 11). After dialysis, the contents of the
tubing were assayed for bacteriocin activity.The bacteriocin LL-1 was sensitive to protease and not
sensitive to alpha-chymotrypsin, trypsin, lipase, pepsin or lysozyme. The bacteriocin was observed
to be most stable from pH 2-8, with some loss in activity at pH 9 and 10. Approximately one-fourth of
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the activity was still Present at pH 11.0. Exposure of the bacteriocin to 121 DEG C did destroy all of
the LL-1 activity. Boiling at 100 DEG C for 10 minutes resulted in 75% loss in activity of the
bacteriocin. This 75% activity loss was also observed at 80 DEG C for 60 minutes.
Example 3
Nutritional Studies
Each of the media listed in Table 1 was prepared in 100 ml quantities.
The media were adjusted to pH 6.8 before autoclaving. The media were inoculated with an 8 hour
culture of NRRL-B-18535 at a rate of 1% and then incubated at 32 DEG C for 24 hours. After 24
hours, 25 ml of the above culture was centrifuged at 24,000 x g for 15 minutes at 4 DEG C. The
supernatant was then filter sterilized using a 0.22 micron filter (Millipore, Bedford, MA) and tested for
the least titer which inhibited Pediococcus pentosaceus FBB63C as the indicator strain.
The results of the nutritional study are depicted in Table 1. The most effective medium for the
production of bacteriocin LL-1 appears to be MRS broth that is unsupplemented. Other media were
not as effective and protein hydrolysate supplements did not stimulate bacteriocin production. Whey
or milk based media were the least effective for the production of LL-1.
Example 4
Production of Dried Bacteriocin LL-1
Lactococcus lactis NRRL-B-18535 was grown in one liter of MRS broth (Difco, Detroit, MI) for 24
hours at 32 DEG C. The cells were pelleted by centrifugation at 16,000 x g at 4 DEG C, and the
supernatant was collected. The supernatant was then filter sterilized with a 0.22 micron pore size
341/1006
filter. Nonfat dry milk powder was added to 10% (weight/volume) to facilitate drying.This mixture was
lyophilized into a dry powder.
Example 5
Minimum inhibitory concentration (MIC) of the lyophilized bacteriocin LL-1 against Pediococcus
pentosaceus FBB63C
The bacteriocin LL-1 powder of Example 4 was dissolved in APT broth or Tryptic Soy Broth and twofold serially diluted to concentrations ranging from 1000 AU/ml to 2.0 AU/ml. Approximately 1 x 10
Pediococcus pentosaceus/ml were added to each of the tubes which were then incubated for 24
hours at 35 DEG C. The MIC value was the lowest concentration tube displaying no visible turbidity.
The results are summarized in Table 2.
Example 6
Minimum inhibitory concentration (MIC) of the lyophilized bacteriocin LL-1 against Staphylococcus
aureus 265
The bacteriocin LL-1 powder of Example 4 was dissolved in APT broth or Tryptic Soy Broth and twofold serially diluted to concentrations ranging from 1000 AU/ml to 2.0 AU/ml. Approximately 1 x 10
Staphylococcus aureus/ml were added to each of the tubes which were then incubated for 24 hours
at 35 DEG C. The MIC value was the lowest concentration tube displaying no visible turbidity. The
results are summarized in Table 3.
Example 7
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Molecular weight determination of the bacteriocin LL-1
The molecular weight of the bacteriocin of Example 1 was determined by gel filtration. 1.5 ml (800
AU/ml) was applied to an ascending Spectra/Gel AcA 202 column (1.6 by 60 cm: Spectrum, Los
Angeles, CA) in 0.05 M sodium citrate buffer (pH 6.0). The elution volume of the bacteriocin was
compared to the elution volumes of standard proteins. Bacteriocin activity was determined as
described above. The protein standards and their molecular weights included the following:
cytochrome C, 12,400; aprotinin, 6,500; melittin, 2,846 (Sigma).
The bacteriocin preparations were examined on 12% SDS-PAGE gel. Samples and molecular weight
standards 1 mg/ml were dissolved in sample buffer and loaded on the gel. The material was then
subjected to electrophoresis for 1 hour at 16 mA and then for 1 hour at 24 mA. The gel was then
stained with silver strain (BioRad, Richmond, CA) or assayed for bcteriocin activity by a direct
detection system (Bhunia, A. K. et al, Appl. Bacteriol. 65:261-268 (1988)).
The molecular weight was observed to be approximately 6,000 daltons from gel filtration. The SDS
gel overlay with Pediococcus pentosaceus confirmed the approximate size observed with gel
filtration.
Example 8
Amino acid profile of purified bacteriocin LL-1
The bacteriocin was purified as previously described in Example 7. An active concentrated fraction
from the Spectra/Gel AcA202 column was subjected to further purification using a C-8 analytical
column. Fractions were assayed for bacteriocin activity. The active fractions were then further
concentrated using the Speed Vac Concentrator TM (Savant Instruments Inc. Farmingdale, NY.) and
343/1006
resuspended in 50 microliters of distilled water. This material was then analyzed for amino acid
content using a modification of the PICO-TAG TM system (Waters Associates, Milford, MA). The
method involves sample hydrolysis followed by derivatization with phenylisothiocyanate and
subsequent analysis by HPLC [Mundt, M. O., W. G. Beattie, and F. R. Wieland, J. Bacteriol. 98:938942 (1969)].Amino acids were identified by comparing the retention times of a known standard to
that of the active fraction hydrolyzate (Figure 1.) The results listed in Table 4 compare the ratios of
the various amino acids observed to glutamic acid, which was observed in the greatest amount.
Example 9
Salad Dressing With Added contaminant Microorganisms
The following Tables 5, 6 and 7 show the use of the bacteriocin LL-1 in salad dressing to which
Lactobacillus fermentum NRRL-B-18586 has been added. The spoilage bacterium strain is one
which is very active in food spoilage.
This Example shows that the bacteriocin LL-1 was effective to inhibit spoilage bacteria introduced
into a salad dressing.
Example 10
Salad Dressing with Natural Contaminant Microorganisms
The following Tables 8 and 9 demonstrate the inhibition of the normal, lactic acid spoilage flora using
the bacteriocin LL-1 in salad dressing:
344/1006
The results show that LL-1 was very effective in inhibiting the growth of the lactic bacteria naturally
present in the salad dressing.
The bacteriocin LL-1 was stable in various environments. The bacteriocin was effective in reducing
the initial contaminant bacteria load and maintaining this protection over a period of several days.
The same results can be achieved in various food systems such as gravies, meats, vegetables and
the like, which can be raw or cooked, and particularly coleslaw, macaroni salad, potato salad and
sausages. The bacteriocin is particularly effective where raw foods are mixed with other ingredients
which promote the growth of Gram-positive bacteria naturally present on food. The bacteriocin can
be effective on such items as bandages, sanitary napkins and ointments (liquids and powders) used
for wound healing. In general the bacteriocin can also be useful in the form of ointments (liquids, or
powders) as disinfectant for animate and inanimate objects where Staphylococcus aureus is a
problem. The bacteriocin can also be used to treat wounds in mammals which can be infected with
Gram-positive bacteria.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
1. A bacteriocin produced by a Lactococcus which comprises: a protein having a molecular weight
of about 6000 daltons, which is inactivated by protease and not inactivated by alpha-chymotrypsin,
trypsin, lipase, pepsin and lysozyme, inhibits growth of bacteria selected from the group consisting
of Staphylococcus aureus, Staphylocococcus epidermidis, Staphylococcus carnosus, Pediococcus
pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc
mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and
Lactobacillus plantarum and has an optimal pH for inhibition between about pH 2 and 8.
2. The bacteriocin of Claim 1 wherein the high pressure liquid chromatographic amino acid profile is
as shown in Figure 1.
3.The bacteriocin of Claim 1 wherein the bacteriocin is produced by Lactococcus lactis NRRL-B18535.
345/1006
4. A method for inhibiting Gram-positive bacteria which can occur with a material which comprises:
providing a bacteriocin with the material in an effective amount which inhibits the Gram-positive
bacteria, wherein the bacteriocin is derived from a Lactococcus lactis and wherein the bacteriocin is
a protein having a molecular weight of about 6000 daltons, is inactivated by protease and not
inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of
bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus
cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus
fermentum, Lactobacillus bifermentans and Lactobacillus plantarum and has an optimal pH for
inhibition between about pH 2 and 8.
5. The method of Claim 4 wherein bacteriocin is derived from Lactococcus lactis NRRL-B-18535.
6. The method of Claim 4 wherein the bacteriocin is derived by growth of the Lactococcus lactis in a
growth medium to produce the bacteriocin and wherein the growth medium with the bacteriocin is
dried to produce a powder.
7. The method of Claim 4 wherein the bacteriocin inhibits the Gram-positive bacteria naturally
present on raw foods in a food system.
8. The method of Claim 7 wherein one of the Gram-positive bacteria in the unspoiled food system is a
Lactobacillus.
9.The method of Claim 4 wherein the bacteriocin is mixed into a food system as the material and
wherein between about 10 AU and 100,000 AU of the bacteriocin is provided per gram of the food
system.
10. The method of Claim 4 wherein the Lactococcus lactis is grown in a growth medium, solids are
separated from the growth medium to produce an aqueous solution of the bacteriocin, the
bacteriocin is precipitated from the aqueous solution by adding excess amount of a salt and the
bacteriocin is isolated from the precipitate.
11. The method of Claim 10 wherein the salt is ammonium sulfate.
12. The method of Claim 4 wherein the material is a salad dressing.
346/1006
13. The method of Claim 4 wherein the material is a food which can be contaminated with gag
forming spoilage microorganisms.
14.A composition which comprises:
(a) an unspoiled food system which is spoiled by Gram-positive bacteria; and
(b) a bacteriocin derived from cells of a Lactococcus lactis, wherein the composition contains an
amount of the bacteriocin to provide between about 10 and 100,000 AU of the bacteriocin per gram
of the food system sufficient for the bacteriocin to inhibit the Gram-positive bacteria and wherein the
bacteriocin is a protein having a molecular weight of about 6000 daltons, is inactivated by protease
and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth
of bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus
epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici,
Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus,
Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillus plantarum and has an
optimal pH for inhibition between about pH 2 and 8.
15. The composition of Claim 14 wherein the food system is salad dressing.
16. The composition of Claim 14 wherein the bacteriocin is derived from Lactococcus lactis NRRL-B18535.
17.A device which comprises:
(a) a material on the device which can become infected with Gram-positive bacteria; and
(b) a bacteriocin provided with the material in an amount sufficient to inhibit the Gram-positive
bacteria, wherein the bacteriocin is from cells of a Lactococcus lactis and is a protein having a
molecular weight of about 6000 daltons, is inactivated by protease and not inactivated by alphachymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of Staphylococcus aureus,
Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus
acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus
bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillus plantarum and
has an optimal pH for inhibition between about pH 2 and 8.
18.The device of Claim 17 which is a wound dressing.
347/1006
19. The device of Claim 17 which is a sanitary napkin.
20. The composition of Claim 14 as in a form for application to a surface which can be infected with
the bacteria.
21. The composition of Claim 14 in a form adapted for application to a wound.
22.A method for producing a bacteriocin which comprises:
incubating live cells of a Lactococcus lactis in a growth medium for the cells so as to produce the
bacteriocin in the growth medium, and wherein the bacteriocin is a protein having a molecular weight
of about 6000 daltons, is inactivated by protease and not inactivated by alpha-chymotrypsin, trypsin,
lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the group consisting of
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus
pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc
mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and
Lactobacillus plantarum and has an optimal pH for inhibition between about pH 2 and 8.
23. A method for producing a bacteriocin which comprises:
incubating live cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells to
produce the bacteriocin in the growth medium, wherein the bacteriocin is a protein having a
molecular weight of about 6000 daltons, is inactivated by protease and not inactivated by alphachymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the
group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus,
Pediococcus pentosaceus, pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis,
Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus
bifermentans and Lactobacillus plantarum and has an optimal pH for inhibition between about pH 2
and 8.
24.A method for producing a bacteriocin which comprises:
(a) growing live cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells; and
(b) isolating the bacteriocin from the growth medium, wherein the bacteriocin is a protein having a
molecular weight of about 6000 daltons, is inactivated by protease and not inactivated by alphachymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of Staphylococcus aureus,
Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus
acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus
348/1006
bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillus plantarum and
has an optimal pH for inhibition between about pH 2 and 8.
25.The method of Claim 24 wherein the bacteriocin is dried to a powder after isolation from the
growth medium.
26. A method for producing a bacteriocin which comprises:
(a) incubating live cells of a Lactococcus lactis NRRL-B-18535 in a growth medium for the cell, so
as to produce the bacteriocin in the growth medium; and
(b) treating the growth medium which has been incubated so as to produce the bacteriocin,
wherein the cells are living or non-living, and wherein the bacteriocin is a protein having a molecular
weight of about 6000 daltons, is inactivated by protease and not inactivated by alpha-chymotrypsin,
trypsin, lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the group
consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus,
Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis,
Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus
bifermentans and Lactobacillus plantarum and has an optimal pH for inhibition between about pH 2
and 8.
27. The method of Claim 26 wherein the growth medium with the cells is dried.
28.The method of Claim 26 wherein the bacteriocin is partially separated from at least some
components of the growth medium.
29. A method for producing a bacteriocin which comprises:
(a) incubating cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells;
(b) passing the incubated growth medium past a microporous filter means which allows the
bacteriocin to pass through as a filtrate and which retains other components of the growth medium
on the filter means, and wherein the bacteriocin is a protein having a molecular weight of about 6000
daltons, is inactivated by protease and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin
and lysozyme, inhibits the growth of bacteria selected from the group consisting of Staphylococcus
aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus,
Pediococcus cidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides,
Lactobacillus bulgaricus, Lactobacillus fementum, Lactobacillus bifermentans and Lactobacillus
plantarum and has an optimal pH for inhibition between about pH 2 and 8.
349/1006
30. The method or Claim 29 wherein the filtrate is dried after being passed through the microporous
filter means to provide the bacteriocin.
31. The method of Claim 29 wherein in addition the bacteriocin in the filtrate is further purified using
liquid chromatography.
32. The method of Claim 29 wherein the growth medium which is to be incubated is pasteurized or
sterilized prior to incubating the cells.
350/1006
31. JP5084092 - 11.09.1991
ULTRAFILTRATION METHOD FOR PURIFICATION OF A PEDIOCOCCAL BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP5084092
Inventor(s):
HENDERSON JAMES T (US); VANDENBERGH PETER A (US); KUNKA BLAIR S (US)
Applicant(s):
MICROLIFE TECHNICS (US)
IP Class 4 Digits: C12N; C07K; C12P; A23J
IP Class:
C12N1/20; C12P21/00; A23J1/00; C07K3/26; C07K3/28
E Class: C07K14/195
Application Number:
EP19900124948 (19901220)
Priority Number: US19900488132 (19900305)
Family: JP5084092
Equivalent:
NZ236443
AU628255; AU7202191; CA2031688; DE445414T; ES2033628T; GR92300017T;
Cited Document(s):
EP0293547; EP0326062
Abstract:
PURIFICATION OF A BACTERIOCIN FROM A PEDIOCOCCUS BY ULTRAFILTRATION OF
SUPERNATANT OF A CULTURE OF THE PEDIOCOCCUS AND HAVING A MOLECULAR WEIGHT
OF BETWEEN ABOUT 4,000 AND 5,000 DALTONS IS DESCRIBED. THE BACTERIOCIN IS USEFUL
IN FOODS TO INHIBIT BACTERIAL GROWTH.Description:
351/1006
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a method for purifying a pediococcal bacteriocin by ultrafiltration. In
particular, the present invention relates to a method wherein the purified bacteriocin retains its
activity after purification.
(2) Prior Art
Proteins can be isolated using a variety of procedures that include precipitation with inorganic salts,
organic solvents, ion exchanges, molecular sieve chromatography and ultrafiltration. Every protein
reacts differently to each of the above methods and the purification achieved and the amount and
activity of the protein recovered varies greatly. Ultrafiltration which was developed in the 1960's has
been successfully utilized to purify a variety of proteins. Different types of protein ultrafiltration
membranes and systems have been developed.
Tangential flow filtration is a separation technique that works by sweeping larger retained molecules
across the surface of the membrane. The process can be used to purify and collect material passing
through the membrane (filtrate) or material retained by the membrane (retentate). Molecules smaller
than the pore size or molecular weight cutoff (MWCO) are able to pass through the membrane and
are thus separated from higher molecular weight molecules.
Molecular ultrafiltration is a form of barrier filtration wherein molecules having a size between about
10 and 10 microns (10 to 1000 angstroms) are separated. Molecules are selectively separated by
molecular weight cutoff. The separation is accomplished at pressures between about 10 and 100 psi.
The filters are generally hollow fiber membrane, plate and frame and spiral tubular. Such apparatus
are well known to those skilled in the art.
352/1006
Chemical and Engineering News 32-54 (April 10, 1989) discuss protein folding and the affects on
activity. A change in shape (configuration) of the protein changes the biological activity. The shape
of a protein molecule can be changed when it is purified. This result was found when purifying
pediococcal bacteriocins from a growth medium. Attempts to purify these bacteriocins resulted in a
loss of activity, even though the amount of protein isolated was increased as a result of the
purification step. It was then realized that the purification step was changing the structure of the
pediococcal bacteriocin in a manner which reduces the activity.
Another problem has been to purify pediococcal bacteriocins so that they do not contribute a flavor
to foods in which they are incorporated. This requires a high activity per unit volume of the
bacteriocin. This purification is essential if these bacteriocins are to be used in foods.
OBJECTS
It is therefore an object of the present invention to provide a method wherein a pediococcal
bacteriocin is produced with enhanced activity per unit volume upon purification. Further, it is an
object to provide a bacteriocin which does not impart any flavor to foods at bacteriocidally effective
levels. Further it is an object of the present invention to provide a method for producing the
bacteriocin which is relatively simple and economical to perform. These and other objects will
become increasingly apparent by reference to the following description.
GENERAL DESCRIPTION
The present invention relates to a method for producing a bacteriocin which comprises: culturing a
Pediococcus in a liquid and solid growth medium to produce the bacteriocin in the liquid; removing
the solids from the liquid of the growth medium; and removing impurities present in the liquid by
ultrafiltration to produce a retentate containing the bacteriocin in the liquid, wherein the retentate
containing the bacteriocin has a higher activity per unit volume than the liquid growth medium
containing the bacteriocin.
353/1006
Further the present invention relates to a bacteriocin for use in foods which has been produced by a
method comprising culturing a Pediococcus in a mixed liquid and solid growth medium to produce
the bacteriocin in the liquid growth medium and removing impurities present in the liquid by
ultrafiltration to produce a retentate containing the bacteriocin in the liquid, wherein the retentate
containing the bacteriocin has a higher activity per unit volume than the liquid growth medium
containing the bacteriocin and wherein the bacteriocin has a molecular weight between about 4,000
and 5,000 daltons, is stable in boiling water and is tasteless in foods at levels which inhibit the growth
of bacteria present in the food.
It has been found that even though the preferred bacteriocin has a molecular weight of between
about 4,000 to 5,000 daltons, it separates as if it was more than three times this size (about 16,500
daltons). Thus the ultrafiltration is conducted so that the retentate contains the bacteriocin even
though it could be expected to pass through the ultrafiltration filter.
The preferred pediococcal bacteriocin is produced by a strain of Pediococcus acidilactici, preferably
NRRL-B-18050. This strain is described in U.S. Patent No. 4,883,673 and has been deposited under
the Budapest Treaty with the Northern Regional Research Laboratory in Peoria, Illinois.
The preferred pediococcal bacteriocin produced by Pediococcus acidilactici NRRL-B-18050 has a
molecular weight of between about 4,000 and 5,000 daltons. The purified bacteriocin is stable in
boiling water and retains its activity. Other pediococcal bacteriocins can be isolated by the method
of the present invention.
The ultrafiltration filters separate the bacteriocin in the retentate so that it is not impaired by the
filtration. Preferably the filter used has a cutoff between about 4,000 and 16,000 daltons so that the
retentate contains the bacteriocin.
The growth medium for the Pediococcus includes a hydrolyzed protein, amino acids, a sugar and
mineral supplements which stimulate growth. Such media are well known to those skilled in the art
and are formulated to maximize production of the bacteriocin in the liquid of the growth medium. The
ultrafiltrate can be treated with a precipitating agent, such as ammonia sulfate which essentially "salts
out" the bacteriocin and then separated from the liquid. The ultrafiltrate can be dialyzed in a buffer
through dializer to remove low molecular weight compounds (less than about 10 K daltons).
354/1006
The ultrafiltrate containing the bacteriocin is preferably dried to a powder for incorporation into a
food or for other uses. This prevents unwanted liquid from being introduced into the food and
provides ease of shipping. Preferably the drying is by lyophilization, spray drying, drum drying, tray
drying or thin film evaporation or any method where heat is applied below the level of heat
inactivation of the bacteriocin. The ultrafiltration can be frozen as well and the activity is preserved.
The amount of lyophilized powder used in the food is up to about ten percent (10%) by weight of the
food. Preferably the amount is between about 0.1 and 10 percent by weight of the food.
The resulting ultrafiltrate has an AU of at least about 100 AU per milliliter. Usually the AU is between
about 100 and 16,000 per ml. One AU of bacteriocin defined as 5 microliters of the highest dilution of
culture supernatent yielding a defined zone of inhibition with a layer of a Gram-positive bacteria on
an agar plate (P. pentosaceus FBB-63 (formerly known as Pediococcus cerevisiae FBB-63). U.S.
Patent No. 4,883,673 and application Serial No. 148,044 assigned to a common assignee, describe
the use of the bacteriocin in foods. The bacteriocin in the culture or growth medium was filter
sterilized at 0.22 micron to remove bacteria and other cell contaminants. There is no separation of
various molecules in solution.
SPECIFIC DESCRIPTION
Strains: The bacterial isolate Pediococcus acidilactici NRRL-B-18050 was stored in liquid nitrogen
and routinely cultured at 35 DEG C on MRS Agar (Difco, Detroit, MI).
Medium: The Pediococcus acidilactici NRRL-B-18050 was grown in different quantities of the
following medium. The medium contained: 4% corn steep (minerals and peptides), 5% glucose
(sugar), 3% yeast extract (proteins and vitamins) and 1% Hycase TM (Sheffield, Norwich, New York)
(an acid hydrolyzed casein protein which liberates amino acids and proteins). The medium was
adjusted to pH 7.0 and sterilized.
Bacteriocin Assay: Production of bacteriocin was assayed as previously described by Gonzalez and
Kunka (Applied and Environmental Microbiology 53: 2534-2538, 1987). Samples were filter sterilized
using an 0.22 um (pore size) (Millipore Corp., Bedford, MA) filter. MRS Agar plates were overlaid with
355/1006
soft agar (0.8%) seeded with indicator cells. The filter sterilized samples were diluted in sterile
dilution water and spotted (5 ul) onto the surface of the overlays.
Protein Assays: The micro-biuret protein determinative method of Koch and Putnam (Analytical
Biochemistry 44: 239-245, 1971) was utilized. The protein standard was bovine serum albumin.
Examples 1 to 3 show various types of ultrafiltration. Example 4 shows the use of the bacteriocin.
Example 5 shows the use of ammonium bicarbonate as a buffer for dialysis. The resulting lyophilized
powder had a better taste when incorporated into food.
Example 1
Comparison of Ultrafiltration and Ammonium Sulfate precipitation on the concentration of PA-1.
Pediococcus acidilactici NRRL-B-18050 was grown for 18 hours at 35 DEG C in 2 liters of the
previously described medium. After 18 hours the broth culture was centrifuged at 8,000 x g for 20
minutes at 4 DEG C to remove cells and other cellular impurities. The supernatant (1.0 1) was
retained and subjugated to different types of purification. Ultrafiltration was accomplished using an
Amicon TM Model 8200 Ultrafiltration cell (Division of W. R. Grace Co., Danver, MA). The
supernatant was filtered through an ultrafiltration membrane YM10 (10,000 daltons molecular weight
cutoff (MWCO)). The retentate was collected and assayed for PA-1 activity (Table 1). The remaining
supernatant (1.0 1) was then subjected to ammonium sulfate precipitation (50% wt/vol), dialyzed
against 0.01 M Tris-maleate pH 6.0 and assayed for PA-1 activity (Table 1).
Example 1 shows that the 10,000 MWCO ultrafiltration produces a large recovery of bacteriocin
which has a high activity in the retentate. This is true even though the protein has a size of between
about 4,000 and 5,000 daltons. This indicates that the molecular weight of the bacteriocin is larger
for ultrafiltration possibly because of aggregation of the molecules.
Example 2
356/1006
Filtration of PA-1 using tangential flow ultrafiltration.
Pediococcus acidilactici NRRL-B-18050 was grown for 18 hours at 35 DEG C in 1 liter of the
previously described medium of Example 1. After 18 hours the broth culture was centrifuged at 8,000
x g for 20 minutes at 4 DEG C. The supernatant (1.0 1) was retained and subjected to different types
of purification. Ultrafiltration was accomplished using a Minitan TM tangential flow system (Millipore
Corp., Bedford, MA). Two membrane plate sets were utilized, 100,000 (MWCO) and 10,000 (MWCO).
The results are set forth in Table 2.
This Example shows a large amount of the bacteriocin is recovered. Again the bacteriocin remained
in the retentate even though its size was less than the MWCO.
Example 3
Filtration of PA-1 using spiral cartridge ultrafiltration.
Pediococcus acidilactici NRRL-B-18050 was grown for 18 hours at 35 DEG C in 300 gallons of the
previously described medium. After 18 hours the broth culture was centrifuged, the supernatant was
retained and subjected to different types of purification. Ultrafiltration was accomplished using the
PUF-15 TM pilot system (Millipore Corp., Bedford, MA) which provides tangential flow. The
supernatant was filtered through a 100,000 (MWCO) polysulfone spiral cartridge. The 100,000
(MWCO) permeate was then filtered through a 10,000 (MWCO) cellulosic spiral cartridge. The results
are observed in Table 3.
This example shows the best recovery of the protein. The bacteriocin did not pass through the
10,000 MWCO even though it was smaller in size.
Example 4
Use of a lyophilized bacteriocin PA-1 produced by P. acidilactici B-NRRL-18050 that had been
concentrated by ultrafiltration.
357/1006
The bacteriocin PA-1 was produced by fermentation in the previously described medium. The
bacteriocin was then concentrated by the method described in Example 3. The material was then
lyophilized. The lyophilized material had an activity of 16,000 AU/g.
Commercially sterile canned chicken was inoculated with Listeria monocytogenes at a rate of 5 x 10
cfu/g of meat which is a heavy overload of this bacteria. The bacteriocin PA-1 was then added to the
chicken at a rate of 1600 AU/g meat. The chicken was then stored at 7 DEG C and aliquots were
then sampled for the growth of L. monocytogenes using standard plate count procedures followed
by plating on McBride's Agar (Difco, Detroit, MI). The results are depicted in Table 4.
The results show that the lyophilized bacteriocin from the bacteriocin protected the canned chicken
and therefore is useful in the extension of shelf life of the processed food. It was determined that the
bacteriocin has no taste in the food.
Example 5
Use of ammonium bicarbonate in the diafiltration and concentration procedures to replace
undesirable low molecular weight components.
Pediococcus acidilactici was grown for 18 hours at 35 DEG C in 1 liter of the previously described
medium of Example 1. After 18 hours the broth culture was centrifuged at 8,000 x g for 20 minutes at
4 DEG C. The supernatant (1 liter) was filtered (.45 micron) and then ultrafiltered using the tangential
flow system with 100,000 MWCO plates. The permeate was concentrated using 10,000 MWCO
plates until 20% of the original volume remained (200 ml) as the retentate. Dialysis was performed by
adding an equal volume of ammonium bicarbonate buffer (0.1 M, pH 7.8) to the permeate and then
reducing the volume by one-half. This operation was done three times to give a final buffer
concentration of 0.0875 M ammonium bicarbonate.
Diafiltered medium was lyophilized and the powder was tested by organoleptic evaluation. The bitter
flavor present in cell-free broth culture and in concentrates thereof was absent in the ammonium
bicarbonate treated sample because the ammonium bicarbonate is volatilized during lyophilization.
358/1006
Other methods of purifying the bacteriocin were tried with very limited success. Included were: (1)
ethanol precipitation and separation; (2) dialysis, centrifugal filtration, and ultrafiltration at 1,000
MWCO rather than 10,000 MWCO. None of these methods provided more than about four times
increase in the activity in AU per ml. Thus the pediococcal bacteriocin requires separation by the
method of the present invention to produce a high activity.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
1. A method for producing a bacteriocin which comprises:
(a) culturing a Pediococcus in a liquid and solid growth medium to produce the bacteriocin in the
liquid;
(b) removing the solids from the liquid of the growth medium; and
(c) removing impurities present in the liquid by ultrafiltration to produce a retentate containing the
bacteriocin in the liquid, wherein the retentate containing the bacteriocin has a higher activity per unit
volume than the liquid growth medium containing the bacteriocin.
2. The method of Claim 1 wherein the bacteriocin is dried to a dry powder.
3. The method of Claim 1 wherein the growth medium included a protein, an amino acid, a sugar and
mineral supplements which stimulate the production of the bacteriocin.
4.The method of Claim 1 wherein the liquid of the retentate is treated with a precipitating agent to
precipitate the bacteriocin which is separated from the liquid.
5. The method of Claim 4 wherein the precipitation agent is ammonium sulfate.
6. The method of Claim 4 wherein the precipitated bacteriocin is dried to a dry powder.
7. The method of Claim 1 wherein the liquid of the retentate is treated with a precipitating agent to
precipitate the bacteriocin which is separated from the liquid and then the bacteriocin is
resuspended in a buffer and dialyzed to remove impurities in the bacteriocin.
359/1006
8. The method of Claim 1 wherein the Pediococcus is Pediococcus acidilactici.
9. The method of Claim 1 wherein the Pediococcus is Pediococcus acidilactici NRRL-B-18050.
10.A bacteriocin for use in foods which has been produced by a method comprising:
(a) culturing a Pediococcus in a mixed liquid and solid growth medium to produce the bacteriocin
in the liquid growth medium; and
(b) removing impurities present in the liquid by ultrafiltration to produce a retentate containing the
bacteriocin, wherein the retentate has a higher activity per unit volume than the liquid growth medium
containing the bacteriocin in the liquid, and wherein the bacteriocin has a molecular weight between
about 4,000 and 5,000 daltons, is stable in boiling water and is tasteless in foods at levels which
inhibit the growth of bacteria present in the food.
11. The bacteriocin of Claim 10 wherein the bacteriocin is in the form of a dry powder.
12.The bacteriocin of Claim 10 which is effective in an amount up to 10 percent by weight of the food
without imparting a taste to the food.
13. The bacteriocin of Claim 10 wherein in the method the ultrafiltration separates the bacteriocin
from the liquid at a molecular weight cutoff for the ultrafiltration of between 4,000 and 16,000 daltons
to provide the retentate containing the bacteriocin.
14. The bacteriocin of Claim 10 wherein in the method the growth medium includes a protein
hydrolysate, an amino acid, a sugar and mineral supplements which stimulate the production of the
bacteriocin.
15. The bacteriocin of Claim 10 wherein in the method the liquid of the retentate is treated with a
precipitation agent to precipitate the bacteriocin which is separated from the liquid.
16. The bacteriocin of Claim 15 wherein the precipitation agent is ammonium sulfate.
17. The bacteriocin of Claim 15 wherein in the method the precipitated bacteriocin is lyophilized to a
dry powder.
360/1006
18. The bacteriocin of Claim 10 with an AU per ml of at least about 100 AU/ml when assayed against
an indicator strain of Pediococcus pentosaceus grown on an agar plate.
19. The bacteriocin of Claim 10 wherein in the method the liquid of the retentate is treated with a
precipitating agent to precipitate the bacteriocin and then separated from the liquid and then the
bacteriocin is resuspended in a buffer and dialyzed to remove impurities in the bacteriocin.
20. The bacteriocin of Claim 19 wherein the buffer includes ammonium bicarbonate.
21. The method of Claim 7 wherein the buffer includes ammonium bicarbonate.
22. The bacteriocin of Claim 10 produced by a Pediococcus acidilactici.
23. The bacteriocin of Claim 10 produced by Pediococcus acidilactici NRRL-B-18050.
361/1006
32. JP54107596 - 23.08.1979
PREPARATION OF BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP54107596
Inventor(s):
others: 01 (--); KAGEYAMA MAKOTO (--)
Applicant(s):
MITSUBISHI CHEM IND LTD (--)
IP Class 4 Digits: C12D
IP Class:
C12D9/20
Application Number:
JP19780014795 (19780210)
Family: JP54107596
Equivalent:
JP1339310C; JP61001040B
Abstract:
PURPOSE:TO PREPARE A FLEXUOUS ROD-SHAPED BACTERIOCIN HAVING BACTERICIDAL
ACTIVITY AGAINST PSEUDOMONAS AERUGINOSA, BY CULTURING PSEUDOMONAS
AERUGINOSA, IN A MEDIUM CONTAINING MITOMYCIN C.
CONSTITUTION:A FLEXUOUS ROD-SHAPED BACTERIOCIN IS PREPARED BY CULTURING
PSEUDOMONAS AERUGINOSA, E.G. P15-40 STRAIN (FERM-P NO. 4387) IN A MEDIUM
CONTAINING MITOMYCIN C, PURIFYING THE CULTURED MEDIUM BY AMMONIUM SULFATE
FRACTIONATION, DIALYSIS, ETC., AND FURTHER PURIFYING THE PRODUCT BY
CHROMATOGRAPHY.
362/1006
33. JP61056091 - 20.03.1986
PREPARATION OF BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP61056091
Inventor(s):
FUKUSHIMA HISANORI (--); others: 01 (--)
Applicant(s):
YAKULT HONSHA CO LTD (--)
IP Class 4 Digits: C12P; A61K
IP Class:
A61K35/74; A61K37/02; C12P21/00
Application Number:
JP19840174102 (19840823)
Family: JP61056091
Equivalent:
JP1909341C; JP6038754B
Abstract:
PURPOSE:TO PREPARE BACTERIOCIN USEFUL AS A PREVENTIVE FOR DENTAL CARIES, IN HIGH
CONCENTRATION, BY CULTURING STREPTOCOCCUS MUTANS RM-10 IN A MEDIUM
CONTAINING YEAST EXTRACT, ETC.
CONSTITUTION:A MEDIUM CONTAINING TRYPTICASE SOY BROTH AS A BASE COMPONENT IS
INCORPORATED WITH >=1WT% YEAST EXTRACT AND TWEEN 80. STREPTOCOCCUS MUTANS
RM-10 IS CULTURED IN THE MEDIUM WITHOUT AGITATION UNTIL THE CYTOLYSIS REACHES
ABOUT >=70%. THE OBJECTIVE BACTERIOCIN IS SEPARATED FROM THE CULTURE LIQUID AND
PURIFIED.
363/1006
34. JP62096429 - 11.03.1987
BATERIOCIN-TARGETED COMPOUNDS FOR CANCER THERAPY
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP62096429
Inventor(s):
PICKER DONALD H (--); SERINO ANTHONY JR (--); HENSON GEOFFREY W (--)
Applicant(s):
JOHNSON MATTHEY INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K35/74; A61K47/00; A61K49/00; A61K43/00
E Class: A61K41/00P; A61K41/00Z4; A61K47/48R; A61K49/00H; A61K51/08Z
Application Number:
EP19860306115 (19860807)
Priority Number: US19850766360 (19850816)
Family: JP62096429
Cited Document(s):
EP0049486; WO8302946; US4452774
Abstract:
COMPOSITIONS COMPRISING A BACTERIOCIN OR ACTIVE FRAGMENT THEREOF LINKED
DIRECTLY OR INDIRECTLY TO AN ANTITUMOR COMPOUND, RADIOISOTOPE, BORON-10
CONTAINING COMPOUND, RADIOSENSITIZER, OR OTHER THERAPEUTIC OR ANALYTICAL
AGENT.Description:
BACTERIOCIN-TARGETED COMPOUNDS FOR CANCER THERAPY
This invention relates to novel bacteriocin-conjugates for the detection, localization, and/or treatment
of tumor cells and tissues.
364/1006
BACKGROUND OF THE INVENTION
The use of a variety of organic and inorganic compounds in the chemotherapeutic treatment of
cancer is now an established clinical technique. Nonetheless, efforts continue to find and develop
new and improved compounds. The problem with most compounds when administered as a
composition together with an inert carrier or diluent is that they are absorbed generally into the
systemic circulation from where they have a toxic effect on normal cells and tissues as well as on
diseased cells and tissue which they are designed to treat. In practice, the maximum dose that can
be administered is limited not by pharmaceutical effectiveness but by toxicity, with the result that the
patient suffers unpleasant or even severe side effects.
In an attempt to render antitumor compounds specific and less toxic for certain types of tumor cells,
many investigators have linked these antitumor compounds to carriers, with varying therapeutic
success, such as DNA, liposomes, antibodies, hormones and various proteins. See
Rowland, G.F., (1983) Clinics in Immunol. & Allergy, 3, 235-257; Trouet, A. (1972) Nature, New Biol.
239, 110-112; Rustum, Y.M. (1979) Cancer Res. 39, 13901395; Ghose, T. (1978) J. Natl. Cancer Inst.
61, 657-677; Kaneko, Y. (1981) Horm. Met. Res. 13, 110114; and Ryser, H.J.P. (1978) Proc. Natl.
Acad. Sci.
75,3867-3870.
OBJECTS OF THE INVENTION
It has been demonstrated that a variety of bacteriocins recognize tumor cells but do not recognize
normal cells. See:
Farkas-Himsley, H. (1983) IRCS Med. Sci. 11, 236237. It is an object of this invention, therefore, to
target a large variety of anti tumor compounds to tumor cells and tissues by the covalent and/or ionic
attachment of such compounds to bacteriocins or active fragments thereof, or bacterial
proteinaceous products. See Farkas-Himsley, H. (1976) Cancer Res.
36, 3561-3567.
A further object of this invention is to provide bacteriocin-antitumor conjugates that are stable in vivo
and which will release enough of the active antitumor compound at the target site to achieve a
therapeutic effect.
365/1006
A further object of this invention is to provide bacteriocin-conjugates of radioisotopes which will
allow for the detection and localization of a tumor mass or metastastes.
A further object of this invention is to provide bacteriocin-conjugates of boron-l0 containing
compounds which can be used for tumor therapy in conjunction with thermal neutron irradiation.
A further object of this invention is to provide bacteriocin-conjugates of radiosensitizers which can
be used for tumor therapy in conjunction with ionizing radiation.
Upon examination of the specifications and appended claims, further objects and advantages of this
invention will be apparent to those skilled in the art.
DESCRIPTION OF THE INVENTION
Accordingly, the bacteriocin or active fragment may be linked to the antitumor compound directly or
indirectly through a spacer which can be a degradable or non-degradable linker or biopolymer. As
will be appreciated, the bacteriocin delivers the anti tumor compound to the target site where either
the anti tumor compound is liberated, or the conjugate is internalized, for therapeutic use.
Any of the available bacteriocins which have the characteristic of selectively recognizing tumor cells
may be used for present purposes but this can include active fragments as well. Typical examples
include Vibriocin 41-S, Vibriocin 506,
Pyocin P-l, Pyocin P-4, Colicin HSC 10
Mycobacteriocin, Colicin HSC 4, etc. which are formed from bacteria such as Vibrio Cholerae, Vibrio
eltor, Echerichia coli, Pseudomonas aeruginosa and
Pseudomonas pyocyanea. See Farkas-Himsley and
Musclow (1985) submitted to Cell. Mol. Biol.
The antitumor compound can be any of the known antineoplastic agents. Representative examples
of such compounds for use herein include
Daunomycin, Adriamycin, Mitomycin-C, Methotrexate, cis-Platin, Chlorambucil, Trenimon, Phenylene
diamine mustard, Bleomycin, Vindesine, Daunorubin, 5
Fluorouridine, Cytosine arabinoside,
Neocarzinostatin, Vincristine, Etoposide,
Cyclophosphamide, Phospholipase C, Glucose Oxidase, or any other antitumor compound,
including functionalized active derivatives.
366/1006
The bacteriocin or active fragment and the anti tumor compound may be linked directly or indirectly
utilizing conventional techniques provided that these do not significantly alter the characteristic
properties of the two components.
The linkage can involve either covalent or ionic attachment brought about by the appropriate
chemical reaction. The attachment will usually involve linkage through carboxyl groups, amino
groups, sulfhydryl groups, or sugar residues on any of the reacting species. A spacer molecule,
which may be cleavable or non-cleavable, can be advantageously employed. Such spacers includes,
among others, the aconityl molecule, protein modification reagents, e.g. glutaraldehyde, and
polypeptides. Additionally a carrier, e.g. dextran or a biopolymer such as poly-glutamic acid or a
protein or protein fragment, can be modified by the attachment of the antitumor compound and then
attached, either directly or indirectly and with or without a spacer molecule as described above, to
the bacteriocin or active fragment.The reaction conditions for the above described attachments are
such that (i) the binding specificity of the bacteriocin is retained (i.e.
that the bacteriocin or active fragment is able to recognize tumor cell surface receptors and not
recognize normal cells), (ii) that the bacteriocinconjugate remains soluble under physiological
conditions, and (iii) that the bacteriocin-conjugate can be localized in vivo.
Among the radioisotopes which may be used to radiolabel bacteriocins or active fragments for the
detection and localization of a tumor mass or metastases are gamma emitters, positron emitters,
xray emitters, and fluorescence emitters. A preferred method of radiolabeling, however, utilizes either
Iodine-131 or Iodine-125 in an oxidative procedure such as reported by Greenwood, et al (1963)
Biochem. J. 89, 114, and later modified by
McConahey, et al (1969) Int. Arch. Allergy Appln.
Immunol. 29, 185. Isotopes other than those of
Iodine which can be detected by gamma cameras and which may be utilized above include Gallium67,
Technetium-99m, and Indium-lll. See: Khaw, et al (1980) Science 209, 295; Scheinberg, et al (1982)
Science 215, 1511; Hantowich, et al (1982) Int. J.
Appl. Radiat. Isot. 33, 327; Crockford, et al (198?)
U.S. Pat. No. 4,323,546; and Wong, et al (1978) Int.
367/1006
J. Appl. Radiat. Isot. 29, 251.
Bacteriocin-conjugates of Boron-lO containing compounds of at least the 19.6% natural abundance
of the Boron-lO isotope for use in a
Neutron-Capture Therapy of tumor tissue may be prepared and utilized according to the teachings of
the invention. The Boron-lO containing compound can be any of the known boron compounds but is
preferably a cluster molecule bearing a functionality suitable for coupling to protein or carrier
molecules by any of a variety of welldocumented procedures. See: Lipscomb, W.N., et al (1974) J.
Med. Chem. 17, 785; Lipscomb, W.N., et al (1974) J. Med. Chem. 17, 792.
Additionally, the bacteriocin-boron-l0 conjugate may be radiolabeled by one or more of the well
known procedures for radiolabeling proteins, as described above, or by incorporating the radiolabel
into the boron cluster by chemical synthesis prior to coupling, for the simultaneous or consecutive
localization and therapy of tumor tissue. See:
Hawthorne, M.F., et al (1985) Inorg. Chem. 24, 19111916.
It has been shown that certain organic and inorganic compounds are effective radiosensitizers in
hypoxic cells. Representative examples include metronidazole, misonidazole, and cis-Platin. See:
Double and Richmond (1978) Br. J. Cancer 37 (Suppl.
III) 98-102; and Adams, et al (1976) Radiat. Res.
67, 9-20; Chibber, et al (1984) Int. J. Radiation
Oncology Biol. Phys. 10, 1213-1215; and Coughlin, et al (1985) Int. J. Radiation Oncology Biol. Phys.
11, 915-919. Bacteriocin-conjugates of radiosensitizers may be prepared and utilized according to
the teachings of the invention. The radiosensitizer may be any of the known organic, inorganic, or
organometallic compounds useful in the treatment of hypoxic tumors in conjunction with ionizing
radiation, including functionalized active derivatives.
The invention is illustrated by the following examples:
Example 1
Preparation of the bacteriocin
The bacteriocins were prepared and titrated as previously described for Vibriocin by Jayawardene
and Farkas-Himsley (1969) Microbios, 4, 325-333.
368/1006
Briefly, the cultures were induced with Mitomycin-C, incubated and then resuspended in 0.58 NaCl
solution at pH 7.8. Upon further incubation, a cell lysate in the NaC1 solution containing the
bacteriocin was obtained. The cell debris was then removed by centrifugation and the bacteriocincontaining supernatant was concentrated by filtration with a PM 10 membrane, and sterilized with
the use of
Millipore filters (0.45 pm pore size). Further purification is done by the method of Farkas-Himsley and
Yu (1985) Cytobios. 42, 193-207. Briefly, this involves an ammonium sulfate precipitation of the
filtered lysate, followed by resuspension in 30 mM
Tris buffer (pH 7.5), chromatography on Sephadex-ASO using a gradient of 0.1 M to 0.4 M with the
purified fraction eluting in the vicinity of 0.2 M Tris.HCl.
Example 2
The bacteriocin of example 1 was linked directly to an antitumor compound as follows:
To 0.1 ml of Diaminecarboxypentylmalonatoplatinum (II) in dimethyladetamide (DMA) at a
concentration of 120 mg/ml was added 0.1 ml of triethylamine in DMA (at a concentration of 0.037
ml/ml). After cooling to 40C. 0.1 ml of isobutylchloroformate in DMA (at a concentration of 0.035
ml/ml) was added, stirred 1 hour, then added in one portion to 2.7 ml of the bacteriocin in 0.1 M
phosphate, pH 8.6 (at a concentration of 5.3 mg/ml). The reaction was stirred overnight then
desalted on Sephadex G25 and dialyzed extensively against water. Further purification can be
achieved using gel filtration media, such as LKB AcA 54. The appropriate fractions are pooled,
assayed, and lyophilized.Platinum content is quantified by either an ICP or graphite furnace AA
analysis and protein content is determined by the Bradford dyebinding assay.
Example 3
The bacteriocin of example 1 was linked to a boron-l0 containing compound through a carrier
molecule as follows:
Dextran (average molecular weight = 40000) is oxidized by a 30-fold molar excess of sodium metaperiodate in 100mM sodium acetate at pH 5.6 for 16 hours. The oxidized dextran is dialyzed against
distilled water and lyophilized, then reacted with a 25-fold molar excess of CAmminecarbaundecaborane at pH 6.5 in distilled water for 6 hours. The resulting Schiff bases are
reduced with an equimolar amount of sodium cyanoborohydride at pH 6.0 for 3 hours, and the
product dialyzed against distilled water and lyophilized.Subsequent reaction with the bacteriocin at a
molar ratio of 10:1 is performed in phosphate-buffered saline (PBS) at pH 6.5 for 6 hours at 40C,
369/1006
followed by reduction by a 10% molar excess of sodium cyanoborohydride at pH 6.5 for 3 hours at
40C, and dialysis against PBS, pH 7.4.
Boron content was quantified by both ICP and promptgamma analyses, and protein concentration
determined by the Bradford dye-binding assay.
Example 4
The bacteriocin of example 1 was linked to an anti tumor compound through a spacer molecule as
follows:
Daunomycin hydrochloride, 1.0 ml (at a concentration of 5 mg/ml) in water was basified to pH 8.5 at
40C by the dropwise addition of 0.1N sodium hydroxide, An equimolar amount of cisaconitic
anhydride was added while maintaining the pH at 8.5. The solution was stirred 5 minutes at 40C,
then 1N hydrochloric acid was added dropwise to form a precipitate which was collected by
centrifugation. The pellet was resuspended in 0.5 ml phosphate-buffered saline (PBS) at pH 7.3 and
added as a 30-fold molar excess to the bacteriocin in PBS (at a concentration of 5 mg/ml). A 50-fold
molar excess of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide in PBS was then added and the
mixture stirred 16 hours while protected from light.The bacteriocin-conjugate was desalted on
Sephadex G25 then dialyzed against distilled water. Drug-bacteriocin ratios were determined by
measuring the concentration of duanomycin by its absorption at 495 nm (E=10000) and bacteriocin
concentration by the Bradford dye-binding assay.
Any one of the bacteriocin-conjugates described in this invention can be used in a variety of
pharmaceutical formulations and can be administered by a variety of conventional routes, such as
intramuscular, intravenous, subcutaneous, and intraperitoneal. When administering the bacteriocinparenterally, pharmaceuticallyacceptable forms for injection may include sterile aqueous solutions of
dispersions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
Various antibacterial and antifungal agents, e.g. parabens, phenol, sorbic acid, chlorobutanol, and
the like may be used.
Sugars, sodium chloride, and isotonic agents may also be desirable additives. Prolonged absorption
of the injectable dose may be accomplished by agents which delay absorption, e.g. aluminum
monostearate and gelatin. Appropriate doses of the bacteriocinconjugate are administered to
achieve the desired diagnostic and/or therapeutic effect.
370/1006
In the testing of Bacteriocin-conjugates, verification of binding and activity of the bacteriocinconjugate can be performed by one or more of the following assays;
1. Tritiated-thymidine uptake inhibition. See: Farkas-Himsley (1980) Microbios.
Lett. 15, 89-96; and Farkas-Himsley and Musclow (1980) IRCS Med. Sci. 8, 497-498.
2. Fluorescence assay. See: Farkas
Himsley and Musclow (1985) Cell. Molec. Biol., submitted.
3. Iodine-125 assay. Farkas-Himsley, personal communication.
4. Flow cytometry. Farkas-Himsley and
Musclow (1980) Cell. and Mol. Biol. 26, 597-603.
5. Cell survival by counting. Farkas
Himsley and Cheung (1976) Cancer Res. 36, 3561-3567.
It will be appreciated by those skilled in the art that various other modifications are contemplated
according to and implied by the invention. Accordingly, the scope of the invention is defined in the
following claims wherein: Claims:
CLAIMS:
1. A composition comprising a bacteriocin or active fragment linked to an agent selected from the
group consisting of antitumor compound, radioisotope, boron10 containing compound,
radiosensitizer, or other therapeutic or analytical agent.
2. A composition according to claim 1 wherein the bacteriocin or active fragment and the agent are
linked directly together by means of a covalent or ionic bond.
3. A composition according to claim 1 wherein the bacteriocin or active fragment and the agent are
linked together through a spacer and/or a carrier molecule, either of which may be cleavable or noncleavable in vivo.
371/1006
4. A composition according to claim 1 wherein the agent is an anti-tumor compound.
5. A method of detecting, localizing, and/or treating a tumor or metastatic cells which comprises
administering a composition according to claim 1.
372/1006
35. JP63079837 - 02.12.1987
BACTERIOCINS AND COMPOSITIONS THEREOF IN ANTI-VIRAL TREATMENT
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=JP63079837
Inventor(s):
FARKAS-HIMSLEY HANNAH (--)
Applicant(s):
UNIV TORONTO (CA)
IP Class 4 Digits: A01N; A61K; C12Q
IP Class:
A01N63/02; A61K35/74; C12Q1/18
E Class: A61K35/74; G01N33/50D4; G01N33/569K; G01N33/569K2
Application Number:
EP19870304731 (19870528)
Priority Number: US19860868250 (19860528)
Family: JP63079837
Equivalent:
AU7360387; DK273087; FI872363; MC1822; NO872223
Abstract:
IT HAS BEEN FOUND THAT BACTERIOCINS ARE ABLE TO KILL VIRALLY-INFECTED MAMMALIAN
CELLS, INCLUDING VIRALLY INFECTED WHITE BLOOD CELLS. ACCORDINGLY, VIRAL
INFECTIONS CAN BE DETECTED AND ULTIMATELY TREATED USING THE BACTERIOCINS AND
THE METHODS DESCRIBED HEREIN. THE INVENTION IS PARTICULARLY SUITED TO DETECTION
AND TREATMENT OF AIDS INFECTION AND INFECTIOUS MONONUCLEOSIS INFECTION.
373/1006
36. KR123946 - 25.11.1997
LACTOCOCCUS SP. WHICH PRODUCES NOVEL BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR123946
Inventor(s):
JOON (KR)
KIM SANG-KYO (KR); BAEK YOUNG-JIN (KR); OH SE-JONG (KR); LEE SANG-
Applicant(s):
HANKUK YAGHURT CO LTD (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19940022715 (19940909)
Priority Number: KR19940022715 (19940909)
Family: KR123946
374/1006
37. KR131136 - 11.04.1998
NOVEL BACTERIOCIN PRODUCING ENTEROCOCCUS AND PRESERVATION METHKIMCHI
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR131136
Inventor(s):
HA DUK-MO (KR)
Applicant(s):
DOOSAN INJAE TECH RESEARCH COO (KR); DOOSAN BEVERAGE CO LTD (KR)
IP Class 4 Digits: C12N; A23L
IP Class:
C12N1/20; A23L1/218
Application Number:
KR19940028669 (19941102)
Priority Number: KR19940028669 (19941102)
Family: KR131136
375/1006
38. KR139397 - 15.06.1998
BACTERIOCIN POLYPEPTIDE PRODUCED BY LACTOCOCCUS SPECIES HY49
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR139397
Inventor(s):
JOON (KR)
KIM SANG-KYO (KR); BAEK YOUNG-JIN (KR); OH SE-JONG (KR); LEE SANG-
Applicant(s):
KOREA YAKULT CO LTD (KR)
IP Class 4 Digits: C07K; C12P
IP Class:
C07K14/195; C12P21/00
Application Number:
KR19940027413 (19941026)
Priority Number: KR19940027413 (19941026)
Family: KR139397
376/1006
39. KR139398 - 15.06.1998
CULTURE MEDIUM FOR LACTOCOCCUS WHICH PRODUCE BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR139398
Inventor(s):
JOON (KR)
KIM SANG-KYO (KR); BAEK YOUNG-JIN (KR); OH SE-JONG (KR); LEE SANG-
Applicant(s):
KOREA YAKULT CO LTD (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19940033013 (19941207)
Priority Number: KR19940033013 (19941207)
Family: KR139398
377/1006
40. KR148340 - 15.10.1998
METHOD OF ANTIBIOTIC BACTERIOCIN USING LACTOBACILLUS ACIDOPHILUS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR148340
Inventor(s):
JO (KR)
CHA SUNG-KWAN (KR); HONG SUK-SAN (KR); KIM WANG-JOON (KR); KOO OU-
Applicant(s):
KOREA INST OF FOOD RESEARCH (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19950014279 (19950531)
Priority Number: KR19950014279 (19950531)
Family: KR148340
378/1006
41. KR2000021520 - 25.04.2000
LACTOCOCCUS LACTICS(KFCC-11047) PRODUCING NATURAL ANTIBACTERIAL BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2000021520
Inventor(s):
PYUN YU RYANG (KR); CHOE HAK JONG (KR); CHOE CHAN IK (KR); LEE HAN
SEUNG (KR); KIM TAE SEOK (KR); YEO IK HEON (KR); AN CHEOL (KR); BAEK HYEON DONG (KR);
HYUN GYUNG HWAN (KR)
Applicant(s):
PULMUONE CO LTD (--)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19980040669 (19980930)
Priority Number: KR19980040669 (19980930)
Family: KR2000021520
379/1006
42. KR2000047065 - 25.07.2000
NOVEL LACTOBACILLUS SP. MT-1077 (KCTC 8903P) AND NOVEL BACTERIOCIN PRODUCED
THEREFROM
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2000047065
Inventor(s):
MIN TAE IK (KR); AHN JONG SUK (KR); LEE HUN JOO (KR); PARK CHAN SUN
(KR); KIM SEUNG HO (KR); LEE HYUN SUN (KR); JOO YOON JUNG (KR)
Applicant(s):
KOREA INST SCIENCE TECHNOLOGY (--)
IP Class 4 Digits: C12N
IP Class:
C12N1/20; C12N15/31
Application Number:
KR19980063818 (19981231)
Priority Number: KR19980063818 (19981231)
Family: KR2000047065
380/1006
43. KR2001001200 - 05.01.2001
COMPOSITION CONTAINING BACTERIOCIN EXTRACT FOR PREVENTION AND CURING OF
COMEDONES
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2001001200
Inventor(s):
(KR)
CHOI SEUNG MAN (KR); HAN SANG GIL (KR); KIM MIN JU (KR); KIM YUN SEOK
Applicant(s):
LG CHEMICAL LTD (KR)
IP Class 4 Digits: A61K
IP Class:
A61K7/48
Application Number:
KR19990020268 (19990602)
Priority Number: KR19990020268 (19990602)
Family: KR2001001200
381/1006
44. KR2001038225 - 15.05.2001
BACILLUS POLYFERMENTICUS KD33 HAVING IMPROVED BACTERIOCIN PRODUCTIVITY AND
METHOD FOR PRODUCING BACTERIOCIN THEREBY
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2001038225
Inventor(s):
PAIK HYUN DONG (KR); LEE GWANG HO (KR)
Applicant(s):
PAIK HYUN DONG (KR); PROCO BIOTECH CO (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19990046115 (19991022)
Priority Number: KR19990046115 (19991022)
Family: KR2001038225
382/1006
45. KR2001054081 - 02.07.2001
BACTERIOCIN PRODUCING MICROORGANISM STRAIN AND METHOD FOR PRODUCING
BACTERIOCIN USING THE SAME
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2001054081
Inventor(s):
JUN SONG AE (KR); KOO GYEONG MO (KR); PAIK HYUN DONG (KR)
Applicant(s):
PAIK HYUN DONG (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19990054721 (19991203)
Priority Number: KR19990054721 (19991203)
Family: KR2001054081
383/1006
46. KR2001096066 - 07.11.2001
ORAL COMPOSITION CONTAINING BACTERIOCIN FOR PREVENTING AND TREATING
PERIODONTITIS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2001096066
Inventor(s):
HA JAE HWAN (KR)
Applicant(s):
KOOKBO PHARMA CO LTD (KR)
IP Class 4 Digits: A61K
IP Class:
A61K7/16
Application Number:
KR20000019977 (20000417)
Priority Number: KR20000019977 (20000417)
Family: KR2001096066
384/1006
47. KR2002052564 - 04.07.2002
PURIFICATION METHOD OF BACTERIOCIN USING EXPANDED BED ADSORPTION-ION
EXCHANGE CHROMATOGRAPHY
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2002052564
Inventor(s):
CHOI CHAN IK (KR); CHOI HAK JONG (KR); KIM GEON (KR)
Applicant(s):
PYUN YU RYANG (KR)
IP Class 4 Digits: C07K
IP Class:
C07K1/18
Application Number:
KR20000081953 (20001226)
Priority Number: KR20000081953 (20001226)
Family: KR2002052564
385/1006
48. KR2003075003 - 22.09.2003
USE OF BACTERIOCIN PRODUCED BY BACILLUS POLYFERMENTICUS FOR CONTROL OR
ERADICATION OF HELICOBACTER PYLORI
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2003075003
Inventor(s):
KANG JAE SEON (KR); KIM HYE JIN (KR); KIM WON SEOK (KR); LEE BAEK CHUN
(KR); PARK YU SU (KR); SON CHEOL HUN (KR)
Applicant(s):
BINEX CO LTD (KR); LEE BAEK CHUN (KR)
IP Class 4 Digits: A61K
IP Class:
A61K35/74
Application Number:
KR20020014075 (20020315)
Priority Number: KR20020014075 (20020315)
Family: KR2003075003
386/1006
49. KR2003076765 - 29.09.2003
PROCESS FOR PREPARING RAW RICE WINE AND REFINED RICE WINE USING BACTERIOCIN
PRODUCED BY LACTIC ACID BACTERIA
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR2003076765
Inventor(s):
CHO EUN KYUNG (KR); CHO SEOK CHEOL (KR); KOOG MU CHANG (KR); PARK
HYUN JEONG (KR); PYUN YU RYANG (KR); SONG JAE CHUL (KR)
Applicant(s): CHO EUN KYUNG (KR); PARK HYUN JEONG (KR); PYUN YU RYANG (KR); SONG
JAE CHUL (KR)
IP Class 4 Digits: C12G
IP Class:
C12G3/04
Application Number:
KR20020015257 (20020321)
Priority Number: KR20020015257 (20020321)
Family: KR2003076765
387/1006
50. KR209787 - 15.07.1999
A NOVEL LACTOCOCCUS SP WHICH PRODUCE NOVEL BACTERIOCIN AND BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR209787
Inventor(s):
LEE HUN JOO (KR); MIN TAE-IK (KR); AHN JONG-SEOG (KR); PARK CHAN-SUN
(KR); KIM SEUNG-HO (KR); LEE HYUN-SUN (KR); PARK YONG-HA (KR); JOO YUN-JUNG (KR);
PARK BONG-KEUN (KR)
Applicant(s):
KOREA INST SCIENCE TECHNOLOGY (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19970015638 (19970425)
Priority Number: KR19970015638 (19970425)
Family: KR209787
388/1006
51. KR9405543 - 30.10.1991
BACTERIOCIN PEPTIDE
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR9405543
Inventor(s):
HENDERSON JAMES T (US); VAN WASSENAAR PIETER DIRK (NL)
Applicant(s):
MICROLIFE TECHNICS (US)
IP Class 4 Digits: C07K; C12P
IP Class:
C12P21/02; C07K7/10
E Class: C07K14/195
Application Number:
EP19910101895 (19910211)
Priority Number: US19900514102 (19900425)
Family: KR9405543
Equivalent:
AU631302; AU7297891; CA2035389; DE453719T; DE69106201D; DE69106201T;
DK453719T; ES2039312T; GR92300016T; JP2005671C; JP6239890; JP7039437B; NZ237028
Cited Document(s):
EP0293547
Abstract:
A PEPTIDE WHICH IS DERIVED FROM A BACTERIOCIN IS DESCRIBED. THE PEPTIDE HAS A
MOLECULAR WEIGHT OF 4629 WHICH IS ABOUT ONE-THIRD THE APPARENT MOLECULAR
WEIGHT OF THE NATURALLY OCCURRING BACTERIOCIN OF 16,500. THE PEPTIDE CAN BE
DERIVED BY PURIFICATION OF THE NATURAL BACTERIOCIN OR CAN BE DERIVED CHEMICALLY.
THE BACTERIOCIN HAS A HIGH ACTIVITY AGAINST LISTERIA MONOCYTOGENES AND OTHER
BACTERIA.Description:
389/1006
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a peptide which is a bacteriocin. In particular, the present invention
relates to the peptide in chemically pure form having a specific identified amino acid sequence and
which is derived from a bacteriocin isolated from Pediococcus acidilactici NRRL-B-18050.
(2) Prior Art
Bhunia, et al (Bhunia, A. K., et al, J. Indust. Micro. 2 319-322 (1987); and Bhunia, A. K., et al., J. Appl.
Bact. 65 261-268 (1988)) reported that Pediococcus acidilactici strain H produced a bacteriocin,
pediocin H with a molecular weight estimated by SDS-PAGE to be about 2700 Da (Bhunia, 1988).
The reported spectrum of activity, however, includes Staphylococcus aureus which is not a
characteristic of the bacteriocin of the present invention.
Tagg et al classify bacteriocins (Tagg, J. R., et al, Bacteriol. Rev. 40, 722-756 (1976)). The
lantibiotics are a category of bacteriocins of gram-positive bacteria. They are characterized by the
presence of lanthionine bridges and dehydroalanine residues. They are also small, basic proteins.
The bacteriocin of the present invention isolated from culture medium shows no evidence of posttranslational modification of any of the amino acid side chains and therefore cannot be classified as a
lantibiotic although the size and charge are characteristic of lantibiotic proteins. Also the amino acid
sequence of some of the lantibiotics is known and is not similar to the bacteriocin of the present
invention.
Hoover, et al (Hoover, D. G., et al., J. Food Prot. 51 29-31, (1988)) reported bacterocin activity
associated with a 5.5 MDa plasmid in several strains of Pediococcus isolated from fermented
390/1006
sausage including Pediococcus acidilactici NRRL-B-5627 which is essentially the same strain as
NRRL-B-18050 described hereinafter. Graham and McKay (Graham, D. C. et al., Appl. Env. Micro.
50, 532-534, (1985)) linked bacteriocin production to a 10.5 MDa plasmid in Pediococcus
pentosaceus FBB-63. Daeschel and Klaenhammer (Daeschel, M.A. and Klaenhammer, T. R., Appl.
Env. Micro. 50, 1538-1541, (1985)) reported that bacteriocin production in Pediococcus pentosaceus
FBB-61 and L7230 was encoded by a 13.6 MDa plasmid. None of the bacteriocins were purified and
sequenced. Purification is difficult since the bacteriocins are sensitive to structural changes caused
by the purification.
Bacteriocins from Pediococcus acidilactici NRRL-B-18050 is known as described in U.S. Patent No.
4,883,673 to Gonzalez. The bacteriocin produced was an impure form in a growth medium or
derived from the growth medium by ammonium sulfate precipitation. The purified bacteriocin had an
activity of up to about 32,000 Au per ml. This bacteriocin was determined not to be pure. Also it was
determined that the bacteriocin had an apparent molecular weight of about 16,500 as described in
U.S patent application Serial No. 07/148,044 filed January 25, 1988 by Vandenbergh. The problem
was that since the bacteriocin was still impure it could not be synthesized by synthetic chemical
means. The presence of the impurities produced variable results in use of impure bacteriocin.
OBJECTS
It is therefore an object of the present invention to provide a pure, chemically identified bacteriocin
which is a peptide. Further, it is an object of the present invention to provide a peptide which can be
derived by chemical synthesis. These and other objects will become increasingly apparent by
reference to the following description and the drawings.
IN THE DRAWINGS
Figure 1 shows the amino acid sequence of the tryptic fragments of the peptide of the present
invention.
391/1006
GENERAL DESCRIPTION
The present invention relates to a peptide consisting of an amino acid sequence and disulfide
bonds between Cys 9 and Cys 14 and between Cys 24 and Cys 44.
The peptide of the present invention has a molecular weight of 4629 daltons which is about 1/3rd the
apparent molecular weight of the impure bacteriocin as it is derived from the growth medium. The
bacteriocin exhibits an Au of about 50,000 Au per ml, which is significantly higher than the Au of the
impure bacteriocin.
The peptide of the present invention can be synthesized by chemical means. Solid phase peptide
synthesis (SPPS) is a method of preparing peptides by chemically coupling the individual amino
acids in a specified sequence. It is described in Chem. Anal. Solid Phase Biochem; Chapter 101,
pages 507-534 (1983). Synthesis begins at the C-terminal amino acid which is covalently attached to
a solid support. The next amino acid is activated at the C-terminal and is blocked from reactivity at
the N-terminal; therefore covalent coupling (formation of a peptide bond) occurs between the Cterminal of the second amino acid and the N-terminal of the first amino acid. After coupling is
complete, the N-terminal of the second amino acid is deprotected and the cycle is begun again with
the third amino acid.After addition of the final amino acid (the N-terminal of the peptide) the peptide
is cleaved from the solid support and the various side chain protecting groups are removed.
In the original Merrifield method of SPPS, the solid support is typically a chloromethylated copolymer
of styrene and divinylbenzene. Cleavage from this support gives a free acid group at the C-terminus.
For the production of peptide amides, a 4-methyl-benzylhydramine resin is used. N-terminal
protection during coupling is provided by a t-butoxycarbonyl (tBOC) group. The protecting group is
removed with anhydrous hydrochloric acid and the resulting amine hydrochloride is neutralized with
triethylamine. The next t-BOC amino acid is activated with dicyclohexylcarbodiimide or
diisopropylcarbodiimide and coupled to the prior amino acid. The cycle is repeated for each
subsequent amino acid. Peptide-resin cleavage and side chain deprotection is performed
simultaneously with anhydrous hydrofluoric acid to give the peptide or peptide amide.
Improvements to the Merrifield method deal with solutions to specific problems which may result in
reduced yield. Insufficient yield is a primary limiting factor in the production of long sequences. An
example of such an improvement is the use of the 9-fluorenylmethoxycarbonyl(Fmoc) protecting
392/1006
group. The Fmoc protectant provides an acid stable group which is removed by treatment with an
amine such as piperidine. The use of Fmoc can improve yield since it provides for the use of base for
deprotection rather than acid. The use of acid deprotectant in the original Merrifield procedure has
been associated with premature cleavage of the peptide-resin bond resulting in lowered yield.
SPECIFIC DESCRIPTION
The bacteriocin PA-1 was purified to homogeneity by gel filtration and ion exchange
chromatography, and reversed-phase liquid chromatography after considerable effort and numerous
failures. The amino acid sequence of PA-1 was found to correspond to an open reading frame of the
DNA sequence of a 6.2 megaldalton plasmid associated with bacteriocin activity in Pediococcus
acidilactici PAC 1.0. From the sequence data, the conclusion was made that PA-1 was a basic
protein with a molecular mass of 4629 daltons. The presence of four cysteine residues gave rise to
the possibility of two disulfide bridges. Analysis of tryptic fragments showed that a disulfide bond
existed between Cys-9 and Cys-14 and between Cys-24 and Cys-44. The purified bacteriocin was
active against the bacterium, Listeria monocytogenes and other bacteria as set forth hereinafter.
Example 1
Growth Medium, Bacteriocin Production, And Assay Of Activity. One liter of MRS broth (Difco, Detroit,
Michigan) with 4% yeast extract (Oxoid TM , Basingstoke, England) was inoculated at 1% by volume
with an 8 hour old culture of strain PAC 1.0 grown in MRS broth and was grown statically at 35 DEG
C for 18 hours. Cells were removed by centrifugation at 16,300 x g for 15 minutes at 4 DEG C. The
supernatant was filtered through a 0.20 micron filter (Millipore) and was kept frozen at -20 DEG C
until use. Bacteriocin production was assayed by spotting 5 ul of a serial twofold dilution series onto
MRS plates overlaid with soft agar seeded with Pediococcus pentosaceus FBB63 indicator cells.
One AU is defined as the highest dilution yielding a definite zone on the indicator lawn.
Purification of PA-1. Supernatant was neutralized to pH 6.0 with sodium hydroxide prior to gel
filtration. A 450 ml aliquot of neutralized supernatant was applied to a 5 cm x 55 cm column of
Spectra/Gel AcA 202 TM (Spectrum, Los Angeles, CA) gel filtration resin which had been
393/1006
equilibrated against 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES) (Research Organics,
Cleveland, OH), pH 6.0. Activity was eluted using the same buffer. Activity was obtained beginning
at the void volume and continued to elute throughout six void volumes. Active fractions were pooled
and applied to a 2.5 cm x 90 cm CM-Sepharose TM column (Pharmacia, Piscataway, NJ) reequilibrated against 0.05 M MES, PH 6.0. Activity was eluted with a linear gradient to 0.05 M MES
containing 1 M sodium chloride, pH 6.0.Active fractions were pooled and dialyzed against a 10 fold
excess of water using 1000 Da molecular weight cut-off dialysis tubing (Spectra-Por 6 TM , Spectrum,
Los Angeles, CA). Dialysate volume was reduced 12 fold by applying the dialysis tubing directly to
solid 20 KDa polyethylene glycol (Carbowax, Union Carbide) and was then further reduced 3.5 fold
by vacuum centrifugation (Speed-Vac TM , Savant, Farmingdale, NY). Concentrated PA-1 was
applied to a 1.0cm x 25cm C18 reversed-phase column (Vydac TM , Hesperia, CA) equilibrated with
0.1% trifluoroacetic acid. Activity was eluted with a linear gradient to 45% acetonitrile over 30
minutes at 1.5 ml/min. Active fractions were determined by directly spotting aliquots of column
effluent on MRS plates overlaid with soft agar containing Pediococcus pentosaceus FBB63
cells.Active fractions were dried by vacuum centrifugation and stored at -20 DEG C.
Example 2
Analysis of Purified Bacteriocin
Amino Acid Analysis. Samples for hydrolysis were dried by vacuum dehydration, reconstituted in 0.1
ml 6N HCl, dried again, and hydrolyzed in vacuo at 100 deg C in 8mm x 60mm hydrolysis tubes
(Pierce, Rockford, IL) for 24 hours in a Reacti-Therm TM heating block (Pierce, Rockford, IL). Phenyl
isothiocyanate (PITC) derivatives were made using the directions supplied with a Pico-Tag
derivatization system (Waters, division of Millipore, Milford, MA) and the PITC derivatives were
detected at 254 nm using the suggested Pico-Tag protocol except that the reversed-phase column
was a 0.45cm x 25cm C18 column (Vydac, Hesperia, CA) instead of the proprietary Pico-Tag column.
Amino Acid Sequencing.Sequence information was obtained with a gas phase sequencer ABI 475A
using protocols supplied by ABI (Applied Biosystems, Inc., Foster City, California). To obtain the
sequence of the C terminal region, the protein was first cleaved at Met-31 by mising with a 2 mg/ml
solution of cyanogen bromide in 88% formic acid overnight at room temperature. Sequence
information was then obtained simultaneously beginning with residues 1 and 32.
394/1006
SDS Polyacrylamide Gel Electrophoresis. Gels were prepared at either a fixed acrylamide
concentration of 12.5% or as a linear gradient from 10% to 25%. The crosslinking agent was
piperazine diacrylamide (BioRad, Richmond, CA).Gels were stained with Coomassie Brilliant Blue G
followed by silver staining (BioRad, Richmond, CA) or were unstained in order to perform an activity
analysis by overlaying the gel with soft agar containing indicator FBB-63 cells (Bhunia, 1987).
Disulfide bond assignment. The presence of four cysteine residues in the sequence of PA-1 gives
rise to the possibility that two disulfide bonds exist. Since determination of free sulfhydryl residues
using Ellman's reagent (DTNB, Sigma) has indicated the absence of free sulfhydryl groups, the
conclusion was made that two disulfide bonds are present in PA-1. The identity of the particular
cysteine residues involved in these disulfide linkages is set forth hereinafter.
The distribution of lysine residues in the PA-1 sequence is such that a tryptic digest will provide three
peptide fragments containing a single cysteine residue. Disulfide bonded peptides produced by
tryptic digestion are reduced to give rise to the component fragments. Amino acid analysis of each
fragment is compared to calculated values from sequence data to identify the fragment with its
tryptic peptide. In this manner, an unambiguous assignment of the disulfide bonding arrangement is
possible.
Tryptic cleavage of PA-1 is expected to give rise to three peptide chains and two single amino acids
(see also Figure 1) as shown in Table 1: Tryptic digestion was done by reaction of 50 ug of purified
PA-1 with 1% by weight trypsin (TPCK, Sigma) overnight at 37 DEG C in 50 ul pH 8 ammonium
bicarbonate buffer containing 0.1 mM calcium chloride. The reaction mix was applied to a 4.5 x 25
cm C-18 reversed phase column (Vydac, Hesperia, CA). Peaks were detected by monitoring
absorbence at 220 nanometers.
Amino acid analysis was performed after overnight vapor phase hydrolysis with 6 N HCl in vacuo at
110 DEG C. PTC amino acid derivatives were produced by reacting with 10% PITC (Pierce) in 70%
ethanol. Analysis of PTC amino acids was accomplished with a 0.45 x 15 cm reversed phase C-18
column (Vydac) using a sodium acetate - acetonitrile gradient. Amino acid hydrolysis standards were
purchased from Pierce (Rockford, IL).
The crude tryptic digest was applied to a reversed phase HPLC column and peptides were eluted
with an acetonitrile gradient. As shown in Table 2, two major peaks were obtained (peaks 7 and 10)
and isolated for further examination. Material from each of the two peaks was reduced and the
reduction products were separated by reversed phase HPLC. Two peaks were obtained upon
395/1006
reduction of the peptide peak 7, indicating that it had been composed of two peptides linked by a
disulfide bond. Results of amino acid analysis showed that the composition of peak 7a matched the
calculated composition of peptide T1 and peak 7b similarly matched T2. Further, the unreduced
peak 7 agreed with the sum of T1 and T2 as well as with the sum of 7a and 7b.Since T1 and T2 are
connected by a disulfide bond, this bond must be between Cys-9 from T1 and Cys-14 from T2.
Peak 7-14.36 is most likely T1, 7-16.03 is T2 and 10-21.38 is T3. Disulfide bond assignments: Cys-9
and Cys-14, Cys-24 and Cys-44. nd = not determined.
A second disulfide bond must then exist between Cys-24 and Cys-44. Reduction of this second bond
would yield only a single peak in the chromatogram since the peptide bond to Cys-44 was disrupted
by tryptic cleavage. Detection at 220 nm is sensitive to peptide bonds, not single amino acids. Peak
10 was reduced and rechromatographed, giving rise to a single peak. Amino acid analysis indicated
a strong similarity to tryptic fragment T3. Since fragment T3 contains Cys-24, a disulfide bond to Cys9 or Cys-14 would have been detected by the appearance of multiple peaks upon reduction of this
fragment. Since a single peak was obtained after reduction, Cys-24 is presumed disulfide bonded to
Cys-44.
Peptide 7a, corresponding to T1, contains an excess over the one lysine residue predicted.
Unreduced peptide 7 also displays this apparent anomaly, with 2.6 moles lysine instead of 2. An
explanation of these results is that the N-terminal lysine residue is resistant to the action of trypsin.
This is not a really surprising result since the N-terminal is often a freely mobile region although
folding might hinder mobility. The N-terminal lysine residue might present difficulty in binding to
trypsin due to this mobility. In addition, the amino terminal amino group might be somewhat positively
charged at pH 8 and might also interfere with trypsin binding.
Crude bacterial culture medium was applied to the AcA 202 gel filtration resin and bacteriocin
activity eluted beginning at the column void volume and continuing for several column volumes.
Since the exclusion volume of this gel filtration medium is 20 KDa, the apparent molecular weight of
PA-1 activity in crude medium is nearly 20 KDa. This result has also been observed during tangential
filtration using a 10 KDa membrane and during hollow fiber diafiltration using 6KDa fiber pore size. In
all cases, PA-1 activity in crude medium is associated with an apparent molecular weight of greater
than 10 KDa. In a previous gel filtration study, purified PA-1 eluted from a calibrated gel filtration
column at an apparent molecular weight of 16,500. After elution from the cation exchange resin and
dialyzing, PA-1 activity elutes much later from the AC202 column.Table 3 shows the activity of the
various fractions. Characterization. An amino acid analysis of PA-1 is shown in Table 4 along with
396/1006
the amino acid content obtained by sequencing. A titration of free cysteine residues with Ellman's
reagent (DTNB, Sigma) reveals no free titratable sulfhydryl groups.
Disulfide bond assignment.
Separation of PA-1 tryptic fragments by HPLC gave four major peaks. The first eluting peak, when
reduced, gave rise to two peaks by HPLC. Amino acid analysis of these two peaks showed strong
similarity to the amino acid composition of two tryptic fragments T1 and T2. This is good evidence
that a disulfide bond exists between Cys-9 on T1 and Cys-14 on T2. Analysis of free sulfhydryl
groups using Ellman's reagent gave a negative result so a second disulfide bond between Cys-24
and Cys-44 is postulated to account for the lack of free sulfhydryl groups.
Sequence analysis. Primary sequence analysis by Chou-Fasman routines indicate a structure as
follows: consisting mostly of beta turns and random coil. Residues 21-25 show a propensity for beta
sheet.
Example 3
Molecular Weight
The purified bacteriocin is derived from the gene product of plasmid-borne bacteriocin activity
expressed in Pediococcus acidilactici strain NRRL-B-18050. A DNA sequence corresponding to the
amino acid sequence reported here lies in an open reading frame of a 7.5 MDa plasmid in an area of
the plasmid which has been shown necessary for expression of bacteriocin activity. From the
sequence data, a molecular weight of 4629 was calculated. This value is lower than the apparent
molecular weight calculated by using standard proteins with a mobility versus log molecular weight
curve from the SDS gel.
Example 4
397/1006
Inhibitory Activity of Bacteriocin
The bacteriocin was effective in inhibiting the bacterial strains listed in Table 5.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
1. A peptide consisting of an amino acid sequence and disulfide bonds between Cys 9 and Cys 14
and between Cys 24 and Cys 44.
398/1006
52. KR9615891 - 23.11.1996
LACTOCOCCUS SP. BL1 AND NEW BACTERIOCIN, AND PROCESSING OF NEW BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=KR9615891
Inventor(s):
YU JIN-YOUNG (KR); JUNG KUN-SUB (KR); CHOE SHIN-YANG (KR); KOO
YOUNG-JO (KR)
Applicant(s):
KOREA FOODS DEV I (KR)
IP Class 4 Digits: C12N
IP Class:
C12N1/20
Application Number:
KR19930010815 (19930614)
Priority Number: KR19930010815 (19930614)
Family: KR9615891
Abstract:
THE NOVEL BACTERIOCIN WHICH IS A KIND OF ANTIBIOTICS, TERMED KOFRIJIN-1, IS
PREPARED BY CULTURING LACTOCOCCUS SP. BL1(KFCC-10790), WARMING THE CULTURE
MEDIA UP IN A DOUBLE BOILER, COOLING, CENTRIFUGING, REMOVING A PRECIPITATION,
REMOVING A PRECIPITATION 2-3 TIMES AFTER ADDING A SOLVENT IN THE SUPERNATANT,
DISSOLVING AN ACTIVE FRACTION OF A FINAL PRECIPITATION IN HCL SOLUTION, FREEZEDRYING, AND HOMOGENIZING TO OBTAIN THE FINAL PRODUCT. THE KOFRIJIN-1 SHOWS A
STRONG ANTIBIOTIC EFFECT TO GRAM POSITIVE MICROORGANISMS, AND DON'T SHOWS ANY
HARMFUL EFFECT TO EUKARYOTES. ITS TITER IS 2.2=G105IU/G AND A MOLECULAR WEIGHT IS
5,600 DALTON.
399/1006
53. NZ225979 - 02.08.1989
METHOD FOR INHIBITING LISTERIA MONOCYTOGENES USING A BACTERIOCIN
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ225979
Inventor(s):
VANDENBERGH PETER A (--); PUCCI MICHAEL J (--); KUNKA BLAIR S (--);
VEDAMUTHU EBENEZER R (--)
Applicant(s):
MICROLIFE TECHNICS (US)
IP Class 4 Digits: A23L; C12P; A01N; A23C
IP Class:
C12P1/04; A01N63/00; A23C3/08; A23C9/12; A23L3/34
E Class: A23L3/3571; C07K14/195
Application Number:
EP19890101125 (19890123)
Priority Number: US19880148044 (19880125)
Family: EP0326062
Equivalent:
AU2288888; AU610649; CA1319850; DE326062T; DE68902320D; DE68902320T;
ES2011234T; GR3006077T; GR90300010T; JP1766259C; JP2005845; JP4051154B; US4929445
Cited Document(s):
EP0265884; DE2714415
Abstract:
A METHOD FOR INHIBITING LISTERIA MONOCYTOGENES IN A FOOD OR OTHER MATERIAL
WHICH CAN BE CONTAMINATED WITH THIS PATHOGEN USING A BACTERIOCIN PRODUCED BY
DNA IN PEDIOCOCCUS ACIDILACTICI IS DESCRIBED. THE BACTERIOCIN IS PARTICULARLY
PRODUCED BY PEDIOCOCCUS ACIDILACTICI CONTAINING A 6.2 MDAL (9.4 KILOBASE PAIRS)
PLASMID ENCODING FOR THE BACTERIOCIN.Description:
400/1006
METHOD FOR INHIBITING Listeria monocytogenes USING A BACTERIOCIN
BACKGROUND OF THE INVENTION
(1) Summary of the Invention
The present invention relates to a method for inhibiting Listeria monocytogenes, a foodborne
pathogen, in a food or other materials which can be contaminated by this pathogen using a
bacteriocin. In particular the present invention relates to the use of a bacteriocin derived from
Pediococcus acidilactici to inhibit the Listeria monocytogenes in the food or other materials which
can be contaminated by this pathogen.
(2) Prior Art
The term "bacteriocin" refers to a protein of the colicin type, characterized by lethal biosynthesis by
the producing bacterium, intraspecific activity in related species of bacteria, and adsorption to
specific receptors on the sensitive bacteria (Tagg, J. R., A. S. Dajani, and L. W. Wannamaker,
Bacteriol. Rev. 40:722-756 (1976)). Bacteriocins have been described as being produced by many
bacteria, however the bacterial strains inhibited by the bacteriocin are usually related to the strain
which produces the bacteriocin (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 53:25342538 (1987)).
In U.S. application Serial No. 012,619 (corresponding to EP 88 101 624.0), filed February 9, 1987 by
Carlos Gonzalez, which is assigned to a common assignee, the preparation and use of a bacteriocin
derived from Pediococcus acidilactici, particularly Pediococcus acidilactici NRRL-B-18050, to inhibit
spoilage bacteria, particularly Lactobacillus fermentum and Lactobacillus bifermentum, is described.
These spoilage bacteria are lactic acid producing strains of the genus Lactobacillus and
Pediococcus.No activity was found against Lactococcus lactis, Lactococcus lactis subsp.
diacetylactis, Lactococcus cremoris (previously in the genus "Streptococcus") or Streptococcus
401/1006
thermophilus, Staphylococcus aureus, Micrococcus varians, Micrococcus sodonensis,
Staphylococcus xylosus, Staphylococcus epidermidis, Staphylococcus carnosus, Lactobacillus
acidophilus, Lactobacillus lactis and Lactobacillus bulgaricus. It was concluded that the bacteriocin
had a limited range of inhibitory activity related to gram positive, lactic acid bacteria.
Listeria monocytogenes has been demonstrated to be transmitted in contaminated food. (J. Applied
Bact. 63:1-11 (1987)). While the culture is sensitive to pH and an acid environment will usually inhibit
the growth of this microorganism, there are many instances of foods where the pH is not sufficiently
acidic.
Listeria monocytogenes produces severe illness in animals and humans. The characteristics of the
disease and this species are described in J. Applied Bact. 63:1-11 (1987). Listeria monocytogenes
grows well at refrigeration temperatures and thus the usual means of inhibiting the growth of Listeria
monocytogenes by refrigeration is ineffective. Because of this there are problems in the marketplace,
an example of which is the recently published recall of several brands of Listeria contaminated ice
cream bars. According to Bergey's Manual of Systematic Bacteriology, Vol 2: 1235-1245, (1986), the
taxonomic position of the genus Listeria with regard to other genera is still not resolved. However, it is
clear that Listeria monocytogenes is quite distinct from Lactobacillus, Staphylococcus, Micrococcus,
Pediococcus and Streptococcus (Lactococcus).
OBJECTS
It is therefore an object of the present invention to provide a method for inhibiting Listeria
monocytogenes in foods and other materials which can be contaminated by this pathogen using a
bacteriocin. Further, it is an object of the present invention to provide a method which is simple and
economical to perform. These and other objects will become increasingly apparent by reference to
the following description and the drawings.
IN THE DRAWINGS
Figure 1 shows the effect of addition of a powder containing the bacteriocin, designated PA-1, from
Pediococcus acidilactici to an exponentially growing Listeria monocytogenes culture. The PA-1
402/1006
powder was added to a L. monocytogenes culture at 200 U(units)/ml or 500 U/ml and turbidity (which
is an indicator of the number of cells) was monitored over time at 660 nanometers.The symbols used
are : cirf& , control (no PA-1 added); cir& , 200 U/ml PA-1; @ , 500 U/ml PA-1.
Figure 2 shows a restriction endonuclease map of plasmid pSRQ11 which encodes for the
production of the bacteriocin PA-1 in Pediococcus acidilactici NRRL-B-18050 (also known as PAC
1.0). Plasmid pSRQ11, having a size of 9.4 kilobases, is shown with the locations of several
restriction endonuclease sites. The approximate location of the coding region of bacteriocin PA-1
gene is shown. Four restriction sites were found within the PA-1 gene:
HindIII, Xba I, Cla I, and PvuII.
GENERAL DESCRIPTION
The present invention relates to a method for inhibiting growth of Listeria monocytogenes in a
material which is a food or other material which can be contaminated with the Listeria
monocytogenes which comprises: providing a bacteriocin obtained from DNA which encodes for the
bacteriocin in Pediococcus acidilactici in the material in an effective amount which inhibits the
Listeria monocytogenes..
The present invention relates to a method for inhibiting growth of Listeria monocytogenes in a food
which can contain the Listeria monocytogenes as a contaminant which comprises: adding a
bacteriocin obtained from a bacterium containing DNA which encodes for the bacteriocin in
Pediococcus acidilactici into the food in an effective amount which inhibits the Listeria
monocytogenes. A preferred bacteriocin is PA-1
The preferred bacteriocin PA-1 used in the present invention is produced by Pediococcus acidilactici
NRRL-B-18050, which is deposited with the Northern Regional Research Laboratory in Peoria, Illinois
and is also known herein as PAC 1.0. Pediococcus acidilactici is a commercially available species
used in meat fermentations. This preferred species contains a 9.4 kilobase (6.2 Mdal) plasmid which
encodes for the bacteriocin. Using well known recombinant genetic techniques, the DNA gene
segment encoding for the bacteriocin can be cut and combined with vector DNA and then inserted
into another microorganism which then produces the bacteriocin.
403/1006
The easiest method for providing the bacteriocin, such as PA-1, is to dry the growth medium
containing the bacteriocin after cell growth to produce a powder. The solid materials can be
removed by filtration or centrifugation from the growth medium. Low molecular weight compounds
can be removed by membrane filtration, particularly reverse osmosis. Food grade drying aids such
as non-fat dry milk (NFDM) can be used to dry the solution containing the bacteriocin. The
bacteriocin is a proteinaceous material and can also be separated from the growth medium by
precipitation or by other well known techniques such as reverse osmosis and it can then be dried in
a pure form.The bacteriocin has a molecular weight of about 16,500 daltons and is inactivated by in
vitro mixing with protease, papain or alpha-chymotrypsin and is unaffected by phospholipase C,
lysozyme, Dnase and RNase or heating to 100 DEG C in water and inhibits Listeria monocytogenes
in a pH range between about pH 4 to 9. The preparation of the bacteriocin and its characteristics are
described in Serial No. 12,619.
The bacteriocin is preferably used in the food system in an amount between 1 and 100,000 Arbitrary
Units (AU) of bacteriocin, such as PA-1, per gram of the food. One AU of bacteriocin was defined as
5 microliters of the highest dilution of culture supernatant yielding a definite zone of growth inhibition
with a lawn of an indicator strain of a gram-positive bacteria on an agar plate (Pediococcus
pentosaceus FBB-63 formerly known as Pediococcus cerevisiae FBB-63).
The foods most often associated with contamination by Listeria monocytogenes are milk based
cheeses, ice cream or ice milk and Cottage cheese. Foods that are handled by machinery and are
not heat-treated in final package are particularly vulnerable. Meats, such as beef, pork or poultry,
can be contaminated during or after slaughtering. Fish can also be contaminated in processing.
Medicinal and veterinary products including packaging, lubricants, bandages, culture media and the
like can be contaminated with Listeria monocytogenes. Further, cosmetics and other related
products can be contaminated with this pathogen. The bacteriocin derived from Pediococcus
acidilactici is useful for inhibiting this pathogen in these products, although the risk is greatest in
foods and other products taken orally. All of these products can come in contact with living tissue in
vitro or in vivo and can cause disease.
SPECIFIC DESCRIPTION
404/1006
The methods and materials used for the production of the bacteriocin were as follows:
Bacterial Strain. Pediococcus acidilactici NRRL-B-18050, was routinely grown at 35 DEG C and
cultivated on MRS broth (Difco, Detroit, MI).
Bacteriocin assay. Production of bacteriocin was assayed by spotting cells or filter sterilized broth
samples onto MRS agar (Difco Laboratories, Detroit, Mich.). Filter sterilized broth samples were
diluted by serial dilution (1:1, 1:2, 1:4, 1:8, 1:16, in sterile water) to titer the level of activity. Assay
plates were overlaid with soft agar (0.75%) seeded with indicator cells (Pediococcus pentosaceus
FBB63C). Plates were incubated at 32 DEG C for 18 hours.
Example 1 - Nutritional Studies
Each of the media listed in Table 1 was prepared in 100 ml quantities.
The media were adjusted to pH 6.8 before autoclaving. The media were inoculated with an 8 hour
culture of NRRL-B-18050 at a rate of 1% and then incubated at 35 DEG C for 18 hours. After 18
hours, 25 ml of the above culture were centrifuged at 12,000 xg for 10 minutes at 4 DEG C. The
supernatant was then filter sterilized using a 0.22 microns filter (Millipore, Bedford, MA) and tested
for the least dilution (titer) which inhibited Pediococcus pentosaceus FBB63C as the indicator strain.
The results of the nutritional study are summarized in Table 1. The most effective medium for the
production of bacteriocin PA-1 appears to be MRS broth supplemented with 2% yeast extract. Other
media were not as effective, however protein supplements generally appear to stimulate bacteriocin
production. Whey based media were the least effective for the production of bacteriocin PA-1.
Example 2 - Production of Dried Bacteriocin PA-1.
Pediococcus acidilactici NRRL-B-18050 was grown overnight at 35 DEG C in 1 liter MRS broth
supplemented with 2% yeast extract (Difco, Detroit, MI). The cells were pelleted by centrifugation
405/1006
and the supernatant was collected. Nonfat dry milk powder was added to 10% (weight/volume) to
facilitate drying. This mixture then was lyophilized into a dry powder.
Example 3
Activity of the bacteriocin (PA-1) from Pediococcus acidilactici NRRL-B-18050 against Listeria
monocytogenes in Dressed Cottage cheese.
Listeria monocytogenes was added to dressed Cottage cheese (pH 5.1) at a rate of 6.5 x 10
cfu/gram of dressed Cottage cheese. The bacteriocin powder of Example 1 (PA-1) was added to the
Cottage cheese at a rate of 10 or 50 U (units)/g dressed Cottage cheese. The samples were mixed
and incubated at 4 DEG C and aliquots were removed and plated onto McBride's Agar (Difco,
Detroit, Michigan) and incubated at 32 DEG C and examined for growth of Listeria monocytogenes.
The results are shown in Table 2.
Columns=3
Head Col 1: Sample
Head Col 2: Contents
Head Col 3: Growth of Listeria at 24 h
A-0
BLM
7.5 x10 cfu/g
CPA-1, 50 U/g0
DLM
; PA-1, 10U/g 0
ELM
; PA-1, 50U/g 0
LM= Listeria monocytogenes at 6.5 x 10 cells per gram.
406/1006
Example 4
Activity of the Bacteriocin (PA-1) against Listeria monocytogenes in Half and Half Cream.
Listeria monocytogenes was added to the cream (pH 6.7) at a rate of 4.3 x 10 cfu/ml of cream. The
bacteriocin powder PA-1 was added to the cream at a rate of 10 or 50 U/ml cream. The samples
were mixed and incubated at 4 DEG C and aliquots were removed and examined for growth of
Listeria monocytogenes.
The results are shown in Table 3.
Columns=3
Head Col 1: Sample
Head Col 2: Contents
Head Col 3: Growth of Listeria at 24 h
A-0
BLM
, 2.2x10 cfu/g
CLM
, PA,1, 50 U/ml 0
LM= Listeria monocytogenes at 4.3 x 10 cells per ml.
Example 5
407/1006
Activity of Bacteriocin (PA-1) against Listeria monocytogenes in cheese sauce.
Listeria monocytogenes was added to the cheese sauce (pH 5.25) at a rate of 5.3 x 10 cfu/g of
cheese sauce. The bacteriocin powder PA-1 was added to the cheese sauce at a rate of 10 or 50
U/g of sauce. The samples were mixed and incubated at 4 DEG C and aliquots were removed and
examined for growth of Listeria monocytogenes. The results are shown in Table 4.
Columns=3
Head Col 1: Sample
Head Col 2: Contents
Head Col 3: Growth of Listeria at 24 h
A-0
BLM
, 2.0x10 cfu/g
CLM
; PA-1, 10U/g 4.0x10 cfu/g
DLM
; PA-1, 50U/g 0
LM= Listeria monocytogenes at 5.3 x 10 cells per gram.
As can be seen from Examples 3 to 5, the bacteriocin PA-1 was effective in controlling the growth of
Listeria monocytogenes in food systems at various pH values. In the cream, Example 4, the pH was
initially 6.7 and the Listeria were inhibited. In the cheese sauce Example 5, the pH was slightly acidic
at a pH of 5.25. In both of the above examples and the Cottage cheese example, Listeria
monocytogenes was inhibited.
Example 6
408/1006
Effect of the bacteriocin powder PA-1 on an exponential culture of Listeria monocytogenes.
Sterile PA-1 bacteriocin powder was added to an exponential culture of Listeria monocytogenes at a
rate of 200 U/ml of broth and 500 U/ml of broth. Listeria monocytogenes was grown in APT broth at
32 DEG C. Absorbance was followed over time at 660 nm. Figure 1 shows that when the sterile
powder was added to the culture after 3.75 hours, the absorbance at 660 nm began to decrease,
whereas the control flask continued to grow and increase in turbidity thus indicating that bacteriocin
PA-1 was not only inhibitory but also bacteriocidal for Listeria monocytogenes.
Example 7
Minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC) of the
bacteriocin PA-1 against Listeria monocytogenes
Bacteriocin PA-1 powder was dissolved in APT broth and two-fold serially diluted to concentrations
ranging from 1000 U/ml to 2.0 U/ml. Approximately 1 x 10 Listeria monocytogenes/ml were added to
each of the tubes which then were incubated for 24 hours at 32 DEG C. The MIC value was the
lowest concentration tube displaying no visible turbidity. The MBC value was the lowest
concentration tube which when plated onto APT agar showed no colony forming units (CFU's). The
results are summarized in Table 5.
Columns=3
Head Col 1: Strain
Head Col 2: MIC
Head Col 3: MBC
Listeria monocytogenes LM01<2.0 U/ml 7.8 U/ml
Pediococcus pentosaceus FBB63C7.8 U/ml 31.3 U/ml
409/1006
As indicated in Table 5, Listeria monocytogenes is quite sensitive to PA-1 bacteriocin, even more so
than the indicator P. pentosaceus strain FBB63C.
Example 8
Plasmid DNA was isolated from Pediococcus acidilactici NRRL-B-18050, and DNA samples were
subjected to agarose gel electrophoresis as previously described (Gonzalez, C.F., and B.S. Kunka,
Appl. Environ. Microbiol. 46:81-89 (1983)). A restriction map as shown in Figure 2 of the plasmid
pSRQ11 was obtained by combinations of the following procedures: (i) analysis of DNA fragments
obtained after digestion with two enzymes and (ii) analysis of the fragments on 0.7% agarose and
5.0% polyacrylamide gel electrophoresis.
Based on the approximate size of the bacteriocin PA-1 of 16,500 daltons, the gene for the
bacteriocin PA-1 appears to be encoded on a 450 bp segment on the plasmid pSRQ11, between the
Ndel site at 1.6 kb and the 3.2 kb Clal site (Figure 2).
The bacteriocin (PA-1) was effective even at pH values which were near neutrality. The observation
that the bacteriocin (PA-1) inhibits Listeria monocytogenes was unexpected because (i) Listeria
monocytogenes is a human pathogen (ii) the ecological niche of Listeria monocytogenes is not
similar to Pediococcus acidilactici.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
-1- A method for inhibiting growth of Listeria monocytogenes in a material which is a food or other
material which can be contaminated with the Listeria monocytogenes which comprises:
providing a bacteriocin obtained from DNA which encodes for the bacteriocin in Pediococcus
acidilactici in the material in an effective amount which inhibits the Listeria monocytogenes.
410/1006
-2- A method for inhibiting growth of Listeria monocytogenes in a food which can contain the Listeria
monocytogenes as a contaminant which comprises::
adding a bacteriocin obtained from a bacterium containing DNA which encodes for the bacteriocin
in Pediococcus acidilactici into the food in an effective amount which inhibits the Listeria
monocytogenes.
-3- The method of Claim 2 wherein the bacteriocin which is added to the food is in an aqueous
solution derived from a growth medium incubated with the Pediococcus acidilactici.
-4- The method of Claim 2 wherein an aqueous solution containing the bacteriocin derived from a
growth medium incubated with the Pediococcus acidilactici is dried to a flowable powder with or
without a food grade drying aid and wherein the flowable powder is added to the food.
-5- The method of Claim 2 wherein the food contains between about 1 and 100,000 AU of the
bacteriocin per gram of food.
-6- The method of Claim 1 wherein the DNA is a plasmid 9.4 kilobase pairs in molecular size which
encodes for the bacteriocin.
-7- The method of Claim 1 wherein the Pediococcus acidilactici is Pediococcus acidilactici NRRL-B18050.
-8- The method of Claim 1 wherein the bacteriocin has a molecular weight of about 16,500 daltons
and is inactivated by in vitro mixing with protease, papain or alpha-chymotrypsin and is unaffected
by phospholipase C, lysozyme, DNase and RNase or heating to 100 DEG C in water and wherein the
bacteriocin inhibits the Listeria monocytogenes in a pH range between about pH 4 to 9.
-9- The method of Claim 2 wherein the food contains milk or a milk derivative.
-10- The method of Claim 2 wherein the food is a prepared salad.
-11- The method of Claim 2 wherein the food is or contains meat.
-12- The method of Claim 2 wherein the food is or contains cheese.
411/1006
-13- The method of Claim 2 wherein the food is or contains Cottage cheese.
-14- The method of Claim 2 wherein the food is selected from an ice milk or ice cream.
-15- The method of Claim 1 wherein the bacteriocin is produced in an aqueous solution derived from
a growth medium for the Pediococcus acidilactici and wherein the growth medium contains an amino
acid source which enhances the production of the bacteriocin.
-16- The method of Claim 15 wherein the amino acid source in the growth medium is yeast extract.
-17- The method of Claim 15 wherein the aqueous solution is dried in the presence of a food grade
drying aid to a flowable powder which is added to the food.
-18- The method of Claim 17 wherein the drying aid is non-fat dry milk.
-19- The method of Claim 18 wherein the flowable powder contains between about 1 and 100,00 AU
per gram.
-20- The method of Claim 19 wherein the Pediococcus acidilactici is Pediococcus acidilactici NRRLB-18050.
-21- The method of Claim 17 wherein the Pediococcus acidilactici is Pediococcus acidilactici NRRLB-18050.
-22- The method of Claim 17 wherein the Pediococcus acidilactici contains the DNA as a plasmid 9.4
kilobase pairs which encodes for the bacteriocin.
-23- The method of Claim 15 wherein the Pedioccocus acidilactici is Pediococcus acidilactici NRRLB-18050.
-24- The method of Claim 1 wherein the bacteriocin is in the form of a powder which is derived by
drying an aqueous solution containing the bacteriocin derived from a growth medium incubated with
the Pediococcus acidilactici.
412/1006
-25- The method of Claim 1 wherein the bacteriocin is derived by membrane filtration of a growth
medium containing the bacteriocin from growth of the Pediococcus acidilactici to remove low
molecular weight compounds from the medium.
-26- The method of Claim 25 wherein the bacteriocin is in the form of a lyophilized powder.
-27- The method of Claim 1 wherein the bacteriocin is separated from an aqueous growth medium
containing the bacteriocin from growth of the Pediococcus acidilactici and dried.
-28- The method of Claim 1 wherein the DNA is a plasmid which encodes for the bacteriocin.
413/1006
54. NZ229674 - 28.12.1989
NISIN COMPOSITIONS FOR USE AS ENHANCED, BROAD RANGE BACTERICIDES
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ229674
Inventor(s):
BLACKBURN PETER (US); GUSIK SARA-ANN (US); POLAK JUNE (US); RUBINO
STEPHEN D (US)
Applicant(s):
NEW YORK HEALTH RES INST (US)
IP Class 4 Digits: A61K; A23C
IP Class:
A61K37/02; A23C19/11
E Class: A23L3/3463; A23C19/11; A23L3/3463A; A23L3/3571; A01N37/46+M; A01N63/02+M;
A61K7/16D13; A61K8/64; A61Q11/00; A23C9/158B; A61K8/37C; A61K8/41L; A61K8/44; A23L3/3535
Application Number:
WO1989US02625 (19890616)
Priority Number: CS19890006897 (19891206); US19880209861 (19880622); US19890317626
(19890301)
Family: WO8912399
Equivalent:
AU3843089; AU631803; DE68913189D; DE68913189T; DE68927189D;
DE68927189T; DK171069B; DK45690; EP0382814WO8912399; FI98880B; FI98880C; HU204980;
HU53795; IE63998; IE77643; IE892015L; IL90700; JP3500051T; JP8009525B; NO179354B;
NO179354C; NO895147
Cited Document(s):
GB738655
Abstract:
BACTERIOCIN COMPOSITIONS COMPRISING LANTHIONINE CONTAINING BACTERIOCINS AND
NON-BACTERICIDAL AGENTS. WHEN THE BACTERIOCIN COMPOSITIONS ARE COMBINED WITH
414/1006
A SUITABLE CARRIER WITH EACH COMPONENT PRESENT IN SUFFICIENT QUANTITIES SUCH
THAT THE COMPOSITION IS EFFECTIVE AGAINST GRAM NEGATIVE BACTERIA IN ADDITION TO
GRAM POSITIVE BACTERIA, THEY BECOME ENHANCED, RAPID ACTING, BROAD RANGE
BACTERICIDES SUITABLE FOR A VARIETY OF APPLICATIONS.Description:
Description
Nisin Compositions For Use As
Enhanced, Broad Range Bactericides
Background of the Invention
This is a continuation-in-part application of
Serial No. 209,861 filed June 22, 1988. Nisin is a polypeptide with antimicrobial properties which is
produced in nature by various strains of the bacterium Streptococcus lactis. It is a known food
preservative which inhibits the outgrowth of spores of certain species of Gram positive
Bacilli.
Although sometimes mistakenly and imprecisely referred to as an antibiotic, nisin is more correctly
classified as a bacteriocin, i.e. a proteinaceous substance produced by bacteria and which has
antibacterial activity only towards species closely related to the species of its origin. Nisin is a
naturally-occurring preservative found in low concentration in milk and cheese, and is believed to be
completely non-toxic and non-allergenic to humans.
Nisin has recently been recognized as safe by the
FDA as a direct food ingredient in pasteurized cheese spread, pasteurized processed cheese
spread, and pasteurized or pasteurized processed cheese spread with fruits, vegetables, or meats.
Furthermore, since it is a polypeptide, any nisin residues remaining in foods are quickly digested.
A summary of nisin's properties appears in
Hurst, A., Advances in Applied Microbiology 27:85-123 (1981). This publication describes what is
generally known about nisin. Nisin, produced by Streptococcus lactis, is available commercially as
an impure preparation, Nisaplin", from Aplin & Barrett Ltd., Dorset, England and can be obtained
by isolating naturally-occurring nisin from cultures of Streptococcus lactic and then concentrating the
nisin according to known methods. There are also reported methods for producing nisin using
altered strains of
415/1006
Streptococcus. See Gonzalez et al., U.S. Pat. No.
4,716,115, issued December 29, 1987. It should also be possible to produce nisin by recombinant
DNA technology.
Nisin has been applied effectively as a preservative in dairy products, such as processed cheese,
cream and milk. The use of nisin in processed cheese products has been the subject of recent
patents. See U.S. Pat. Nos.
4,584,199 and 4,597,972 The use of nisin to inhibit the growth of certain Gram positive bacteria has
been well documented. However, its complete success and acceptance as a food preservative has
heretofore been hampered by the belief that nisin was ineffective against Gram negative and many
Gram positive bacteria. Gram negative bacteria are almost always present in conjunction with Gram
positive bacteria and are a major source of food spoilage and contamination. See Taylor, U.S. Pat.
No. 5,584,199, issued
April 22, 1986 and Taylor, U.S. Pat.No. 4,597,972, issued
July 1, 1986; Tsai and Sandine, "Conjugal Transfer of Nisin
Plasmid Genes from Streptococus Lactis 7962 to Leuconostoc
Dextranicum 181, Applied and Environmental Microbiology,
Feb. 1987, p. 352; "A Natural Preservative," Food
Engineering Int'l,, May 1987, pp. 37-38; "Focus on Nisin,"
Food Manufacture, March 1987, p. 63.
Summary of the Invention
It has now been found that contrary to prior teaching, compositions comprising nisin, in combination
with various non-bactericidal agents have enhanced, broad range bactericidal activity against Gram
negative bacteria as well as enhanced activity against a broader range of Gram posi tive bacteria
than nisin alone. The enhanced bactericidal activity against Gram positive bacteria occurs in a pH
range broader than previously taught.The invention provides bacteriocin compositions of nisin or
other, lanthionine containing bacteriocins, in combination with various nonbactericidal agents for
example chelating agents or surfactants. The invention further provides the compositions dissolved
or suspended in a suitable carrier to yield enhanced broad range bactericides.
Detailed Description of the Invention
Specifically, it has been found that a solution of about O.lHg/ml to 300 < g/ml of nisin in the
presence of about 0.1 mM to 20 mM of a chelating agent, for example
416/1006
EDTA, virtually eliminates the growth of Gram negative bacteria such as Salmonella typhimurium,
Escherichia coli,
Pseudomonas aeruginosa, Bacterioides gingivalis,
Actinobacillus actinomycetescomitans, and Klebsiella pneumoniae and is more active towards Gram
positive bacteria such as Staphylococcus aureus, Streptococcus mutans,
Listeria monocytogenes Streptococcus agalactiae and
Coryneform bacteria than nisin alone.Although the enhancement of nisin activity by chelator was
concentration dependent, contrary to expectations, concentrations of EDTA in excess of 20mM were
inhibitory to the bactericidal activity of nisin. However, in the presence of a proteinaceous carrier,
and polyvalent polymers such as serum albumin, collagen, gelatin, casein and keratin, the inhibition
of nisin by concentrations of EDTA above 20mM was significantly reduced, thereby extending the
useful range of
EDTA enhancement of nisin.
It has also been found that a solution of about O.1CAg/ml to 300Rg/ml nisin and about 0.1 mM to
20 mM of a chelating agent will further enhance the effectiveness of nisin against Gram negative and
Gram positive bacteria in the presence of about 0.01% to 1.0% of surfactant.
Additionally, it has been found that, in the presence of surfactant alone, nisin has enhanced activity
against Gram positive bacteria.
In the present invention, suitable chelating agents include, but are not limited to, EDTA, CaEDTA,
CaNa2EDTA, and other alkyldiamine tetraacetates, EGTA and citrate. Surfactants, valuables
cleansing agents, suitable for combination with nisin, with or without EDTA, include, but are not
limited to, the nonionic surfactants
Tweens, Tritons, and glycerides, ionic surfactants such as fatty acids, quaternary compounds,
anionic surfactants such as sodium dodecyl sulphate and amphoteric surfactants such as
cocamidopropyl betaine and emulsifiers.
Since Gram positive and Gram negative bacteria are almost always found together in foods, the
effectiveness of the nisin compositions towards Gram negative bacteria such as Salmonella
typhimurium, Escherichia Coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides
gingivalis,
Actinobacillus actinomycetescomitans, and other Gram negative pathogens and Gram positive
bacteria will be of great use. The bactericides are particularly suited for the control and prevention of
417/1006
contamination of raw ingredients, processed foods and beverages by bacterial pathogens and other
microbial spoilage organisms. Potential food related uses include treatment of meats, especially
poultry, eggs, cheese and fish and treatment of food packaging and handling equipment. Further
uses include as food preservative, such as in processed cheese, cream, milk, dairy products and in
cleaning poultry, fish, meats, vegetables, and dairy and food processing equipment. The use of the
nisin compositions should not be limited to food related uses and the nisin compositions should be
useful in any situation in which there is a need or desire to eliminate Gram negative and Gram
positive bacteria.
The compositions can be dissolved in a suitable carrier for example an aqueous solvent or buffer or
suspended in any suitable liquid, colloidal or polymeric matrix to create bactericides. The
compositions or bactericides can be incorporated into ointments or coatings for medicinal uses such
as the treatment of infections, wound dressings or surgical implants and as a broad spectrum
disinfectant for skin or oral rinses, disinfectant scrubs, wipes or lotions. The bactericides can be used
for cleaning medical instruments, in pre-operative surgical scrubs and the like. The bactericides are
particularly useful in circumstances where environmental disinfection is desired but where chemical
germicidals are precluded because of the risks of corrosive or otherwise toxic residues.
Unlike the activity of most broad spectrum germicidals which is compromised by the presence of
complex organic matter, the compositions of the present invention are effective as bactericides in the
presence of organic matter, such as milk or serum.
Nisin was known to optimally inhibit the growth of a few closely related Gram positive bacteria,
particularly certain Gram positive spore forming bacilli at pH 5.0. The bactericidal activity of nisin in
solution with a chelating agent was surprisingly rapid and greatly enhanced towards a broad range
of Gram positive bacteria at pH values greater than pH 5.0, and, moreover, was activated towards
Gram negative bacteria at both acidic and basic pH, preferably in the range pH 5.0 to 8.0. This
unexpectedly rapid and broadranged bactericidal activity of chelator-activated nisin makes it
suitable for use as, among other things, a disinfectant.
Nisin belongs to the class of peptide bacteriocins containing lanthionine. Also included among that
class are subtilin, epidermin, cinnamycin, duramycin, ancovenin and
Pep 5. These bacteriocin peptides are each produced by different microorganisms. However,
subtilin obtained from certain cultures of Bacillus subtilis, and epidermin obtained from certain
418/1006
cultures of Staphylococcus epidermidis, have been found to have molecular structures very similar to
that of nisin (see Hurst, pp. 85-86, and
Schnell et al., Nature, 333:276-278). It is therefore believed that because of the molecular similarities,
other lanthionine containing peptide bacteriocins will be equally as effective as nisin in combination
with chelating agents and non-ionic surfactants in eliminating Gram negative and
Gram positive bacterial contaminations.
The effectiveness of the nisi, and by extension other lanthionine containing peptide bacteriocin,
compositions as bactericides against Gram negative bacteria is surprising, since the prior art
generally teaches away from this activity of nisin. The enhanced activity of nisin against Gram
positive bacteria in the presence of EDTA at a pH greater than 5.0 is unexpected since it was
previously believed that nisin activity is optimal at pH 5.0. Furthermore, the discovery of such
effectiveness of the nisin and lanthionine containing peptide bacteriocin compositions as
bactericides fulfills a long-felt need in the science of food preservation, which has suffered from the
absence of an acceptable, natural, non-toxic agent effective against a broad range of bacteria.
In order to demonstrate the superior and unexpected rapid activity of the composition containing
nisin, EDTA and/or various surfactants against both Gram negative and Gram positive bacteria, a
number of experiments were conducted with the bactericides. These experiments are meant as
illustration and are not intended to limit this invention. - It is to be expected that other, lanthionine
containing peptide bacteriocins would be effective substitutes for nisi and that chelating agents
other than
EDTA will be effective substitutes for EDTA.
All tests in the following examples were performed at 37 0C. The efficacy of the enhanced broad
range bactericides was determined by assaying bactericidal activity as measured by the percent
bacterial survival after treatment with the bactericide. Generally, after incubation of a 107 cell per ml
suspension of target species with the novel bactericide for specified lengths of time, bacteria were
collected by centrifugation for 2 minutes. The bacterial pellet was washed free of the bactericide with
a rescue buffer, termed herein Phage buffer (50mM Tris-HCl buffer pH 7.8, lmM MgSO4, 4mM
CaC12, 0.1 M Nacl, and 0.1% gelatin), resuspended and serially diluted into Phage buffer, and
100ml of the suspended bacteria were spread on nutrient agar plates.Surviving bacteria were
determined by scoring colony forming units (CFU) after incubation for 2448 hours at 370C. An
effective bactericide according to this invention is one which allows less than 0.1% of the initial viable
count of the bacteria to survive.
419/1006
Example 1
Activity of Nisin and a
Chelating Agent Against
Gram Negative Bacteria (S. typhimurium)
As shown in Table 1, two tests were conducted in 20mM Tris, pH 8.0 at 370C to show the effect of
the bactericide containing nisin and the chelating agent EDTA alone. Test ;1, a control, was
conducted without EDTA and shows the effect of nisin alone toward the Gram negative bacterium S.
typhimurium. The increased concentrations of nisin do exhibit some activity, but even the activity of
the higher concentrations in the absence of EDTA, 1.6% survival at 100 2 g/ml nisin, is wholly
inadequate for a food preservative. The level of bactericidal activity obtained from nisin and EDTA is
significant.
TABLE 1
Initial
Viable
Test Bacteria EDTA Nisin ( g/ml)
; Count (mM) 0 10 30 50 100 300
Percentage S. typhimurium
Survival at 3 hours 1 3.0 x 106 0 100 - 51.3 - 7.0 1.6 2 5.7 x 106 20 2.5 < 10 4 < 10-4 < 10
Test ;2 (Table 1), conducted using nisin plus 20mM
EDTA, demonstrates the surprising activity of the nisin composition in eliminating the target Gram
negative bacteria.
Table 1 shows that in test ;;2 at a concentration of 20mM EDTA and 30 Ag/ml of nisin, the
bactericide has a marked bactericidal activity towards S. typhimurium, while at nisin concentrations
of 100 g/ml and greater, the nisin and EDTA bactericide virtually eliminates the bacteria (percentage
survival less than 10 4 which indicates no surviving bacteria in the assay). Thusr the combination of
EDTA and nisin demonstrates a synergistic activity of greater than 1000 times that of nisin alone.
Example 2
Activity of Nisin, a Chelating
Agent and a Surfactant Against
Gram Negative Bacteria (S. typhimurium)
Four tests (Table 2) were conducted to determine the effect on S. typhimurium of the bactericide
containing nisin and both EDTA and the surfactant Triton X-100 in 20mM
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Tris, pH 8.0 at 370. The control (Test ;1) is identical to the control of Example 1 (Table 1).
TABLE 2
Initial
Viable Triton
Test Bacteria EDTA X-100 Nisin (g/ml) ; Count (mM) (%) 0 10 30 50 100 300
Percentage S. typhimurium
Survival at 3 hours 1 3.0x106 0 0 100 51.3 - 7.0 1.6 2 3.0x106 0 1.0 37.4 93.0 - 64.0 47.0 3 5.7x106
20 0.1 0.03 - < 10-3 - - 4 5.7x106 20 1.0 < 10-4 - < 10-4 - < 10-4 < 10-4
Test ;2 (Table 2) was conducted using nisin and 1.0% Triton X-100, but without EDTA. The presence
of the detergent alone inhibits the activity of the nisin towards the Gram negative bacteria and nisin
was ineffective.
However, in tests ;3 and ;4 (Table 2), which represent the invention, the presence of 20mM EDTA in
combination with
Triton X-100 is a bactericide which markedly increases the bactericidal activity of nisin towards S.
typhimurium.
Indeed the combination of Triton X-100 with EDTA but without nisin was effective, although to a lesser
degree than in the presence of nisin. While in both tests ;3 and ;4 (Table 2) the nisin combinations
were very effective, the concentration of 1.0% Triton X-100 (test ;4, Table 2) was most effective.
The presence of the non-ionic surfactant, Triton X-100, in combination with EDTA, enhances the
activity of nisin toward Gram negative bacteria even more than the bactericide containing nisin and
EDTA alone (Example 1).
Example 3
Activity of Nisin, a Chelating Agent
and a Surfactant Against Gram Negative
Bacteria (S. typhimurium)
Table 3 shows the enhanced activity toward
S. typhimurium of the bactericide containing nisin, 20mM of the chelating agent EDTA and the nonionic surfactant Tween 20 in 20 mM Tris, pH 8.0 at 370C. As with Triton X-100 (Example 2) the
combination of nisin and EDTA with (1%) of
Tween 20 is most effective.
421/1006
TABLE 3
Initial
Viable
Test Bacteria EDTA Tween20 Nisin kg/ml)
; Count (mM) (%) 0 10 30 50 100 300
Percentage S. typhimurium
Survival at 3 hours 1 3.0x106 0 0 100 51.3 - 7.0 1.6 2 5.7x106 20 0 2.5 - < 10 3 - < 10 < 10-4 3
4.3x106 20 1.0 < 10-2 2 < 10 4 - < -4 < 10-4
Example 4
Activity of Nisin, a Chelating Agent
and a Surfactant Against Gram Negative
Bacteria (Escherichia coli)
The effect of the bactericide containing nisin and
EDTA towards the Gram negative bacteria E. coli was demonstrated, as shown in Table 4.
TABLE 4
Initial
Viable - Triton
Test Bacteria EDTA X-100 Nisin (;g/ml)
; Count (mM) (g) 0 30 100 300
Percentage E. coli
Survival at 2 hours 1 1.0 x 107 0 0 100 27 25 8.5 2 1.0 x 107 20 0 14.5 0.86 0.01 0.001 3 1.0 x 107
0 1.0 100 - 30 - - 4 1.0 x 107 20 1.0 - 1.2 0.8 0.05 < 10-4
The tests, with and without EDTA, were performed in 20mM Tris buffer solution, pH 8.0 at 37 C, with
an initial viable count of 1 x 107 E. coli cells/ml. The effects of the bactericide were measured as a
function of percentage bacteria survival after 2 hours.
In test ;1, (control, Table 4) without EDTA, nisin exhibited little meaningful activity toward the
elimination of E. coli. In test ;2 (Table 4), however, where 20mM EDTA was present, the bactericidal
composition exhibited substantial activity towards the E. coli bacteria. The activity increased in
effectiveness as the concentration of nisin was increased. The combination of nisin with EDTA as a
bactericide demonstrates a 1000 fold synergistic increase in effectiveness towards E. coli. In tests X3
and ;4 (Table 4), it can be seen that Triton X-100 has no significant bactericidal activity towards E.
coli. In fact,
422/1006
Triton X-100 appears to inhibit nisin activity towards Gram negative bacteria as was found with S.
typhimurium (Table 2).However, the overall enhancement of nisin by EDTA substantially reverses the
inhibitory effects of Triton X100 as seen in Tables 2 and 4.
It thus appears that the bactericide containing nisin and a chelating agent, such as EDTA, is an
effective food preservative towards various types of Gram negative bacteria even in the presence of
surfactants.
Example 5
Activity of Nisin and a Chelating
Agent Against Gram Negative
Bacteria (Klebsiella pneumoniae)
The effect of the bactericide containing nisin and
EDTA alone towards the Gram negative bacteria K. pneumoniae was demonstrated, as shown in
Table 5.
TABLE 5
Initial
Viable Triton No sin g/ml
Bacteria EDTA x-100
Test Count (mM) (%) 0 30 100 300
% Survival at 2 hours
1 107 0 0 100 - 50 38
2 107 20 0 22 0.5 1.1 0.085
The two tests, one with and one without EDTA (control), were performed in 20 mM Tris buffer, pH 8.0
at
37 C with an initial viable count of 107 cells/ml of K.
pneumoniae. The effect was measured as a function of percentage bacterial survival after 2 hours.
In test ;1, (control, Table 5) without EDTA, nisin exhibited little meaningful bactericidal activity toward
K.
pneumoniae. In test ;2 (Table 5), however, where 20mM EDTA was present, the bactericide exhibited
substantial activity towards K. pneumoniae. The activity increased in effectiveness as the
concentration of nisin was increased.
423/1006
Example 6
Nisin Activity Against Gram Negative
Bacteria (Salmonella typhimurium) is Dependent
on Chelator Concentration
The data in Table 6 demonstrate that the enhanced activation of nisin towards Gram negative
bacteria (S. typhimurium) is dependent on the concentration of EDTA in either 50 sodium acetate,
pH 5.0, or 20 mM Tris, pH 8.0 at 370C.
TABLE 6
Initial
Viable
Test Bacterial Nisin EDTA(mM) ; pH Count g/ml 0 0.2 2.0 10 50 100 % Survival at 2 hours 1 5.0
3x106 0 100 - 38.7 15.2 3.5 2 5.0 3x106 100 0.6 10-4 10-4 0.004 0.02 3 8.0 5x106 0 100 - 8.7 14 11.4
45 4 8.0 5x106 100 4 10-4 10-4 10-4 0.6 30
In tests ;1 and X3, (controls, Table 6) using EDTA concentrations up to 100 mM without nisin
exhibited little meaningful activity towards S. typhimurium at either pH 5.0
(;1) or pH 8.0 (;3). In tests ;2 and ;4 (Table 6), however, where 100 Hg/ml nisin was present in
combination with EDTA, the bactericides exhibited substantial activity towards
S. typhimurium. The activity of the bactericides was similar at both acidic pH (5.0) and basic pH (8.0),
despite the fact that the activity of nisin alone towards Gram positive bacteria is optimal at pH 5.0.
The enhancement of nisin by EDTA was concentration dependent, being optimal in the range 0.2
mM to 10-mM at pH values 5.0 and 8.0. Surprisingly, at concentrations greater than 10 mM EDTA,
the enhancement of nisin by EDTA becomes reduced; the reduction of activation is significantly
greater at pH 8.0 than at pH 5.0.
Example 7
Nisin and a Chelating Agent Against
Gram Negative Bacteria (S. typhimurium)
The enhancement of the activity of nisin by EDTA towards Gram negative bacteria in the presence of
biological tissue was demonstrated with S. typhimurium on chicken muscle, and is shown in Table 7.
TABLE 7
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Test Nisin EDTA(mM) ; pH g/ml 0 0.1 0.3 1 3 10 20 30 100 % Survivala at 2 hours 1 5.0 0 11.8 - - - 6.4 - - 2 5.0 300 0.1 0.2 0.05 0.01 0.003 0.016 0.03 0.02 0.07 3 8.0 0 100 - - - - 5.2 - - 4 8.0 300 7.5
0.1 0.02 0.02 0.09 0.47 0.5 - 2.2 5 8.0 300b 0.02 0.09 0.0002 < 10-4 0.0004 0.003 - 0.03 0.09 a
Unadhered cells b Contains 1% Bovine serum albumin (BSA)
Incubations were performed in either 50mM sodium acetate, pH 5.0,or 20 mM Tris, pH 8.0 at 370C.
Cubes of chicken muscle were cleansed with sodium hypochlorite and povidone iodine prior to use.
To inoculate the tissue, the cubes of chicken muscle were dipped into a 108 cells/ml suspension of S.
typhimurium in 20 mM Tris HC1, pH 8.0. Excess moisture was removed from dipped cubes by
tapping. The chicken samples were placed into sufficient buffer containing the nisin composition to
cover the tissue and incubated for 2 ;ours at 37 C after which the tissue was removed to sufficient
Phage buffer to cover the tissue. The bacteria remaining in the test solution were collected by
centrifugation, washed with Phage buffer, and combined with bacteria washed from the tissue by
Phage buffer. The combined samples (termed "unadhered" cells) were serially diluted and 100 &
aliquols were plated for determination of surviving bacteria.
In tests and ;3 (Table 7), in the absence of nisin at either 5 or pH 8, EDTA alone has no significant
effect on the survival of S. typhimurium. In tests ;2 and ;4 (Table 7), however, where 300 g/ml nisin
was present, the bactericides exhibited substantial activity towards
S. typhimurium on chicken muscle at both pH 5.0 and pH 8.0.
The enhancement of nisi by EDTA was concentration dependent, the optimal concentration being in
the range 0.3mM to lOmM EDTA at both pH values 5.0 and 8.0. At concentrations greater than
lOmM EDTAat pH 8.0, the activation of nisin by EDTA is reduced. However, as is shown in test ;5
(Table 7), in the presence of 1.0 % bovine serum albumin at pH 8.0, the efficacy of nisin towards S.
typhimurium on chicken muscle is expressed throughout the range of EDTA concentrations up to
l00mM.
Thus, bactericides containing nisin and low concentrations of chelating agent, such as EDTA in the
range 0.ism to 20mM, can be extremely effective for the elimination or prevention of contamination
of food by Gram negative bacteria.
Example 8
Titration of Nisin Activity Against
425/1006
Gram Negative Bacteria (S. typhimurium)
At the optimal concentration of chelating agent, the efficacy of the bactericide in Tris buffer towards
Gram negative bacteria was demonstrated to be substantial, as is shown in Table 8.
TABLE 8
Initial
Viable
Test Bacterial EDTA BSA Nisin g/ml ; Count (mM) % 0 0.1 0.3 1.0 3.0 10 30 100 % Survival at 2
hours 1 6x106 0 0 100 - - - - 51.3 - 1.6 2 6x106 1.0 1.0 63 0.7 0.08 0.01 0.05 0.01 < 10-4 In test X2 (Table 8), it can be seen that as little as 0.3 8 g/ml of nisin, with 1.0 mM EDTA in 20mM
Tris at pH 8.0 in the presence of 1% bovine serum albumin (BSA), significantly reduced the survival
of S. typhimurium. The bactericide is as active towards Gram negative bacteria as nisin alone is
towards Gram positive Streptococci.
Example 9
Titration of Nisin Activity Against
Gram Negative Bacteria (S. typhimurium)
At the optimal concentration of chelating agent, the efficacy of a bactericide towards Gram negative
bacteria in the presence of biological tissue was demonstrated with
S. typhimurium on chicken muscle, and is shown in Table 9.
TABLE 9
Initial
Viable
Test Bacterial EDTA BSA Nisin A < g/ml
; pH Count (mM) ( %) 0 10 100 200 300
% Survival at 2 hours
1 8.0 3x107 0 0 100 - - 7
2 8.0 3x10 1.0 1.0 27 0.26 0.008 0.007 0.006
Cubes of chicken muscle were cleansed with sodium hypochlorite and povidone iodine prior to use.
To inoculate the tissue, the cubes of chicken muscle were dipped into a 108 cells/ml suspension of S.
typhimurium in 20 mM Tris HC1, pH 8.0. Excess moisture was removed from dipped cubes by
tapping. The tissue was placed into sufficient buffer containing the nisin compositions to cover the
tissue, and incubated for 2 hours at 37 0C after which the tissue was removed to sufficient Phage
426/1006
buffer to cover the tissue.The bacteria remaining in the test solution were collected by centrifugation,
washed with Phage buffer, and combined with bacteria washed from the tissue by Phage buffer. The
combined samples (termed "unadhered" cells) were serially diluted and 100 1 aliquots were plated
for determination of surviving bacteria.
Example 10
Nisin EDTA and Methyl Paraben
Activity Against Gram Negative
Bacteria (S. typhimurium)
A bactericicontaining nisin and EDTA, when combined with a known food preservative, methyl
paraben, was demonstrated to be exceptionally effective towards Gram negative bacteria, as shown
in Table 10.
TABLE 10
Initial
Viable b
Test Bacterial Nisin EDTAb % Methyl Paraben
; Count - g/ml (mM) O 0.1 1.0
% Survival C at 2 hours
1 3x106 0 10 11.8 1.0 10-4
2 3x106 300 10 0.03 < 10-3
50 mM Na acetate buffer, pH 5.0
c Unadhered cells
Cubes of chicken muscle were cleaned with sodium hypochlorite and povidone iodine prior to use.
To inoculate the tissue, the cubes of chicken muscle were dipped into a 108 cells/ml suspension of S.
typhimurium in 50 mM sodium acetate buffer, pH 5.0. Excess moisture was removed from dipped
cubes by tapping The tissue was placed into sufficient buffer containing nisin compositions to cover
the tissue, and incubated for 2 hours at 370C after which the tissue was removed to sufficient Phage
buffer to cover the tissue.The bacteria remaining in the test solution were collected by centrifugation,
washed with Phage buffer, and combined with bacteria washed from the tissue by Phage buffer. The
combined samples (termed "unadhered" cells) were serially diluted and lOO2 aliquots were plated
for determination of surviving bacteria.
In test ;1 (Table 10), methyl paraben in the presence of 10 mM EDTA was shown to be effective
towards
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S. typhimurium only at a concentration of 1.0%. In test ;2 (Table 10), however, in the presence of
3004 g/ml nisin, the effectiveness of methyl paraben and nisin towards
S. typhimurium was substantially improved.
The compositions containing nisin and EDTA significantly improve the utility of the food preservative
methyl paraben. Furthermore, the bactericides may lead to substantial reductions in the
concentrations, or eliminate the need for these commonly recognized, though less desirable, food
preservatives such as methyl paraben.
Example 11
Nisin and Chelating Agent Activity
Against Gram Positive Bacteria
(Staphylococcus aureus)
The activation of nisin by a chelating agent is pH-dependent. The data in Table 11 confirm that at pH
5.0, nisin is somewhat more bactericidal towards S. aureus than is nisin at pH 8.0. At pH 5.0, EDTA
does not enhance nisin activity towards S. aureus and at concentrations of EDTA greater than 10 mM,
EDTA is inhibitory to the bactericidal activity of nisin. However, the bactericidal activity of nisin
activated by EDTA at pH 8.0 is significantly greater than the bactericidal activity of nisin alone, or in
combination with EDTA at pH 5.0.
Table 11
Influence of pH on the Effects of EDTA on Nisin
Bactericidal Activity towards Staphyloccus aureus
Nisin - EDTA mM
pH g/ml 0 0.1 0.3 1.0 3.0 10 30 100
% Survival 2 hra
8.0 0 100 - 100 81 100 100 100
8.0 3.0 7.4 0.03 0.01 0.2 0.4 3 56
5.0 - 0 - 100 - - - 100 - 5.0 3.0 0.6 1.0 - 1.3 - 1.4 1.8 - 34 80
a Initial viable count: 8.0 x 106 cfu/ml
Incubations were performed in 50 mM sodium acetate buffer,
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pH 5.0 or 20 mM Tris-HCl buffer, pH 8.0 at 370C.
The bactericidal activity of nisin alone is reported (see Hurst) to be greatest at pH 5.0 or lower, and
data presented in Table 11 support this. On the basis of this information it was believed that the
bactericidal activation of nisin by EDTA towards S. aureus would likewise be greatest at lower pH.
However, as can be seen in
Table 11 and contrary to expectations (see Table 6), EDTA was not observed to enhance nisin
activity towards Gram positive bacteria at pH 5.0. However, inhibition of nisin activity by high
concentrations of EDTA was still observed at pH 5.0. Thus, the activation of nisin by a chelating
agent occurs only within a range of chelator concentrations and, with respect to Gram positive
bacteria, is dependent upon pH with the preferred pH range greater than pH 5.0.
Example 12
Nisin and Chelating Agent Activity
Against Gram Positive Bacteria
The effects of EDTA on the bactericidal activity of nisin at pH 8.0 are not limited to S. aureus, an
important human pathogen, but are also observed with Streptococcus mutans, responsible for dental
plaque (Table 12A), Listeria monocytogenes, a foodborne pathogen (Table 12B), and with a mixed
population of axillary Coryneform bacteria, contributors to body odor (Table 12C).
Table 12A
The Effects of EDTA on the Bactericidal
Activity of Nisin towards Streptococcus mutans
Nisin EDTA mM
pH 6(g/ml 0 0.01 0.1 0.3 1.0 3.0 10 30 100
% Survival after 2 hra
8.0 0 100 8.0 0.1 4.3 1.8 0.04 0.02 0.06 1 25 100 100
a Initial viable count: 6.0 x 106 cfu/ml
Incubations were performed in 20 mM Tris-HCl, pH 8.0 at
37 C.
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Table 12B
The Effects of EDTA on the Bactericidal
Activity towards Listeria monocytogenes
Nisin EDTA mM
pH g/ml 0 0.1 0.3 1.0 3.0 10 30 100
% Survival after 2hra
8.0 0 100 - - 84 - - 8.0 3.0 0.71 0.04 0.04 0.02 0.1 0.64 10 14
a Initial viable count: 6.0 x 106 cfu/ml
Incubations were performed in 20 mM Tris-HCl, pH 8.0 at
37 C.
Table 12C
The Effects of EDTA on Nisin Bactericidal
Activity towards Coryneform bacteria
Nisin EDTA mM
pH g/ml 0 - 0.1 0.3 1.0 3.0 10
% Survival 2hra
8.0 0 100 - 4.6 3.6 8 36
8.0 3 0.22 0.03 0.0009 0.1 -- 0.16
a Initial viable count: 1.0 x 106 cfu/ml
Incubations were performed in 20 mM Tris-HCl, pH 8.0 at
370C.
Example 13
Rapid Bactericidal Activity of
Nisin Activated by Chelator
The bactericide comprising nisin with EDTA is rapidly bactericidal as is illustrated by the data
presented in Table 13A. Suspensions of the Gram positive bacterium
430/1006
S. mutans at 107 cells/ml were incubated in 20 mM Tris buffer, pH 7.3 at 370C with a range of
concentrations of nisin activated by 1 mM EDTA. The suspensions were incubated for various times
ranging from 0.5 to 60 minutes with the bactericides. The bactericidal efficacy of the bactericides
was estimated by determining the percent survival of bacteria. Enhanced by EDTA, as little as 10
(g/ml of the nisin in this formulation is able to reduce the bacterial load by 6 logs within 1 minute.
Rapid bactericidal activity is a prerequisite for effective disinfection. Thus, the compositions are
predicted to be effective bactericides particularly as demonstrated here, as a component of a
mouthwash, rinse, toothpaste, or other similar dentrifice active against plaque forming S. mutans.
The activity of nisin enhanced by EDTA against
Gram negative bacteria after 2-3 hours was shown in Examples 1-7. Rapid bactericidal activity of
nisin enhanced by EDTA is also seen towards Gram negative bacteria and this is illustrated by the
data in Table 13B.
Table 13A
Kinetics of Bactericidal Activity towards
Streptococcus mutans of Nisin Enhanced by EDTA
Incubation Nisin /(g/ml with 1.0 mM EDTA
Time 0 0 1 3 - 10 30 100
(Minutes)
96 Survivala
0.5 - - - - - < 10
1 - - - < 10-4 < 10-4 < 10
3 100 0.5 0.002 < 10-4 < 10-4
15 - 0.03 < 10-4 < 10-4 30 - - < 10-4 - 60 100 0.003 - - - a Control viable cell count: 1.0 x 107 cfu/ml
Incubations were performed in 20 mM Tris-HCl, pH 7.3 at
37 C.
TABLE 13B
431/1006
Rapid Bactericidal Activity towards Escherichia coli
of Nisin Enhanced by EDTA
Nisin kg/ml
mM EDTA 0 0.3 1.0 3 10 30 100
% survival at 1 mina
1.0 100 100 56 0.37 0.013 0.015 0.008
a Initial viable count: 1.0 x 107 ofu/ml
Incubations were performed in 20 mM Tris, pH 7.0 at 370C.
Example 14
Effect of Divalent Cations on EDTA
Enhancement of Nisin Activity
Divalent cations bind to EDTA and other chelating agents and would be expected to neutralize the
activation of nisin by EDTA. However, as can be seen by the data in Table 14, the bactericidal
activity of nisin against S. mutans is enhanced by 1 mM EDTA even in the presence of 1 mM Ca ion;
only above 3 mM was Ca 2+ ion inhibitory to EDTA-activated nisin. This is particularly important in
mouthwash applications where calcium ion concentrations are relevant.
TABLE 14
Rapid Bactericidal Activity towards
Streptococcus mutans of Nisin Activated by
EDTA in the presence of Divalent Cation
CaCl3 mM
Nisin 0 0.1 0.3 1.0 3 10
% survival at 1 min.a
0 - 100
3 2.9
3E 0.0042 0.0042 0.052 18
30E 0.0019 0.0003 0.0004 0.06 6.8
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100E < 10-4 < 10-4 < 10-4 0.0001 1.5
E 1 mM Na2EDTA
a Initial viablc count 1.0 x 102
a Initial viable count 1.0 X 10 cfu/ml.
Incubations performed in 10% Fetal Calf Serum at
370C.
Example 15
Nisin and Surfactant Activity
Against Gram Positive Bacteria
The bactericidal activity of nisin can also be significantly enhanced when combined with a surfactant
alone. This is best illustrated at a limiting nisin concentration (0.2 g/ml) as shown in Table 15A. At
concentrations up to 0.1%. the food grade surfactant monolaurin has little significant bactericidal
activity towards Streptococcus agalactiae in the complex medium milk.
Nisin, at concentrations up to 0.2 t(g/ml, likewise does not exhibit significant bactericidal activity in
milk. However, the combination of the two agents, 0.1% monolaurin and nisin 0.2 g/ml, is extremely
potent towards S. agalactiae. This bactericide is over 100 times more active than what would be
expected for the additive effect and 10,000 times more active than either of the components
individually. Thus, when the application of nisin is limited by its available activity, a bactericide
comprising nisin with a surfactant can be expected to be more useful.
An example of where the application of nisin is limited by its available activity is illustrated by the
data in Table 15B. Although nisin, and particularly the bactericide comprising nisin and EDTA, is
bactericidal towards L. monocytogenes, the data in Table 15B demonstrate that in a complex
medium like milk the available nisin activity towards this organism is restricted. However, the
bactericide comprised of nisin with the glyceride, monooleate, is effective in milk towards this
foodborne pathogen even though monooleate by itself had no bactericidal activity towards this
organism.
Table 15A
Nisin Bactericidal Activity towards
Streptococcus agalactiae in milk at 37 C
433/1006
(Activation of nisin by monolaurin)
Nisin Monolaurin
( g/ml) (%)
o 0 0.01 0.1
% survival at 2ha
0 - 100 100 4.5
0.02 100 100 0.2
0.2 2.2 0.05 0.0008
a Initial visable counts 6.0 X 107 cfu/ml.
Incubations were in milk at 370C.
Table 15B
Nisin Bactericidal Activity towards
Listeria monocytogenes in milk at 37 C (Activation of nisin by monooleate)
Nisin % Monooleate
g/ml 0 0.1 1.0
% Survival 2 hra
0 100 67 63
100 0.56 10-3 10-4
a Initial viable count 5.0 x 107 cfu/ml
Incubations were in milk at 37 C. Claims:
Claims
1. A composition comprising a lanthionine contain
434/1006
ing bacteriocin and a chelating agent.
2. A composition comprising a lanthionine contain
ing bacteriocin and a surfactant.
3. A composition comprising a lanthionine contain
ing bacteriocin, a chelating agent and a
surfactant.
4. The composition as defined in claim 1, 2 or 3
wherein the lanthionine containing bacteriocin
is selected from the group consisting of nisin,
subtilin, epidermin, cinnamycin, duramycin,
ancovenin and Pep 5.
5. The composition as defined in claim 1 or 3
wherein the chelating agent is selected from
the group consisting of alkyldiamine tetra
acetates, CaEDTA, Na2CaEDTA, EGTA and citrate.
6. The composition as defined in claim 5 wherein
the alkyldiamine tetraacetate is EDTA and the
bacteriocin is nisin 7. The composition as defined in claim 2 or 3
wherein the surfactant is selected from the
group consisting of Tritons, Tweens,
glycerides, fatty acids, emulsifiers,
quaternary compounds, amphoteric and anionic
surfactants.
8. The composition as defined in claim 1 also
containing a food perservative.
9. An enhanced broad range bactericide comprising
a carrier, a lanthionine containing bacteriocin
and a chelating agent.
435/1006
10. An enhanced broad range bactericide comprising
a carrier and a lanthionine containing bacteriocin and a surfactant.
11. An enhanced broad range bactericide comprising
a carrier, a lanthionine containing
bacteriocin, a chelating agent and a surfac
tant.
12. The enhanced broad range bactericide as in
claim 9, 10 or 11 wherein the lanthionine
containing bacteriocin selected from the group
consisting of nisin, subtilin, epidermin,
cinnamycin, duramycin, ancovenin and Pep 5 and
the chelating agent selected from the group
consisting of alkyldiamine tetraacetates, EGTA
and citrate are present in quantities such that
the bactericide has enhanced effectiveness
against at least one of the bacteria from the
group consisting of Staphylococcus aureus,
Streptococcus mutans, Listeria monocytogenes,
Streptococcus agalactiae, Cornyeform bacteria,
Salmonella typhimurium, Escherichia coli,
Klebsiella pneumoniae, Pseudomonas aeruginosa,
Bacterioides gingivalis and Actinobacillus
actinomycetescomitans.
13. The enhanced broad range bactericide as in
claim 12 wherein the alkyldiamine tetraacetate
is EDTA.
14. The enhanced broad range bactericide as in
claim 10 or 11 wherein the surfactant is
selected from the group consisting of Tritons,
Tweens, glycerides, fatty acids, quaternary
compounds, emulsifiers, amphoteric and anionic
436/1006
surfactants and is present in an amount suffi
cient such that the bactericide has enhanced
effectiveness against at least one of the
bacteria from the group consisting of Gram
negative and Gram positive bacteria.
15. The enhanced broad range bactericide as in
claim 12 wherein the concentration of nisin is
between about 0.lA(g/ml and 300.0 Ag/ml and the
concentration of chelating agent is between
about 0.1 mM and 20mM.
16. The enhanced broad range bactericide as in
claim 14 wherein the concentration of surfac
tant is between about 0.01% and 1.0%.
437/1006
55. NZ236730 - 02.01.1992
METHOD AND COMPOSITION FOR EXTENDING THE SHELF LIFE OF MEATS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ236730
Inventor(s):
BOUDREAUX DONALD P (US); MATROZZA MARK A (US)
Applicant(s):
MICROLIFE TECHNICS (US)
IP Class 4 Digits: A23B
IP Class:
A23B4/22; A23B4/20; A23B4/023; A23B4/08; A23B4/12
E Class: A23B4/22; A23B4/12; A23B4/023; A23B4/08; A23B4/20
Application Number:
EP19910100327 (19910111)
Priority Number: US19900514681 (19900424)
Family: NZ236730
Equivalent:
AU624038; AU7297991; CA2033853; DE463284T; DE69100877D; ES2032191T;
GR92300018T; JP4349846; MX171529
Cited Document(s):
GB1562568; US3899594; US4883673
Abstract:
A METHOD FOR INHIBITING THE GROWTH OF BACTERIA IN RAW OR PROCESSED MEAT
PRODUCTS HAVING A PH BETWEEN ABOUT 6.0 AND 6.5 STORED AT ABOVE FREEZING
TEMPERATURES USING AN INORGANIC PROPIONATE SALT WHICH EXTENDS THE SHELF LIFE
OF THE MEAT IS DESCRIBED. THE SALT IS PREFERABLY SODIUM PROPIONATE OR CALCIUM
PROPIONATE AND IS USED IN AN AMOUNT LESS THAN ABOUT 1% BY WEIGHT AND
PREFERABLY BETWEEN ABOUT 0.05 AND 0.5 PERCENT BY WEIGHT OF THE MEAT SUCH THAT
NO FLAVOR IS IMPARTED TO THE MEAT. PREFERRED DRIED COMPOSITIONS CONTAINING A
438/1006
BACTERIOCIN FROM PEDIOCOCCUS ACIDILACTICI AND A PROPIONATE SALT ARE ALSO
DESCRIBED.Description:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a method for inhibiting the growth of bacteria in a raw or cooked
processed meat at temperatures above freezing and at near neutral pH's with an inorganic
propionate salt thereby extending the shelf life of the meat. The present invention relates to preferred
compositions including a bacteriocin which are useful in the method. In particular the present
invention relates to the use of low levels of a sodium propionate or calcium propionate salt, and
preferably the bacteriocin, in the processed meat, such as beef, poultry or fish and mixtures thereof,
for this purpose.
(2) Prior Art
The prior art has used propionate salts in various processed foods. Examples are U.S. Patent No.
3,899,594 to Nickerson et al and British Patent application No. 1,562,568, filed March 12, 1980, and
British Patent Application No. 1,275,480. The propionates are used in low pH foods, less than pH 6.0.
British Patent No. 1,275,480 indicates that the propionate salts require a low pH to be effective. It had
not been thought that the propionate salts would be useful against bacteria at low levels in higher pH
processed meats at temperatures above freezing probably because propionates, considered to be
mycostats, are not effective against mold at high (greater than 5.3) pH. British Patent No. 1,562,568
describes the use of sodium propionate in frozen meats as a mycostat at pH 5.5 to 7.0 without any
specific amounts being disclosed.Freezing greatly reduces the risk of microbial growth and also
reduces the taste of the meat. Woolford and Anderson, Food Industries 17:622 (1945) describes the
439/1006
use of propionates in various foods for inhibiting various bacteria and molds, but not in meats at
temperatures above freezing.
U.S. Patent No. 4,883,673 to Gonzalez describes the use of bacteriocins in foods. The bacteriocins
are not described as useful with propionates.
The shelf life of packaged (canned or fresh packaged) processed meat products is limited by
bacterial spoilage. This spoilage is caused primarily by gram-negative bacteria and secondarily by
lactic acid producing bacteria. One solution to this problem is to freeze the product during
distribution to inhibit spoilage. This practice detracts from the fresh concept of the product and taste,
increases the product cost and is a problem if there is a failure in the freezing. It would be preferred
to refrigerate the meat preferably at between about 4 DEG C to 12 DEG C. A method is needed for
slowing the growth of pathogens at these temperatures in meat.
OBJECTS
It is therefore an object of the present invention to provide a method for inhibiting the growth of
bacteria in a processed meat to extend shelf life. Further, it is an object of the present invention to
provide a method which is simple and economical and which does not impart an off taste to the meat.
Further still, it is an object of the present invention to provide preferred compositions incorporating a
bacteriocin and a propionate adapted for use in the processed meat. These and other objects will
become increasingly apparent by reference to the following description.
GENERAL DESCRIPTION
The present invention relates to a method for protecting an unspoiled processed meat having a pH
between about pH 6.0 and 6.5 which comprises: inoculating the meat with a source of an inorganic
propionate salt in an amount less than about 1% by weight which inhibits bacteria present in the
meat without contributing a flavor to the meat; and storing the meat at temperatures above freezing,
wherein growth of the bacteria in the meat is inhibited by the propionate salt.
440/1006
The present invention further relates to a preferred composition which comprises an inorganic
propionate salt and a bacteriocin from Pediococcus acidilactici, wherein the composition inhibits
Pediococcus, Lactobacilli, Streptococcus, Listeria, Pseudomonas, Salmonella, Enterobacter, and
Serratia in meats.
As used herein the term "meat" means animal flesh alone or in combination with various fillers. Such
meats include, for instance, beef, poultry and fish. The term "processed" means handling of the flesh
after the animal is killed, including slaughter, cutting, mixing, cooking and packaging. All of these
steps introduce bacteria.
Preferably the inorganic propionate salt is an alkali metal or alkaline earth metal salt. The propionate
salt can be in a pure form or provided as a component of a dried fermentation broth as described by
Anderson (U.S. Patent No. 4,497,833) and Ahern et al (U.S. Patent Nos. 4,743,453 and 4,676,987).
The Propionibacterium shermanii is used for this purpose. Other transition metal propionate salts can
be used but are not preferred so long as they do not impart a taste to the meat.
The propionate salt is used in an amount of less than about 1% by weight in the meat, preferably
between about 0.05 and 0.5% by weight. The composition can also contain chloride salts (e.g.
sodium chloride) or nitrite or nitrate salts (e.g. sodium nitrite) or other such salts which inhibit the
growth of bacteria. The chloride salt is used in amounts up to 4% by weight of the raw meat. The
nitrite salt is limited to 2% by weight of the meat by law in the United States. The composition can
also contain various fillers or dispersing liquids.
The bacteria inhibited in the processed meat by the propionate salt include psychrotrophs such as
Pseudomonas sp, Salmonella sp such as Salmonella newport; and Listeria sp such as Listeria
monocytogenes, Enterobacter sp, such as Enterobacter agglomerans, Serratia sp such as Serratia
liquefaciens and other bacteria which occur in processed meat, because of the processing steps
including slaughtering.
The Pediococcal bacteriocin is preferably derived from Pediococcus acidilactici NRRL-B-18050 as
described in U.S Patent No. 4,883,673 to Gonzalez. The bacteriocin is provided at levels between
about 1,500 and 5,000 AU of bacteriocin per gram of the composition which is introduced into the
meat in a composition with the propionate salt. The result of introducing the composition into the
meat is that the meat contains between about 15 and 45 AU of the bacteriocin per gram of meat.
Pediococci, Lactobacilli and Streptococcus as well as Listeria are inhibited by the bacteriocin.
441/1006
Preferably the propionate salt alone or with the bacteriocin are provided as a dried composition. The
dried composition preferably contains between about 10 and 100% propionate salt by weight when
used alone. When used with the bacteriocin the dried composition contains between about 10 and
99% propionate depending upon the weight of the bacteriocin. The propionate salts and optionally
the bacteriocin can also be introduced into the meat as a liquid.
The propionate salts are effective in inhibiting the growth of spoilage organisms when incorporated
into a marinade or sauce which dresses a packaged meat product. Additionally, incorporation of
propionate salts in the basting solution used to pump meat products by injection provides an
appropriate method for delivery of effective concentrations of the propionate salt to fresh meat and
poultry products before cooking. The propionate salt can also be mixed with ground or comminuted
processed meats.
The meats are usually packaged using flexible films for refrigerated meats or in cans, jars and the
like. These packages can allow growth of the bacteria if not perfectly sealed.
SPECIFIC DESCRIPTION
The following are illustrative Examples of the method of the present invention.
Example 1
A fresh, ground, pork sausage formulation was prepared which contained:
3000.0 g pork (30% fat)
60.0 g water
75.0 g salt
16.8 g spice mix (Paprika, white pepper, caraway seeds, cayenne pepper and ground anise seeds)
0.09 g ea. BHA/BHT
0.09 g sodium citrate
442/1006
The meat mixture was divided into 4 equal portions. One portion was retained as a control. Sodium
propionate was added at 0.08%, 0.15% and 0.30% and the samples were incubated at 5 DEG C.
Total aerobic plate count and Gram-negative plate counts were conducted during 12 days
incubation at 5 DEG C. As seen in Table 1, as little as 0.08% sodium propionate effectively controlled
the growth of Gram-negative spoilage bacteria. The total aerobic bacterial population was reduced
10 fold by the added sodium propionate.
Example 2
A fresh sausage was prepared as in Example 1. The sausage was divided into 5 equal portions. One
portion was retained as control. The remaining portions were treated with either sodium propionate
(0.30% or 0.43%) or calcium propionate (0.29% or 0.42%). The five samples were incubated at 5
DEG C and evaluated for total aerobic count and Gram-negative count during 17 days storage.
The results presented in Table 2 demonstrate that calcium propionate is equally effective as sodium
propionate in inhibiting the growth of spoilage organisms in the meat system and provides less
sodium in the meat.
Example 3
Fresh sausage was prepared as in Example 1 and was treated with either sodium lactate at 0.38% or
sodium propionate at 0.30% which resulted in equivalent concentrations of lactate and propionate.
The samples were stored at 5 DEG C and evaluated for total aerobic count and Gram-negative count.
As seen in Table 3, 0.30% sodium propionate inhibited the total aerobic population for 13 days and
prevented the increase in the Gram-negative population for 17 days. Conversely, sodium lactate
treatment failed to affect the total aerobic count and reduced the Gram-negative count only modestly
during the 17 day incubation period.
443/1006
This demonstrates that the inhibitory activity of propionate salts can be observed at a much lower
concentration than might be needed for sodium lactate as set forth in U.S. Patent No. 4,798,729 to
Anders et al.
Example 4
Inhibition of Salmonella newport in cooked chicken by sodium propionate.
Commercially sterile, canned, cooked chicken was obtained from a local market. The product
contained white and dark chunked chicken, salt and water. The salt concentration was determined to
be approximately 0.75%. The chicken was inoculated with Salmonella newport to deliver
approximately 5000 CFU/gram. The inoculated chicken was divided into 4 portions. Sodium
propionate was added to three portions at 0.22%, 0.30% or 0.42%. The fourth portion had no
additions and served as the control.
As seen in Table 4, the sodium propionate effectively inhibited the growth of Salmonella newport in
the inoculated chicken.
Example 5
Inhibition of Salmonella newport by calcium propionate in chicken.
Canned chicken was inoculated with Salmonella newport as in Example 4. The inoculated chicken
was divided into two portions. Calcium propionate (0.42%) was added to one portion and the second
was used as an uninoculated control. The samples were incubated at 5 DEG C.
As noted in Table 5, growth of Salmonella newport was completely inhibited for the entire 27 day
incubation. Thus, the calcium propionate inhibits the growth of S. newport as effectively as does the
sodium salt (Example 4).
444/1006
Example 6.
Inhibition of Listeria monocytogenes by calcium propionate in cooked chicken.
Commercially sterile canned chicken was obtained from a local market, as in Example 4. The
product contained white and dark chunked chicken, salt and water. The salt concentration was
determined to be approximately 0.75%. The chicken was inoculated with a stock culture of Listeria
monocytogenes to deliver approximately 4000 CFU/g. The chicken was divided into two portions.
Calcium propionate was added to one portion at a rate of 0.42%. The second portion functioned as
the control.
As seen in Table 6, 0.42% calcium propionate effectively reduced the growth rate of Listeria
monocytogenes in the processed chicken.
Example 7
A fresh sausage prepared as in Example 1 was divided into two portions. One portion was retained
as the control. The second portion was treated with 1.32% by weight of a dried fermentation broth
that contained 38% calcium propionate (0.50 percent by weight of the sausage). The broth was
prepared as per Ahern et al (U.S. Patent 4,743,453). The samples were incubated at 5 DEG C Total
aerobic counts were conducted on APT agar during 18 days incubation at 5 DEG C. The dried
fermentation broth effectively reduced the level of bacterial growth in the sample.
Example 8
A propionate containing powder was prepared. One broth was prepared and dried by the method of
Ahern et al (U.S. Patent 4,743,453). The second broth was prepared by fermenting a dextrose yeast
extract medium by a strain of Pediococcus acidilactici (NRRL-B-18050) by the method described by
Gonzalez (U.S. Patent 4,883,673) to produce a bacteriocin. The dried propionate as a powder was
added to the bacteriocin containing broth and then dried. Eighty-five percent (85%) by weight of the
445/1006
dried product is the propionate salt containing material and fifteen percent (15%) by weight is the
dried bacteriocin containing material. The bacteriocin was present in an amount of about 1600 AU
per ml in the broth. The propionate salt was present in an amount of about 32% by weight calcium
propionate in the powder. The AU of the powder was about 3000 AU per gram.The bacteriocin
provided inhibition of spoilage against various Pediococci, Lactobacilli and Streptococcus, as well as
Listeria.
This fermented powder was incorporated into a lemon herb dressing (27% solids) at a rate of 1%.
The dressing has a near neutral pH. Chicken breast quarters were dredged through the marinade
and vacuum packaged. A control chicken breast was coated with an identical marinade that did not
contain the propionate containing solids and similarly vacuum packaged.
The samples were incubated at 5 DEG C for 21 days at which time the samples were evaluated for
the increase in the concentration of gram-negative bacteria. The control sample contained 4 x 10
gram-negative bacteria per gram. Conversely, in the treated sample the gram-negative count was
less than 100.
Example 9
The calcium propionate and bacteriocin powder described in Example 8 was evaluated in a canned
meat spread which contains nitrite which has a near neutral pH. The commercially available meat
spread (SPAM TM , Hormel) was inoculated with the hereinafter specified test bacteria at a rate of
5,000 viable cells per gram. The inoculated product was divided into two portions. The calcium
propionate and bacteriocin powder was added to one portion at a rate of 1% (contains 0.32%
calcium propionate). The second portion of meat served as the control. The samples were incubated
at 7 DEG C for 19 days at which time the bacterial population was enumerated.
As seen in Table 8, the propionate powder effectively eliminated Listeria monocytogenes and
Salmonella newport. Staphylococcus aureus was significantly reduced.
As can be seen from the foregoing description, the propionate salts at near neutral pH's and at
refrigeration temperatures inhibited the growth of spoilage bacteria. Taste tests confirmed that the
meats with the propionate salts at the low levels (0.05 to 1.0% and preferably 0.5% or less by weight
446/1006
of the meat or meat formulation) did not impart any taste. The bacteriocin is tasteless at the levels
used.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
1. A method for protecting an unspoiled processed meat having a pH between about pH 6.0 and 6.5
which comprises:
(a) inoculating the meat with a source of an inorganic propionate salt in an amount less than about
1% by weight which inhibits bacteria present in the meat without contributing a flavor to the meat;
and
(b) storing the meat at temperatures above freezing wherein growth of the bacteria in the meat is
inhibited by the propionate salt.
2. The method of Claim 1 wherein the salt is selected from the group consisting of calcium
propionate and sodium propionate.
3. The method of Claim 1 wherein the propionate salt is used in an amount between about 0.05% to
0.5% by weight of the meat.
4. The method of Claim 1 wherein the bacteria are psychrotrophic bacteria.
5. The method of Claim 1 wherein the bacteria include psychrotrophic bacteria.
6.The method of Claim 1 wherein the bacteria include Salmonella newport.
7. The method of Claim 1 wherein the bacteria include Listeria monocytogenes.
8. The method of Claim 1 wherein the meat contains sodium chloride.
9. The method of Claim 8 wherein the sodium chloride is present in an amount up to about 4% by
weight.
447/1006
10. The method of Claim 1 wherein the meat is raw which is refrigerated.
11. The method of Claim 1 wherein the meat is canned meat.
12. The method of Claim 1 wherein the meat contains a nitrite salt.
13. The method of Claim 1 wherein along with the inorganic propionate salt, the meat is inoculated
with a bacteriocin derived from Pediococcus acidilactici into the meat in an amount which inhibits
Pediococci, Lactobacilli and Streptococcus.
14.The method of Claim 13 wherein the propionate salt and the bacteriocin are provided together as
a dried product derived from combining fermentation broths from Propionibacterium shermanii and
from the Pediococcus acidilactici and drying the combined broths to provide the dried product.
15. A composition which comprises:
an inorganic propionate salt and a bacteriocin derived from Pediococcus acidilactici, wherein the
composition inhibits Pediococci, Lactobacilli, Streptococcus, Listeria, Pseudomonas, Salmonella,
Enterobacter, and Serratia in meats.
16. The composition of Claim 15 wherein the composition is effective at levels of less than about 1%
by weight in the meat.
17. The composition of Claim 15 wherein the inorganic propionate salt is provided by fermenting
Propionibacterium shermanii in a broth with neutralization to provide a fermented broth and mixing
the Propionibacterium shermanii derived fermented broth with a fermented broth from the
Pediococcus acidilactici and drying the mixture to produce a dried product which can be introduced
into the meat.
18. The composition of Claim 17 wherein the dried product is effective at levels less than about 1%
by weight in the meat.
19. The composition of Claim 15 as a dried product wherein the composition contains between about
1500 and 5000 AU per gram of the bacteriocin and wherein the propionate salt is present in an
amount between about 10 and 99 percent by weight of the composition.
448/1006
20. The composition of Claim 15 wherein the inorganic propionate salt is a chemically pure form.
449/1006
56. NZ238515 - 26.12.1991
NOVEL BACTERIOCIN FROM A STRAIN OF LACTOCOCCUS LACTIS SUBSP. CREMORIS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ238515
Inventor(s):
HOLO HELGE (NO); NES INGOLF F (NO)
Applicant(s):
NORWEGIAN DAIRIES ASS (NO); HOLMES MICHAEL J (GB)
IP Class 4 Digits: C12N; C12P; A23C
IP Class:
C12N15/31; A23C19/00; C12P7/06
E Class: A23C19/11; C07K14/315; A23C9/13E; C12C11/00B; C12H1/14
Application Number:
WO1991EP01109 (19910612)
Priority Number: GB19900013577 (19900618); GB19900023380 (19901026)
Family: WO9119802
Equivalent:
AU8081691; CA2085580; EP0535039WO9119802; IE912035
Abstract:
THE INVENTION PROVIDES A POLYPEPTIDE HAVING OR INCLUDING THE AMINO ACID
SEQUENCE (I) AND DERIVATIVES AND FRAGMENTS THEREOF HAVING BACTERIOCIN ACTIVITY
AND A POLYPEPTIDE HAVING OR INCLUDING THE AMINO ACID SEQUENCE (II) AND
DERIVATIVES AND FRAGMENTS THEREOF HAVING BACTERIOCIN IMMUNITY
ACTIVITY.Description:
Novel bacteriocin from a strain of Lactococcus lactis subsp. cremoris
This invention relates to a novel bacteriocin and its isolation, synthesis and use.
450/1006
We have isolated a novel bacteriocin from a strain of
Lactococcus lactis subsp. cremoris. This strain has never been described in the literature.
The above microorganism is autolytic when cell concentrations are high and we have discovered that
it produces a bacteriocin which is found extracellularly in growth media, for example M17 medium.
The autolysis of the bacteria can be shown to be due to the lytic properties of the bacteriocin. Lysis is
prevented by the addition of proteases that degrade the bacteriocin.
Term "bacteriocin" is used herein to include substances released by bacteria which kill not only the
productive organism itself, but also other strains of bacteria, by any mechanism, including lysis. Thus,
the new bacteriocin here concerned has been shown to inhibit the growth of more than 120 strains of
lactococci, including strains producing the known bacteriocins diplococcin and nisin. However, the
productive organism itself carries a gene coding for an immunity factor providing resistance to the
bacteriocin to prevent indiscriminate lysis and the bacteriocin only seems to lyse its productive
organism at high cell concentrations.
We have determined the amino acid sequence of the new bacteriocin. According to one aspect of
the present invention, we provide a polypeptide having or including the amino acid sequence: 1 -Lys
Leu Thr Phe lie Gln Ser Thr Ala Ala Gly Asp Leu Tyr Tyr 1 SAsn Thr Asn Thr His Lys Tyr Val Tyr Gin
Gln Thr Gln Asn Ala 31-Phe Gly Ala Ala Ala Asn Thr lie Val Asn Gly Trp Met Gly Gly 4SAla Ala Gly
Gly Phe Gly Leu His His and derivatives and fragments thereof having bacteriocin activity.
The above structure is different from that of nisin and has no meaningful sequence homology with
other known polypeptide sequences in the SWISS PROT data bank. The lytic activity of the
bacteriocin of the invention is substantially greater than that of previously known L.lactis bacteriocins.
The novel bacteriocin is heat stable and retains activity after boiling in water for 30 minutes. This is of
value in permitting its use in industrial processes using L.lactis organisms for example cheese and
yoghurt manufacture.
The bacteriocin appears to kill lactococci by lysis.
Accelerated lysis of lactococci is beneficial in accelerating cheese ripening and the new bacteriocin
is thus of particular application in the production of cheese.
451/1006
The lytic activity of the bacteriocin may also be of use in production of cell wall preparations or for
liberation of nucleic acid material.
Since certain bacteria, for example Gram-negative bacteria, are resistant to bacteriocins, negative
selection is possible by using the bacteriocin according to the invention to remove certain cells, for
example
L.lactis, from mixed cell populations e.g. in starter cultures for fermentation.
Where the productive strain of L.lactis is used as the sole or principle organism in an industrial
process such as cheese or yoghurt production, addition of the bacteriocin of the invention to the
starter culture serves to eliminate foreign organisms and may be effective against, for example,
spore forming clostridia or unwanted strains of L.lactis.
The bacteriocin may advantageously be added to a cheese or yoghurt fermentation at a relatively
late stage, after lactic acid, protease and flavour production by the L.Lactis organism has already
taken place.
By keeping the productive strain pure, either in the starter culture or in the milk or other medium,
unformity of production can be improved.
The bacteriocin may also be used to kill selectively strains of lactic acid producing bacteria in beer
and distillery fermentations, since these are attributed in the literature to be the major causes of
spoilage in unpasteurised beers and give rise to the greatest proportion of infections during
fermentation.
The invention particularly includes starter cultures of microorganisms containing the bacteriocin as
an inhibitor of contaminating lactococcus species. Such microorganisms may, for example, be
strains of L.lactis resistant to the bacteriocin e.g. the producing organism, so that only unwanted
microorganisms are removed from the starter culture, or yeasts of use in beer or distillery
fermentations. Such starter cultures will normally be in lyophilised form.
In view of its high specificity, the bacteriocin may be used as a taxonomic tool in the identification of
Lactococcus species.
452/1006
The new bacteriocin may be isolated from cultures of
Lactococcus lactis subsp. cremoris by fractionation of the growth medium whereby fractions
enriched in the bacteriocin are collected. By applying known fractionation techniques it is possible to
obtain the bacteriocin in electrophoretic purity. Thus for example, the organism may be grown in a
suitable culture medium, e.g. M17 broth, and the supernatant subjected to fractional precipitation
e.g. with ammonium sulphate, followed by chromatography e.g. on carboxymethyl agarose with
elution with phosphate buffer and/or on phenylsuperose with gradient elution with phosphate buffer
containing increasing concentrations of ethanol.
We have been able to clone and sequence a two gene operon from L. lactis cremoris which codes
for the bacteriocin in the pro-form as well as a further protein which is believed to be the immunity
factor providing resistance against self-destruction by the bacteriocin.
The full sequence of the operon is shown in Fig. 1 which also shows the sequence of the
corresponding proteins, i.e. the bacteriocin and the immunity factor. The operon starts with a
regulating region for expression of the genes. This is believed to comprise promoters at regions -35
and -10 and a ribosomal binding site. The gene coding for the pro-sequence runs from base 312 to
base 374. The gene coding for the bacteriocin runs from base 375 to base 536. The gene coding for
the putative immunity factor runs from base 554 to base 847 in a different reading frame. Three
putative promoter sequences are indicated as P1, P2 and P3 in regions -35 and -10 and ribosome
binding sites are indicated as RBS.
According to a further feature of the invention we provide DNA coding for the bacteriocin and for the
immunity factor respectively. It will be appreciated that knowledge of the overall amino acid
sequence shown in claim 1 and/or the DNA sequence coding for the probacteriocin does not provide
an indication of the position of the first codon coding for the mature bacteriocin.
The invention thus includes not only the DNA sequences shown in Fig. 1 but also sequences which
due to the degeneracy of the code, are also capable of coding for the proteins concerned.
The invention also includes cloning and expression vectors containing the DNA coding for the
mature bacteriocin and/or for the immunity factor. Expression vectors appropriate to L. lactis are
particularly preferred.
The invention also includes strains of L. lactis transformed with such vectors.
453/1006
The immunity factor according to the invention may be of use in combating the effects of L. lactis
bacteriocins, for example, in controlling the effects of the bacteriocin according to the invention.
The gene coding for the immunity factor may be used as a selective marker in future construction of
food grade cloning vector, for example instead of an antibiotic matter.
The operon shown in Fig. 1 was obtained from the fragmented plasmid DNA of L. lactis cremoris,
using a probe comprising all or part of the non-coding DNA strand corresponding to the mature
bacteriocin coding portion of the DNA strand shown in Fig. 1. The DNA coding for the mature
bacteriocin or immunity factor may be incorporated into any convenient cloning vector for
amplification and into an expression vector for transformation of host microorganisms such as L.
lactis, for example cloning vector pIL253 (A. Chopin, Biochimie 70, 1988, 59-566). Growth under
suitable culture conditions will provide the bacteriocin in the growth medium, from which it can be
isolated by the techniques described above.
Furthermore, strains of L. lactis may be transformed with multiple copies of a plasmid or other vector
containing the required DNA sequence to provide an improved strain giving rise to enhanced
production of the bacteriocin. Such improved strains may provide more rapid lysis and hence
accelerated cheese ripening when used in cheese manufacture. In particular, the strain of L.lactis
which produces the bacteriocin and which thus also carries a resistance gene, may be provided with
such multiple copies of the vector; this will thus be able to proliferate without premature destruction
by the bacteriocin.
The new bacteriocin may also be prepared by chemical synthesis, for example using solid phase
synthesis, advantageously using a polypeptide synthesis apparatus, as commercially available. In
such a synthesis, active side chain groupings (e.g. amino or carboxyl groups) of the respective
amino acids will be protected and the final step will be deprotection and/or removal from the inert
support to which the polypeptide is attached during synthesis.
In building up the peptide chains, one can in principle start either at the C-terminal or the N-terminal
although only the C-terminal starting procedure is in common use.
Thus, one can start at the C-terminal by reaction of a suitable derivative of, for example histidine with
a suitable protected derivative of leucine. The histidine derivative will have a free a-amino group
454/1006
while the other reactant will have either a free or activated carboxyl group and a protected amino
group. After coupling, the intermediate may be purified for example by chromatography, and then
selectively N-deprotected to permit addition of a further N-protected and free or activated amino acid
residue. This procedure is continued until the required amino acid sequence is completed.
Carboxylic acid activating substituents which may, for example, be employed include symmetrical or
mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2,4,5, trichlorophenylester,
N-hydroxybenzotriazole ester (OBt), N-hydroxysuccinimidylester (OSu) or pentafluorophenylester
(OPFP).
The coupling of free amino and carboxyl groups may, for example, be effected using
dicyclohexylcarbodi- imide (DCC). Another coupling agent which may, for example, be employed is
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).
In general it is convenient to effect the coupling reactions at low temperatures, for example, -20'C up
to ambient temperature, conveniently in a suitable solvent system, for example, tetrahydro- furan,
dioxan, dimethylformamide, methylene chloride or a mixture of these solvents.
It may be more convenient to carry out the synthesis on a solid phase resin support. Chloromethylated polystyrene (cross-linked with 1% divinyl benzene) is one useful type of support; in this
case the synthesis will start the C-terminal, for example by coupling
N-protected histidine to the support.
A number of suitable solid phase techniques are described by Eric Atherton, Christopher J. Logan,
and
Robert C. Sheppard, J. Chem. Soc. Perkin I, 538-46 (1981); James P. Tam, Foe S. Tjoeng, and R. B,
Merrifield J. Am. Chem. Soc. 102, 6117-27 (1980); James
P. Tam, Richard D. Dimarchi and R. B. Merrifield Int. J.
Peptide Protein Res 16 412-25 (1980); Manfred Mutter and
Dieter Bellof, Helvetica Chimica Acta 67 2009-16 (1984).
A wide choice of protecting groups for amino acids are known and are exemplified in Schrdder, E.,
and Ltibke,
455/1006
K., The Peptides, Vols. 1 and 2, Academic Press, New
York and London, 1965 and 1966; Pettit, G.R., Synthetic
Peptides, Vols. 1-4, Van Nostrand, Reinhold, New York 1970, 1971, 1975 and 1976; Houben-Weyl,
Methoden der Organischen Chemie, Synthese von Peptiden, Band 15,
Georg Thieme Verlag Stuttgart, NY, 1983; The Peptides,
Analysis, synthesis, biology 1-7, Ed: Erhard Gross,
Johannes Meienhofer, Academic Press, NY, San Fransisco,
London; Solid phase peptide synthesis 2nd ed., John M.
Stewaet, Janis D. Young, Pierce Chemical Company.
Thus, for example amine protecting groups which may be employed include protecting groups which
may be employed include protecting groups such as carbobenzoxy (Z-), t-butoxycarbonyl (Boc-), 4methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr-), and 9-fluorenylmethoxycarbonyl (Fmoc-). It will be
appreciated that when the peptide is built up from the C-terminal end, an amine protecting group will
be present on the a-amino group of each new residue added and will need to be removed selectively
prior to the next coupling step. One particularly useful group for such temporary amine protection is
the
Fmoc group which can be removed selectively by treatment with piperidine in an organic solvent.
Carboxyl protecting groups which may, for example be employed include readily cleaved ester
groups such as benzyl (-OBZ1), p-nitrobenzyl (-ONB), or t-butyl (-tOBu) as well as the coupling on
solid supports, for example methyl groups linked to polystyrene.
It will be appreciated that a wide range of other such groups exists as, for example, detailed in the
above-mentioned literature references, and the use of all such groups in the hereinbefore described
processes fall within the scope of the present invention.
A wide range of procedures exists for removing amineand carboxyl-protecting groups. These must,
however, be consistent with the synthetic strategy employed. The side chain protecting groups must
be stable to the conditions used to remove the temporary a-amino protecting groups prior to the next
coupling step.
456/1006
Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be
removed simultaneously by acid treatment, for example with trifluoro acetic acid. In building up the
peptide chains, one can in principle start either at the C-terminal or the
N-terminal although only the C-terminal starting procedure is in common use.
Thus, one can start at the C-terminal by reaction of a suitabe derivative of, for example histidine with
a suitable protected derivative of leucine. The histidine derivative will have a free a-amino group
while the other reactant will have either a free or activated carboxyl group and a protected amino
group. After coupling, the intermediate may be purified for example by chromatography, and then
selectively N-deprotected to permit addition of a further N-protected and free or activated amino acid
residue. This procedure is continued until the required amino acid sequence is completed.
Carboxylic acid activating substituents which may, for example, be employed include symmetrical or
mixed anhydrides, or activated esters such as for example p-nitrophenyl ester, 2,4,5,trichlorophenylester,
N-hydroxybenzotriazole ester (OBt), N-hydroxy succinimidylester (OSu) or pentafluorophenylester
(OPFP).
The coupling of free amino and carboxyl groups may, for example, be effected using
dicyclohexylcarbodi-imide (DCC). Another coupling agent which may, for example, be employed is
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).
In general it is convenient to effect the coupling reactions at low temperatures, for example, -20 C up
to ambient temperature, conveniently in a suitable solvent system, for example, tetrahydro-furan,
dioxan, dimethylformamide, methylene chloride or a mixture of these solvents.
It may be more convenient to carry out the synthesis on a solid phase resin support. Chloromethylated polystyrene (cross-linked with 1% divinyl benzene) is one useful type of support; in this
case the synthesis will start the C-terminal, for example by coupling
N-protected histidine to the support.
A number of suitable solid phase techniques are described by Eric Atherton, Christopher J. Logan,
and
Robert C. Sheppard, J. Chem. Soc. Perkin I, 538-46 (1981); James P. Tam, Foe S. Tjoeng, and R. B,
Merrifield J. Am. Chem. Soc. 102, 6117-27 (1980); James
457/1006
P. Tam, Richard D. Dimarchi and R. B. Merrifield Int. J.
Peptide Protein Res 16 412-25 (1980); Manfred Mutter and
Dieter Bellof, Helvetica Chimica Acta 67 2009-16 (1984).
A wide choice of protecting groups for amino acids are known and are exemplified in Schroder, E.,
and Ltlbke,
K., The Peptides, Vols.-l and 2, Academic Press, New
York and London, 1965 and 1966; Pettit, G.R., Synthetic
Peptides, Vols. 1-4, Van Nostrand, Reinhold, New York 1970, 1971, 1975 and 1976; Houben-Weyl,
Methoden der
Organischen Chemie, Synthese von Peptiden, Band 15,
Georg Thieme Verlag Stuttgart, NY, 1983; The Peptides,
Analysis, synthesis, biology 1-7, Ed: Erhard Gross,
Johannes Meienhofer, Academic Press, NY, San Fransisco,
London; Solid phase peptide synthesis 2nd ed., John M.
Stewaet, Janis D. Young, Pierce Chemical Company.
Thus, for example amine protecting groups which may be employed include include protecting
groups such as carbobenzoxy (Z-), t-butoxycarbonyl (Boc-), 4-methoxy-2,3,6-trimethyl-benzene
sulphonyl (Mtr-), and 9-fluorenylmethoxycarbonyl (Fmoc-). It will be appreciated that when the
peptide is built up from the
C-terminal end, an amine protecting group will be present on the a-amino group of each new residue
added and will need to be removed selectively prior to the next coupling step. One particularly useful
group for such temporary amine protection is the Fmoc group which can be removed selectively by
treatment with piperidine in an organic solvent.
Carboxyl protecting groups which may, for example be employed include readily cleaved ester
groups such as benzyl (-OBZ1), p-nitrobenzyl (-ONB), or t-butyl (-tOBu) as well as the coupling on
solid supports, for example methyl groups linked to polystyrene.
It will be appreciated that a wide range of other such groups exists as, for example, detailed in the
above-mentioned literature references, and the use of all such groups in the hereinbefore described
processes fall within the scope of the present invention.
458/1006
A wide range of procedures exists for removing amineand carboxyl-protecting groups. These must,
however, be consistent with the synthetic strategy employed. The side chain protecting groups must
be stable to the conditions used to remove the temporary a-amino protecting groups prior to the next
coupling step.
Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu may be
removed simultaneously by acid treatment, for example with trifluoro acetic acid.
The following Example is given by way of illustration only:
Example 1
Bacterial strains, media, plasmids, and enzymes. The bacterial strains, plasmids and phases used
are listed in Table 1. All lactococcal strains were grown in M17 broth (44) and maintained as frozen
stocks at -800C in
M17 broth containing 10% glycerol. Escherichia coli
DH5a was used for propagating pUC18 and its derivatives.
M13 vectors and clones were propagated in 2x YT (2a) with E. coli JM101 as the host.
Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and DNA molecular weight
standards were purchased from Bethesda Research
Laboratories, Inc. (Gaitherburg, Md.). Calf intestinal alkaline phosphatase, sequence-grade trypsin,
and endoprotease glu-C were purchased from Boehringer GmbH (Mannheim, Germany). Sequenase
was obtained from United
States Biochemical Corp. (Cleveland, Ohio).
Plasmid curing. L. lactis subsp. cremoris LMG 2130 was grown in M17 broth supplemented with 1%
glucose at 38"C in the presence of 0.1 pg of novobiocin per ml. Diluted aliquots from this culture
were spread on M17 broth-1% glucose plates and incubated at 30"C. Colonies were scored for
bacteriocin production.
Bacteriocin assays. Three methods were used to determine bacteriocin activity. (i) Colonies of
possible bacteriocin-producing bacteria were grown on agar plates overnight. A lawn of 3 ml of M17
soft agar (0.7%) containing 100 1 of a fresh culture of the indicator organism was poured over a
plate. After incubation overnight at 30, the colonies were examined for zones of growth inhibition. (ii)
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In M17 agar plates, wells with a diameter of 4 mm were made and filled with bacteriocin solutions.
After the liquid had been completely absorbed by the gel. M17 soft agar containing the indicator
organism was overlaid on the plates to demonstrate bacteriocin activity as described above. (iii)
Bacteriocin activity was quantified as described by Geis et al. (15), except that microtiter plates with
wells containing 200 1 of M17 broth were used. One unit of bacteriocin activity (BU) was arbitrarily
defined as the amount of bacteriocin required to produce 50% growth inhibition (50% of the turbidity
of the control without bacteriocin) of L.
lactis subsp. cremoris IMN C18 in this assay.
Table 1 : Strains, plasmids and phaqes used in this studv
Strains, plasmids Relevant phenotype Source of
or phages reference
Strains
L.lactis subsp.
cremoris
LMG 2130 LCN-A-producing strain G. Vegarud
LMG 2131 lcnA derivative of LMG This study
2130
IMN C18 D. Lillehaug
BC 101 51
L.lactis subsp.
lactis
NIZO 4.25 biovar diacetylactis J. Narvhaus
IL 1403 6
2130
IMN C18 D. Lillehaug
BC 101 5
E. Coli
DH5a 17
JM101 31
Plasmids
pIL253 41
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pUC18 53
M13mpl8 31
M13mpl9 31
pON1 pUC18 with 4-kb HindIII This work
fragment containing lcnA
pON2 pUC18::pIL253 with 4-kb This work
HindIII fragment
containing lcnA
pON7 pUC18::pIL253 with This work
1.2-kb Rsal-HindIII
fragment containing lcnA
Purification of LCN-A. The bacteriocin was purified from 1-liter cultures of L. lactis subsp. cremoris
LMG 2130. The various steps of the purification procedure were carried out at 4'C-unless otherwise
stated.The cells were grown to the early stationary phase, and the bacteria were removed by
centrifugation at 10,000 x g for 10 min. The bacteriocin was precipitated from the culture
supernatant by the addition of 280 g of ammonium sulfate per liter. Following centrifugation at 10,000
x g for 30 min, the pellet was dissolved in water and adjusted to pH 7.3 by the addition of 0.5 M
Na2HPO4.
This solution was applied to a 10-ml CM-Sepharose column (Pharmacia Uppsala, Sweden)
equilibrated with mM sodium phosphate (pH 7.3). The column was washed with 40 ml of 20 mM
sodium phosphate (pH 7.3) before the bacteriocin was eluted with 20 ml of the same buffer
containing o.3
M NaCl. The bacteriocin was subjected to reverse-phase liquid chromatography at room temperature
with fast protein liquid chromatography equipment (Pharmacia).
The eluate from the cation exchanger was applied to a 1ml Phenyl-Superose column (Pharmacia)
equilibrated with 10 mM sodium phosphate (pH 7.3). Following washing with 10 mM sodium
phosphate (pH 7.3), elution was carried out with a linear gradient of 0 to 60% ethanol at a flow rate
of 0.3 ml/min. Purified LCN-A was stored in 60% ethanol-2.5 mM sodium phosphate (pH 7.3) at -20 C.
Protein concentrations were determined spectrophotometrically at 280 nm.
Amino acid sequencing. An Applied Biosystems (Foster
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City Calif.) 477A sequencer was used for amino acid sequencing. The phenylthiohydantionderivatized amino acid residues were determined on-line with an Applied
Biosystems 120 phenylthiohydantoin analyzer. The Cterminal part of the sequence was obtained
after cleavage of the Asn-Gly bond with hydroxylamine at pH 9 as described by Bornstein and Galian
(3).
DNA isolation, analysis and manipulations. Plasmid DNA was isolated from L. lactis as described by
Klaenhammer (23). Small-scale preparation of E. coli plasmid DNA was performed with GeneClean
(BIO 101, La Jolia,
Calif.). Large-scaled isolation of plasmids from E.
coli was performed by the alkaline lysis method described by Maniatis et al. (25). The M13 plusstrand
DNA template for sequencing was prepared from infected 1.5 ml cultures as described previously
(2a).
Enzymes for DNA manipulations were used in accordance with manufacturer's specifications.
Plasmid DNA from strain LMG 2130 used for cloning was purified by CsCl isopycnic centrifugation
(33).
Restriction fragments of the desired size for cloning were isolated and purified from 0.7% agarose
gels with
Gene-Clean.
DNA cloned in E. coli was subcloned in lactococci as follows. pUC18 plasmids with inserts were
fused to pIL253 by EcoRI digestion and ligation. The resultant constructs were transformed into E.
coli. Clones were obtained by selection for erythromycin (300 pg/ml) and ampicillin (50 gXml)
resistance. Plasmid DNA extracted from the clones was used to transform lactococci by
electroporation as described by Holo and Nes(20).
Transformation of E. coli was performed by the method of
Hanahan (17).
Nucleic acid hybridizations and nucleotide sequencing.
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On the basis of the sequence extending from amino acid 25 in LCN-A, the following 64-folddegenerated synthetic oligodeoxynucleotide probe was made (with an Applied
Bio-systems 381A DNA synthesizer); 3'-ATIGT(T/C)GT(T/C)
TG(I/C)TG(T/C)TTICG(I/C)AAICC-5'. Colony hybridization was performed as described by Hanahan
and Meselson (18).
Southern blots were made by vacuum transfer (2016
Vacugene; Pharmacia) of-restriction endonucleasedigested DNA (fractionated on 0.7t agarose gels)
to
GeneScreen Plus membranes (NEN Research Products;
Dupont, Boston, Mass,) (42), Hybridization with the 26mer oligodeoxynucleotide was performed as
described by
Church and Gilbert (7). Nucleotide sequencing by the didoxynucleotide method (37) was carried out
on restriction fragments cloned into M13mpl8 and M13mpl9 [a-35S]dATP (600 Ci/mmol; Amersham
International,
Amersham, United Kingdom) was used for labelling.
Nucleotide sequence accession number. The nucleotide sequence presented in this article has been
assigned
EMBL accession number M63675.
Table 2 : Purification of LCN-A
Fraction Vol A28o Total Sp act Purif- Yield
(ml) activity Bus/ml/A28o cation (Z) (105BUs) (fold)
(10 Bus) (foLd)
Culture supernatant 1,000 14.6 15 102.8 1 100 Amnoniun sulfate 100 5.35 13 2,428 23 87 precipitate
Cation-exchange 12 0.17 9.6 4.6 x 105 4,485 64 chromatography
Reverse-phase 2 0.51 2.4 2.4 x 105 2,281 16 chromatography
Purification of LCN-A L. lactis subsp. cremoris LMG 2130 was found to produce a bacteriocin
constitutively during growth in M17 medium. A procedure for purifying the bacteriocin from the
culture supernatant was developed.
The purification scheme is shown in Table 2. The protein was about 95% pure, as judged by amino
acid sequence analysis. The amino acid sequence of the purified bacteriocin is es shown above.
The bacteriocin was found to contain 54 amino acid residues with a calculated molecular weight of
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5,778 which has been confirm by mass spectrography, thus showing the substantial absence of
glycosylation or methylation. No significant sequence similarly was found to any protein or putative
gene product in the Swiss-Prot or NBRF data bases.
We have named the new bacteriocin LCN-A. The protein is rich in alanine residues (8 of 54) and
glycine residues (8 of 54) and contains only three charged amino acid residues. The calculated
isoelectric point of the bacteriocin was 9.2. The extinction co-efficient of
LCN-A at 280 nm was estimated to be 1.2 z 104 cm~1 W1 from its content of tryptophan and
tyrosine (4). Thus, pure LCN-A had a specific activity of about 4.9 x 105
BUs/mg. Assuming that the activity of LCN-A was not reduced during purification, strain LMG 2130
produced about 3 mg of LCN-A per liter. By comparison, L. lactis subsp. cremoris 346 was found to
produce 6 mg of diplococcin per liter (10). The pure bacteriocin was not very soluble in water. Upon
storage in aqueous buffers at 4"C, the bacteriocin formed an inactive precipitate.Pure LCN-A could,
however, be stored longer than 6 months at -20C in 60% ethanol containing 2.5 mM sodium
phosphate (pH 7.3) without a detectable loss of activity.
Effect of proteases, LCN-A lost its activity when exposed to various proteases, including the highly
specific endoprotease glu-C and trypsin. In phosphate buffer (pH 7.8), endroprotease glu-C could
cleave the bacteriocin at one site, between amino acid residues 12 (Asp) and 13 (Leu); trypsin could
cleave the bacteriocin at the carboxyl side of its two lysing residues (1 and 21).
Inhibitory spectrum and mode of action. By means of the agar diffusion assay, more than 120 strains
of L. lactis subsp. lactis and L. lactis subsp. cremoris were found to be sensitive to purified LCN-A.
Sensitive strains were rapidly killed by the bacteriocin. The viable count of an exponentially growing
culture of strain IMN
C28 dropped from 2 x 108l/ml after 5 min of exposure to 200 BUs/ml in M17 medium at 30"C.
Table 3 : Sensitivities of some lactococcal strains to
LCN-A
Strain* Sensitivity (ssUS/ml
L.lactis subsp. cremoris
IMN C18 5
LMG 2141 1,000
NCDO 607 1.3
NCDO 924 1,000
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NCDO 1198 0.4
BC 101 50
BC 101(pON2) 5,000
BC 101(pON7) 5,000
L.lactis subsp. lactis
NCDO 604 30
IL 1403 0-4
IL 1403(pON2) 1,500
IL 1403(pON7) 1,500
NCDO 176 (biovar diacetylactis) 20 L.varvisae NCDO 2155 5,000
Table 3 shows the sensitivities of various lactococcal strains to LCN-A. Wide variations in sensitivity
were found. The most sensitive strains tested appeared to be more sensitive to the bacteriocin when
grown in lactic broth (14) than in M17 medium.In lactic broth, 50% growth inhibition of strain NCDO
1198 was observed at a calculated LCN-A concentration of 40 pg/ml, or 7 pM. This amount
corresponds to about 400 molecules of
LCN-A per CFU in the assay.
Of the strains tested, only two, the bacteriocin producer itself (LMG 2130) and L. lactis subsp. lactis
biovar diaceylactis NIZO 4-25, were resistant. This latter strain, however, was not found to produce
the bacteriocin. The nisin (L. lactis subsp. lactis NCDO 496 and NCDO 1403) and diplococcin (L.
lactis subsp.
cremoris NCDO 893)-producing strains tested were all sensitive to LCN-A and were inhibitory to LMG
2130. In addition, the bacteriocin showed weak inhibition of L.
aarvieae NCDO 2155 (Table 3).
Identification and cloning of the genetic determinant for LCN-A. An oligodeoxynucleotide probe
based on the amino acid sequence of LCN-A was used in Southern hybridization analysis to localize
the gene. When plasmid DNA from strain LNG 2130 was probed, one signal, corresponding to a 55kb plasmid, was observed. Strain
LMG 2130 was exposed to plasmid curing. One isolate,
LMG 2131, which did not produce LCN-A was found both to be deprived of the 55-kb plasmid and to
give no signal on a Southern blot. Furthermore, Southern analysis of
LMG 130 plasmid DNA digests revealed signals from a 4-kb
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HindIII fragment a 1.2-kb HindIII-RsaI fragment and a 0.6-kb DraI fragment. The 4-kb fraction of
HindIIIdigested LMG 2130 plasmid DNA was cloned in E. coli with pUC18 as the vector.Of 1,400
clones, 10 were found to be positive after screening with the oligodeoxynucleotide probe. The
recombinant plasmid (pON1) from one of these 10 clones was further restricted with DraI and RsaIII.
The fragments that hybridized to the probe, the 4-kb HindIII fragment, the 1.2-kb HindIII-RsaI
fragment, and the 0.6-kb DraI fragment were subcloned into M13Mp18 and M13mpl9 to yield inserts
in both orientations.
Nucleotide sequence of lcnA. The Hind III-Rsal fragment was sequenced. The nucleotide sequence
of the two consecutive DraI fragments of 625 and 292 nucleotides is shown in Fig. 1. The entire lcnA
gene was contained with the 0.6-kb DraI fragment. Computer analysis of the six possible open
reading frames (ORFs) revealed long
ORFs only on one of the DNA strands. Mature LCN-A of 54 amino acid residues is encoded by the
DNA segment from nucleotide positions 316 to 477. The only possible initiation codon was found at
nucleotide position 253, implying that LCN-A is synthesized as a 75-amino-acid precursor containing
a 21-amino-acid N-terminal extension. The initiation codon is preceded by the possible ShineDalgarno sequence 3' AGGAGA 5' (40).
Three putative promoter elements, all showing considerable similarity to the E. coli a70 consensus
and streptococcal promoters, were found just upstream of this ribosome binding site (RBS) (Fig. 1)
(27,35).
Downstream of lcnA a second ORF, ORF2, was found.
Assuming that there is a translation start site at the
ATG at nucleotide position 495, this ORF encodes a 98amino-acid polypeptide. A possible RBS
sequence, 5'
GAGGATTGA 3', occurs 7 nucleotides from the Met codon.
Downstream of ORF2, extending from nucleotide positions 803 and 896, at two regions of dyad
symmetry, which could form stem-loop structures with AG values of 144.8 and -102.1 kJmol,
respectively (45). The uridine content in their distal-stems suggests that these structures constitute
Rho-independant terminators of the lcnA transcript. No putative terminator or promotor sequences
were found between lcnA and ORF2, indicating that 1cnA and ORF2 may constitute an operon.
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No DNA sequence in the EMBL data base showed a high degree of DNA homology to the DNA
sequence presented here. The best score found was a 57.4% identity to a 122-bp sequence in the
data base.
Cloning in L. lactis. The lcnA gene was cloned in L.
lactis. The PIL.253::pUC18 constructions carrying the 4-kb HindIII fragment and the 1.2-kb HindIIIRsaI fragment were named pON2 and pON7, respectively.
Neither of these two plasmids caused detectable bacteriocin production in L. lactis subsp. cremoris
BC 101. However, when present in BC 101, both pON2 and pON7 conferred resistance to LCN-A.
With either plasmid, the LCN-A concentration causing 50% growth inhibition increased from 50 to
5,000 BUs/ml (Table 3); this result was not seen with transformants containing the cloning vector
alone. Similar results were observed with other strains of L. lactis (data not shown). The only strain
tested that showed bacteriocin production after transformation with the lcnA gene was L. lactis subsp.
lactis IL 1403. When carrying pON2 or pON7, L.
lactis subsp. lactis IL 1403 produced about 60 BUs/ml.
By comparison, the LCN-A-producing strain, LMG 2130, produces about 1,500 BUs/ml.
Sensitivity to LCN-A appears to be general among strains of L. lactis. Since this bacteriocin also is
highly specific, it may be used for the identification of L.
lactis strains. LCN-A is a hydrophobic protein. Its hydrophobic character was demonstrated by its
high affinity for phenyl-Superose. This matrix is intended for use in hydrophobic interaction
chromatography, and most proteins bind to it only at high salt concentrations. LCN-A bound to the
column in the absence of salt and could only be eluted as an active bacteriocin by solvents less
polar than water.
The toxic effects of nisin have been ascribed to its ability to form pores in cytoplasmic membranes
(36).
The hydrophobic character of LCN-A suggests that the cytoplasmic membrane may also be the
target for this bacteriocin. Calculations made as described by Rao and
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Argos (34) predicted that the stretch from amino acids 30 to 52 in LCN-A can form a membranespanning helix (data not shown). The idea that LCN-A acts on the membrane is further supported by
the finding that this bacteriocin causes leakage of intracellular components even in hypertonic
sucrose-containing media (unpublished data).
Secreted proteins are usually synthesized as precursors with a short N-terminal extension called the
signal peptide, which promotes secretion and which is removed by specific enzymes (1, 43, 49, 50,
52). Comparison of the gene-derived sequence for mature LCN-A with the direct amino acid
sequencing data shows that the LCN-A is synthesized as a 75-amino-acid precursor. The LCN-A
leader peptide of 21 amino acids has a positively charged N-terminus followed by a hydrophobic
stretch typical of signal peptides of gram-positive bacteria (1). Mature LCN-A has a lysine as its Nterminal amino acid. The sequence Ala-Asn-Gly-Gly precedes this lysine in the LCN-A precursor.
According to the "-3, -1" rule of Von Heijne (49,50), a signal peptidase could cleave the precursor
between the two glycines (-2,-1) but not between the glycine and the lysine (-1, +1).This theory may
suggest a stepwise processing of the LCN-A precursor in which a 20-amino-acid peptide and then a
glycine are removed from the N terminus to yield mature
LCN-A of 54 amino acids
Three putative promoter elements were found upstream of the 1cnA gene (Fig. 1). Conceivably,
transcription initiation could occur 5 to 9 nucleotides downstream of any of the putative Pribnow
boxes, yielding leaders of 17 to 33 nucleotides. Overlapping the -10 regions of the putative promoter
elements is an inverted repeat sequence that could form a stem-loop structure (Fig. 1).
This structure, with a calculated aG value of -9.6 kcal/mol (- 40.2 kJ/mol) (45), could represent a
Rhodependent terminator of ORF1.
Strain LMG 2131, which had lost the lcnA gene, was sensitive to LCN-A. This result suggests that the
producing organism harbors a gene(s) encoding immunity to the bacteriocin. Strain IL 1403 carrying
recombinant plasmid pON7 produced LCN-A and was (by necessity) resistant to the bacteriocin.
Thus, the l.2-kb (RsaI
HindIII fragment appears to carry not only the gene encoding LCN-A but also a genetic determinant
for resistance. The DNA sequence of this fragment shows only one complete'ORF in addition to the
lcnA gene.
this is ORF2, located downstream of and in the same operon as lcnA. Hence, the apparently
cotranscribed
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ORF2 is the likely candidate to encode an LCN-A immunity function. A very similar organization of
bacteriocin genes and their corresponding immunity genes has been shown for several E. coli
bacteriocins (2, 26). ORF2 with Met at nucleotide position 495, preceded by the possible RBS
sequence 5' GGATTAG 3', encodes a hypothetical polypeptide of 98 amino acids.
Altenatively, there could be an ORF2, with Leu at nucleotide position 540, preceded by the possible
RBS sequence 5' AAGAAG 3', with the capacity to encode a hypthetical 83-amino-acid polypeptide.
However, codon usage in the 15 N-terminal amino acids of the 98-aminoacid polypeptides correlates
well with the compiled codon usage pattern of the rest of the ORF2 polypeptide and of LCN-A,
indicating that the ORF2 encodes a 98 amino-acid polypeptide. Its six N-terminal residues (Met-LysLys-Gln-Ile) show great similarity to signal peptides of gram-positive bacteria. Despite the presence
of Glu in positions 7, 9 and 11, the putative signal sequence retains a hydrophobic character
extending from amino acid positions 5 to 20. According to the -3, -1 rule of Von Heijne, there is a
possible signal peptidase cleavage site after Ala-Thr-Ala at amino acid position 20.Of the 14 grampositive signal sequences compiled by Abrahams et al. (1), 7 contained
Ala-X-Ala at their cleavage sites. Moreover, Ala-Thr
Ala was found to be the -3, -1 amino acid sequence of the signal peptidase cleavage site of Bacillus
subtilis
B-glucanase (29), possibly suggesting a mature ORF2 protein of 79 amino acids. It remains to be
shown whether the ORF2-encoded polypeptide is secreted or anchored within the membrane.
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Levine, O.C. Uhlenbeck, D.M. Crothers and J. Gralla, 1973. Improved estimation of secondary
structure in ribonucleic acids. Nature (London) New Biol. 246:40-41.
46. van Belkum, M.J., B.J. Ilayema A., Geis, J. Kok and
G. Venema. 1989. Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid. Appl.
Environ.
Microbiol. 55:1157-1191.
47. van Belkum, J., B.J. Hayema, R.E. Jeeninga, J. Kok, and G. Venema. 1991. . Organization and
nucleotide seuqences of two lactococcal bacteriocin operons. Appl.
Environ. Microbiol. 57:492-498.
48. van der Eizen, P.J.M., J. Maat, H.H.B. Walters, E.
Veltkamp and H.J.J. Nijkamp. 1982. The nucleotide sequence of bacteriocin promoters of plasmids
Clo DF13 and Col El: role of 1cxA repressor and cAMP in regulation of promoter activity. Nucleic
Acids Res.
10:1913-1928.
49. Von Heijne, H. 1983. Patterns of amino acids near signal-sequence cleavages sites. Eur. J.
Biochem.
133:17-21.
50. Von Heijne, G. 1984. How signal sequences maintain cleavage specificity. J. Mol. Biol. 173:243251.
51. Walsh, P.M., and L.L. McKay. 1981. Recombinant plasmid associated with cell aggregation and
highfrequency conjugation of Streptococcus lactis M13. J.
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Bacteriol. 146:937-944.
52. Wong, S.L., and R.H. Dol. 1986. Determination of the signal peptides cleavage site in the
preprosubtilisin of Bacillus subtilis J. Biol. Chem.
261:10176-10181. Claims:
Claims
1. A polypeptide having or including the amino acid sequence: 1 -Lys Leu Thr Phe lie Gln Ser Thr
Ala Ala Gly Asp Leu Tyr Tyr 1 6Asn Thr Asn Thr His Lys Tyr Val Tyr Gln Gln Thr Gln Asn Ala 31-Phe
Gly Ala Ala Ala Asn Thr lie Val Asn Gly Trp Met Gly Gly 46-Ala Ala Gly Gly Phe Gly Leu His His and
derivatives and fragments thereof having bacteriocin activity.
2. A polypeptide having or including the amino acid sequence:
Glu Lys Asp lie Ser Gln Glu Glu Arg Asn Ala Lai Asn lie Ala Glu
Lys Ala Lai Asp Asn Ser Glu Tyr Lai Pro Lys lie lle Leu Asn Leu Arg Lip Ala Leu
Thr Pro Leu Ala lie Asn Arg Thr Leu Asn Ths Asp Leu Ser Glu Leu Tyr Lys Phe lie
Thr Ser Ser Lys Ala Ser Ans Lys Asn Leu Gly Gly Gly Lei lle Met Ser Trp Gly Arg Leu Phe and
derivatives and fragments thereof having bacteriocin immunity activity.
3. A starter culture of microorganisms for use in a microbiological process comprising a polypeptide
as claimed in claim 1, said microorganisms being resistant to said polypeptide.
4. A starter culture as claimed in claim 1 in which the microorganisms are lactic acid bacteria or
yeasts.
5. A method of cheese or yoghurt production in which a polypeptide as claimed in claim 1 is added
to effect lysis of lactic acid bacteria.
476/1006
6. A method of fermentation for production of ethanol wherein a polypeptide as claimed in claim 1 is
used to kill selectively contaminating strains of lactic acid bacteria.
7. A method of isolation of a polypeptide as claimed in claim 1 wherein a culture of a microorganism
expressing said polypeptide is subjected to fractionation whereby fractions enriched in said
polypeptide are collected.
8. A method as claimed in claim 7 in which the microorganism is Lactococcus lactis subsp. cremoris.
9. A DNA sequence coding for a polypeptide as claimed in claim 1 and/or claim 2.
10. Strains of L. lactis transformed with a vector containing
DNA coding for a polypeptide as claimed in claim 1 and/or claim 2.
11. A process for the preparation of a polypeptide as claimed in claim 1 or claim 2 in which a
corresponding protected or immobilised polypeptide is subjected to deprotection or removal from an
inert support.
477/1006
57. NZ243159 - 07.01.1993
NOVEL BACTERIOCIN FROM LACTOCOCCUS LACTIS SUBSPECIES LACTIS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ243159
Inventor(s):
VEDAMUTHU EBENEZER R (US); HENDERSON JAMES T (US); MARUGG JOHN D
(NL); VAN WASSENAAR PIETER D (NL)
Applicant(s):
QUEST INT (NL)
IP Class 4 Digits: C12N; A23L; C07K; C12P; A01N; A23C
IP Class:
C12N1/20; C12P21/02; A01N63/02; A23L3/3526; C12N15/31; A23L3/3571;
C07K13/00; A23C9/13; C07K3/12; C12N15/10
E Class: A23C19/11; C07K14/315; A23L3/3526; A23L3/3571; A23C9/13E
Application Number:
EP19920103287 (19920226)
Priority Number: US19910721774 (19910701)
Family: NZ243159
Equivalent:
AU1609992; AU639638; CA2061177; DE69224237D; DE69224237T; DK521240T;
JP2637879B2; JP6009690; US5173297
Cited Document(s):
WO9218633
Abstract:
A BACTERIOCIN OR POLYPEPTIDE (LL-2 SEQ ID NO:2) DERIVED FROM LACTOCOCCUS LACTIS
SUBSPECIES LACTIS NRRL-B-18809 IS DESCRIBED. SEQUENCED DNA AND POLYPEPTIDE
PRECURSOR ENCODED THEREBY (SEQ ID NO: 1) ARE DESCRIBED. METHODS OF USE AND
PRODUCTION OF THE POLYPEPTIDE LL-2 ARE DESCRIBED.Description:
478/1006
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a novel polypeptide, also referred to as a bacteriocin, produced by
Lactococcus lactis subspecies lactis which is inhibitory against selected Gram-positive bacteria, to a
method of inhibiting the Gram-positive bacteria with the bacteriocin and to methods for producing
the bacteriocin. In particular, the present invention relates to a bacteriocin encoded by DNA from
Lactococcus lactis subspecies lactis NRRL-B-18809 and to a precursor polypeptide which is post
translationally modified after expression of the DNA.
(2) Prior Art
Lactic acid bacteria comprise a group of Gram-positive spherical and rod-shaped bacteria that are
usually non-pathogenic and fermentative. The genera included in this group are Lactococcus,
Streptococcus, Lactobacillus and Pediococcus. Many members of these genera are traditionally
used in food fermentations and are considered "generally regarded as safe" (GRAS). Production of
antagonistic substances and bacteriocins by lactic acid bacteria has been widely reported
(Klaenhammer, T. R., Biochimie. 70:337-349 (1988)) . Some of the bacteriocins elaborated by these
bacteria have a rather narrow spectrum (for example, closely related species) while others exhibit a
broader spectrum crossing even the genus grouping. Members of the genus Lactococcus commonly
used as starter cultures in fermented foods are Lactococcus lactis subsp. lactis, and Lactococcus
lactis subsp. cremoris.Both these species produce bacteriocins. A well characterized and
nomenclaturally recognized bacteriocin from Lactococcus lactis subsp. lactis is nisin. Lactococcus
lactis subsp. cremoris produces diplococcin. Nisin has a wide antibacterial spectrum (Hurst, A.,
Advances Appl. Microbiol. 27:85-123 (1981)) while diplococcin is primarily active against
Lactococcus lactis subsp. lactis and other non-bacteriocin producing Lactococcus lactis subsp.
479/1006
cremoris strains (Foster, E. M., F. E. Nelson, M. L. Speck, R. N. Doetsch, and J. C. Olson, Jr., Dairy
Microbiology, Prentice-Hall Inc., Englewood Cliffs, N.J. p.15 (1957)). Because of the wide
antibacterial activity of nisin and its potential applications (Hurst, A., Advances Appl. Microbiol.
27:85-123 (1981); Delves-Broughton, J.Food Technology, 44:100-112, 117 (1990)), a search for other
strains within this group that elaborate different bacteriocin(s) with wide antibacterial spectra was
made.
Structural characteristics of nisin are well documented in several review articles (Klaenhammer, T. R.,
Biochimie, 70:337-349 (1988); Geis, A., Kieler Milchwirtschaftliche Forschungsberichte 41:97-104
(1989)). The nucleotide sequence of the nisin gene is known (Buchman, G. W., et al., J. Biol. Chem.
263:16260-16266 (1988)) and descriptions of its post-translational modifications (Kaletta, C. and K-D
Entian., J. Bacteriol. 171:1597-1601 (1989)) and degradation products (Chan, W. C., et al., FEBS Lett.
252:29-36 (1989); Barber, M., et al., Experentia 44:266-270 (1988)) have appeared. The solution
conformation of nisin was determined (Slijper, M., et al., FEBS Lett. 252:22-28 (1989)) as well as other
physical and chemical properties (Liu, W. and J. N. Hansen, Appl. Environ. Microbiol. 56:2551-2558
(1990)).
Nisin is similar to other known lanthionine containing bacteriocins, including epidermin (Schnell, N.,
et al., Nature 333:276-278 (1988)), pep 5 (Weil, H-P., et al., Eur. J. Biochem. 194:217-223 (1990)),
lanthiopeptin (Wakamiya, T., et al., Tet. Lett. 29:4771-4772 (1988)), ancovenin (Wakamiya, T., et al.,
Tet. Lett. 29:4771-4772 (1988)), subtilin (Buchman, G. W., et al., J. Biol. Chem. 263:16260-16266
(1988)), and gallidermin (Kellner R. and G. Jung., Proc. 20th Europ. Peptide Sym. 366-368 (1988)).
The aforementioned lantibiotics share characteristics of small (3500 Da), basic (pI >10) posttranslationally modified proteins with an inhibitory spectrum limited to gram positive bacteria. Nisin
has been attributed the ability to limit sporulation of Bacilli and Clostridia.
U.S. Patent No. 4,883,673 to Gonzalez describes a pediocin. U.S. Patent No. 4,929,445 to
Vandenbergh et al describes the DNA encoding the pediocin. U.S. Patent Nos. 4,740,593 and
4,716,115 to Gonzalez et al describe microorganisms containing DNA from a nisin producing
microorganism transferred to a non-nisin producing microorganism using various techniques for
manipulating the DNA. The prior art also describes the production of antifungal substances from
Lactococcus and Pediococcus which are unrelated to the present invention.
480/1006
U.S. Patent Application Serial No. 079/492,969 assigned to a common assignee describes
bacteriocin LL-1 from Lactococcus lactis NRRL-B-18535. This strain produces a bacteriocin which is
related to nisin whereas the bacteriocin LL-2 of the present invention is different from nisin.
OBJECTS
It is therefore an object of the present invention to provide a novel bacteriocin derived from DNA of
Lactococcus lactis subspecies lactis NRRL-B-18809. Further, it is an object of the present invention
to provide the DNA encoding a precursor polypeptide to the bacteriocin. Further still, it is an object of
the present invention to provide a method for inhibiting selected Gram-positive bacteria using the
bacteriocin. Finally, it is an object of the present invention to provide a method for producing the
bacteriocin. These and other objects will become increasingly apparent by reference to the following
description and the drawings.
IN THE DRAWINGS
Figures 1A and 1B show high performance liquid chromatography (HPLC) chromatographs of the
LL-2A, LL-2B and nisin. They show that LL-2A and LL-2B are different from nisin.
Figure 2 shows inhibition zones produced on overlay gels with HPLC purified bacteriocins LL-2A and
LL-2B, using a sensitive Pediococcus pentosaceus FBB63C as an indicator strain.
Figure 3 is a graph showing the effects of various percentages of crude bacteriocin in yogurt.
GENERAL DESCRIPTION
The present invention relates to a polypeptide having an inhibitory activity against sensitive Grampositive bacteria and having the amino acid sequence wherein Xaa is selected from Ser, Ala-, or
Dha, and Xab is selected from Abu-, or Dhb, and Xac is selected from Ala-S-, wherein Ala- is 3-
481/1006
substituted Alanine (-(CO)CH(NH-)CH2), Abu- is 3 substituted 2-aminobutanoic acid (-(CO)CH(NH)CH(CH3)-), Ala-S- is (3-alanyl)-thio, Abu is 2-amino butanoic acid, Dha is 2-amino propenoic acid
and Dhb is 2-aminobut-2-enoic acid, and subunits thereof having an essentially equivalent inhibitory
activity to the sensitive bacteria.
The present invention particularly relates to a polypeptide produced by Lactococcus lactis
subspecies lactis NRRL-B-18809 in a growth medium and having inhibitory activity against sensitive
Gram-positive bacteria.
The present invention further relates to a method for producing DNA comprising the gene encoding a
precursor polypeptide of a polypeptide, the latter polypeptide having inhibiting activity against
selected Gram-positive bacteria, which method comprises: providing DNA obtained from
Lactococcus lactis subspecies lactis NRRL-B-18809 comprising a DNA sequence encoding a
precursor polypeptide of the polypeptide described above; and subjecting the DNA to a polymerase
chain reaction wherein the primers are: 5'-CGCGAGCATAATAAACGGCT-3' as a sense primer and
5'-GGATAGTATCCATGTCTGAAC-3' as an antisense primer to produce a DNA sequence comprising
the gene encoding said precursor polypeptide.
The present invention particularly relates to the DNA sequence encoding a precursor polypeptide,
said DNA sequence being the polynucleotide 1-321 Further the present invention relates to a
precursor polypeptide having the sequence The present invention also relates to a method for
producing a polypeptide having activity against sensitive Gram-positive bacteria which comprises:
providing a lactic acid bacterium containing DNA as carried in Lactococcus lactis subspecies lactis
NRRL-B-18809 in a growth medium containing a carbon source, a nitrogen source and minerals
under conditions to express the polypeptide having the inhibitory activity against the selected
bacteria; and optionally isolating the polypeptide from the growth medium.In particular the present
invention relates to a process for purifying a polypeptide produced by a culture of Lactococcus lactis
subspecies lactis NRRL-B-18809, in which the purification is performed by passing a broth
fermented by the culture through a chromatography column, preferably using an anion exchange
resin, and the polypeptide containing product in purified form is collected from the chromatography
column, preferably by controlled elution from the column.
The present invention further relates to a method wherein the polypeptide is used to inhibit sensitive
Gram-positive bacteria. In particular the present invention relates to a method for inhibiting sensitive
482/1006
Gram-positive bacteria which comprises: exposing the bacteria to an inhibitory amount of the
polypeptide described previously to thereby inhibit the bacteria.
Lactococcus lactis subspecies lactis NRRL-B-18809 is deposited under the Budapest Treaty with the
Northern Regional Research Laboratory in Peoria, Illinois and is available upon request by name and
accession number. Plasmid pSRQ 400 which encodes for the bateriocin LL-2 is carried by this
deposit. The strain has the following salient fermentation characteristics: sucrose-positive, lactosepositive, milk coagulated, and arginine deaminase positive.
The plasmid pSRQ 400 can be transferred to other Lactococcus species by mating or by various
known techniques for transferring plasmids between microorganisms. The DNA encoding a
precursor polypeptide to the bacteriocin can also be transferred to various microorganisms using
recombinant genetic techniques with vectors as is well known to those skilled in the art.
When the bacteriocin is produced in culture, it is preferably isolated and purified. The bacteriocin
aggregates to about 100,000 daltons and thus can be initially purified by ultrafiltration where the
bacteriocin is the retentate. The bacteriocin can then be purified by HPLC.
The bacteriocin is used to inhibit selected Gram-positive bacteria including:
Lactobacillus plantarum;
Lactobacillus casei;
Lactobacillus brevis;
Lactobacillus bulgaricus;
Lactobacillus fermentum;
Pediococcus acidilactici;
Pediococcus pentosaceus;
Streptococcus mutans;
Bacillus subtilis; and
Lactococcus lactis.
The crude LL-2 is not as effective as the bacteriocin purified by HPLC which is to be expected since
the purified bacteriocin has a higher activity per unit volume. The LL-2 is separable into LL-2A and
LL-2B. The former polypeptide is derived from the latter.
483/1006
The activity of the bacteriocin is expressed in Arbitrary Units (AU) per ml. The AU is defined as five (5)
microliters of the highest dilution of culture supernatant yielding a definite zone of growth inhibition
against the indicator strain which in this case is Pediococcus pentosaceus FBB63C. The titer is
expressed as the reciprocal of the highest dilution showing inhibition.
The culture supernatant contains about 1600 AU per ml of the bacteriocin. The HPLC purified
bacteriocin exhibited about 2,500,000 AU per ml. In general, between about 15 and 100 AU per
gram of a material being treated is sufficient to provide inhibition.
The materials being treated to provide inhibition are preferably foods. Other non-food material can
also be treated with the bacteriocin.
SPECIFIC DESCRIPTION
Example 1
This Example shows the isolation and characterization of Lactococcus lactis subspecies lactis NRRLB-18809 and the bateriocin produced therefrom.
Isolation of lactic acid bacteria. To obtain a wide selection of naturally occurring "wild" strains of lactic
acid bacteria, samples of natural habitats of these bacteria such as raw milk, fermenting vegetable
matter and fruits, and fresh vegetables were plated on suitable media with or without enrichment
(Speck, M. L., Compendium of Methods for the Microbiological Examination of Foods, American
Public Health Association, Washington, D. C., pp. 184-194 (1984)). Enrichment generally involved
using high concentrations of common salt (NaCl) - from 2% - 6% in broth cultures followed by plating
on solid media containing the same levels of NaCl.
Screening for antimicrobial activity. Colonies appearing on the agar plates were screened for
antibacterial activity against a selected indicator, which usually predicted a wide antibacterial
spectrum. The indicator chosen was Pediococcus pentosaceus FBB63C (Gonzalez, C. F., and B. S.
Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987) and U.S. Patent No. 4,883,673 to Gonzalez.
484/1006
Antibacterial activity was determined by picking colonies to replicate agar plates containing 1.9%
sodium beta-glycerophosphate as buffer and after appearance of colonies overlaying with indicator.
Characterization of antimicrobial isolates. Cultures showing antibacterial activity were examined for
Gram-staining reactions, cell morphology and arrangement, key carbohydrate fermentations using
purple base broth with specific carbohydrate, and for ability to coagulate milk.
Bacteriocin assay. To ascertain if the antibacterial substance is secreted into liquid medium, the
cultures were grown in MRS broth and cell free filtrates of turbid cultures were made and the filtrate
posted on a seeded semisolid agar overlay of the indicator. Antibacterial activity was quantitated by
assigning arbitrary units. One arbitrary unit (AU) was defined as 5 mu l of the culture supernatant
yielding a definite, clear zone of inhibition on the indicator lawn. The titer was expressed as the
reciprocal of the highest dilution of supernatant showing inhibition.
Preliminary characterization of crude bacteriocin. Preliminary characterization of the bacteriocins with
respect to size, molecular weight, pH optimum, effect of enzymes, and effect of nutritive supplements
on bacteriocin titer were done using procedures described by Gonzalez and Kunka (Gonzalez, C. F.,
and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)).
Plasmid isolation, electrophoresis and curing. Plasmid contents of the cultures were examined using
the procedure described by Gonzalez and Kunka (Gonzalez, C. F. and B. S. Kunka, Appl. Environ.
Microbiol. 46:81-89 (1983)).
Plasmid curing. Plasmid curing was done using high temperature and curing agents according to
previously described procedures (Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol. 46:8189 (1983)).
Large scale culturing and processing for purification of bacteriocin(s). Four liters of MRS broth (Difco,
Detroit, MI) was inoculated at 1% with an 8 hour old culture of strain NRRL-B-18809 grown in MRS
broth and was grown statically at 32 DEG C for 24 hours. Cells were removed by centrifugation at
16,300 x g for 15 minutes at 4 DEG C. The supernatant was filtered using a Minitan tangential
filtration apparatus (Millipore, Bedford, MA) equipped with a 0.2 mu m pore size polyvinylidene
difluoride (PVDF) membrane. Bacteriocin production was assayed as previously described. Medium
pH was less than 5.0.
485/1006
Purification of LL-2. Filtrate from tangential filtration was adjusted to pH 4.0 using 10% hydrochloric
acid and then was concentrated approximately 2-fold using a spiral-wound cellulose-based
ultrafiltration cartridge with a 1 ft surface area and a 3000 dalton molecular weight cutoff (Amicon
SlY3, Beverly, MA). Concentration was performed at 4 DEG C using a peristaltic pump (Cole-Parmer,
Chicago, IL) to maintain a 20 lb/in differential across the membrane.
A 1600 ml aliquot of concentrated supernatant was applied to a 10 cm x 20 cm column (1.57 liters) of
DEAE-650M anion exchange resin (Toso-Haas, Philadelphia, PA) equilibrated with 0.1 M sodium
acetate buffer, pH 4.0 at a flow rate of 10 ml/min. Absorbance of the eluent at 280 nanometers was
monitored and eluent was collected from the first increase from baseline absorbance until baseline
absorbance was again reached. The eluent volume was 3150 ml and the activity was 1600 AU/ml.
Preconcentration and anion exchange chromatography are necessary to achieve maximal binding of
the bacteriocin to a cation exchange resin.
The entire volume of eluent from anion exchange chromatography was applied to a 10 cm x 35 cm
column (2.75 liters) of CM-650M cation exchange resin (Toso-Haas, Philadelphia, PA) which had
been equilibrated against 0.1 M sodium acetate buffer, pH 4.0. Activity was eluted using the same
buffer containing 1 M sodium chloride at pH 4.0. Eluent was collected from the first increase in
conductivity from baseline conductivity Collection was terminated when absorbance at 280
nanometers returned to baseline absorbance. The eluent volume was 2000 ml and the activity was
1600 AU/ml.
The eluent from cation exchange chromatography was concentrated approximately 20-fold by
ultrafiltration until 110 ml remained (Amicon SlY3, Beverly, MA). Sodium chloride content was then
reduced approximately 5-fold by adding 500 ml deionized water and then concentrating to 110 ml
again. The cartridge was emptied and then washed with 50 ml deionized water. The concentrate was
combined with the wash solution to obtain 150 ml bacteriocin with 25,600 AU/ml activity.
Volume of the bacteriocin concentrate was further reduced using vacuum centrifugation (Savant,
Farmingdale, NY) until 12 ml remained. Aliquots of this concentrate were applied to a 2.5 cm x 25 cm
ODS column (Vydac, Hisperia, CA) equilibrated with 0.1% trifluoroacetic acid (TFA) in water. Activity
was eluted using a gradient which typically used a linear change over 30 minutes to 45% acetonitrile
containing 0.1% trifluoroacetic acid. A flow rate of 10 ml/min was used. Fractions were collected at
0.5 minute intervals and activity was located in the chromatogram by directly spotting 5 mu l from
486/1006
each fraction onto an P. pentosaceus FBB 63C indicator plate. Protein elution was monitored using a
UV detector (Beckman 166,SanRamon,CA) at 230 nanometers wavelength.
Two zones of activity were consistently observed, the first eluted at 27.5-28.5 minutes and the
second eluted at 32-37 minutes. All fractions were dried using vacuum centrifugation, active fractions
were reconstituted in 0.1 ml deionized water and were combined as follows. Active fractions eluting
between 27.5 and 28.5 minutes were combined as LL-2A and active fractions eluting between 32.5
and 33 minutes were combined as LL-2B.
The LL-2A component was further purified by applying to a 2.5 cm x 25 cm ODS column (Vydac,
Hisperia, CA) as before. A gradient beginning at 20% and linearly progressing to 40% acetonitrile
containing 0.1% TFA was performed over a 30 minute time frame. Activity was observed to elute
between 26.5 and 28.0 minutes. Fractions within these elution limits were dried by vacuum
centrifugation and then were combined as purified LL-2A.
Analytical HPLC. Purified protein was characterized by the appearance of peaks in the HPLC
chromatogram using a 0.45 x 25 cm ODS column (Vydac, Hisperia, CA) with the UV detector set to
monitor absorbance at 230 nanometers. Elution profiles were obtained using a gradient from 0.1%
TFA to 45% acetonitrile containing 0.1% TFA.
Amino Acid Analysis. Samples for hydrolysis were dried by vacuum dehydration, reconstituted in 0.1
ml 6N HCl (Pierce, Rockford, IL), dried again, and hydrolyzed in vacuo at 110 DEG C in 8mm x
60mm hydrolysis tubes (Pierce, Rockford, IL) for 24 hours in a Reacti-Therm heating block (Pierce,
Rockford, IL). PITC derivatives were made using the directions supplied with a Pico-Tag
derivatization system (Waters, Milford, MA) and PTC derivatives were detected at 254 nm using the
suggested Pico-Tag protocol except that the reversed-phase column was a 0.45cm x 250cm C18
column (Beckman, SunRamoin, CA) instead of the proprietary Pico-Tag column.
Enzymatic Reactions. Trypsin and chymotrypsin susceptibility of crude and purified bacteriocin was
explored. Enzymes were obtained from Sigma (St. Louis, MO) and were stored lyophilized at -20
DEG C until use. Enzymatic reactions were done in a 0.1 M ammonium bicarbonate buffer, pH 7.8,
containing 0.1 mM calcium chloride (Deutcher, M. P., Meth. Enzymol. 182:611-612 (1990)).
Susceptibility was defined as inability of the bacteriocin to inhibit the growth of a lawn of indicator
bacteria when applied to the surface of an agar plate containing the bacteria at a rate of 16 units
activity in a volume of 5 mu l.
487/1006
Amino Acid Sequencing. Sequence information was obtained with a gas phase sequencer (Applied
Biosystems, Foster City, CA) using protocols supplied by the manufacturer.
SDS Polyacrylamide Gel Electrophoresis. Gels were prepared at either a fixed acrylamide
concentration of 12.5% or as a linear gradient from 10% to 25%. The crosslinking agent was
piperizine diacrylamide (BioRad, Richmond, CA). Gels were stained with Coomassie Brilliant Blue G
followed by silver staining (BioRad, Richmond, CA) or were unstained in order to perform an activity
analysis by overlaying the gel with soft agar containing indicator P. pentosaceus FBB-63C cells
(Bhunia, J. Ind. Micro. Biol. 2:319-322 (1987)). The unstained gels were calibrated with pre-stained
standards (BioRad, Richmond, CA).
Isolation and Characterization of Bacteriocin-Producing Lactococci. During the course of screening,
a colony showing good antibacterial activity was observed. The colony was streaked and an isolated
colony was propagated, retested for inhibitory activity and was stocked. The culture was found to be
Gram-positive cocci arranged in short chains and the cell-morphology was oval and characteristic of
lactic streptococci. The culture coagulated milk and was positive for sucrose fermentation. The
culture was identified as Lactococcus lactis subsp. lactis and was designated as NRRL-B-18809.
The culture had a single, large plasmid pSRQ 400 (69 Kb in size). Curing of the plasmid employing
high temperature incubation (42 - 43 DEG C) resulted in the loss of inhibitory activity and ability to
ferment lactose. Several repeated trials employing curing agents (acriflavin, proflavin, novobiocin)
failed to yield colonies unable to ferment sucrose. From these results it was concluded that
bacteriocin-production (Bac) and lactose-fermentation phenotype (Lac) were coded on the same
plasmid and that sucrose fermentation (Suc) was chromosomally determined. Bacteriocin-negative
(Bac) and lactose negative (Lac) derivatives, however were resistant (Bac) to the bacteriocin
produced by the parent. Hence, Bac (or immunity) was coded on the chromosome.
The results of the preliminary characterization of crude bacteriocin in the cell-free filtrates are
summarized in Tables 1 through 6.
From Table 1, it is evident that the crude bacteriocin in cell-free filtrate is greater than 100,000 Da
indicating aggregation of multiple protein molecules.
Id=TABLE 1 Columns=5 MOLECULAR WEIGHT OF CRUDE BACTERIOCIN FROM CELL-FREE
FILTRATE OF NRRL-B-18809
488/1006
Head Col 1: MEMBRANE MWCO
Head Col 2: RETENTATE VOLUME (ml)
Head Col 3: PERMEATE VOLUME (ml)
Head Col 4 to 5: BACTERIOCIN AU/ML
SubHead Col 1:
SubHead Col 2:
SubHead Col 3:
SubHead Col 4: RETENTATE
SubHead Col 5: PERMEATE
2,0002038800None
10,0002034800None
30,0001745800None
100,000--1600None
When purified, the bacteriocin has a molecular weight of about 6,600.
For maximal production of bacteriocin, MRS broth fortified with 1% yeast extract and 7% whey
containing 0.5% yeast extract were suitable as can be seen from Table 2. Maximum bacteriocin titer
was obtained at 24 DEG C and 32 DEG C; at 37 DEG C incubation, the titer was lower.
Id=TABLE 2 Columns=4 EFFECT OF DIFFERENT MEDIA AND NUTRITIVE SUPPLEMENTS ON
CRUDE BACTERIOCIN TITER IN CELL-FREE FILTRATES OF NRRL-B-18809.
Head Col 1: MEDIUM
Head Col 2: SUPPLEMENT
Head Col 3: pH
Head Col 4:TITER (AU/ml)
MRS-4.7800
MRS1% yeast extract4.71600
APT-4.3800
Skim Milk-4.7200
Peptonized Milk-4.8800
7% Whey
489/1006
-4.3400
7% Whey
0.2% yeast extract4.0800+
7% Whey
0.5% yeast extract4.01600
7% Whey
1.0% yeast extract4.21600
Whey powder contained 10% maltodextrin
The crude bacteriocin was stable at pH 4.1 at all the temperature treatments used including
autoclaving (121 DEG C for 15 min.) as shown in Table 3.
Id=TABLE 3 Columns=3 EFFECT OF HEAT ON CRUDE BACTERIOCIN IN CELL-FREE FILTRATE
(pH 4.1) of NRRL-B-18809.
Head Col 1: Temperature of heating ( DEG C)
Head Col 2: Duration (min)
Head Col 3: Titer (AU/ml)
Control (iced)601600
21601600
37601600
60601600
100101600
121151600
The crude bacteriocin was stable at pH 2.0-3.0 and progressively lost its activity at higher pH values
as shown in Table 4.
Id=TABLE 4 Columns=2 THE STABILITY OF CRUDE BACTERIOCIN FROM CELL-FREE FILTRATE
OF NRRL-B-18809 DIALYZED AGAINST BUFFERS WITH DIFFERENT pH
Head Col 1: pH
Head Col 2: Titer AU/ml
21600
31600
490/1006
4800
5800
6200
7400
8200
9400
10400
11400
As can be seen from Table 5, the crude bacteriocin was active against a wide range of Grampositive bacteria with the exception of Listeria monocytogenes and Staphylococcus aureus. It had no
activity against E. coli, a Gram-negative species.
Id=TABLE 5 Columns=6 SPECTRUM OF ANTIMICROBIAL ACTIVITY
Head Col 1: Indicator
Head Col 2: LL2 Crude
Head Col 3: Nisin Crude
Head Col 4: LL2A Pure
Head Col 5: LL2B Pure
Head Col 6: Nisin Pure
SubHead Col 1: Titer AU/ml
SubHead Col 2: 1600
SubHead Col 3: 1600
SubHead Col 4: 3200
SubHead Col 5: 3200
SubHead Col 6: 3200
SubHead Col 7:Indicator Strain
L. plantarum 346+++++
L. plantarum 355++-++
L. casei 326++-++
L. casei 842++-++
L. brevis 329+++++
L. brevis 888++-++
L. fermentum 342++-++
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L. fermentum 701+++++
P. acidilactici PAC 1.0++-++
P. acidilactici A++-++
P. acidilactici B+++++
P. acidilactici C-++-+
P. acidilactici D-++-+
P. pentosaceus FBB63C+++++
L. monocytogenes 04--+++
L. monocytogenes 08--+++
L. monocytogenes 36--+++
L. monocytogenes 38--+++
L. monocytogenes 59--+++
L. monocytogenes 62--+++
L. monocytogenes 69-++++
S. mutans GS5+--++
S. mutans V262----B. subtilis RM125+ND+++
B. subtilis amylase+ND-++
L. lactis LLA 1.0---++
L. lactis LLA 2.0-+-++
L. lactis 367+NDNDNDND
L. bulgaricus (4 strains)+NDNDNDND
S. aureus Z-88-ND
E. coli HB101-ND
ND = not determined; + = inhibition; - = no inhibition
As can be seen from Table 6, the crude bacteriocin was resistant to treatment with 5% trypsin and
alpha-chymotrypsin. Trypsin and chymotrypsin sensitivities of purified LL-2 bacteriocins to purified
nisin were compared. Compared to nisin, LL-2 is more sensitive to chymotrypsin and more resistant
to trypsin. Titer 200 is resistant.
Chymotrypsin digestion was done by 2 hour incubation at 40 DEG C with 3200-6400 AU/ml
bacteriocin concentration.
492/1006
Trypsin digestion was done by 20 hour incubation at 40 DEG C with 3200-6400 AU/ml bacteriocin
concentration.
The use of purified bacteriocin in enzyme sensitivity testing is preferred since the crude media can
contain an unknown protease inhibition activity or other phenomena which would tend to confound
results.
As shown by Table 7, purified LL-2 was obtained in good yield and high purity. The LL-2B
component as shown in Table 7 accounted for more than 90% of the activity. 25% of the activity
initially present was recovered. The two bacteriocins LLB-2A and LLB-2B obtained as a result of
HPLC purification were active on overlay gels with activity centered about a region corresponding to
molecular weight 6500 daltons as shown by Figure 2.
Analytical HPLC results show that LL-2A and LL-2B are related. Purified LL-2B will produce LL-2A
over time, although purified LL-2A does not produce LL2-B. Nisin is also known to consist of several
molecular species in addition to the parent protein. The most predominant is the parent structure
lacking two carboxy terminal residues and containing a terminal Val-NH2. These results are
consistent with the hypothesis that LL-2A is a product of degradation of LL-2B and that this
degradation product is formed in a similar manner to that of the nisin degradation product. The
degradation product of nisin however was reported inactive as a bacteriocin, whereas LL-2A retains
a wide spectrum of bacteriocin activity.
Analytical HPLC results of the mixture of LL-2A, LL-2B and nisin illustrate that molecular differences
are present between the molecules. These differences provide a small but significant difference in
HPLC elution time which is readily apparent in chromatograms of mixtures of LL-2A, LL-2B and nisin
as shown in Figures 1A and 1B. The elution of LL-2B before nisin shows a more hydrophilic nature of
LL-2B compared to nisin.
Example 2
A DNA sequence was found in NRRL-B-18809 cells which produced a polypeptide corresponding to
the sequence of nisin except that His27 is replaced by Asn27 resulting from a change from CAT to
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AAT in the DNA. Amino acid composition results shown in Table 8 confirm the loss of one His residue
from the amino acid make-up of LL-2B. Results from amino acid sequencing of LL-2B indicate an
amino terminal isoleucine residue is followed by no other residues presumably due to an N-terminal
blockage at the second residue. This result is also found for nisin, since the second residue, a
dehydrobutyrine residue, has been found to cyclize after Edman degradation of the previous residue
thus blocking further sequencing.
The following data shows how the results were obtained.
Id=TABLE 8 Columns=5 AMINO ACID COMPOSITION OF LL-2B COMPARED TO NISIN
Head Col 1:
Head Col 2: LL-2B
Head Col 3: Theo. LL-2
Head Col 4: Nisin
Head Col 5: Theo. Nisin
D Asp/Asn1.420.51
E Glu/Gln0.800.00
S Ser1.010.81
G Gly2.733.43
H His0.812.42
R Arg0.000.00
R Thr0.000.00
A Ala1.821.82
P Pro1.011.21
Y Tyr0.000.00
V Val1.411.31
M Met0.621.22
C Cys0.000.00
I Ile4.733.13
L Leu1.922.12
F Phe0.000.00
K Lys2.833.23
21.021.021.021.0
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Total DNA isolation. Total genomic DNA from Lactococcus lactis NRRL-B-18809 was isolated
according to methods described elsewhere (van der Vossen, J.M. B. M., van der Lelie, D., and
Venema, G., Appl. Environ. Microbiol. 53:2452-2457 (1987)).
Polymerase Chain Reaction (PCR). Deoxy-oligonucleotide PCR primers were based on the
nucleotide sequence flanking the structural gene for the precursor of the bacteriocin nisin produced
by Lactococcus lactis F15876 (Dodd, H. M., Horn, N., and Gasson, M. J., J. Gen. Microbiol. 136:555566 (1990)). The two primers were synthesized on a DNA-synthesizer (Applied Biosystems 380A,
Foster City, CA) using the Phospho-amidit technique (Barone, A. D., et al., Nucleic Acid Research,
12:4051-4061 (1984)). The 20-base primer Ns-A (5'-CGCGAGCATAATAAACGGCT-3', sense primer)
starts 100 bases upstream of the ATG start codon, while the 21-base primer Ns-B (5'GGATAGTATCCATGTCTGAAC-3', antisense primer) starts 47 bases downstream of the TAA stop
codon.
AmpliTaq Recombinant Taq DNA polymerase (Perkin-Elmer/Cetus Corp., Norwalk, CT) was used to
carry out the PCR using a Perkin Elmer Cetus DNA Thermal Cycler (Perkin-Elmer/Cetus Corp.,
Norwalk, CT). The manufacturer's recommendations were followed with slight modifications. Each
100- mu l reaction mixture included 100 pmol of each primer and approximately 10 ng of template
DNA. To minimize the synthesis of regions caused by low-stringency annealing of primers the
reaction mixtures were incubated at 94 DEG C for 5 minutes before cycle 1. Each of the 30 cycles
consisted of 1 minute at 94 DEG C, 1.5 minute at 55 DEG C, and 2 minutes at 72 DEG C. After the
last cycle the polymerization step was extended by 5 minutes at 72 DEG C to complete synthesis of
all strands.
Electrophoresis. PCR products (10- mu l portions) were analyzed by electrophoresis on 1.5%
agarose gels in Tris-borate-EDTA buffer (Maniatis, T., Fritsch, E. F., and Sambrook, J., Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y., (1982)).
Sequence Analysis. Prior to sequencing PCR products were purified via an electro-elution step
(Maniatis, T., Fritsch, E. F., and Sambrook, J., Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor, N.Y., (1982)). PCR products were sequenced by the dideoxy chain termination procedure
(Sanger, F., Nicklen, S., and Coulson, A. R., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977)), using
the Sequenase 2.0 kit (U.S. Biochemicals, Cleveland, Ohio) with the following modifications. The
heat-denaturation step (3 minutes at 95 DEG C) was directly followed by a short annealing phase at 70 DEG C. The labelling reaction mixture was incubated at 37 DEG C for 30 to 45 seconds. The
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sequencing reaction products were separated on a denaturing polyacrylamide gel with a buffer
gradient as described by Biggin et al., (Biggin, M. D., et al., Proc. Natl. Acad. Sci. USA, 80:39633965 (1983)).
Determination of the nucleotide sequence of a gene encoding a bacteriocin precursor from
Lactococcus lactis NRRL-B-18809. To determine whether bacteriocin LL-2 had homology with nisin,
a bacteriocin which is produced by several other Lactococcus lactis strains (Dodd, H. M., Horn, N.,
and Gasson, M. J., J. Gen Microbiol. 136:555-566 (1990)), a PCR (Polymerase Chain Reaction)
analysis on total DNA from Lactococcus lactis NRRL-B-18809 was performed. Two nisin-specific
primers (Ns-A and Ns-B) based on the nucleotide sequence surrounding the gene for the precursor
of nisin (Dodd, H. M., Horn, N. and Gasson, M. J., J. Gen. Microbiol. 136:555-566 (1990)) were
synthesized and used in the PCR using a total DNA preparation of Lactococcus lactis NRRL-B-18809
as template. The PCR generated a molecule which had the predicted size (i.e. 321 bp), as
determined by agarose gel electrophoresis.The molecule was similar in size to a DNA molecule that
was generated on total genomic DNA of a Lactococcus lactis strain NRRL-B-18535 as described in
Serial No. 07/492,969 using the same primers. Controls, which lacked either primers or template
DNA, yielded no DNA molecules. Sequencing of the PCR-generated molecule from Lactococcus
lactis NRRL-B-18809, using both Ns-A and Ns-B as sequencing primers resulted in nucleotide
sequence similar to the sequence of the gene encoding precursor nisin and its flanking regions
(Dodd, H. M., Horn, N., and Gasson, M. J., J. Gen. Microbiol. 136:555-566 (1990)), except for one
base difference within the structural gene. At position 2362 (map position of Lactococcus lactis
F15876 (Dodd, H. M., Horn, N., and Gasson, M. J., J. Gen.Microbiol. 136:555-566 (1990)) an
adenosine (A) was present instead of a cytosine (C) at the corresponding position in the gene for
precursor polypeptide (SEQ ID NO: 1). The nucleotide sequence of the corresponding region in the
NRRL-B-18535 PCR-generated molecule did not show the alteration. Due to the alteration in the
genomic DNA of NRRL-B-18809, the amino acid, asparagine (Asn) is predicted at position 27 of
mature bacteriocin. In nisin, instead, histidine (His) is encoded at this position. This indicates that
bacteriocin LL-2 is similar to, but not identical to nisin. These results confirm the data on the amino
acid composition of bacteriocin LL-2 in which the ratio of asparagine:histidine is 2:1, rather than 1:2
in mature nisin as shown in Figure 8.
Table 9 shows the amino acids for the Xaa residues shown in SEQ ID No: 2. There are a limited
number of possibilities for Xaa. (Cys is Xac; Ser is Xaa and Thr is Xab except as written in SEQ ID
NO:2)
Id=TABLE 9 Columns=3
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Head Col 1: Translated Amino Acid
Head Col 2: Sequence Position
Head Col 3: Final form of amino acid
Cys7,11,19,26,28Ala-SSer3,5,29,33Ala or Dha or Ser
Thr2,8,13,23,25Abu or Dhb
As can be seen in Table 9, the Xaa at each sequence position SEQ ID NO:2 represents one of only
two or three choices based on the types of post-translational modification presumed to occur in this
cell line. Amino acid analysis shows no cysteine, no threonine and a single serine residue. Amino
acid analysis of nisin is identical for these three residue types. Xaa arising from Cys and Thr
represent one of only two possibilities, since the unmodified amino acid is not a possibility in LL-2.
The polypeptide has a tentative formula wherein Dha is 2-aminopropenoic acid, Dhb is 2-aminobut2-enoic acid, and the amino acid names between brackets below the partial chemical formulae
indicate the original amino acids from which these atypical amino acid residues were formed. The
sequences are shown in SEQ ID NO.:2 with Xaa as discussed previously.
Example 3
Effect of adding different levels of the crude LL-2 bacteriocin of Example 1 lyophilized to a powder
on post-acidification (spoilage) of yogurt held at 12C:
Yogurt was made by inoculating sterile 11% reconstituted non-fat dried milk with a yogurt starter
culture and incubating at 35 DEG C for 16 hr. At the end of incubation, the yogurt was chilled in an
ice-bath. After homogeneous mixing, 100 gm. portions were transferred into five wide-mouth screwcap bottles which were labelled 0 (Control), 2%, 3%, 4%, and 5%. Nothing was added to the bottle
marked 0. Crude lyophilized LL-2 bacteriocin powder (containing bacteriocin titer of 8000 AU/gm.)
was added in the amounts of 2 gm., 3 gm., 4 gm., and 5 gm. to bottles labelled 2%, 3%, 4% and 5%,
respectively. After uniform mixing of the powder, the yogurt pH was measured and the bottles were
placed in an incubator adjusted to hold at 12 DEG C. After 6 and 13 days, the pH values of the
individual bottles were measured and recorded. The results were plotted and are shown in Figure 3.
It is intended that the foregoing description be only illustrative of the present invention and that the
present invention be limited only by the hereinafter appended claims. Claims:
497/1006
1. A polypeptide having an inhibitory activity against sensitive Gram-positive bacteria and being
obtainable from a polypeptide precursor comprising the sequence
2. A polypeptide according to Claim 1 having at the 27th position an Asn.
3. A polypeptide having an inhibitory activity against sensitive Gram-positive bacteria and having the
amino acid sequence wherein Xaa is selected from Ser, Ala-, or Dha, and Xab is selected from Abu-,
or Dhb, and Xac is selected from Ala-S-, where Ala- is 3-substituted Alanine (-(CO)CH(NH-)CH2-),
Abu- is 3-substituted 2-aminobutanoic acid (-(CO)CH(NH-)CH(CH3)-), Ala-S- is (3-alanyl)-thio, Abu is
2-amino butanoic acid, Dha is 2-amino propenoic acid and Dhb is 2-aminobut-2-enoic acid, and
subunits thereof having an essentially equivalent inhibitory activity to the sensitive bacteria and
wherein there are sulfur bridges between the amino acids which provide the activity.
4. A polypeptide as produced by Lactococcus lactis subspecies lactis NRRL-B-18809 in a growth
medium and having inhibitory activity against sensitive Gram-positive bacteria.
5. The polypeptide of Claim 4 in an essentially pure form.
6. The polypeptide of Claim 5 wherein the polypeptide has been purified by ultrafiltration and wherein
the polypeptide is a retentate in the ultrafiltration.
7. The polypeptide of Claim 5 wherein the polypeptide has been purified chromatographically using
an anion exchange resin.
8. The polypeptide of Claim 7 wherein in addition the polypeptide has been purified by ultrafiltration.
9.The polypeptide of Claim 4 or a product containing the polypeptide in freeze dried form.
10. A method for inhibiting sensitive Gram-positive bacteria which comprises:
exposing the bacteria to an inhibitory amount of a polypeptide as claimed in Claim 1 to thereby
inhibit the bacteria.
498/1006
11. A method for inhibiting sensitive Gram-positive bacteria which comprises:
exposing the bacteria to an inhibitory amount of a polypeptide as claimed in Claim 2 to thereby
inhibit the bacteria.
12. A method of inhibiting sensitive Gram-positive bacteria which comprises:
exposing the bacteria to an inhibitory amount of a polypeptide derived from a precursor
polypeptide encoded by DNA obtained from Lactococcus lactis subspecies lactis NRRL-B-18809 to
thereby inhibit the bacteria.
13.The method of Claim 10 wherein the DNA is present on a plasmid and wherein the plasmid is
pSRQ 400.
14. The method of Claim 12 wherein the polypeptide is in a formulation containing between about
1600 and 2,500,000 arbitrary units of polypeptide per gram of the formulation.
15. The method of Claim 12 wherein the precursor polypeptide is produced by expression of a DNA
sequence present in a lactic acid bacterium, which precursor polypeptide is subsequently converted
by the lactic acid bacterium to the polypeptide.
16. The method of Claim 15 wherein the polypeptide is in an essentially pure form.
17. The method of Claim 15 wherein the polypeptide has been purified by ultrafiltration and wherein
the polypeptide is a retentate in the ultrafiltration.
18. The method of Claim 15 wherein the polypeptide has been purified chromatographically using an
anion exchange resin.
19.The method of Claim 15 wherein in addition the polypeptide has been purified by ultafiltration.
20. The method of Claim 12 wherein the polypeptide is in a solution which has a minimum of 1600
arbitrary units per ml.
21. The method of Claim 12 wherein the polypeptide or a product containing the polypeptide is
freeze-dried.
499/1006
22. A method for producing a polypeptide having activity against sensitive Gram-positive bacteria
which comprises:
(a) providing a Lactococcus lactis subspecies lactis NRRL-B-18809 in a growth medium containing
a carbon source, a nitrogen source and minerals under such conditions that the Lactococcus lactis
expresses the polypeptide having the inhibitory activity against the selected bacteria; and optionally
(b) isolating the polypeptide from the growth medium, preferably in the form of a concentrate.
23.A method for producing a polypeptide having activity against sensitive Gram-positive bacteria
which comprises:
(a) providing a lactic acid bacterium containing DNA as carried in Lactococcus lactis subspecies
lactis NRRL-B-18809 in a growth medium containing a carbon source, a nitrogen source and
minerals under conditions to express the polypeptide having the inhibitory activity against the
selected bacteria; and optionally
(b) isolating the polypeptide from the growth medium.
24. The method of Claim 23 wherein the polypeptide is purified.
25. The method of Claim 23 wherein the polypeptide is purified by ultrafiltration wherein the
polypeptide is the retentate.
26. The method of Claim 23 wherein the polypeptide is purified chromatographically using an ion
exchange resin.
27. The method of Claim 25 wherein in addition the polypeptide is purified by ultrafiltration.
28.A process for purifying a polypeptide produced by a culture of Lactococcus lactis subspecies
lactis NRRL-B-18809, in which the purification is performed by ultrafiltration of a broth fermented by
the culture, and a resulting retentate of an ultrafiltration is collected to provide the polypeptide in a
purified form.
29. A process for purifying a polypeptide produced by a culture of Lactococcus lactis subspecies
lactis NRRL-B-18809, in which the purification is performed by passing a broth fermented by the
culture until pH is less than about 5 through a chromatography column, preferably using a cation
exchange resin, and the polypeptide containing product in purified form is collected from the
500/1006
chromatography column, preferably by controlled elution from the column using a higher ionic
strength buffer.
30.The process as claimed in Claim 29, in which the polypeptide from the chromatography column is
subsequently subjected to an ultrafiltration and the polypeptide containing product in purified form
so produced is collected as a retentate of the ultrafiltration.
31. The process as claimed in Claim 28 in which the ultrafiltrate retentate is further concentrated and
purified to homogeneity by eluting from a reversed phase high performance liquid chromatography
column.
32. A method for producing DNA comprising the gene encoding a precursor polypeptide of a
polypeptide, the latter polypeptide having inhibiting activity against selected Gram-positive bacteria,
which method comprises:
(a) providing DNA obtained from Lactococcus lactis subspecies lactis NRRL-B-18809 comprising
a DNA sequence encoding a precursor polypeptide of the polypeptide given in Claim 1; and
(b) subjecting the DNA to a polymerase chain reaction wherein the primers are:
5'-CGCGAGCATAATAAACGGCT-3' as a sense primer and 5'-GGATAGTATCCATGTCTGAAC-3' as
an antisense primer to produce a DNA sequence comprising the gene encoding said precursor
polypeptide.
33.A method for producing DNA comprising the gene encoding a precursor polypeptide of a
polypeptide, the latter polypeptide having inhibiting activity against selected Gram-positive bacteria,
which method comprises:
(a) providing DNA obtained from Lactococcus lactis subspecies lactis NRRL-B-18809 comprising
a DNA sequence encoding a precursor polypeptide of the polypeptide given in Claim 2; and
(b) subjecting the DNA to a polymerase chain reaction wherein the primers are:
5'-CGCGAGCATAATAAACGGCT-3' as a sense primer and 5'-GGATAGTATCCATGTCTGAAC-3' as
an antisense primer to produce a DNA sequence comprising the gene encoding said precursor
polypeptide.
34.A method for producing DNA encoding a polypeptide having inhibiting activity against selected
Gram-positive bacteria which comprises:
(a) providing DNA from Lactococcus lactis NRRL-B-18809 encoding a precursor polypeptide of a
polypeptide;
501/1006
(b) subjecting the DNA to a polymerase chain reaction wherein the primers are:
5'-CGCGAGCATAATAAACGGCT-3' as a sense primer and 5'-GGATAGTATCCATGTCTGAAC-3' as
an antisense primer to produce the DNA.
35. The method of Claim 34 wherein the DNA is incorporated into another lactic acid bacterium
which is cultured under conditions for producing the precursor polypeptide and converting the latter
to the polypeptide.
36. A DNA sequence encoding a precursor polypeptide capable of being converted to the
polypeptide as claimed in Claim 1.
37. A DNA sequence encoding a precursor polypeptide capable of being converted to the
polypeptide as claimed in Claim 3.
38. A DNA sequence encoding a precursor polypeptide comprising the amino acid sequence
39. A DNA sequence encoding a precursor polypeptide, said DNA sequence being the
polynucleotide 1-321
40. A polypeptide precursor comprising the amino acid sequence
41. A polypeptide precursor having the sequence
42. A biologically pure culture of Lactococcus lactis subspecies lactis NRRL-B-18809.
502/1006
58. NZ299034 - 22.07.1993
PHARMACEUTICAL BACTERIOCIN COMPOSITIONS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ299034
Inventor(s):
BLACKBURN PETER (US); PROJAN STEVEN J (US); GOLDBERG EDWARD B (US)
Applicant(s):
APPLIED MICROBIOLOGY INC (US)
IP Class 4 Digits: A61K
IP Class:
A61K31/19; A61K37/02
E Class: A61K38/16B; A61K38/16B+M
Application Number:
WO1993US00377 (19930115)
Priority Number: US19920822433 (19920117); US19920866135 (19920409)
Family: NZ299034
Equivalent:
AU3475093; AU672384; BR9305754; CA2127987; CZ282676; CZ9401661;
DE69310346D; DE69310346T; DK623024T; EP0623024WO9313793; ES2102010T; FI943261;
GR3024049T; HU219234; HU70839; IL104364; JP7508499T; MX9300207; NO314221B; NO942644;
NZ249007; RU2104710; SK282523B; SK83794
Cited Document(s):
WO8912399
Abstract:
COMPOSITIONS COMPRISING LANTHIONINE-CONTAINING BACTERIOCINS SUCH AS NISIN ACT
AS BACTERICIDES UNDER CONDITIONS SUCH AS THOSE FOUND IN THE GASTROINTESTINAL
TRACT. IN A PREFERRED EMBODIMENT PHARMACEUTICAL PREPARATIONS CONTAINING THE
COMPOSITIONS ARE USED FOR THE CONTROL OF BACTERIA RESPONSIBLE FOR DISORDERS
OF THE GASTROINTESTINAL TRACT.Description:
503/1006
PHARMACEUTICAL BACTERIOCIN COMPOSITIONS
Background and Summarv of the Invention
The antimicrobial activity of the lanthioninecontaining bacteriocin nisin is restricted towards certain
gram positive organisms and is optimal at pH 5.0.
The antimicrobial activity of nisin is enhanced when used in combination with a chelator such as
EDTA. The activity of the nisin-chelator compositions have been found to be significantly greater or
optimal at a pH greater than 5.0. For example, it has been determined that the antimicrobial activity
towards Stahvlococcus aureus of a nisin and EDTA composition is significantly greater at pH 8.0
than the activity of the same composition against S. aureus at pH 5.0. The combination of a chelator
with nisin was also found to result in activity towards gram negative bacteria, an activity which is not
normally attributed to nisin itself.
The present invention concerns lanthioninecontaining bacteriocin compositions which are active in
acidic pH below 5.0 and display considerable activity against gram negative bacteria. These low-pHactive compositions may be useful for example in methods of treating or preventing infections or
growth of microorganisms in the gastrointestinal tract of humans and animals. These compositions
when introduced into the gastrointestinal tract will act as bactericides even in the low-pH
environment of the stomach. This antibacterial activity may be useful in containing the growth of
infections caused by gastrointestinal pathogens such as species of Helicobacter, Escherichia,
Salmonella,
Bacillus, Clostridia, Bacteroides, Camnvlobacter and
Yersinia.Such-low-pH active bacteriocin compositions would therefore be useful in the treatment of
various diseases or symptoms due to the presence of such pathogenic bacteria.
Various gastrointestinal diseases or symptoms including diarrhea, gastritis, peptic and duodenal
ulcer, and gastric carcinoma are due to the presence of pathogenic microorganisms in the
gastrointestinal tract.
Escherichia and Salmonella, in particular, but also certain species of Clostridia, Bacillus, Bacteroides,
Campylobacter and Yersinia can be responsible for diarrhea especially in nednatal farm animals.
(R.E.
504/1006
Holland, 1990, Clin. Microbiol. Rev. 3:345, "Some infectious causes of diarrhea in young farm
animals.")
Helicobacter pylori are implicated in gastritis, duodenal and peptic ulcer disease. (Peterson, W.L.,
1991, New Ena. J. Med. 324: 1043, "Helicobacter pylori and peptic ulcer disease") and are also
associated with gastric carcinoma. (Henderson, B.E., Ross, R.K., and Pike, M.C., 1991, Science
254:1131, "Toward the primary prevention of cancer," Nomura, A. Stemmermann, G.A., Chyou, P.H.,
Kato,I., Perez-Perez, G. and Blaser, M.J. (1991) New Ena.
J. Med. 325: 1132 "Helicobacter Dvlori infection and gastric carcinoma among Japanese Americans
in Hawaii.";
Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Chang,
Y., Vogelman, J.H., Orentreich,N, and Sibley, R.K. (1991)
New Ens. J. Med. 325:1127; Forman, D., Sitas, F., Newell,
D.G., Stacey, A.R., Boreham, J., Peto, R., Campbell,
T.C., Li, J. and Chen, J. (1990) Int J. Cancer 46:608 "Geographic association of Helicobacter pYlori
antibody prevalence and gastric cancer mortality in rural China").
Many gastrointestinal pathogens are gram negative bacteria, organisms against which nisin would
be expected to be inactive. (Hurst, A., 1981, "Nisin," Adv. in App.
Micr. V. 27, p. 85-121.) For example, Helicobacter pylori (which has also been identified in the prior
art as Campylobacter pylori) is a gram negative microaerophilic bacillus that colonizes the gastric
mucosa. Since 1983, when first reported in association with histologic gastritis, a relationship
between suppression of H. pylori infection and improvement of gastric disorders has been noted.
However, although numerous antibiotics have been tried against H. pylori infection, none have so far
proved acceptable and no agent or regimen has been approved for use against this organism. Long
term eradication of the organism has seldom been achieved and antibiotics themselves can produce
unacceptable side effects. (Peterson, W.L., 1991, New Eng. J.Med. 324:1043 "Helicobacter pylori
and peptic ulcer disease; Warren, J.R., 1983, Lancet 1:1273, "Unidentified curved bacilli on gastric
epithelium in active chronic gastritis"; Morgan et al., 1988, Gastroenteroloov 95:1178, "Nitrofurans in
the treatment of gastritis associated with Campylobacter Dvlori";
Glupczynski, Y. et al., 1988, Am. J. Gastroenterol.
505/1006
83:365 "Campylobacter pylori-associated gastritis: a double-blind placebo controlled trial with
amoxycillin";
Rauws, E.A. et al., 1988, Gastroenteroloav 94:33, "Campylobacter Pvlori-associated chronic antral
active gastritis"; Glupczynski, Y. 1990 in Helicobacter pylori, gastritis, and peptic ulcer";
Malfertheiner, P.,
Ditschuneit, H., Eds. Springer-Verlag, Berlin, Germany pp 49-58; Rauws, E.A. and Tytgat, G.N. 1990
Lancet 335:1233 "Cure of duodenal ulcer associated with eradication of
Helicobacter pylori. O'Riordan, T. et al., 1990, Gut 31:999 "Adjuvant antibiotic therapy in duodenal
ulcers treated with colloidal bismuth subcitrate"; Weil, J. et al., 1990, Aliment. Pharmacol.Ther.,
4:651 "Helicobacter pylori infection treated with a tripotassium dicitrato bismuthate and
metronidazole combination"; Coghlan, J.G.,
Gilligan, D., Humphries, H., et al., 1987, Lancet 2:1109 "Campylobacter pylori and recurrence of
duodenal ulcer a 12-month follow-up study"; Marshall, B.J. Goodwin,
C.S., Warren, J.R. et al., 1988, Lancet 2:1437, "Prospective double-blind trial of duodenal ulcer
relapse after eradication of Campylobacter pylori").
The present invention concerns pharmaceutical compositions comprising a lanthionine-containing
bacteriocin such as nisin and a chelating agent with a suitable carrier for use in low-pH environments
as bactericides. These compositions are stable and active at acidic pH and are useful for their
antibacterial activity against gram negative bacteria in low-pH environments such as encountered in
the gastrointestinal tract. Pharmaceutical nisin compositions according to the invention act quickly;
so that when delivered into the stomach and gastrointestinal tract their activity should not be limited
by the clearance rate of the stomach contents. Furthermore, unlike antibiotics, nisin compositions
can be safely ingested. The pharmaceutical compositions may be used alone in treatment regimens
or in combination with other pharmaceutical agents or drugs.
The invention also concerns methods of using and making such compositions.
Detailed Description of the Invention
The efficacy of the present invention has been demonstrated on E. coli bacteria which are found in
the mammalian gut and are frequently responsible for gastrointestinal disorders. The survival of E.
coli is unaffected by exposure to EDTA or citrate by themselves or by exposure to nisin by itself. In
addition, suspensions of E. coli exposed to acid survive well in an acidic environment until the pH
drops below pH 2.5.
506/1006
However, as is set forth below, when nisin is combined with EDTA at a range of acidic pH values,
significant reduction in the viability of the bacteria was seen after only 1 minute of exposure to the
nisin compositions. At pH 3.5, a reduction by more than 6 logarithms in the viable count of bacteria
can be attributed to nisin after only 1 minute exposure to the nisin-chelator composition.
Below pH 3.5 some reduction of the potency of the nisin compositions is apparent but, nevertheless,
an approximately 1000-fold enhancement of nisin activity remains even at pH 2.5 (Table 1).
EDTA-activated nisin is bactericidal towards E. coli in the presence of various acid vehicles
including acetate, citrate, lactate, and succinate, as shown in
Tables 2-5. As illustrated by results obtained at pH 3.5, the rapid bactericidal activity of the nisin
compositions can be influenced by the choice of acid vehicle. In all the illustrated cases, as the
concentration of each acid anion is increased, the bactericidal activity of the nisin compositions is
observed to decrease. Nevertheless, each of these acid vehicles is suitable for the expression of
chelatorenhanced nisin activity. Exposure of the bacteria to these nisin compositions for a longer
period than 1 minute is effective in reducing the number of bacteria even when the formulations
contain the less effective concentrations of the acid vehicles.
Evaluation of Germicidal Activity of Chelator-Enhanced
Nisin in Acid Vehicles towards Gram Negative Bacteria.
The rapid activities of various chelator-enhanced nisin formulations were evaluated in acid vehicles
in a germicidal suspension assay.
E. coli cells from an overnight Trypticase soy nutrient agar (TSA) were resuspended to a density
measured as an absorbance of 1.0 at 600 nm in sterile ddH20. The reaction of the cells with each of
the bactericidal test formulations analyzed was started by addition of 30 1 of cells to 970 p1 of test
formulation.
The reaction mixture was incubated at 370C for at least 1 minute and then centrifuged in a microfuge
for 1 minute.
The cell pellet was washed by resuspension in 1 ml of neutralization buffer. (The neutralization buffer:
50 mM
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Tris-HC1, pH 7.0, 5 mM MgSO4, 20 mM CaC12, 0.1 M NaCl and 0.1% gelatin was prepared by first
making Tris buffer and adjusting the pH. The salts and gelatin were then added and the solution
stirred with heat until the solution was clear. The solution was then autoclaved for 20 min. The
neutralization buffer was used without dilution.) The cells were centrifuged in a microfuge for 1
minute and resuspended in 1 ml of neutralization buffer.The viable count was determined by
spreading 100 p1 of bacterial suspension and serial dilutions thereof in neutralization buffer on
nutrient agar and scoring surviving colonies after 24 h at 370C. Percent survival relative to untreated
controls was calculated from the scored values.
EDTA-enhanced activity of nisin is expressed at low pH. Below pH 3.5 the degree of enhancement is
reduced, presumably as the carboxyl groups of the chelator are titrated. Nevertheless, an
approximately 1000-fold enhancement of nisin by EDTA was observed even at pH 2.5.
See results presented in Table 1. In Table 1 and subsequent Tables 2, 4 and 8-13 values preceded
by " < " represent the percent survival figures, based on the initial concentration of viable bacteria
(expressed as colony forming units (cfu) /ml) in the assayed bacterial suspension and the aliquot
volume of said suspension taken for assay, corresponding to the detection of no surviving colonies.
Table 1
EDTA activation of Nisin towards Escherichia coli
Dependence with respect to acidic pH
EDTA Nisin pH value
mM gg/ml
7.0 2.0 2.5 3.0 3.5 4.0
% survival at 1 mina
0 0 100b - - - 100
0 100 - 0.28 3.37 4.43 100 100
1.0 0 - 0.07 - 5.41 1.0 100 - 0.01 0.005 0.0007 < 104 < 10
a Incubations performed at 370C in 20 mM Na acetate buffer
adjusted to pH b Initial viable count 4 x 107 colony forming units /ml
Table 2
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Chelator Activation of Nisin-Acetate towards Escherichia coli
Dependence with respect to Acetate concentration
EDTA Nisin (% w/v) Acetate pH 3.5
mM g/ml
0 0.1 0.3 1.0 3.0
% survival at 1 mina
0 0 100b 55 80 90 10.1
0 100 - 9.6 70 100 100
1.0 0 - 40 25 25 10.7
1.0 100 - 0.0005 < 10-4 0.02 0.4
a Incubations performed at 370C b Initial viable count 2 x 107 colony forming units /ml
Table 3
Chelator Activation of Nisin-Citrate towards Escherichia coli
Dependence with respect to Citrate concentration
EDTA Nisin (% w/v) Citrate pH 3.5
my all
0 0.1 0.3 1.0 3.0
survival at 1 mina
0 0 100b 100 56.3 72.9 100
0 100 - 0.003 0.0007 0.56 14.6
1.0 0 - 100 50.0 47.9 100
1.0 100 - 0.0006 0.0002 0.69 20.8
a Incubations performed at 370C b Initial viable count 5 x 107 colony forming units /ml
Table 4
Chelator Activation of Nisin-Lactate towards Escherichia coli
Dependence with respect to Lactate concentration
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EDTA Nisin (% w/v) Lactate nH 3.5
mM ga/ml
0 0.1 0.3 1.0 3.0
% survival at 1 mina
0 0 100b 22.2 46.7 35.6 4.2
0 100 - 4.4 0.24 17.8 26.7
1.0 0 - 5.1 0.46 0.91 1.58
1.0 100 - < 104 0.02 0.20 2.84
a Incubations performed at 37 C b Initial viable count 5 x 107 colony forming units /ml
Table 5 chelator Activation of Nisin-Succinate towards flscherichia coli
Dependence with respect to Succinate concentration
EDTA Nisin (t w/v) Succinate DH 3.5
mM g/ml
0 0.1 0.3 1.0 3.0
% survival at 1 mina
0 0 100b 85.5 29.9 36.7 13.9
0 100 - 18.3 66.6 83.4 28.4
1.0 0 - 56.8 63.6 46.2 43.2
1.0 100 - 0.02 6.21 14.2 4.73
a Incubations performed at 370C b Initial viable count 3.4 x 106 colony forming units /ml
At pH 3.5 EDTA-enhanced nisin activity was observed in the presence of all acid anions tested (see
Tables 25). At pH 3.5, lactate at 0.3% was somewhat inhibitory to EDTA-enhanced nisin
activity.Nevertheless the activity is still enhanced more than 1000-fold over 0.3% lactate alone and
0.16 lactate is not inhibitory to EDTAenhanced nisin.
At pH 3.5, up to 0.3% acetate and 0.36 citrate appear compatible with chelator-enhanced nisin
germicidal activity towards E. coli suspensions. These anions appear to show the most promise as
acid vehicles to be formulated with EDTA-enhanced nisin. Citrate is a most suitable acid vehicle for
EDTA-enhanced nisin compositions. Citrate is a naturally occurring food substance and intermediary
metabolite and an effective enhancer of nisin bactericidal activity in its own right (Table 3). Nisincitrate compositions can be expected to be safe and effective for containing or eliminating the
growth of undesirable microorganisms in the gastrointestinal tract of humans and animals.
510/1006
Citrate is a metabolite, it does not inhibit the growth of bacteria and bacteria grow well on nutrient
agar supplemented with citrate. However, nisin in the presence of citrate is active against gram
negative bacteria. Thus, it is possible to demonstrate the activity of nisin towards gram negative
bacteria by performing growth inhibition assays on nutrient agar supplemented with various
concentrations of citrate.
This nisin-citrate agar assay has much more general applicability. The assay provides a method for
screening potential agents other than citrate in combination with nisin for their potential properties as
enhancers of nisin's inherent bactericidal activity. Examples of other organic acids in combination
with nisin would include acetate, propionate, lactate, succinate, fumarate, malonate, adipate,
sorbate, phosphate and ascorbate.
Other agents that potentiate nisin activity include nonionic and amphoteric surfactants and
emulsifiers, quaternary compounds, monoglycerides, and fatty acids.
The nisin-citrate agar assay is performed as follows. E. coli is resuspended to an optical density of
1.0 at Ag. A 100 pl sample of the bacterial suspension is spread uniformly on Trypticase Soy
nutrient agar (TSA) supplemented with various concentratins of citrate (eg.
0.1%, 0.3%, 1.0%, 3.0%) and incubated for 1 hour at 370C.
A nisin stock solution and serial dilutions thereof in 0.18 bovine serum albumin (BSA), are prepared
and 5 Al are taken from each and deposited onto the growing bacterial lawn. The TSA plates are
then incubated for 24 hours at 370C After 24 hours at 370C, E. coli grown on
TSA supplemented with citrate form a confluent lawn. The activity of nisin towards bacteria grown in
the presence of citrate is demonstrated by clear zones in the bacterial lawn where the serially diluted
nisin samples were deposited. The effectiveness of nisin against the gram negative bacteria can be
assessed from determining the minimum amount of nisin required to produce a clear zone of growth
inhibition.As the concentration of citrate in the nutrient agar is increased, less nisin is required to
inhibit the growth of E. coli, as is illustrated by the data shown in Table 6.
TABLE 6
The activity of Nisin towards E. coli grown
on Nutrient Aaar in the presence of Citrate
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% Citrate Nisin NIC1
0% 3,333 Ag/ml
0.1% 370 g/ml
0.3% 123 Zg/ml
1.0% 13.7 Sg/ml
3.0% 0.06 Mg/ml 1 Nominal inhibitory concentration of nisin minimally
required to prevent growth of E. coli strain
ATCC8739 grown on Trypticase Soy Agar supplemented
with the various concentrations of citrate as
indicated.
The activity of nisin enhanced with EDTA, citrate or other chelators has also been demonstrated
towards several strains of Helicobacter pylori as well as related species, particularly Campvlobacter
nenuni, by the germicidal suspension assay. Examples are shown in
Tables 7 - 13. Freshly grown H. pylori cells, grown on a nutrient agar plate (Trypticase Soy Agar, BBL
11043, supplemented with 5% defibrinated sheep blood), were harvested and subsequently grown
at 370C for 72-96 hours in a BBL Gaspaks System chamber with BBL Campy PakTM
Microaerophilic System envelopes and using a Campylobacter microaerophilic gas generator
(BBL71034).
The cells were then resuspended in sterile, deionizeddistilled water to a cell density of 1.0 A to
provide a suitable stock suspension. The assay was started by addition of 50 pl of bacterial
suspension to 950 pl of test solution, incubated at 370C for 5 minutes and then centrifuged for 1
minute in a microfuge. The cell pellet was washed by resuspension in 1 ml of the sterile neutralization
buffer described previously and centrifuged for 1 minute in a microfuge. The cells were then
resuspended in Brucella-Albimi broth (BBL) and serially diluted in same prior to plating on nutrient
agar.The viable count was determined by spreading 100 pl of bacterial suspension and dilutions
thereof on the nutrient agar described above and scoring surviving colonies after 72-96 hours'
incubation at 370C in the modified atmosphere described above. Percent survival relative to
untreated controls was calculated from the scored values.
The concentration dependence of the activity of nisin towards Helicobacter pylori in the presence
and absence of 0.1% citrate at pH 5.0 is illustrated by the data presented in Table 7. Although H.
pylori is a gram negative bacterium, nisin, considered active only against gram positive bacteria,
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surprisingly exhibits some bactericidal activity towards this organism. However, the activity of nisin
towards H. pylori is significantly enhanced by the presence of citrate.
The concentration dependence of the activity of nisin towards H. pylori by citrate at pH 5.0 and pH
7.0 is illustrated by the data presented in Table 8. In general, citrate by itself has little effect on the
viability of this bacterial species at pH 5.0, although at pH 7.0 the viability of the organism is
somewhat reduced at higher concentrations of citrate. The effects of citrate alone are surprising
since citrate is a metabolite. The enhanced activity attributable to nisin in the presence of citrate is
sufficient to completely kill a 105 cfu/ml suspension of H. pylori within 5 minutes at 370 C.
Data presented in Table 9 illustrate that the activity of nisin towards H. pylori at pH 5.0 and pH 7.0 is
also significantly enhanced in the presence of the chelator EDTA. The chelator itself has little effect
on the viability of these organisms except at higher concentrations. However, the EDTA-enhanced
activity attributable to nisin is sufficient to completely kill a 105 cfu/ml suspension of H. pylori within 5
minutes at 370C.
The bactericidal activity of nisin towards H. pylori in the presence or absence of citrate or EDTA over
a range of pH values is illustrated by the data presented in Table 10 and Table 11, respectively.
Despite the fact that H. pylori is isolated from the stomach, the lumen of which is acidic, the viability
of this organism is surprisingly poor after exposure to low pH conditions.
H. pylori colonizes the stomach mucosal epithelia, a less acidic microenvironment than that of the
stomach lumen.
Despite the limiting viability of H. pYlori at low pH in these experiments, the data indicate that nisin
with citrate or EDTA can be expected to be bactericidal towards H. pylori under conditions similar to
those that prevail in the stomach and its mucosal epithelium where
H. pylori is able to thrive.
Campylobacter renuni is a gram negative bacterium that colonizes the intestines of birds and
mammals and has been associated with food poisoning. The bactericidal activity of nisin, in the
presence and absence of citrate or EDTA, towards C. renuni is illustrated by the data presented in
Table 12 and Table 13, respectively. Nisin by itself is extremely effective towards this gram negative
bacterium. At pH 5.0, higher
concentrations of citrate also proved to be toxic towards
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this bacterium. Thus, combinations of nisin with citrate
or EDTA can be expected to be effective towards C. jejuni
as is illustrated by the data in Tables 12 and 13.
Table 7
Bactericidal activity of Nisin towards Helicobacter pylori
Activity with respect to nisin concentration
Strain Citrate Nisin (ug/ml)
ATCC; pH 5.0
0 10 30 100 300
% survival at 5 mina
ATCC o 100b 1.41 0.39 0.085 0.0056
43579
0.1% 86.2 0.21 0.008 0.001 1.89 x 10-5
a Incubations performed at 370C b Initial viable count 3.19 x 107 cfu/ml
Table 8
Bactericidal activity of Nisin towards Helicobacter pylori
Activity with respect to citrate at pH 5.0 and pH 7.0
Strain pH Nisin (% wlv) Citrate
ATCC; g/ml
0 0.1 0.3 1.0
% survival at 5 mina
ATCC 5.0 0 100b 100 100 100
43579
100 9.62 < 0.01 < 0.01 < 0.01
ATCC 5.0 0 100C 83.6 42.9 22.1
43504
0 100d 25.8 4.39 50.9
100 n.a. 0.033 0.066 0.14
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100 0.043 < 3.1x10-3 0.92 2.44
ATCC 7.0 0 100e 12.1 0.26 0.02
43579 100 12.1 0.2@ 0.02
100
0.78 < 8.1 x 10-3 < 8.1 x 10-3 < 8.1 x 10-3
ATCC 7.0 0 lOOf 17.7 8.94 0.82
43504
100
2.08 < 0.01 < 0.01 < 0.01
a Incubations performed at 370C for 5 minutes b Initial viable count 1.04 x 10 cfu/ml c Initial viable
count 1.40 x 105 cfu/ml d Initial viable count 3.26 x 105 cfu/ml e Initial viable count 1.24 x 105 cfu/ml
f Initial viable count 9.62 x 104 cfu/ml
Table 9
Bactericidal Activity of Nisin Towards Helicobacter pylori
Activity with respect to EDTA at pH 5.0 and pH 7.0
Strain pH Nisin (mM) EDTA
ATCC; g/ml
0 1.0 10 100
% survival at 5 mina
ATCC 5.0 0 100b 13.63 8.82 0.43
43579
100 4.7 < 5.0 x 10-3 < 5.0 x 10-3 < 5.0 x 10-3
ATCC 7.0 0 100C 94.1 21.0 1.62
43526
100 5.88 < 0.03 < 0.03 < 0.03
a Incubations performed at 370C for 5 minutes b Initial viable count 2.04 x 10@ cfu/ml c Initial viable
count 3.40 x 104 cfu/ml
Table 10
Bactericidal Activity of Nisin Towards Helicobacter pylori
Dependence with respect to citrate in pH range 2.5 to 5.0
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Strain Citrate Nisin pH value
TCC; % g/ml
2.5 3.0 3.5 4.0 5.0
% survival at 5 mina
TCC O 0 - - - - 100
43579
0 100 - - - - 8.6x10-3 e
0 100 - - - - 0.30c
0.1 0 < 5.0 x 10-4 1.1 x 10-3 0.18 9.56 97.2b
0.1 100 < 5.0 x 10-4 1.1 x 10-3 1.1 x 10-3 2.0 x 10-3 2.7 x 10-3 b
ATCC
43504 0 0 - - - - 100d
0 0 - - - - 100e
0 100 - - - - 0.69d
0 100 - - - - 4.86e
0.1 0 0.14 < 0.004 < 0.004 0.36 68.5d
0.1 100 < 0.004 < 0.004 < 0.004 < 0.004 0.022d
0.3 0 0.027 < 0.027 29.7 17.6 100e
0.3 100 < 0.027 < 0.027 2.7 0.32 0.18e
a Incubations for 5 min at 370C in citrate adjusted to pH,
or 20 mM acetate, pH 5.0.
b Initial viable count 1.85 x 106 cfu/ml c Average of 5 experiments d Initial viable count 2.7 x 105
cfu/ml e Initial viable count 3.7 x 104 cfu/ml
Table 11
Bactericidal activity of Nisin Towards Helicobacter pylori
Activity with respect to EDTA in the pH range 2.5 to 5.0
Strain EDTA Nisin
ATCC; mM g/ml
2.5 3.0 3.5 4.0 5.0
% survival at 5 mina
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ATCC 0 0 - - - - 100b
43504
0 0 - - - - 100c
0 100 - - - - 0.021d
0 100 - - - - 7.03e
1.0 0 0.023 < 0.021 94.2 11.6 10.0b
1.0 0 0.013 0.001 0.015 16.5 100c
1.0 100 < 0.021 < 0.021 0.012 < 0.021 0.53b
1.0 100 < 0.001 < 0.001 0.005 0.54 0.32c
a Incubations for 5 min at 370C in citrate adjusted to pH,
or 20 mM acetate, pH 5.0.
b Initial viable count 8.6 x 104 cfu/ml c Initial viable count 9.82 x 105 cfu/ml d Incubated in presence
of 0.18 citrate e Incubated in presence of 20 mM acetate
Table 12
Bactericidal Activity of Nisin Towards Campylobacter jejuni
ATCC29428
Dependence with respect to citrate at pH 5.0 (strain ATCC29428)
Nisin (%w/v) Citrate nH 5.0
g/ml
I 0 0.1 0.3 1.0
% % survival at 5 mina
0 0 100b 2.9 < 6.4x10-3 < 6.4x10-3
100 < 6.4 x 10-3 < 6.4 x 10-3 < 6.4 x 10-3 < 6.4 x 10-3
a Incubations performed at 370C for 5 minutes
b Initial viable count 1.57 x 105 cfu/ml
Table 13
Bactericidal Activity of Nisin Towards Campylobacter jejuni ATCC29428
Dependence with respect to EDTA at pH 5.0 (strain ATCC29428)
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Nisin mM EDTA pH 5.0
g/ml
0 1.0 10 100
% survival at 5 mina
0 100b 56.5 76.1 76.1
100 < 5.0 x 10-4 < 5.0 x 10-4 < 5.0 x 10-4 < 5.0 x 10-4
a Incubations performed at 370C for 5 minutes b Initial viable count 1.84 x 106 cfu/ml
The activity of nisin enhanced with EDTA, citrate or other chelators can also be demonstrated
towards species of Salmonella by germicidal suspension assays. Freshly grown S. tvphimurium cells
are taken from a nutrient agar plate (Trypticase Soy Agar, BBL11043,) grown at 370C for 24 hours.
The cells are resuspended to a cell density of 1.0 A to provide a suitable stock suspension. The
assay is started by addition of 30 Al of bacterial suspension to 970 pl of test solution and incubated
for at least 1 minute and then centrifuged for 1 minute in a microfuge. The cell pellet is washed by
resuspension 1 ml of sterile neutralization buffer, resuspended again and then serially diluted in
neutralization buffer.The viable count is determined by spreading 100 ul of bacterial suspension and
dilutions thereof on nutrient agar and scoring surviving colonies after 24 hours incubation at 370C.
Percent survival relative to untreated controls is calculated from the scored values.
The low-pH-active bacteriocin compositions of the invention are preferably administered orally in the
form of a pharmaceutical preparation which contains an effective amount of the lanthioninecontaining bacteriocin and a pharmaceutically acceptable carrier.
The carrier may also include an effective amount of a chelator and/or an acidic vehicle and/or a
surfactant or emulsifier, monoglyceride, or fatty acid. The lanthionine-containing bacteriocin may be
selected from the group consisting of nisin, subtilin, epidermin, Pep 5, ancovenin, gallidermin,
duromycin or cinnamycin.
Suitable chelating agents include, but are not limited to, EDTA, CaEDTA, CaNa2EDTA and other
alkyldiamine tetracetates as well as citrate. In certain instances the chelator and the acidic vehicle
can be the same, such as when the acidic vehicle and the chelator are both citrate. Suitable acidic
vehicles for use in the compositions of this invention are acetate, propionate, citrate, lactate,
succinate, fumarate, malonate, adipate, sorbate, phosphate and ascorbate.
518/1006
The compositions of the invention are also effective at slightly acid pH levels, (e.g., pH 5.0) and even
higher pH levels, (e.g., pH 8.0), against pathogenic bacteria which may inhabit the gastrointestinal
tract, such as E. coli and S. tsphimurium as disclosed in issued
U.S. Patent No. 5,135,910. The disclosure of U.S. Patent
No. 5,135,910 is hereby incorporated by reference in its entirety.
The pharmaceutical compositions of the invention may thus also be formulated as antacid
compositions or administered in combination with an antacid wherein the administration would result
for instance in a higher stomach pH environment than that existing prior to administration. The nisin
chelator compositions would still be effective against the pathogenic bacteria under such conditions.
The pharmaceutically acceptable carrier may be in the form of a solid, semi-solid or liquid diluent or
a capsule. In certain embodiments of the invention the acidic vehicle and the pharmaceutical carrier
may be the same. Other pharmaceutically acceptable carriers may be cellulose derivatives, gelatin,
lactose, starch, etc.
The pharmaceutical compositions may be in the form of solutions, colloids or emulsions, powders,
tablets, capsules or gels.
The dry forms of the compositions active at low pH may be pressed into tablets which may be
coated with a concentrated solution of sugar and which may contain other pharmaceutically
acceptable substituents such as gum arabic, gelatin, talc, or titanium dioxide and may be also
coated with various dyes. Hard gelatin capsules may be prepared which contain granules of the
bacteriocin, acid vehicle and chelating agent in combination with a solid carrier such as lactose,
potato starch, corn starch, cellulose derivatives or gelatin.
Liquid preparations for oral administration may be prepared in the form of syrups or suspensions
comprising the peptide bacteriocin, the chelating agent, the acid vehicle, and sugar, water and
glycerol or propylene glycol. If desired, such liquid preparations may contain coloring agents,
flavoring agents, sweeteners such as saccharin and thickening agents such as cellulose derivatives.
Delivery of a dosage could obviously be achieved by modifications of the simple aqueous
formulations by inclusion of thickeners, emulsifiers, or particulates to effect a colloidal suspension.
Alternatively, osmotically balanced solutions containing a suitable dosage could be administered in
519/1006
volumes as little as 10 ml or as large as 4 liters. Osmotically balanced solutions such as those used
as gastrointestinal lavage solutions would be suitable (Di Palma, J.A. and Brady,
C.E., 1989, Am. J. Gastroenterol. 84:1008, "Colon
Cleansing for Diagnostic and Surgical Procedures:
Polyethylene glycol Lavage Solution"; Di Palma, J.A. and
Marshal, J.B., 1990, Gastrointestinal Endoscopy 1990, 36:285, "Comparison of a new Sulfate-free
Polyethylene glycol Electrolyte Lavage Solution versus a Standard
Solution for Colonoscopy Cleansing"; Fordtran, J.S., et al., 1990, Gastroenterol. 98:11, "A low-Sodium
Solution for Gastrointestinal Lavage"). The performance of gastrointestinal lavage solutions used to
cleanse the gastrointestinal tract would be expected to be improved by inclusion of the germicidal
compositions described herein.
The typical daily dose of the inventive compositions may vary according to the pathogenic
microorganism infection being treated, the site of infection, and the symptoms of the disease being
treated. In general, it is expected that oral dosages would range from 0.1 mg per dose to 300 mg per
dose of lanthionine-containing bacteriocin substance, and 0.1 g per dose to 30 g per dose of
chelator.
For example, since the volume of stomach contents varies as a function of the time lapsed after the
last meal, simple aqueous formulations suitable for gastrointestinal use may be prepared as follows:
For a final concentration to be achieved in the stomach at 0.16 citrate + 0.001% nisin (10 ug/ml)
delivered in 10 ml and assuming approximately 100 ml in stomach:
Dosage 1: 1.0 mg nisin and 0.1 g citrate Na citrate 1.0%
nisin 0.01%
saccharin 0.005%
polysorbate 20 1.0%
glycerol 10.0%
water 87.985%
For a final concentration to be achieved in the stomach at 3.0% citrate + 0.03% nisin (300 ug/ml)
delivered in 10 ml and assuming 100 ml in stomach:
Dosage 2: 30 mg nisin and 3.0 g citrate Na citrate 30.0%
nisin 0.3%
saccharin 0.005%
polysorbate 20 1.0%
glycerol 0.0%
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water 58.695%
For a final concentration to be achieved in the stomach at 0.1% citrate + 0.001% nisin (10 ug/ml)
delivered in 10 ml and assuming 1000 ml in stomach:
Dosage 3: 10 mg nisin and 1.0 g citrate Na citrate 10.0%
nisin 0.1%
saccharin 0.005%
polysorbate 20 1.0%
glycerol 10.0%
water 78.895%
For a final concentration to be achieved in the stomach at 3.0% citrate + 0.03% nisin (300 ug/ml)
delivered in 100 ml and assuming 1000 ml in stomach:
Dosage 4: 300 mg nisin and 30 g citrate Na citrate 30.0%
nisin 0.3%
saccharin 0.005%
polysorbate 20 1.0%
glycerol 10.0%
water 58.695%
For a final concentration to be achieved in the stomach at 3.0% citrate + 0.03% nisin (300 ug/ml)
delivered in 100 ml and assuming 100 ml in stomach:
Dosage 5: 60 mg nisin and 6.0 g citrate Na citrate 6.0%
nisin 0.06%
saccharin 0.005%
polysorbate 20 1.0%
glycerol 10.0%
water 82.935%
It is also contemplated that depending on the type of pathogenic microorganism and disease being
treated, the treatment regimen may comprise other drugs and pharmaceutical agents either as part
of the pharmaceutical composition being administered or in treatment regimens which combine both
the low-pH-active bacteriocin composition and another drug effective for treating the gastrointestinal
tract. For example, in the treatment of diarrhea which may be caused by infections of a pathogenic
microorganism such as one of the species of Salmonella, the bacteriocin composition active at low
pH may be administered in a pharmaceutical preparation which also contains kaolin, pectin, or some
other binding agent.Such symptoms may also be treated by the concurrent administration of the
bacteriocin composition active at low pH and the binding agent. In addition, antacid formulations
521/1006
may be used in such treatment regimens and it is not expected that the antacid will affect the activity
of the nisin-chelator composition.
In treating infections of the pathogenic microorganism Helicobacter pylori, the low-pH-active
bacteriocin composition may be administered in connection with another pharmaceutically active
substance against H.
Pylori such as a bismuth salt, e.g., bismuth subcitrate or bismuth subsalicylate. The inventive
compositions may be administered in connection with other agents such as cimetidine, ranitidine,
omeprazole, antacids, urease inhibitors or combinations thereof in order to treat some of the
diseases and symptoms associated with the presence of H. pylori in the gastrointestinal tract. It is
contemplated that in these therapies the active pharmaceutical agents may be administered
concurrently or intermittently with the inventive pharmaceutical compositions and the mode of
administration may be varied during the course of the treatment as required.
H. pylori has been isolated from dental plaque which may constitute a reservoir for recurrent
infection of the stomach (Desa; H.G., Gill, H.H., Shankaran, K., Mehta,
P.R., and Prabha, S.R. (1991) Dental Plaque: a permanent reservoir of Helicobacter pylori? Scand. J.
Gastroenterol. 26: 1205 and Shames, B., Krajden, S.,
Fukasa, M., Babida, C. and Penner, J.L. (1989) Evidence for the Occurrence of the Same Strain of
Campylobacter pylori in the Stomach and Dental Plaque. J. Clin.
Microbiol. 27: 2849.)
It, therefore, is anticipated that bacteriocin compositions suitable for use against H. pylori in dental
plaque may be used in conjunction with the bacteriocin compositions active at low pH against H.
pylori in the gastrointestinal tract. Claims:
We claim:
1. A pharmaceutical composition comprising a lanthionine-containing bacteriocin and a
pharmaceutically acceptable carrier.
522/1006
2. The composition of claim 1 further comprising a chelator.
3. The composition of claim 2 wherein the chelator is citrate.
4. The composition of claim 2 wherein the chelator is EDTA.
5. The composition of claim 2 wherein the chelator comprises citrate and EDTA.
6. The composition of claim 1 or 2, wherein the bacteriocin is selected from the group consisting of
nisin, subtilin, epidermin, Pep 5, ancovenin, gallidermin, duromycin or cinnamycin.
7. The composition of claim 1 wherein the bacteriocin is nisin.
8. The composition of claim 2 wherein the bacteriocin is nisin and the chelator is citrate or EDTA.
9. The composition of claim 1 or 2, further comprising an acidic vehicle.
10. The composition of claim 9, wherein the acidic vehicle is selected from the group consisting of
acetate, propionate, citrate, lactate, succinate, fumarate, malonate, adipate, sorbate, phosphate, or
ascorbate.
11. Use of a composition according to any of claims 1-10 for preventing or treating gastrointestinal
disorders caused by pathogenic microorganisms in the gastrointestinal tract.
12. Use according to claim 11 wherein the disorders are attributable to gram negative bacteria.
13. Use according to claim 11, wherein the microorganism is a species of Helicobacter, Salmonella,
Escherichia, Clostridia, Bacillus, Bacteroides, Campvlobacter or Yersinia.
14. Use according to claim 13 wherein the microorganism is Helicobacter pylori or Campylobacter
ieiuni.
15. Use of a composition according to claim 7 for preventing or treating gastrointestinal bacterial
infection resulting from Helicobacter pylori or Campylobacter eiuni.
523/1006
59. NZ305941 - 27.03.2001
BACTERIOCINS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=NZ305941
Inventor(s):
ROSS REYNOLDS PAUL (IE); HILL COLIN (IE); REA MARY CLARE (IE); RYAN
MARIE PHILIPPA (IE)
Applicant(s):
TEAGASC (US)
IP Class 4 Digits: C12N
IP Class:
C12N15/00
E Class: A23C19/11; A23L3/3463A; C07K14/315; A23C19/032B; A61K7/16F; A61K7/48C26F
Application Number:
US19980945081 (19980413)
Priority Number: IE19950000269 (19950412); WO1996IE00022 (19960412)
Family: WO9632482
Equivalent:
AU5408196; AU712143; EP0821736WO9632482; IE70514; IE950269;
JP11511648T; WO9632482
Abstract:
THE PRESENT INVENTION RELATES TO A NOVEL ANTI-MICROBIAL AGENT, MORE
PARTICULARLY, A NOVEL BACTERIOCIN WITH NISIN-LIKE PROPERTIES. THE BACTERIOCIN IS
DESIGNATED LACTICIN 3147 AND HAS THE FOLLOWING PROPERTIES: A MOLECULAR WEIGHT
OF APPROXIMATELY 2.8 KDA; INHIBITING ACTIVITY AGAINST LACTOCOCCI, LACTOBACILLI,
ENTEROCOCCI, BACILLI, LEUCONOSTOCS, PEDIOCOCCI, CLOSTRIDIA, STAPHYLOCOCCI AND
STREPTOCOCCI; SENSITIVITY TO THE PROTEASES TRYPSIN, ALPHA-CHYMOTRYPSIN,
PROTEINASE K AND PRONASE E BUT NOT PEPSIN; HEAT-STABILITY; ACTIVITY AT ACID PH; AND
THE CAPABILITY OF INHIBITING NISIN-PRODUCING BACTERIAL STRAINS.Description:
524/1006
This application claims priority to PCT/IE96/00022, filed Apr. 12, 1996 and Irish patent application
S950269, filed Apr. 12, 1995.
The present invention relates to a novel anti-microbial agent, more particularly, a novel bacteriocin
with nisin-like properties.
Recent years have seen major advances in the development of microbial metabolites with
antagonistic activities towards spoilage and pathogenic micro-organisms associated with food.
Although, there now exists in excess of seven thousand known antibiotic compounds of microbial
origin, very few have been evaluated for food use. The danger exists that antibiotic-resistant
microorganisms of clinical importance will appear with repeated exposure to antibiotics in food, thus
compromising the clinical usefulness of the antibiotics (although, of course, clinically important
antibiotics are not used for food applications). Additionally, consumer emphasis is now on minimally
processed foods which are natural and preservative free. Because of this considerable and
justifiable resistance to the use of chemical additives and antibiotics as food preservatives, other
biological inhibitors produced by microorganisms are currently being investigated for use in foods.
Of particular interest are those inhibitory substances produced by the Lactic Acid Bacteria (LAB)
which include hydrogen peroxide, diacetyl, bacteriocins, as well as secondary reaction products
such as hypothiocyanite. Bacteriocins are potentially very attractive natural preservatives as they are
produced as normal by-products of microbial metabolism. The LAB are industrially important
microorganisms and include the genera Lactococcus, Streptococcus, Pediococcus, Leuconostoc,
Lactobacillus and Carnobacterium. They have been used for the production of fermented foods
which have been consumed safely for hundreds of years. Given their status as "safe" organisms, they
are a particularly suitable source for natural antimicrobials such as bacteriocins for use in foods.
In 1925, the first prototype bacteriocin was discovered by Gratia et. al. from a strain of Escherchia
coli. Bacteriocins are always proteins which can be broad or narrow spectrum and are not lethal to
the cells which produce them. Bacteria protect themselves from the lethal effects of their own
bacteriocins by such mechanisms as post-translational modification or the production of an immunity
protein(s). In any case, bacteriocins are potent antibacterial substances produced by a large and
diverse assortment of species. They form a heterogeneous group with respect to their producer,
inhibitory spectrum, mode of action and chemical properties. There are four distinct classes of LAB
bacteriocins:
525/1006
A. Lantibiotics--small peptides of less than 5 kDa which contain unusual amino acids such as
lanthionine, .beta.-methyllanthionine and dehydrated residue, e.g. nisin, lacticin 481, carnocin U149
and lactocin S.
B. Non-Lanthionine containing Peptides--small peptides of 10 kDa or less and can be subdivided as
follows: (i) Listeria-active peptides e.g. Pediocin PA-1 and Sakacin A. (ii) Poration complexes
consisting of two proteinaceous peptides e.g. Lactacin F. (iii) Thiol-activated peptides requiring
reduced cysteine residues for activity e.g. Lactococcin B.
C. Large Heat-Labile Proteins--larger proteins, generally having a molecular weight greater than 31
kDa e.g. Helveticin V-1829.
D. Complex Bacteriocins--composed of a protein with one or more chemical moieties which may be
of a lipid or carbohydrate nature e.g. Pediocin SJ-1.
Lactococci are widely used as starter cultures in the dairy industry and several strains of dairy
species can produce bacteriocins. L. lactis subspecies can produce diplococcin, lactococcin,
lactostrepcins or nisin. Diplococcin and lactococcins are small molecular weight proteins, active
against other lactococci while nisin is a lantibiotic with a broad spectrum of activity against many
Gram positive bacteria.
Nisin is the most extensively characterised bacteriocin of the antimicrobial proteins produced by
LAB and has found widespread application in the food industry. Nisin was the first "antibiotic"
compound to be used on a commercial scale in the food industry. It is used to prevent spore
outgrowth and toxin production by Clostridium botulinum in processed cheese and cheese spreads.
In some countries, it has been used to extend the shelf-life of dairy products and to prevent the
spoilage of canned foods by thermophiles.
Nisin is a pentacyclic, subtype A lantibiotic and it displays an amphiphathic character, with a
hydrophobic residue (Ile) at its N-terminus and a hydrophilic residue (Lys) at its C-terminus. It is a
peptide of 34 amino acids and is inactivated by proteases including chymotrypsin, pancreatin and
subtilopeptidase. It is insensitive to carboxypeptidase A, elastase, erepsin, pepsin and trypsin. It
contains one lanthionine residue, four .beta.-methyllanthionines, a dehydroalanine and a
dehydrobutyrine. The thioether amino acids, (lanthionine and .beta.-methyllanthionine) account for
the high sulphur content of nisin. The usual amino acid residues are thought to be responsible for the
important functional properties of nisin e.g. the associated acid tolerance and thermostable
properties of nisin are attributed to the stable thioether linkages while the specific bactericidal activity
526/1006
is thought to be due to the very reactive double bonds. Nisin has a molecular mass of 3.5 kDa (Gross
& Morell, 1971) and often forms dimers and oligomers.
Nisin has a very broad spectrum of activity against Gram positive vegetative bacterial cells. The
closely related lactococci are especially sensitive but nisin is also inhibitory to several strains of
bacilli and clostridia (particularly their spores), lactobacilli, leuconostocs, micrococci, pediococci,
streptococci and actinonycetes. Other sensitive strains include Mycobacterium tuberculosis,
Staphylococcus pyogenes, S. aureus, S. epidermidis and Listeria monocytogenes (de Vuyst &
Vandamme, 1994). Nisin does not display activity against Gram negative bacteria, except for three
Neisseria strains (Mattick & Hirsch, 1947) and three Flavobacter strains (Ogden & Tubb, 1985), nor
does it inhibit yeasts or viruses. However, Salmonella subspecies and other Gram negative bacteria
can be made sensitive by using a chelating agent in combination with nisin (Stevens et. al. 1992).
The site of action of nisin appears to be the cytoplasmic membrane. The outer membrane of Gram
negative bacteria is thought to exclude the bacteriocin making contact with the cytoplasmic
membrane. Hence, by incorporating a chelating agent such as EDTA with nisin, the structure of the
outer membrane undergoes alteration, resulting in destabilization of the lipopolysaccharide (LPS)
layer with a corresponding increase in cell permeability. Binding of the bacteriocin to the membrane
leads to the aggregation of similar peptides, thus initiating oligomerization. Such aggregates adopt a
transmembrane orientation so that the hydrophobic portion is exposed to the core of the membrane
and the hydrophilic part forms the aqueous channel. Membrane insertion, pore formation, (both of
which require a transmembrane potential) and subsequent depolarization leads to the efflux of small
cellular constituents and destruction of energy metabolism of the cell (Ruhr & Sahl, 1985). This
results in a deficiency of metabolic intermediates and hence, inhibition of synthesis of DNA, RNA,
proteins and polysaccharides. There appears to be a separate mechanism for the prevention of
spore outgrowth. Unlike vegetative cells, bacterial spores never lyse when treated with nisin. The
dehydro residues of nisin provide possible covalent attachment sites for membrane sulfhydryl groups.
These residues appear to have no such role in membrane pore formation (Morris et. al. 1984).
Nisin is encoded by a conjugative transposon which can insert into either plasmid DNA or
chromosomal DNA (Horn et. al., 1991). Analysis of the nisin operon indicates that the nisin structural
gene, as well as the genes required for processing and maturation are clustered over a polycistronic
region exceeding 8.5 kb (Steen et. al., 1991).
Nisin has certain disadvantages for industrial application, one being that resistance to the
bacteriocin can frequently occur. For example there is a nisin resistance gene (as opposed to an
527/1006
immunity gene) on the conjugative lactococcal plasmid pNP40. Its second disadvantage is that nisinproducing strains are generally not good acid producers, are phage sensitive and are nonproteolytic and therefore are not efficient cheese starter cultures.
It is an object of the present invention to provide a bacteriocin suitable for food use, particularly one
which has a broad spectrum of inhibition and for which there is no detectable spontaneous
resistance thereto. A further object is to provide bacteriocin encoding gene(s) which may be more
easily conjugally mobilized that the nisin-encoding gene.
It is also an object to provide bacteriocin encoding gene(s) which may be easily conjugally
mobilised along with genes which may be attached to them. It is a further object to provide
bacteriocin-encoding gene(s) which are not linked to lactose catabolism genes. There is a further
object to provide bacteriocin-producing strains which are effective cheese starters. A further object
of the invention is to provide phage resistance genes, particularly such genes linked to bacteriocinencoding genes.
According to the present invention there is provided a bacteriocin characterised by a molecular
weight of approximately 2.8 kDa, inhibiting activity against lactococci, lactobacilli, enterococci, bacilli,
leuconostocs, pediococci, clostridia, staphylococci and streptococci, sensitivity to the proteases
trypsin, alpha-chymotrypsin, proteinase K and pronase E but not pepsin, heat-stability, activity at
acid pH, and the capability of inhibiting nisin-producing bacterial strains.
The bacteriocin is designated lacticin 3147. The bacteriocin lacticin 3147-encoding gene does not
cross-hybridise with the nisin-encoding gene.
The invention also relates to L. lactis DPC3147 strain as deposited at the National Collection of
Industrial and Marine Bacteria, Aberdeen, Scotland, on Apr. 11, 1995 under the Accession No.
NCIMB 40716. This strain produces the bacteriocin lacticin 3147.
The present invention also provides a 63 kDa plasmid encoding the bacteriocin as defined above.
The plasmid may be the plasmid pMRC01 which encodes the novel bacteriocin designated lacticin
3147 and lacticin 3147 immunity genes, as deposited in the National Collection of Industrial and
Marine Bacteria, Aberdeen, Scotland on Apr. 11, 1995 under the Accession Number NCIMB 40716.
528/1006
Furthermore, the invention provides isolated genes for the production of, and immunity to, lacticin
3147 as deposited in the plasmid pMRCO1 above.
The present invention also relates to the use of lacticin 3147 or a lacticin 3147 producing host in the
treatment of mastitis in cattle, to prevent clostridial spoilage in cheese, as a food preservative in
pasteurised cheeses and cheese spreads, as a shelf-life extender in milk and milk products, in the
production of alcoholic beverages particularly in the brewing industry, in vegetable fermentations, by
incorporation into canned foods and in meat preservation. Additional uses include incorporation into
oral healthcare products such as toothpaste and mouthwashes and in cosmetic treatments for acne.
The bacteriocin may be used against Gram negative bacteria which have been treated with chelating
agents.
The invention also relates to the use of a plasmid or a gene(s) encoding the lacticin 3147 bacteriocin
to confer bacteriocin producing properties on a host such as a bacterium, particularly a cheesestarter culture. The invention also provides a host such as a bacterium containing a plasmid or a
gene(s) as defined above, encoding lacticin 3147. The invention also provides a method of
producing lacticin 3147 comprising culturing a bacterium containing lacticin 3147-encoding gene(s)
and isolating lacticin 3147 from the culture, and a method of conferring lacticin 3147-producing
properties on a host such as a bacterium, comprising introducing and expressing in the host a
plasmid or gene(s) as defined above encoding lacticin 3147.
The invention also relates to a food-grade genetic marker system comprising genes for immunity to
lacticin 3147 as encoded by plasmid pMRC01. The genetic determinants which encode lacticin 3147
immunity may be introduced into a bacterial strain together with any desirable gene(s) which have
been linked to them. A bacterial cell which has received the genes can be selected from the general
population of cells by plating on medium containing lacticin 3147, since cells containing the lacticin
3147 immunity genes will be able to grow in this environment. Given that spontaneous resistance to
lacticin 3147 occurs at a low frequency in lactococcal strains (undetectable for some strains) this
marker system should provide all the advantages of well known antibiotics such as Ampicillin and
Erythromycin with none of the negative clinical associations. Since these 3147 genes have originated
from a GRAS (Generally Regarded As Safe) organism they are considered to be Food Grade.
The invention also provides phage resistance gene(s), particularly gene(s) encoding total resistance
to the small isometric-headed phage 712 and burst size limitation for the prolate-headed phage c2.
529/1006
The phage resistance gene(s) may be encoded by the plasmid pMRC01, deposited as described
above.
Further the invention relates to a host such as a bacterium containing a plasmid or gene(s) as
defined above encoding phage resistance. Also provided is a method of conferring phage
resistance on a host such as a bacterium and particularly a cheese starter culture, comprising
introducing and expressing therein a plasmid or gene(s) as defined above encoding phage
resistance. The invention also relates to the use of a plasmid or phage resistance gene(s) to confer
phage resistance on a host such as a bacterium, particularly a cheese-starter culture.
In a further aspect the invention provides L. lactis strain DPC2949 as deposited at the National
Collection of Industrial and Marine Bacteria, Aberdeen, Scotland on Apr. 11, 1995 under the
Accession No. NCIMB 40715, the bacterium-encoding gene(s) thereof and the bacteriocin produced
thereby. The invention also relates to methods of preventing late gas-blowing or of controlling nonstarter lactic acid bacteria in Cheddar cheese production comprising either the addition of L. lactis
DPC2949 or the bacteriocin produced thereby to the initial cheese starter culture.
The invention also provides bacteriocin-encoding genes, bacteriocin immunity genes, phage
resistance genes, plasmids and strains substantially homologous to those defined above also
encoding bacteriocin production and immunity and phage resistance. Such homologous genes,
plasmids and strains arise because of the degeneracy of the genetic code, the possibility of
replacing one amino-acid with another without affecting the functional characteristics of a protein and
the possibility of deleting a non-essential portion of a gene or amino-acid sequence while still
producing a functional end-product. The invention also provides genes as defined above linked to
other DNA sequences.
Since the biological activity of the bacteriocin of the present invention is similar to that of nisin, it
could be used for similar applications. Unlike Northern Europe, many fermented dairy products in
Southern Europe are still being made using traditional methods without the use of commercial
starters. One source of these strains in Ireland is Buttermilk plants commonly used by Irish
housewives in the souring of bread for breadmaking. These Buttermilk plants, also known as kefir
grains, are creamy white in colour and resemble cauliflower florets. They are resilient and difficult to
break up due to the presence of kefir in a water soluble polysaccharide produced by the lactobacilli
in the plant. They are composed of lactococci, leuconostocs, lactobacilli, yeasts and acetic acid
bacteria which are held together in a matrix and are recoverable at the end of the fermentation
530/1006
process as a solid mass. Some isolates produce a bacteriocin which may be important in
maintaining the integrity of the grain as it is assumed that all other organisms in the grain are
resistant to it (Rea & Cogan, 1994).
The present invention will now be described in greater detail with reference to the accompanying
drawing in which:
FIGS. 1A and 1B. Cross-Sensitivity Study, a L. lactis DPC3147; b, NCDO496; c. DPC2949; d.
DPC3220; e. DPC33(1) were grown on GM17 agar plates and subsequently overlaid with A.
DPC3147 and B. NCDO496. Zones where no growth has occurred indicate inhibition of the indicator
due to bacteriocin(s) produced by the producer.
FIGS. 2A, 2B, 2C, 2D, and 2E. Protease Sensitivity Assay. Filtered cell-free bacteriocin solution and
enzyme solution (A represents control, no enzyme; B, alpha-chymotrypsin; C, trypsin; D, pronase E
and E represents proteinase K) were spotted 1 cm apart on GM17 agar plates and subsequently
overlaid with the indicator L. lactis strain HP.
FIG. 3. Polymerase Chain Reaction. The 166 bp PCR amplified fragment from genomic DNA isolated
from L. lactis NCDO496 is indicated in lane 1. No amplified products are evident where DNA isolated
from strains DPC3147, MG1614, and DPC2949 were used as templates. Lane 3 corresponds to DNA
molecular weight markers.
FIG. 4. DNA Hybridization. Hind III restricted genome DNA from L. lactis DPC3147, MG1614, and
NCDO496 is shown in lanes 1, 2 and 3 respectively. Lane 4 corresponds to DNA molecular weight
markers. The nis A gene probe, hybridized to a 3.5 kb fragment on the NCDO496 genome (lane 3').
No hybridizing DNA is observed in DNA isolated from DPC3147 (lane 1) or from MG1614 (lane 2).
FIG. 5. Growth of L. lactis DPC3147 in GM17 and TYT30 media.
FIG. 6. Effect of heating at 60 DEG C. to 121 DEG C. for 10 minutes on the stability of lacticin 3147 at
pH5, pH7, pH9. 100% activity is the activity at pH 5, 7 and 9 with no heating.
FIG. 7. Overlaid SDS-PAGE gel. Preparations of lacticin 3147 (A. lanes 2, 3 and 4; B. lane 2) and of
lactococcin A (B. lane 3) were electrophoresed on a 10% SDS-PAGE gel. This was overlaid with an
531/1006
active culture of L. lactis HP. Inhibition of the indicator is seen as zones in the indicator lawn.
Molecular weight markers (2.5 kDa to 17 kDa) are indicated in A. lane 1.
FIG. 8. Analysis of the plasmid complements of a putative transconjugant (lane 2) obtained from a
mating between L. lactis DPC3147 (lane 1) and the plasmid-free strain MG1614. The transconjugant
contain a 63 kDa plasmid acquired from the DPC3147 donor which is not evident in the plasmid-free
MG1614 strain.
FIG. 9. pH profiles during Cheddar cheese manufacture using the DPC3147 strain as cheese starter.
FIG. 10. Growth of starter and NSLAB during ripening of Cheddar cheese using the DPC3147 strain
as cheese starter.
FIG. 11. pH profiles during Cheddar cheese manufacture using a transconjugant of strain 303 which
produces lacticin 3147 as cheese starter.
FIG. 12. Growth of starter and NSLAB during ripening of Cheddar cheese using a transconjugant of
strain 303 which produces lacticin 3147 as cheese starter.
FIG. 13A. Residual Lacticin 3147 activities in Cheddar cheese made with bacteriocin-producing
strains (DPC3147, DPC3256 and DPC3204) sampled after 2 weeks, 4 months and 6 months. The
commercial strain DPC4268 was used as the control starter.
FIG. 13B. Residual lacticin 3147 activity in Cheddar cheese made with the bacteriocin-producing
transconjugant DPC4275. Again, DPC4268 was used as the control.
FIG. 14A. Antimicrobial effectiveness of lacticin 3147 requires the presence of Tween Au/ml) has
been added.
FIG. 14B. Effect of adding lacticin 3147 (10.240 AU/ml) to stationary cells of Streptococcus
dysgalactiae.
MATERIALS AND METHODS
532/1006
A complete list of strains, their growth media and temperature of incubation used throughout this
study is included in Table 1. Both L. lactis DPC3147 and DPC3220 were isolated from kefir grains,
whereas L. lactis strains NCD0496 and NCD0497, known nisin producers, were obtained from the
National Collection of Dairy Organisms (NCDO), National Institute for Research and Dairying,
Shinfield, Reading, Berkshire, RG2 9AT, England. The source of each of the indicator organisms
tested are also listed in Table 1. L. lactis cells were routinely propagated at 30 DEG C. in M17 (Difco
Laboratories, Detroit, USA) supplemented with 0.5% glucose or lactose. Other media used in this
study, as indicated in Table 1, included MRS (Difco Laboratories), BHI (Oxoid Ltd., Hampshire,
England), TYP (Tryptone 16 g/l, Yeast Extract 16 g/l, NaCl 2.5 g/l, K2 HPO4 2.5 g/l). TYT30 (Tryptone
2.5 g/l, Yeast Extract 5 g/l, Tween 20 l g/l, Glucose 10 g/l, .beta.-glycerophosphate 19 g/l. MgSO4
7H2 O, 0.25 g/l, MnSO4 4H2 O 0.05 g/l), TSA, (Becton Dickson Microbiology Systems, Maryland,
USA), Baird-Parker media (Merck, Darmstadt, Germany); RSM (Reconstituted Skim Milk) and
pasteurized whole milk.
To test the sensitivity of a strain to DPC3147, to L. lactis NCD0496 and to L. lactis DPC3220, 10 .mu.l
aliquots of a fresh overnight culture of each were first spotted on GM17 agar plates and incubated
overnight at 30 DEG C. These plates were then gently overlaid with 3 ml of soft agar seeded with
100 .mu.l of the test strain, so as not to disturb the grown producer. The sensitivity of a strain to each
bacteriocin producer was scored according to the diameter of the zone of inhibition surrounding that
producer. The scoring system adopted is as follows: 0 to 1 (mm) (-); 1 to 5 mm (+); 5 to 15 mm (++);
over 15 mm (+++). All strains were stocked in 40% glycerol and stored at -80 DEG C. Working
cultures were stored at 5 DEG C. and transferred periodically.
Plates containing bacteriocin were prepared as follows. A cell-free, sterile bacteriocin solution was
obtained from an overnight culture of L. lactis DPC3147 grown in GM17 by first centrifuging at 8000
g (Sorvall RC-5B) for 10 min. The resulting supernatant was then sterilized by filtering with Millipore
HVLP filters (0.45 um) and tempered to 45 DEG C. An equal volume was then added to double
strength GM17 and the plates were poured. Where needed, streptomycin was added to agar at a
concentration of 500 .mu.g/ml.
To identify lactose metabolizing bacteria, Lactose Indicator Agar (LIA) was used (Tryptone 20 g/l,
Yeast Extract 5 g/l, Lactose 10 g/l, NaCl 4 g/l, Sodium Acetate-anhydrous 1.5 g/l, Ascorbic Acid 0.5
g/l, Gelatin 2.5 g/l, Bromocresol Purple (0.004%). Agar Bacteriological 15 g/l. Sucrose Indicator Agar
plates were prepared similarly except that sucrose was substituted for lactose as the carbon source.
533/1006
Measurement of Bacteriocin Activity:
Bacteriocin activity was estimated using an agar well-diffusion assay essentially as described in
Parente and Hill (1992). In the agar well diffusion assay, molten agar (GM17 for lactococci) was
cooled to 48 DEG C. and inoculated with overnight cultures of the appropriate indicator strain
(200 .mu.l in 25 ml agar). The inoculated medium was rapidly dispensed in sterile Petri-dishes and,
after solidification, dried for 30 min under a laminar flow hood.
Wells of a uniform diameter were bored in the agar and sealed with 15 .mu.l of tempered soft agar.
Sterile culture supernatant fluids (50 .mu.l) were then dispensed in the wells and the plates were
incubated overnight at 30 DEG C. These cell-free solutions of the bacteriocin were obtained as
above. The difference between the area of the zone of inhibition (in mm@2) and the area of the well
was measured to estimate bacteriocin activity.
Protease Sensitivity Assays:
The following enzymes were dissolved in sterile distilled H2 O (SDW) to a final concentration of 50
mg/ml: Trypsin (type II, Sigma Chemical Co., Poole, Dorset, England), alpha-chymotrypsin (type II,
Sigma), Proteinase K (Sigma), Pronase E (type XIV, Sigma), Catalase (Sigma). Pepsin was dissolved
in 0.02N HCl, also to a final concentration of 50 mg/ml. All enzyme solutions were filter sterilised
using disposable filters (Rotrand/Red rim 0.2 .mu.m, Schleicher & Schuell). 20 .mu.l aliquots of
filtered cell-free bacteriocin solution and 20 .mu.l of each enzyme solution were spotted 1 cm apart
on GM17 agar plates and dried for 30 min. All plates were incubated overnight at 30 DEG C. The
plates move subsequently overlaid with the indicator organism. Growth of the producer was evident
as a zone of inhibition. Controls included plates with spots of either bacteriocin solution or enzyme
solution.
Effect of pH and temperature on bacteriocin stability:
A cell-free bacteriocin was exposed to various pH/temperature treatments. Samples were heated to
60, 70, 80, 90, 100, 110 and 121 DEG C. at pH5, pH7 and pH9 for 10 min. Following treatment, all
solutions were rapidly cooled and bacteriocin activity was assayed by the well diffusion method.
Estimation of Molecular Weight:
534/1006
Samples containing 3147 bacteriocin were loaded on urea-SDS polyacrylamide gels, prepared as
described by Swank and Munkres (1971). Molecular weight markers for peptides ranging from 2.5 to
17 kDa (Sigma) were used as a standard. A sample of lactococcin was also included on the gel as
another indicator of molecular weight. After electrophoresis at 22 mA for 16 h the gels were divided.
One half containing the sample and molecular weight markers was stained according to procedures
as recommended by the manufacturers (Hoeffer Scientific Instruments). This essentially involved
staining overnight with 0.125% Coomassie Brilliant Blue R250 stain. The following day, the gel was
destained with "Destain Solution 1" [Methanol 50% (v/v), Acetic Acid 10% (v/v)] for 5 h and then
placed in "Destain Solution 2" [Methanol 5% (v/v), Acetic Acid 7% (v/v)]. The other half of the gel was
fixed immediately for 2 h in a solution of 20% (v/v) isopropanol and 10% (v/v) acetic acid. It was then
washed in several volumes of deionised water for 4 to 6 h. The gel was placed in a large sterile glass
Petri-dish and overlaid with 30 ml of soft tempered agar seeded with 800 .mu.l indicator strain. The
plate was incubated overnight at 30 DEG C. and examined for a zone of inhibition (Bhunia et. al.,
1987)
Isolation of DNA:
Plasmid DNA from lactococci was isolated by the Anderson & McKay method (1983) as follows.
Actively growing lactococci cells were collected as with the previous method by centrifugation for 5
sec. The pelleted cells were then resuspended in 379 .mu.l of solution containing 6.7% sucrose, 50
mM Tris, 1 mM EDTA (pH8) and heated to 37 DEG C. Lysozyme (Sigma), dissolved in 25 mM Tris
pH8 was added (96.5 .mu.l), and incubated at 37 DEG C. for 5 min. 48.2 .mu.l 0.25 M EDTA. 50 mM
Tris pH8 was then mixed in by gentle vortexing, followed by addition of 27.6 .mu.l of the lysis solution
(20% SDS, 50 mM Tris, 20 mM EDTA pH8). The resulting mixture was then incubated for 5 to 10 min
at 37 DEG C. to complete lysis after which it was completely clear. The lysate was rigorously
vortexed for 30 sec and 3 N NaOH (27.6 .mu.l) added. This was mixed in gently by inversion for 10
min. after which, 49.6 .mu.l of 2 M Tris pH7 was added. This was mixed for a further 3 min by
inversion. 71.7 .mu.l of 5 M NaCl was then added and the resulting mixture vortexed. This was
extracted once with phenol and once with chloroform isoamyl alcohol (24:1). The DNA was then
precipitated on addition of 600 .mu.l isopropanol. Following centrifugation for 10 min. the precipitated
DNA was washed once in 70% ethanol, dried and resuspended in 30 .mu.l SDW, all of which was
then loaded on the gel.
Genomic DNA was extracted from lactococci according to a modification of the method outlined by
Hoffman & Winston (1987) which uses shearing with glass beads to lyse the cells. The strains from
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which DNA was extracted were grown overnight in 5 ml volumes of the appropriate medium. The
cells were collected by centrifuging 2 ml volumes of the cultures for 5 sec. The supernatant was
discarded and the remaining pellet was vortexed briefly. The cells were resuspended in 0.2 ml of
sterile "Extraction Solution" which consists of 2% Triton X 100, 1% SDS, 100 mM NaCl, 10 mM Tris
(pH8) and 1 mM EDTA. Phenol-chloroform (0.2 ml) was then added, followed by 0.3 g of acidwashed glass beads of diameter 0.45 to 0.52 mm (Sigma). The suspension was vigorously vortexed
for 2 min and then microcentrifuged for 5 min. The resulting upper aqueous phase was gently
transferred into a sterile micro-centrifuge tube to which 20 .mu.l of 3 M sodium acetate was added.
After a gentle vortex, 600 .mu.l absolute ethanol (-20 DEG C.) was added and the mixed suspension
centrifuged for 10 min. Then the pellet was washed in 70% alcohol, dried and finally resuspended in
50 .mu.l SDW. To estimate DNA concentration, a 5 .mu.l sample was electrophoresed on a 0.7%
agarose gel with ethidium bromide staining. Alternatively, lactococcal genomic DNA was isolated by
a modification of the Anderson & McKay procedure. This was performed identically to the method
outlined above except that the alkaline denaturation step was omitted.
Polymerase Chain Reaction:
Amplification of lactococcal DNA was performed by the following method using a Perkin Elmer DNA
Thermal Cycler. PCR reactions of 100 .mu.l were set up which contained one-tenth volume 10.times.
buffer (Bioline), 5 mM MgCl2, 200 .mu.M of each of the dNTPs. 1 .mu.M of primer(s) and 1.25 units of
Taq DNA polymerase. After overlaying each tube with 100 .mu.l sterilised paraffin oil. 1 .mu.l DNA
(isolated as previously described) was added to the reaction. The Taq enzyme was added during the
first temperature cycle ("Hot Start") and the DNA was amplified for 35 cycles. Each cycle involved 1
min denaturation at 93 DEG C. followed by an annealing step at 55 DEG C. for 1 min and an
extension step of 72 DEG C. for 1 min. Of the final reaction mixture, 10% was analyzed on 1.8% (w/v)
agarose (Sigma) gels with ethidium bromide staining.
Restriction of DNA
The isolated genomic DNA from the three L. lactis strains. DPC3147 (lacticin 3147 producer),
MG1614 (Bac@-) and NCDO496 (nisin producer) were restricted with Hind III enzyme. Restrictions
were carried out in 10.times. "Cuts-All" buffer containing 200 mM Tris-HCl (pH7.5), 70 mM MgCl2, 1
M KCl and 20 mM .beta.-mercaptoethanol and the entire reaction mix was incubated overnight at 37
DEG C. The DNA samples were run on a 1.8% (w/v) agarose gel at 25V overnight. The DNA
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fingerprint thus obtained, was examined under ultra-violet light to ensure that sufficient DNA had
been restricted before proceeding to the next step.
Southern Blotting
After electrophoresis, the restricted DNA was transferred to a Hybond N@+ nylon membrane by
capillary blotting. The procedure used was that described by Maniatis et al. (1989). Initially, the gel
was soaked for 10 min in several volumes of 0.2 N HCl and rinsed briefly in deionised water. The
DNA was then denatured by soaking the gel for 45 min in several volumes of 1.5 M NaCl, 0.5 N
NaOH with constant gentle agitation. Again, the gel was rinsed in deionised water. To neutralise the
gel, it was soaked twice in 1 M Tris (pH7.4), 1.5 M NaCl for 30 min with gentle agitation. After
neutralisation, the DNA was transferred to a Hybond N@+ nylon membrane by capillary action. The
membrane, which had been previously immersed in 10.times. SSC transfer buffer (NaCl 87.65 g/l,
sodium citrate pH7.0 44.1 g/l) for 5 min was placed directly over the gel and transfer was allowed
proceed for 24 h. After this time, the membrane was removed, dried and baked for 2 h at 80 DEG C.
Hybridization:
Hybridizations were performed according to the "Enhanced Chemiluminescence" ECL Gene
Detection System in a Techne Hybridiser HB-1. All hybridizations were carried out at 42 DEG C. as
suggested by the manufacturer. The nylon membrane containing the transferred DNA was placed in
hybridization buffer (supplied with the ECL kit) at 42 DEG C. and a pre-hybridization was carried out
for 15 min. The labelled DNA probe was then added and incubated was allowed to proceed
overnight at 42 DEG C. The membrane was then removed from the hybridization solution and
washed twice at 42 DEG C. for 20 min with primary wash buffer which consists of Urea 36% (w/v),
SDS 0.4% (w/v) and 20.times. SSC 2.5% (v/v). It was then washed for 5 min at room temperature in
secondary wash buffer i.e. 20.times. SSC 10% (v/v).
Amplified DNA (20 .mu.l) from L. lactis NCD0496 was run on a 0.6% (w/v) low melting point Sea
Plaque Agarose (FMC) gel. The 166 bp nisin probe DNA amplified from NCD0496 DNA was carefully
cut from the gel. Labelling this DNA was achieved as follows. 20 .mu.l DNA labelling reagent was
added to an equivalent volume of cooled to 37 DEG C. denatured DNA and mixed thoroughly.
20 .mu.l glutaraldehyde solution was then added and the mixture was incubated for 10 min at 37
DEG C. Labelling reagent and glutaraldehyde solutions were both supplied with the ECL kit.
Detection of hybridization signals was performed according to the manufacturers recommendations.
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Conjugation:
Conjugations were performed as follows. A conjugal mating was set up using L. lactis DPC3147
(bac@+, bac', strep@3) as the donor strain and the plasmid free strain MG1614 (bac@-, bac@3.
strep') as the recipient. Both strains were grown to mid-log phase (OD600nm 0.5 to 1). The DPC3147
strain was cultivated in GM17 containing pronase E (50 mg/ml), while MG1614 was grown in GM17
supplemented with streptomycin (500 .mu.g/ml). 1 ml aliquots of these cultures were then
centrifuged in a microfuge for 30 sec and the resultant pellets were washed once in 1 ml volumes of
GM17. The pellets obtained were resuspended in 25 .mu.l GM17, mixed and spotted on a nonselective GM17 agar plate. Donor and recipient controls were prepared in a similar manner.
Following overnight incubation at 30 DEG C. the cultures were resuspended in GM17 broth
supplemented with 40% glycerol for long-term storage at -80 DEG C. A serial dilution was carried out
on an aliquot of the mating mix which was then plated on selective media. The conjugation frequency
was estimated as the number of transconjugants (appearing on selection plates) divided by the
number of donor cells. Putative transconjugants were checked for lactose metabolizing activity by
streaking on LIA plates. To determine the molecular weight of the plasmid encoding bacteriocin
production, plasmid DNA from the transconjugants was isolated by the Anderson and McKay
method and was run on a 0.7% agarose gel. L. lactis MG1614 was used as a negative control and
the plasmid profile of L. lactis subsp. lactis DRC3 was used as the molecular weight standard.
Preparation of Inocula for Cheese-making Trials:
The selected strains were stored at -80 DEG C., and were sub-cultured once in LM17 broth. At 30
DEG C. overnight and twice in RSM before use. Bulk starters were cultivated in 10% RSM which had
been pre-heated to 90 DEG C. for 30 min and cooled to 21 DEG C. before inoculation. All the strains
were grown separately at 21 DEG C. for 16 h, mixed in equal proportions and inoculated at the levels
shown in Table 2.
Cheesemaking:
Milk was pasteurised (72 DEG C..times.15 s) and cooled to 30 DEG C. Cheese was made in circular
jacketed stainless steel 500 l vats. The milk was inoculated with cultures at the levels summarized in
Table 1. Filter-sterilised rennet (Chr. Hansens Laboratories: 31 ml, diluted in 500 ml sterile distilled
water) was added 30 min after addition of the starter and the coagulum was cut approximately 40
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min later. The curds and whey were cooked to 38 DEG C. and pitched at pH 6.2. The cheddared
curd was milled at approximately pH5.2, salted at a level of 27 g/kg and pressed in 18 kg moulds
overnight at approximately 412 kPa. Cheese were vacuum packed and ripened for 12 months at 8
DEG C.
Analysis of Cheese:
Bacteriological analysis: At intervals, cheese were aseptically sampled. Starter cells were
enumerated on LM17 agar for 3 days after incubation at 30 DEG C. lactobacilli on LBS agar after 5
days at 30 DEG C. enterococci on Kanamycin aesculin agar (Oxoid) after 24 h at 37 DEG C. and
coliform on VRB agar after 24 h at 30 DEG C. Microbiological analyses were single estimations at
each sampling time.
Gross composition:
Grated cheese samples (2 weeks old) were analysed for salt, fat, protein and moisture. The pH was
measured on a paste prepared by macerating 10 g of grated cheese in 10 ml of distilled water. All
values are the average of duplicate analyses.
Proteolysis:
Proteolysis of cheese was monitored by measuring the percentage of total nitrogen soluble in water
at pH 4.6 (WSN) or in 5% phosphotungstic acid (PTA-N).
Free amino acids:
Free amino acids were measured in the 12% TCA-soluble fraction of the WSN on a Beckman 6300
Amino Acid Analyser.
Sensory Evaluation:
Cheeses were assessed for flavour at 3, 6, 9 and 12 months by a sensory evaluation panel based on
a score of 0-8 (0-1 unacceptable; 2-3 poor; 4-5 acceptable; 6-7 good; 8 excellent).
Assaying bacteriocin directly in cheese samples:
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Bacteriocin activity was assayed from cheese samples as follows. Cheese samples were initially
macerated in equal volumes of distilled water in a stomacher (Lab-Blender 400) for 15 min and
heated to 80 DEG C. for 10 min. Then aliquots of 50 .mu.l were dispensed in wells and bacteriocin
activity calculated as the difference in area of the zone of inhibition (in mm2) and the area of the well
(as outlined previously).
Transfer of pMRC01 into lactococcal starter strains:
Initially a conjugation was set up with L. lactis DPC3147 (Lac+, Bac- and Streps) as the donor strain
and the plasmid-free antibiotic sensitive L. lactis MG1363 as the recipient. Both strains were grown
from an overnight culture for 4 hr at 30 DEG C. in L/GM17. The conjugation was carried out in a ratio
of 20:1 of recipient to donor. 1 ml of recipient (MG1363) and 50 .mu.l of donor (DPC3147) were
centrifuged. The donor (Bac+) was washed with LM17 broth and resuspended in 50 .mu.l of LM17.
The donor, recipient and mating mix were spotted onto non-selective GM17 agar plates and allowed
to dry. Following an overnight incubation at 30 DEG C. the cultures were harvested from the agar
plates and resuspended in 500 .mu.l of GM17 supplemented with 40% glycerol (for long-term
storage at -80 DEG C.). A serial dilution of an aliquot of the mating mix was plated onto selective
media (LIA containing lacticin 3147). The conjugation frequency was estimated by dividing the
number of transconjugants appearing on the selection plates by the number of donor cells.
Transconjugants were not readily visible in this case because the donor Lac+ colonies turn the LIA
plate yellow and the Lac- colonies are masked. To overcome this problem colonies were picked off
at random and spotted onto LIA and observed for lactose utilisation. The transconjugant resulting
from this mating was used as a Food Grade donor on further matings with the commercial
lactococcal starters. These matings were carried out similarly to above, but at a ratio if 1:1 (with the
exception of strain AM2). In these matings transconjugants were observed as Lac+ colonies in a
Lac- background. Putative transconjugants were picked from the mating plates and streaked for
purity. They were tested for lacticin 3147 production--which is usually an indicator of the success of
the mating and plasmid profiles were prepared to compare the recipient to the transconjugant.
Phage resistance:
The phage resistance of a culture was determined by comparing the transconjugant to the parent
strain for resistance to a phage homologous to the parent. Plaque assays were carried out as follows.
0.25 ml of an overnight culture, 0.1 ml of 1M CaCl2 and 0.1 ml of the appropriate phage dilution were
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added to 3 ml of L/GM17 sloppy agar (0.7% agar). The contents were mixed, poured onto M17 agar
and incubated at 30 DEG C. for 18 hr.
Detection of Diacetyl, Citrate and Acetolactate.
The assays were conducted on cells grown in 10%RSM (+0.5% tryptone) at 21 DEG C. and 30 DEG
C. for 16 hr and 18 hr (respectively) according to the methods for detection of diacetyl, citrate and
acctolactate as described in Prill & Hammer (1938). Marier & Boulet (1958) and Jordan & Cogan
(1995) respectively. Each assay was carried out in triplicate and the average presented in Table 5.
Concentration and partial purification of lacticin 3147 for incorporation into teat seals:
TY (Tryptone 2.5 g/L, Yeast Extract 5 g/L, Glucose 10 g/L, b-glycerophosphate 19 g/L,
MgSO4.7H2O 0.25 g/L, MnSO4.4H2O 0.05 g/L, pH 6.75) broth was prefiltered (15 filter/liter) with HA
(Millipore) filters to remove proteins in the media which would bind to the filters. L. lactis DPC 3147
was then grown overnight in the filtered TY-broth. The culture was centrifuged at 10,000 rpm for 15
minutes and then filtered through HVLP filters (Millipore). The bacteriocin was then bound to HA
filters (8 filters/liter) and subsequently harvested from the filters using acetone/5 mM PhosphateBuffer, pH 7.0 (2:1). The mix was subsequently centrifuged and the acetone removed by evaporation.
The resultant bacteriocin preparation was freeze-dried and dissolved in sterile distilled water. This
was then assayed as above. To add bacteriocin into the teat seal 17,000 units of the bacteriocin
prepared as above was added to the seal in a sterile petri disc. On mixing, the bacteriocin and seal
formed as emulsion which was then aseptically transferred to a syringe (Cross Vetpharm).
Effectiveness of lacticin 3147 on Streptococcus dysgalactiae M:
10 .mu.l from a overnight culture was added to 490 .mu.l of sterile 50 mM phosphate-buffer. pH 7.0
500 .mu.l lacticin 3147 (10,000 AU/ml) was then added and plate counts were carried out after 0, 15,
30, 60, 90 and 120 minutes at 37 DEG C. to assess cell viability.
Oral Streptococci:
Four cariogenic streptococcal strains isolated from infected patients were grown overnight at 30
DEG C. in Brain Heart Infusion broth. These strains were obtained from Dr. Ger Fitzgerald, University
541/1006
College Cork. The relative sensitivity of each of these strains to the bacteriocin was then determined
in comparison to L. lactis HP.
Results
A number of lactococci which exhibited antimicrobial activities were isolated from kefir grains.
Protease sensitivity assays demonstrated that the antimicrobials were bacteriocins, since they could
readily be degraded by proteinase K. On the basis of cross-sensitivity assays, these bacteriocin
producers could be classified into different groups. Strains from the first group, DPC3147, DPC3153,
DPC3215, DPC3400, DPC3204 and DPC3244 exhibited cross-immunity indicating that they all
produced the same or a very similar bacteriocin (see FIG. 1). Likewise, strains from the second
group, DPC3220 and DPC33(1) also exhibited cross-immunity to each other, but were sensitive to
the bacteriocin(s) produced by the first group. Strain DPC2949 defined a third set of producers
which exhibited cross-sensitivity to members of the first group.
Subsequently, L. lactis DPC3147, L. lactis DPC2949 and L. lactis DPC3220 were chosen as
representatives of each group for further study. Initially, these strains were tested for their ability to
inhibit a wide range of organisms. The results of these experiments are given in Table 1. It can be
seen that the bacteriocin-producers. DPC3147, DPC2949 and DPC3220 do not inhibit themselves
but do however inhibit one another. This indicates that the strains are at least distinct from one
another. To reduce the possibility that they may be previously well characterised bacteriocinproducing strains, cross-sensitivity studies were carried out to some well known bacteriocinproducers which were, L. lactis CNRZ481, the producer of lacticin 481 (Piard et. al., 1991). L. lactis
NCDO496 (FIG. 1) and NCDO497, nisin producers, and L. lactis subsp. cremoris 9B4, the producer
of lactococcin A, B and M. Each of the bacteriocin-producing strains in question inhibit these four
previously characterized strains very well, with DPC3147 being particularly effective against them. In
addition, these four strains effectively inhibited DPC3147, DPC2949 and DPC3220. Based on these
observations, it thus appeared that none of the strains DPC3147, DPC2949 and DPC3220 produced
either nisin, lacticin 481 nor lactococcin A, B and M.
Spectrum of Inhibition:
The range of organisms inhibited by each of the strains was examined. Given the extensive
application which nisin has found in the food industry, the nisin producer NCDO496 was included in
this study for comparative purposes. The relative sensitivities of 54 strains to L. lactis DPC3147,
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NCDO496. DPC2949 and DPC3220 are presented in Table 1. All four producers were very effective
in inhibiting other lactococci which included a number of cheese-making strains. The range of
inhibition exhibited by DPC3220 however, appears limited to lactococci, and as such is described as
having a narrow spectrum. In contrast, the bacteriocin produced by DPC3147 has a very broad
spectrum of inhibition which closely resembles that of the nisin producer, NCDO496. Without
exception, all Gram positive indicator bacteria tested, including lactococci, lactobacilli, enterococci,
bacilli, leuconostocs, pediococci, clostridia, staphylococci and streptococci were sensitive to it.
Notably, the two clostridial strains tested were particularly sensitive to DPC3147. Indeed, C.
sporogenes was so sensitive that the entire overlay was inhibited and no actual zone of inhibition
could be measured. C. tyrobutyricum was also found to be extremely sensitive. The Listeria strains
tested, including the pathogenic strain L. monocytogenes NCTC5348 were also sensitive to
bacteriocin(s) produced by the DPC3147 strain. Overall, the biological activity of the 3147
bacteriocin closely resembled that of nisin. Interestingly, it was found that DPC3147 was significantly
more active than the nisin producer against S. thermophilus HA. The closely related lactococci were
all very sensitive.
In contrast, L. lactis DPC2949 has an intermediate spectrum of inhibition lying somewhere between
that observed for strains DPC3147 and DPC3220. Like DPC3220, this strain was effective in inhibiting
all of the lactococci tested, but in addition, inhibited most of the Lactobacillus and Leuconostoc
species. Thus, its biological activity was quite unlike that of lacticin 481 producers. However, as
stated above, the DPC2949 strain and L. lactis CNRZ481 exhibited cross-sensitivity. Interestingly,
this strain also had slight activity against Escherichia coli and Pseudomonas aeroginosa but it did not
show any inhibition against enterococci, Listeria or staphylococci.
Relationship with Nisin:
Although the cross-sensitivity studies suggest that DPC3147 is not a nisin producer, even though
their biological activity appears remarkably similar, a number of experiments aimed at probing the
relationship between the two bacteriocins were then performed. Protease sensitivity assays
demonstrated that the 3147 bacteriocin is sensitive to trypsin, alpha-chymotrypsin, proteinase K and
pronase E but not to pepsin (FIG. 2). In contrast, nisin is not sensitive to trypsin but is degraded by
alpha-chymotrypsin, pancreatin and subtilopeptidase. As expected, catalase had no effect on the
antimicrobial activity of the 3147 bacteriocin thus eliminating the possibility that the antimicrobial
activity may be due to hydrogen peroxide. In addition, none of the strains producing lacticin 3147
were capable of fermenting sucrose. This provides additional evidence that this bacteriocin is not
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nisin, since the genes encoding nisin (nis A) is linked to genes responsible for sucrose catabolism on
the nisin-sucrose transposon. Tn5276 (Horn et. al., 1991). Thus, all those Nip@+ (nisin-producing)
strains studied to date are in addition Suc@+ (Sucrose-fermenting). As expected, L. lactis NCDO496
was found to be Suc@+ while neither the DPC3220 nor DPC2949 strains could utilize sucrose as a
fermentable substrate. In addition, the minimum concentrations (MIC) of pure nisin required for
inhibition of strains DPC3147 and NCDO496 were determined. It was found that DPC3147 was
inhibited at 100 .mu.g/ml of nisin while NCDO496 was not inhibited until 400 .mu.g/ml of nisin was
added. These differing MIC values of nisin for DPC3147 and NCDO496, together with all of the above
results all strongly suggest that the DPC3147 bacteriocin is distinct from nisin.
Initially, certain strains were investigated for their genetic potential to produce nisin by the
polymerase chain reaction (PCR). Using the published sequence of the nisin structural gene. Dodd
et al (1990), two primers were synthesized which are complementary to sequences occurring
proximal to the 3' and 5' ends of the nis A gene. These should amplify a PCR product of 166 base
pairs from nis A-containing template DNA. Indeed, an amplified product of approximately that size
was consistently amplified from genomic DNA isolated from the NCDO496 (FIG. 3) strain. In contrast,
no amplified product was observed when genomic DNA from DPC3147, DPC2949 and DPC3220
was used. The DNA amplified from L. lactis 496 representing most of the nis A gene was then used
as a gene probe to DNA isolated from lactococcal strains NCDO496, DPC3147, DPC2949 and
DPC3220. These DNAs were first digested with Hind III and electrophoresed on a 1% (w/v) agarose
gel prior to transfer to a nylon membrane. As shown in FIG. 4, the nis A gene probe hybridised to a
3.5 kb Hind III fragment on the 496 genome. In contrast, no hybridizing DNA was observed in DNA
isolated from strains MG1614 nor DPC3147. This suggests that the bacteriocin produced by
DPC3147 is not a close homologue of nisin. Given the inhibition spectrum of the bacteriocin
produced by L. lactis DPC 3147 and the results of the experiments discussed above demonstrating
that it was not nisin, it was concluded that DPC 3147 produced a novel, broad-spectrum bacteriocin
which was designated lacticin 3147.
Growth of L. lactis DPC3147 and production of lacticin 3147 was monitored in GM17 and TYT30
over a 24 hour period. As shown in FIG. 5 higher cell numbers and bacteriocin activity are obtained
in GM17, the richer medium. In both media, lacticin 3147 is produced during exponential phase and
peaks during early stationary phase. Subsequently, the bacteriocin activity declines gradually during
stationary phase. Production was also determined in a variety of different media including MRS, BHI,
TYP, RSM and whole milk. Activity (mm@2) was measured from the filtered supernatant of an
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overnight culture. Production was found to be greatest in MRS with an activity of 170 mm@2. The
activity in RSM and whole milk was 90% and 73% of that found in MRS.
This indicates that lacticin 3147-producing starter cultures could be used to produce lacticin 3147containing dairy products.
Effect of pH and temperature on bacteriocin stability:
The effect of pH and temperature on the stability of lacticin 3147 was investigated and results are
shown in FIG. 6. Three samples of bacteriocin were brought to a pH of 5, 7 and 9 and aliquots of
each were subsequently heated to 60 DEG C., 70 DEG C., 80 DEG C., 90 DEG C., 100 DEG C., 110
DEG C. and 121 DEG C. for 10 minutes. The activity at each pH prior to heat treatment was
measured and was taken to be 100% for that pH value. From the graphical representation in FIG. 6, it
can be seen that lacticin 3147 is heat-stable, particularly at an acid pH. Indeed at pH 5, the
bacteriocin survives autoclaving at 121 DEG C. for 10 minutes and at pH 9, maintains more than 50%
of its activity up to a temperature of 100 DEG C.
This means that lacticin 3147 has a potential for use in both high-acid and low-acid canned foods.
Low-acid foods (pH4.5) should receive sufficient heat treatment to destroy heat resistant spores of
pathogenic C. botulinium. By adding lacticin 3147 to these foods, it should be possible to reduce the
extent of heat-processing required, thus resulting in improved flavour, increased nutritional value and
an overall more economical process. Such applications may be particularly beneficial for products
such as canned milk puddings where heat penetration is often a problem. Lacticin 3147 could also
be potentially used quite successfully in high-acid foods (pH<4.5), given its acid pH optimum. Even
though, a substantial proportion of lacticin 3147 is lost on heating to temperatures exceeding 100
DEG C., its efficiency as a food preservative should not be compromized by heat, as heat-treated
bacterial spores display greater sensitivity to bacteriocins. Hence, inhibition of such spores would
require the same level of active lacticin 3147. For the reasons stated above lacticin 3147 also has a
role in meat preservation.
Molecular weight determination:
The molecular weight of lacticin 3147 was estimated by SDS polyacrylamide gel electrophoresis
according to the method of Swank and Munkres (1971). As a control, a sample of lactococcin A,
which has a molecular weight of 3 kDa was also analyzed. The gel, which was subsequently overlaid
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with agar seeded with L. lactis indicator strain HP is shown in FIG. 7. It can be seen that lacticin 3147
is somewhat smaller than lactococcin A and its molecular weight was estimated at 2.8 kDa.
Molecular weight markers ranging from 2.5 to 17 kDa were used as a standard in the other half of the
gel.
Genetic studies:
During initial genetic work with lacticin 3147, it was observed that the bacteriocin-producing
property was an easily lost trait. Preliminary experiments were attempted to establish if bacteriocin
production by DPC3147 was encoded on a conjugally transmissable piece of DNA. These conjugal
matings involved using DPC3147 as the donor strain and the plasmid-free strain, L. lactis subsp.
lactis MG1614, which is streptomycin resistant as the recipient. The selection of transconjugants from
such matings was achieved using the plasmid-encoded bacteriocin immunity/resistance
determinants as a selectable marker. This involved the incorporation of lacticin 3147 into the
selective media. Even when plated at a high cell density (up to 10@8 -10@9 CFU/ml), L. lactis subsp.
lactis MG1614 sensitive cells failed to grow on such media, indicating a very low level of
spontaneous resistance occurring for this strain to the 3147 bacteriocin. The incorporation of lacticin
3147 into selective media may thus form the basis for a novel food-grade marker system for use in
genetic manipulation of lactococci. In matings involving strains MG1614 and DPC3147, putative
transconjugants (bac@imm.strep'), were isolated at a frequency of 10@-3 per donor cell. These cells
were subsequently found to be lactose deficient and also had acquired the ability to produce
bacteriocin. As expected, these putative transconjugants exhibited cross-immunity to DPC3147 and
also, could inhibit a wide range of Gram positive bacteria. Evidence that these actually represented
true transconjugants was obtained on analysing their plasmid complements. In all cases, the bac@-,
bac@imm.strep' cells had acquired a 63 kDa plasmid, designated pMRCO1. A similar sized plasmid
is clearly evident in plasmid profiles of the DPC3147 strain (FIG. 8). This indicates that the genetic
determinants encoding lacticin 3147 are encoded on the pMRCO1 conjugative plasmid. Furthermore,
similar numbers of colonies were obtained from the mating when plated on media containing
streptomycin and bacteriocin, and when plated on media containing streptomycin solely. Moreover,
when these strep' colonies were overlaid with strain MG1614, all were observed to be bac@+. This
would suggest that all those MG1614 cells which had not received the plasmid during mating had
been killed off by lacticin 3147 produced by DPC3147 in the mating mix. This would negate the
requirement for incorporating bacteriocin to select transconjugants in such matings.
Mastitis:
546/1006
Since lacticin 3147 was shown to be effective in inhibiting S. aureus ATCC25923 and S.
thermophilus HA and ST112 (Table 1), a separate study was initiated to investigate its ability to inhibit
a variety of clinical isolates obtained from mastitic animals. These virulent bacteria were obtained
from the Department of Dairy Husbandry, Teagasc, Moorepark, Fermoy, Co. Cork. In all six
streptococci and ten staphylococci were tested. DPC3147 was most effective against these
pathogenic strains, as shown in Table 3. All the streptococci, except S. dysgalactiae strain M were
quite sensitive to it as were all the staphylococci, except strain 12. A similar outcome was seen with
NCDO496, although in some cases DPC3147 is slightly more successful against the streptococci.
DPC3220 does not display any activity against either the streptococci or the staphylococci.
In general the streptococcal strains were more sensitive to DPC3147 that the staphylococcal strains
whereas the reverse is true for the nisin producers NCDO496 and NCDO497. This suggests that it
would be particularly effective to use lacticin 3147 in combination with another bacteriocin, such as
nisin, as a mastitis treatment. In such a situation it is much less likely that the infecting organisms
would develop resistance to both bacteriocins. Clearly, the use of bacteriocins in the treatment of
mastitis may mean that milk would not have to be withheld as would be the case with an antibioticbased treatment.
Phage Resistance:
The conjugal plasmid, pMRCO1, also conferred an increased level of phage resistance to L. lactis
subsp. lactis MG1614. In contrast to the MG1614 parent, transconjugants containing the plasmid
exhibited total resistance to the small isometric-heated phage 712. In addition, the burst size of
prolate headed phage c2 appeared drastically reduced (as evidenced by pinpoint plaques). Further
studies demonstrated that the resistance mechanism encoded by the pMRCO1 plasmid did not
affect the ability of phage to adsorb to the cells, nor did it appear to inhibit phage DNA replication
once the infecting phage DNA was internalised inside the host. Consequently, the life cycle of the
phage was inhibited at some point after phage DNA replication had occurred. This potent phage
resistance mechanism was thus assumed to be an abortive infection (or Abi) mechanism.
In lactococci catabolism of lactose is usually plasmid-linked. Therefore, the plasmid-free strain L.
lactis subsp. lactis MG1614 cannot ferment lactose. Since MG1614 transconjugants containing the
multifunctional pMRCO1 are also lac deficient, this would suggest that the genes necessary for
lactose catabolism are not encoded by pMRCO1 plasmid. MG1614 containing pMRCO1 was then
547/1006
mated with a lactococcal cheese starter strain HP. Putative transconjugants were selected from such
matings based on their ability to ferment lactose and become resistant to lacticin 3147. These lac@cells also now produced lacticin 3147 and had also become totally resistant to phage which normally
infect the HP strain. Examination of the plasmid complements of these strains revealed that they had
acquired an extra plasmid of 63 kb, the size of pMRCO1. This demonstrates that bacteriocin linked
phage resistance may prove to be a very efficient method in the improvement of commercial cheese
starters.
These results indicate that lacticin 3147 producers may actually be used as cheese starter strains
rather than adding a bacteriocin preparation as with nisin. The primary advantage of this is that by
directly adding the producing strain, food additive legislation may be overcome as there is no
restriction on the use of the strain itself.
Cheese-Making Trials:
Problems associated with nisin producing starters include such undesirable characteristics as an
inability to produce sufficient acid for cheesemaking, that they are usually proteinase deficient and
also that they are phage sensitive. As outlined above the latter characteristics are not associated with
lacticin 3147 producers since bacteriocin functions and phage resistance are linked on pMRCO1. To
test the ability of such strains to act as cheese starters two separate cheese trials were performed. In
the first, three strains were used, one of which was strain DPC3147. The other two, DPC3204 and
3256 are also kefir isolates, both of which exhibit crossimmunity with DPC3147. Consequently, these
are lacticin 3147 producers as well. As illustrated in FIG. 9, these strains produced acid much like
the fast acid commercial strain 303 during cheesemaking. Thus it can be concluded that with regard
to acid production these strains make acceptable starters. During cheddar cheese ripening nonstarter lactic acid bacteria (NSLAB) can reach levels exceeding 10@7 cfu/g. Since these can be
mainly lactobacilli, it was important to investigate their growth or otherwise in cheese made with
lacticin 3147 producers (remembering that lacticin 3147 inhibits all lactobacilli tested). As shown in
FIG. 10, no NSLABs were detected in cheese made with the bacteriocin-producing strains even after
six months (the average duration of cheddar ripening). In contrast, NSLABs had reached levels of
10@7.5 cfu/g after approximately 4 months in controls produced without the bacteriocin. In the
second cheese trial, a transconjugant of strain 303 which produces lacticin 3147 was used as a
starter with 303 again as the control strain. The results shown in FIG. 11 again demonstrate that the
303 transconjugant performed satisfactorily as a starter during cheese manufacture. In addition the
NSLAB levels appearing in the cheese made in this trial (FIG. 12) were significantly lower (in excess
548/1006
of 100-fold) than in the control cheese. Sensory analyses subsequently demonstrated that there were
no major differences in flavour and aroma between the two cheeses.
NSLAB which are found in ripening cheese may contribute to the flavour of the cheese. It would be
possible to use lacticin 3147 or a commercial starter strain producing it to control the entire microbial
population of a cheese, thus allowing the flavour of a cheese to be designed.
Lacticin 3147 was found to be particularly active against Clostridia tyrobutyricum and C. sporogenes.
It is well established that the outgrowth of milk-contaminating anaerobic spore-formers such as C.
tyrobutyricum and C. butyricum is primarily responsible for the problem of late-gas blowing in some
cheeses i.e. the formation of hydrogen gases and carbon dioxide during ripening resulting in the
development of large holes. These bacteria convert lactic acid into butyric acid giving rise to offflavours and aromas. Thus, lacticin 3147 also has a use in such products given its potency against
clostridial strains.
The introduction of this genetic material into lactococcal industrial strains introduces complications
regarding the availability of food-grade selection markers, and the possibility of the loss of industrially
important plasmid-encoded functions such as lactose fermentation, proteolytic activity,
bacteriophage resistance and citrate utilization. In this regard lacticin 3147 production and immunity
may be incorporated as a desirable phenotype of some industrial starter cultures used in many
commercial applications. The results described here demonstrate that incorporation of this
bacteriocin as a selectable marker into the media itself can form the basis for a novel selectable
system. The gene(s) encoding immunity to lacticin 3147 can be linked to desirable traits on plasmids
and transconjugants containing these plasmids would then selectively grow on bacteriocinproducing media. It has been observed that the level of spontaneous resistance to lacticin 3147 by
some commercial cheese making strains is very low and additionally, plasmid maintenance by
pMRC01-containing strains would be assured in fermentations since cells which lose the plasmid
would be killed by the lacticin 3147 produced, both properties which are required for an effective
selectable system. This is a significant advantage as most selectable markers to date are not of foodgrade quality. Indeed, most are actually antibiotics which cannot be used because of the danger of
the occurrence of antibiotic resistant strains of clinical importance.
Strain DPC2949:
549/1006
The biological activity exhibited by L. lactis DPC2949 was quite similar to that of the previously
characterized L. lactis CNRZ481 in that common sensitive strains include lactococci, Clostridium
tyrobutyricum, Leuconostoc and some, but not all lactobacilli. In addition, Bacillus substilis,
Enterococcus faecalis, Listeria innocua and L. monocytogenes were not inhibited by either
bacteriocin producer. However, cross-sensitivity studies indicated that they were actually different
since both inhibited each other. Unexpectedly, DPC2949 gave slight inhibition of the two Gram
negative strains Escherichia coli and Pseudomonas aeroginosa. Strain DPC2949 thus produces an
intermediate spectrum bacteriocin which has applications such as the prevention of late gas-blowing
in Cheddar cheese. It could also be applied to the control of non-starter lactic acid bacteria (NSLABs)
and consequently, to assess their effect on Cheddar cheese quality, which still remains to be
established.
Cheese-Making Trials:
As stated previously, cheese made with lacticin3147-producing starters had significantly less nonstarter lactic acidbacteria (NSLABs) in them when compared to corresponding cheese made with a
commercial starter. Since then we have assayed the bacteriocin in these cheeses. In Cheese trial 1
the presence of lacticin 3147 in take test cheese was confirmed (using L. lactis AM2 as the indicator
strain) at a level of approximately 1.280 AU/ml which remained constant in the cheese over the 28week ripening period (FIG. 13A). In contrast, no anti-microbial activity was detected in the control
cheese. Similarly, the amount of bacteriocin detected in cheese trial 2 was approximately 1.280
AU/ml which also remained constant over the ripening period (FIG. 13B). Thus the level of
bacteriocin observed in the cheese corresponds to the number of NSLABs which occur in the
cheese during ripening.
Genetic studies:
To evaluate the potential usefulness of the lacticin 3147-encoding plasmid (pMRC01) for starter
strain improvement it was transferred into a variety of lactococcal strains including those currently
used for Cheddar cheese and lactic butter manufacture. These transfers were performed in a Food
Grade manner via conjugations (bacterial matings) after which the newly modified strains were
selected based on their immunity to the bacteriocin. Such matings first necessitated the construction
of a Food Grade donor strain for the plasmid which is sensitive to antibiotics. Depending on the
starter recipient used one of three possible results for each of the matings was recorded. In the first,
starters were isolated which had improved phage resistance properties and produced (and were
550/1006
resistant to) bacteriocin (Table 4). Genetic analyses confirmed that they had received the pMRC01
plasmid which could efficiently be mated back out of the strain.
These strains retained their commercially important characteristics for example ability to produce
acid (for cheese starters) and/or diacetyl (for lactic butter production, Table 2). In addition, the
plasmid appeared stable in these strains and was maintained over a number of successive
subcultures. One such strain was subsequently used for pilot-scale Cheddar cheese manufacture.
Another result of these conjugations was the identification of strains which produced the bacteriocin
but had not improved phage resistance. Genetic analysis of one such strain demonstrated that such
a phenotype was associated with plasmid co-integration in this strain. Lastly, a number of strains
were found to be recalcitrant to the pMRC01 plasmid. One possible explanation for this observation
is that the plasmid maybe incompatible with a resident plasmid of the strain (e.g. pMRC01 was found
to be incompatible with the lactococcal plasmid pNP40 in this study). The overall significance of the
results of these matings can be summarized as follows: 1) The genetic determinant(s) encoding
immunity to lacticin 3147 is very useful as a food grade selectable marker for starter strain
improvement. The lactococcal strains tested in this study proved very sensitive to the bacteriocin
incorporated in solid media unless they had received the bacteriocin genes. Indeed, the bacteriocin
is as convenient to use as conventional antibiotics for such studies. 2) A number of new starter
strains have now been generated using the bacteriocin. Many of these have also been improved with
regard to their phage resistance. Importantly all these new strains now produce the bacteriocin and
may be used as starters for products where the bacteriocin might impart a desirable effect.
Examples include reduction of NSLAB numbers in cheese products or elimination of undesirable
bacteria from some fermented meat and fish products.
Mastitis:
As a result of the growing concern over the use of antibiotics for the treatment and prevention of
disease in animals the potential of using lacticin 3147 in the prevention of bovine mastitis was
investigated. To achieve this the bacteriocin was incorporated into teat seals preparations. These
seals are manufactured by Cross Vetpharm (Broomhill Road, Tallaght, Dublin 24. Ireland) and act as
a physical barrier in the cow against infection. The bacteriocin provides an additional anti-microbial
barrier over the physical one provided by the seal itself. Incorporation of the bacteriocin into the teat
seals first required the preparation of the bacteriocin in a highly concentrated and semi-purified form.
The anti-microbial effectiveness of lacticin 3147 in the environment of the seal required the addition
of either Tween 20 or 80 in concentrations of 2% (FIG. 14A). Where the detergent was not present
551/1006
negligible bacteriocin activity was recorded in the treated seals. Having successfully prepared
effective bacteriocin-containing teat seals a number of animal trials were then performed. These
involved exposure of the modified seals to the animal for different time periods after which the seals
were removed. Overall such studies have demonstrated that the animals tolerated the bacteriocincontaining seals as evidenced by low associated somatic cell counts. In addition, seals extracted
from the animal were later shown to retain bacteriocin activity. Since lacticin 3147 was found to inhibit
a range of mastitic streptococci its effectiveness on Streptococcus dysgalactiae M, a strain
previously isolated from an infected animal, was studied. Addition of the bacteriocin (10.240 AU/ml)
to stationary phase cells of this strain resulted in a 99.99% kill in just 2 hours (FIG. 14B). Other
aspects of this study focused on the frequency at which a mastitic strain can develop resistance to
lacticin 3147 since this could limit its practical usefulness as an effective antimicrobial under certain
conditions. However, only 0.003% bacterial cells of the M strain developed increased tolerance to
the bacteriocin after prolonged exposure.
Inhibition of Oral Streptococci:
A number of oral streptococci have also been tested for sensitivity to lacticin 3147 to investigate the
use of the bacteriocin in such applications as mouth washes, dental products etc. The strains tested
include four cariogenic streptococci isolated from infected patients. All four strains tested proved
sensitive to the bacteriocin (12.5% as sensitive as L. lactis HP). Importantly, fermented dairy
products manufactured with lacticin 3147-producing starters may therefore have additional anticariogenic characteristics.
TABLE 1
Inhibition Spectrum of Lactococcus lactic DPC3147,
L. lactis NCDO496 and L. lactis DPC3200
Sensitivity
DPC NCDO DPC DPC
Strain
Medium 3147 496 2949 3220 Source
Acetobacter acetii UB@1 + + + NZ DPC
Acetobacter suboxy- UB@1 NZ NZ NZ NZ DPC
dons
Bacillus cereus GM17@2 + + NZ NZ DPC
ATCC9139
Bacillus subtilis TYP@2 + + NZ NZ DPC
552/1006
BD630
Clostridium sporo- RCM@3 +++ +++ NZ NZ DPC
genes NCFB1791
Clostridium tyro- RCM@3 +++ +++ +++ NZ DPC
butyricum NCFB1755
Enterococcus GM17@2 ++ ++ - NZ NCDO
faecium NCDO942
Enterococcus faecalis
NCDO610
GM17@2 ++ + NZ NZ DPC
10Cl
GM17@2 ++ + NZ NZ DPC
NCDO581
GM17@2 +++ + NZ NZ
Lactobacillus acido- MRS@2 * + + ++ NZ ATCC
philus ATCC4356
Lactobacillus bulgari- MRS@4 * ++ ++ NZ NZ ATCC
cus ATCC11842
Lactobacillus casei MRS@2 ++ ++ - NZ ATCC
ATCC334
Lactobacillus curvatus MRS@2 + ++ NZ NZ CNRZ
CNRZ117
Lactobacillus fermenti- MRS@2 * +++ +++ + NZ ATCC
cum ATCC9338
Lactobacillus
helveticus
NCDO257
MRS@2 * +++ ++ + NZ NCDO
NCDO1209
MRS@2 * +++ ++ ++ NZ NCDO
NCDO1244
MRS@2 * +++ +++ ++ NZ NCDO
ATCC15009
MRS@2 ++ ++ + NZ ATCC
Lactobacillus kefir MRS@3 +++ ++ + NZ NCFB
NCFB2737
Lactobacillus
leichmanii
NCDO299
MRS@2 * +++ ++ ++ NZ NCDO
NCDO302
MRS@2 * +++ +++ + NZ NCDO
Lactobacillus reuteri MRS@2 ++ ++ NZ NZ DPC
DSM20016
553/1006
Lactobacillus sake MRS@3 * +++ +++ ++ NZ NCFB
NCFB2714
Lactococcus lactis
DPC3147
GM17@3 NZ + ++ ++ DPC
NCDO496 (nisin pro- GM17@3 ++ NZ ++ ++ NCDO
ducer)
NCDO497 (nisin pro- GM17@3 +++ NZ ++ ++ NCDO
ducer)
DPC2949
GM17@3 +++ + NZ NZ DPC
DPC3220
GM17@3 +++ + ++ NZ DPC
DPC33(1)
GM17@3 +++ + NZ NZ DPC
AM2
GM17@3 +++ +++ ++ ++ DPC
CNRZ481
GM17@3 +++ +++ ++ +++ CNRZ
303
GM17@3 ++ ++ ++ ++ DPC
HP
GM17@3 +++ +++ ++ +++ DPC
9B4
GM17@3 +++ +++ + ++ DPC
938
GM17@3 +++ +++ + + DPC
DPC147
GM17@3 +++ +++ ++ +++ DPC
DPC712
GM17@3 +++ +++ ++ ++ DPC
SK11G
GM17@3 +++ +++ +++ +++ DPC
290P
GM17@3 +++ ++ ++ ++ DPC
DRC3
GM17@3 +++ ++ ++ +++ DPC
Leuconostoc MRS@2 * ++ +++ + NZ CNRZ
CNRZ1091
Listeria innocua GM17@3 ++ ++ NZ NZ DPC
BD86/26
Listeria monocyto- GM17@2 + + NZ NZ DPC
genes NCTC5348
Pediococcus
pentriceans
NCDO992
GM17@3 ++ ++ NZ NZ NCDO
NCDO1850
GM17@3 + ++ NZ NZ NCDO
Pediococcus pento- GM17@3 ++ ++ + NZ DPC
saceus FBB63
Staphlococcus aureus TYP@2 + + NZ NZ DPC
554/1006
ATCC25923
Streptococcus
thermophilus
HA
GM17@4 ++ + + NZ DPC
ST112
GM17@4 ++ ++ NZ NZ DPC
Salmonella typhi TSA@2 NZ NZ NZ NZ DPC
Escherchia coli GM17@3 NZ NZ + NZ DPC
Pseudomonas GM17@1 NZ NZ + NZ DPC
aeroginosa
[No Zone (NZ); 0 to 1 mm (-); 1 to 5 mm (+); 5 to 15 mm (++); 15 mm over
(+++)]
Temperature of Incubation: @1 21 DEG C.; @2 37 DEG C.;
@3 30 DEG C.; @4 42 DEG C.
DPC3147 produces lacticin 3147; NCD0496 produces nisin; DPC3220 produces a
bacteriocin with a narrow spectrum of inhibition.
TABLE 2
Strains and levels of inocula used in cheesemaking
Country
Inoculation
Vat DPC number of origin Source
rate (v/v %)
1@a 303 -- Chr. Hansens Lab. 1
2@a 3147
Ireland Kefir
0.7
3204
Ireland Kefir
0.7
3256
Ireland Kefir
0.7
1@b 303 -- Chr. Hansens Lab. 1
2@b 303 (pMRC01) Ireland DPC
1
@a Trial 1.
@b Trial 2
TABLE 3
Sensitivity of mastitic strains of Streptococci and Staphylococci to
Lactococcus lactis DPC3147. L. lactis NCDO496 and L. lactis DPC3220
Sensitivity
DPC NCDO NCDO DPC DPC
Strain
Medium 3147 496 497 2949 3220
555/1006
Streptococcus GM17 ++ ++ NZ + NZ
agalactiae (B)
Streptococcus GM17 ++ + ++ NZ NZ
agalactiae (H)
Streptococcus GM17 ++ + + NZ NZ
agalactiae (P)
Streptococcus dysa- GM17 NZ - - NZ NZ
galactiae (M)
Streptococcus faecalis GM17 ++ ++ NZ NZ NZ
(l)
Streptococcus uberis GM17 ++ + + NZ NZ
(L)
Staphylococci
subspecies
2
TSA + ++ ++ NZ NZ
11
TSA + ++ ++ NZ NZ
12
TSA - ++ ++ NZ NZ
13
TSA + ++ ++ NZ NZ
89
TSA + ++ ++ NZ NZ
10
TSA + ++ ++ NZ NZ
1
TSA + ++ ++ NZ NZ
22
TSA + ++ ++ NZ NZ
32(a)
TSA + ++ + NZ NZ
36
TSA + ++ ++ NZ NZ
[No Zone (NZ); 0 to 1 mm (-); 1 to 5 mm (+); 5 to 15 mm (++); 15 mm over
(+++)]
DPC3147 produces lacticin 3147; NCDO496 produces nisin; DPC3220 produces a
bacteriocin with a narrow spectrum of inhibition.
Temperature of Incubation = 37 DEG C.
TABLE 4
Conjugation results for mating pMRC01 into a variety of
Lactococcus lactis starter recipients
Mating Improved
L. lactis Transfer frequency phage Mating
556/1006
Recipient pMRC01 (per donor) resistance back out*
DPC4268 + 2.3 .times. 10@-3 - DPC4272 + 1.06 .times. 10@-5 - +
DPC4273 + 1.03 .times. 10@-4 - DPC4274 + 4.0 .times. 10@-5 + +
DPC220 + 5.4 .times. 10@-4 + +
DPC429 + 1.7 .times. 10@-8 + +
HP
+ 6.0 .times. 10@-6 + +
712
+ 1.0 .times. 10@-5 + +
ML8
+ 5.5 .times. 10@-4 + +
077
+ 1.1 .times. 10@-5 - +
007
+ 1.3 .times. 10@-7 ? +
Transconjugants tested exhibit similar acid production to the parents and
all were lacticin 3147 producing.
*Mating of transconjugant with L. lactis MG1614.
TABLE 5
Diacetyl and acetolactate production by
L. lactis DPC220 and L. lactis DPC220 containing
pMRC01 (220 TcA)
Diacetyl Acectolactate
Production Production
Culture
(mM .+-. SD) (mM .+-. SD)
DPC220
0.218 .+-. 0.001 3.237 .+-. 0.04
220 TcA
0.235 .+-. 0.017 3.616 .+-. 0.283
Citrate utilisation = 100% for all the test cultures.
10% RSM + 0.5% tryptone at 30 DEG C.
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Jordan & Cogan, Irish J. Agric. Food Res. 1995, 34, 39. Claims:
We claim:
1. An isolated naturally occurring bacteriocin designated lacticin 3147 from Lactococcus lactis
DPC3147 characterised by
a molecular weight of approximately 2.8 kDa,
inhibiting activity against lactococci, lactobacilli, enterococci, bacilli, leuconostocs, pediococci,
clostridia, staphylococci and streptococci
sensitivity to the proteases trypsin, alpha-chymotrypsin, proteinase K and pronase E but not pepsin,
heat-stability,
activity at acid pH,
and the capability of inhibiting nisin-producing bacterial strains.
2. L. lactis DPC3147 strain as deposited at the National Collection of Industrial and Marine Bacteria,
Aberdeen, Scotland, on Apr. 11, 1995 under the Accession No. NCIMB 40716.
3. An isolated plasmid comprising gene(s) encoding a bacteriocin as defined in claim 1.
4. An isolated plasmid pMRC01 which comprises gene(s) which encode the bacteriocin designated
lacticin 3147, lacticin 3147 immunity gene(s) and phage resistance genes as deposited at the
National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, on Apr. 11, 1995 under
Accession No. NCIMB 40716.
5. Isolated gene(s) which encode lacticin 3147 as deposited at the National Collection of Industrial
and Marine Bacteria, Aberdeen, Scotland, on Apr. 11, 1995 under Accession No. NCIMB 40716.
6. An isolated gene encoding a protein or polypeptide conferring immunity to lacticin 3147 as
deposited in the plasmid pMRCO1 as deposited at the National Collection of Industrial and Marine
Bacteria, Aberdeen, Scotland, on Apr. 11, 1995 under Accession No. NCIMB 40716.
559/1006
7. An isolated gene encoding a protein or polypeptide which confers resistance to a phage and as
deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, on Apr.
11, 1995 under the Accession No. NCIMB 40716.
8. A host cell into which a plasmid according to claim 3 or 4 has been introduced.
9. A method of producing lacticin 3147 comprising culturing a host cell as claimed in claim 2
containing lacticin 3147-encoding gene(s) and isolating lacticin 3147 from the culture.
10. A method of conferring lacticin 3147-producing properties on a host cell, comprising introducing
and expressing in the host a plasmid as claimed in claim 3 or 4.
11. A food-grade genetic marker system comprising a gene for immunity to lacticin 3147 comprising
a gene of claim 6.
12. A method of conferring phage resistance on a host cell, comprising introducing and expressing
therein a gene as claimed in claim 7.
13. A food-grade genetic marker system comprising a gene for immunity to lacticin 3147 comprising
a gene of claim 6.
14. The isolated bacteriocin-encoding gene of L. lactis strain DPC2949 as deposited at the National
Collection of Industrial and marine Bacteria, Aberdeen, Scotland on Apr. 11, 1995 under the
Accession No. NCIMB 40715.
15. An isolated bacteriocin produced by the L. lactis strain DPC2949 as claimed in claim 13.
16. A host cell comprising the gene as claimed in claim 14.
17. A method of preventing late gas-blowing in Cheddar cheese production comprising introducing
into the starter culture a gene of claim 16.
18. A method of controlling non-starter lactic bacteria in Cheddar cheese production comprising
introducing into the starter culture a gene of claim 14.
560/1006
19. A host cell comprising gene(s) according to any of claims 5, 6 or 7.
20. A method for producing lacticin 3147 which comprises growing the host cell of claim 19 under
conditions which favor the expression of lacticin 3147.
21. A host cell comprising of gene(s) according to any of claims 5, 6 or 7.
22. A method of inhibiting or preventing the growth of a microorganism comprising contacting a
composition with an effective amount of the gene of claim 14 an culturing the composition under
conditions for the expression of the gene.
23. A method of inhibiting or preventing the growth of a microorganism comprising contacting the
microorganism with an effective amount of the bacteriocin of claim 1.
24. A method of treating bovine mastitis in an animal comprising administering to the animal an
effective amount of the compound of claim 1, effective to treat mastitis.
25. A composition comprising the bacteriocin of claim 1.
26. A composition for the treatment of mastitis comprising an effective amount of the bacteriocin of
claim 1 and a carrier.
27. A composition for oral hygiene comprising an effective amount of the bacteriocin of claim 1 and
a carrier.
28. The composition of claim 27, wherein the carrier is an oral or dental product.
29. A composition comprising the gene of claim 14 and a carrier.
30. An isolated plasmid comprising gene(s) according to claim 5.
31. An isolated Lacticin 3147 as produced by the strain of claim 2.
32. An isolated Lacticin 3147 as encoded by the plasmid of claim 4.
561/1006
33. An isolated plasmid comprising gene(s) according to claim 5.
34. An isolated Lacticin 3147 as encoded by the gene of claim 7.
35. An isolated Lacticin 3147 as produced by the cell of claim 8.
36. An isolated Lacticin 3147 as encoded by the gene(s) of claim 5.
562/1006
60. RU2059716 - 10.05.1996
STRAIN OF STREPTOCOCCUS LACTIS - A PRODUCER OF BACTERIOCIN NISINE
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=RU2059716
Inventor(s):
LITVINOVA MIRRA N (RU); KRASNIKOVA LYUDMILA V (RU); BIRYUKOV
VALENTIN V (RU); BITTEEVA MARYAM B (RU); SHCHEBLYKIN IGOR N (RU); SHUSHENACHEVA
ELENA V (RU); SHUMAKOV SERGEJ A (RU)
Applicant(s):
GNII BIOSINTEZA BELKOVYKH VESH (RU)
IP Class 4 Digits: C12N; C12P; C12R
IP Class:
C12N1/20; C12R1/46; C12P1/04
Application Number:
RU19940025647 (19940708)
Priority Number: RU19940025647 (19940708)
Family: RU2059716
563/1006
61. SU771152 - 15.10.1980
LACTABACILLUS PLANTARUM 109 STRAIN AS BACTERIOCIN P 109 PRODUCENT
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=SU771152
Inventor(s):
FILIPPOV VIKTOR A (--)
Applicant(s):
POLTAV MED STOMATOLOG INST (SU)
IP Class 4 Digits: C12D
IP Class:
C12D9/22
Application Number:
SU19782685277 (19781003)
Priority Number: SU19782685277 (19781003)
Family: SU771152
564/1006
62. SU854981 - 15.08.1981
LACTOBACILLUS ACIDOPHILUS 17 STRAIN AS BACTERIOCIN A17 PRODUCENT
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=SU854981
Inventor(s):
FILIPPOV VIKTOR A (--)
Applicant(s):
POLTAV MED STOMATOLOG INST (SU)
IP Class 4 Digits: C12N; C12P; C12R
IP Class:
C12P1/04; C12R1/23; C12N1/00
Application Number:
SU19792774106 (19790528)
Priority Number: SU19792774106 (19790528)
Family: SU854981
565/1006
63. SU877927 - 15.08.1982
STRAIN LACTOBACILLUS ACIDOPHILLUS 79 PRODUCER OF BACTERIOCIN A79
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=SU877927
Inventor(s):
FILIPPOV V A (--)
Applicant(s):
POLTAV MED STOMATOLOG INST (SU)
IP Class 4 Digits: C12N; C12P; C12R
IP Class:
C12R1/23; C12N1/00; C12N1/10; C12P1/02
Application Number:
SU19792881409 (19791122)
Priority Number: SU19792881409 (19791122)
Family: SU877927
566/1006
64. SU896071 - 07.01.1982
LACTOBACILLUS ACIDOPHILUS 578 STRAIN AS BACTERIOCIN A-578 PRODUCENT
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=SU896071
Inventor(s):
FILIPPOV VIKTOR A (--)
Applicant(s):
POLTAV MED STOMATOLOG INST (SU)
IP Class 4 Digits: C12P; C12R
IP Class:
C12P1/04; C12R1/23
Application Number:
SU19802899389 (19800324)
Priority Number: SU19802899389 (19800324)
Family: SU896071
567/1006
65. US4142939 - 06.03.1979
METHOD OF PRODUCING AN R-TYPE BACTERIOCIN AND ITS USE IN THE DETECTION
OF SPECIFIC MICROORGANISMS
URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=US4142939
Inventor(s):
Morse Stephen A. (US); Iglewski Barbara (US)
Applicant(s):
Oregon State Board of Higher Education An agency of the State of Oregon (US)
IP Class 4 Digits: C12D
IP Class:
C12D13/06
Family: US4142939
Abstract:
A METHOD OF DETECTING A SPECIFIC MICROORGANISM COMPRISING CONTACTING SAID
MICROORGANISM WITH BACTERIOCINS FROM A MICROORGANISM OF A GENUS WHICH IS
TAXONOMICALLY UNRELATED TO SAID SPECIFIC ORGANISM. THE RESULT OF SUCH CONTACT
MAY BE UTILIZED TO DETECT THE PRESENCE OF A MICROORGANISM BELONGING TO A
TAXONOMICALLY UNRELATED GENUS. RADIO-LABELED OR FLUORESCEIN-LABELED
BACTERIOCINS CAN BE REACTED WITH SPECIFIC BACTERIA IN A BIOLOGICAL SAMPLE AND
THE PRESENCE OF SUCH SPECIFIC BACTERIA DETECTED BY REMOVING EXCESS
BACTERIOCINS AND DETERMINING THE PRESENCE OF FLUORESCENT OR RADIOACTIVE
BACTERIA IN THE SAMPLE. NEISSERIA GONORRHOEAE IS IDENTIFIED BY SPOTTING
BACTERIOCINS ON A PLATE OF CLINICAL MATERIAL; OR USING A DISK IMPREGNATED WITH
BACTERIOCINS PLACED ON A PLATE INOCULATED WITH THE CLINICAL MATERIAL; OR THE
BACTERIOCINS CAN BE INCORPORATED INTO ONE-HALF OF A SPLIT AGAR PLATE, THE
IDENTIFICATION BEING MADE ON THE BASIS OF A ZONE OF INHIBITION SURROUNDING THE
SPOT WHERE THE BACTERIOCINS WERE APPLIED, OR GROWTH INHIBITION ON THE PORTION
OF THE PLATE TO WHICH THE BACTERIOCINS WERE ADDED.Description:
568/1006
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of diagnostic microbiology and particularly to diagnostic methods
for detecting the presence of, and typing of, a microorganism belonging to a specific genus in a
biological sample, and to methods for identifying antigens which are common to taxonomically
unrelated genera.
2. Brief Description of the Prior Art
The presence of bacteriocin-like activity in isolates of Neisseria gonorrhoeae has been reported by
Flynn and McEnteggart, J. Clin. Path. 25:60-61; (1971). Substances from other organisms have also
been reported to exhibit bacteriocin-like activity against N. gonorrhoeae. Volk and Kraus, Brit. J.
Vener. Dis. 49:511-512 (1973) reported the in vitro inhibition of N. gonorrhoeae by a substance from
N. meningitidis. Geizer, J. Hyg. Epid. 12:241-243 (1968) has reported the inhibition of gonoccocal
growth by unidentified substances produced by strains of a number of organisms including
Pseudomonas aeruginosa. The bacteriocin-like activity exhibited against many strains of N.
gonorrhoeae was attributed to the production of inhibitory levels of free fatty acids and
lysophosphatidyethanolamine as reported by Walstad, et al., Infect. Immunity, 10:481-488 (1974).
(a) Origin and Structure of Bacteriocins
Bacteriocins are a group of specific bacteriacidal substances produced by many bacterial during
growth. They are proteins of varying molecular weight. Bacteriocins have antibiotic properties, but in
contrast to antibiotics which are in clinical use, are much more specific, acting only on members of
the same or closely related species. They are extracellular substances which become bound to
receptor sites of susceptible organisms. Bacteriocins usually remain contained within the producer
strain until released by cell lysis. These extracellular substances can then bind to receptor sites of
susceptible organisms. Some bacteriocins closely resemble parts of bacteriophage when examined
microscopically. Their production can be induced by agents which interfere with metabolism, such
as ultra violet light, mytomicin C, nitrogen mustard and many other agents.
There are two basic types of bacteriocins. One type is a small molecule which is thermo-stable,
which cannot be sedimented in the ultra centrifuge, and is not easily resolved by the electron
569/1006
microscope. The other, R-type, is larger and resembles phage tails. This difference in basic types is
well illustrated by comparing Colicin V with Colicin 15 (colicins being bacteriocins specific to coliform
organisms). Colicin V forms a dialyzable product which has a low molecular weight. Colicin 15 is
sedimentable, has a molecular weight of 200,000 and, on electron microscopy, resembles the tail
structure of a phage.
Small quantities of bacteriocins are released in normal cultures of organisms, and are presumably
released during normal lysis found during degeneration of organisms in culture. Their genetic
determinants exist as an extra chromosomal element which replicate in phase with bacterial
chromosomes, and therefore persist as long as the strain persists. They are released in quantity by
lysis of the bacterial cell whether this occurs by phage infection, the action of bacteriolytic agents,
such as metabolic inhibitors, or other factors.
Chemically, all bacteriocins are macromolecular and contain polypeptide, protein, other radicals
such as carbohydrate, phosphate and lipopolysaccharide which contributes to the ultimate size of
the molecule.
(b) Nomenclature
While classification and nomenclature are necessarily undergoing change as more evidence of their
origin, chemistry and activities accumulate, bacteriocins are named, as a general rule, on the
specific rather than generic name of the originating organisms. For example, E. coli bacteriocins are
termed colicins. Serratia marcescens give marcesins; Enterobacteraerogenes, aerocins;
Pseudomonas aeruginosa (pyocyaneus), pyocins; Listeria monocytogenes, monocins;
Staphylococcus sp., staphylocins, etc.
This classification began after Gratia in Belgium first reported that filtrates of a particular strain of E.
coli inhibited growth of the same species, the inhibiting factor being called a colicin. Some 20
colicins were subsequently recognized and classified as A-V. Each colicin was specific for a small
group of strains of Enterobacteraciae. Each bacteriocin whether from E. coli or other species
appears to be specific in action to the same, or to taxonomically related, species of organisms.
(c) Assay of Bacteriocins
570/1006
The concentration of bacteriocin in a filtrate titrated by placing a drop (10-20 .mu.l) on a lawn culture
inoculated witn indicator bacteria (10.sup.7 /ml) of freshly grown cells. After incubation for 18-24
hours at 37.degree. C the plates are read and scored. Titers are regarded as a reciprocal of the
highest dilution that yields a clear spot. Another method is to add bacteriocin to an enumerated
excess of sensitive organisms, the bacteriacidal activity being proportional to the quantity of
bacteriocin present.
One unit of bacteriocin activity is the lowest concentration which completely inhibits growth of an
indicator strain.
At the present time purification is more a matter of concentration from the original broth or saline
suspensions then isolation of specific fractions. The most commonly used method to remove the cells
by centrifugation after 6-24 hours of incubation. Purification is completed by column chromatography
following ammonium sulfate precipitation followed by dialysis against equilibration buffer.
Purified bacteriocins are stable in a lyophilized state for long periods of time. In solution they are
stable at 4.degree. C and pH 7.0 for 6 weeks. Bacteriocin are not irreversibly denatured by 4M
guanidine thiocyanate or 6M urea, but are completely inactivated at 60.degree. C and pH 7.0 in 60
minutes.
(e) Mode of Action
Bacteriocins act on cells which are in the logarithmic phase of proliferation. The treatment of sensitive
cells rapidly inhibits incorporation of labeled leucine and thymidine into acid insoluble materials. The
time required for inhibition of .sup.14 C and .sup.3 H labeled isotopes is dependent upon the
concentration of bacteriocins. It has been established that both DNA and protein synthesis are
blocked by bacteriocin. When organisms in cultures are exposed to bacteriocin, they do not
incorporate .sup.14 C leucine into the protein or .sup.3 H thymidine into DNA. This is probably due to
interference with the active transport of leucine and thymidine by the specific bacteriocin.
Concurrently the concentration of ATP falls to 10-15% of control value. This is not related to a decline
in macromolecular synthesis, but does help to explain the faltering energy transport mechanisms
which are seen in bacterial cells exposed to bacteriocins. Though ATP activity is inhibited, the
phosphotransferase system is not affected and a-methyl D-glucoside has been shown to accumulate
in coliforms. Apart from these modes of activity, interference with cell membrane integrity may occur
in some species of susceptible organisms.
571/1006
SUMMARY OF THE INVENTION
An object of the present invention is to provide a new and unique method for inhibiting the growth of
microorganisms. Another object is to provide an improved test for detecting the presence in a
biological sample of a microorganism belonging to a particular genus. It is a further object to provide
an improved test for the identification of Neisseria gonorrhoeae. Another object is to provide a means
for typing of Neisseria gonorrhoeae. A still further object is to provide a test for the identification of
common bacterial antigens. A still further object is to provide test means for demonstrating the
presence or absence of common antigens or surface components, mammalian cells. A still further
object is to provide a new and unique pyocin which inhibits the growth of N. gonorrhoeae.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention will be more fully understood by the following description and the attached drawings,
in which:
FIG. 1 is a chart illustrating purification of R-type pyrocin (611 131) by DEAE-cellulose
chromatography. Fractions containing inhibitory activity are indicated by A and B. The insert shows
the induction of pyocin production in Pseudomonas aeruginosa ATCC 29260 by mitomycin C
(1 .mu.g/ml). The arrow indicates the time of mitomycin C addition.
FIG. 2 is an electron micrograph of a negative-stained preparation of R-type pyocin 611 131.
Symbols: uc uncontracted pyocin; c. contracted pyocin; Bar = 0.l .mu.m.
FIG. 3 is a chart showing the effect of R-type pyocin (611 131) on the growth of N. gonorrhoeae strain
72H870. Purified pyocin or mitomycin C were added to exponentially growing cultures (1.4 .times.
10.sup.8 CFU/ml) of strain 72H870. Symbols: 0, no additions; .quadrature. 400 units of
pyocin/ml; .DELTA. 1,000 units pyocin/ml; 4,000 units of pyocin/ml; 20,000 units of pyocin/ml.
FIG. 4 is a micrograph showing interaction of R-type pyocin 611 131 wiht cells of N. gonorrhoeae
strain 72H870. Bar = 0.1 .mu.m.
FIG. 5 is a photograph showing inhibition of Neisseria gonorrhoeae by an R-type pyocin (611 131).
Symbols: a. N. gonorrhoeae strain JW-31; b. N. Gonorrhoeae strain 72H870; c. N. gonorrhoeae strain
572/1006
1138 (colony type T-1); d. N. gonorrhoeae strain 1138 (colony type T-4); e. N. Gonorrhoeae strain
CS-7; f. P. aeruginosa strain ATCC 29260.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the invention, the foregoing and other objects are accomplished as hereinafter
described.
One embodiment of the invention is represented by a method for inhibiting the growth of a
microorganism belonging to a specific genus which method comprises contacting that organism with
a bacteriocin from a microorganism of a taxonomically unrelated genus, which binds to the first said
microorganism, that method being hereinafter described as follows:
Bacteriocin, Organisms and Media
The bacteriocin was an R-type pyocin (611 131) obtained from a strain of P. aeruginosa (ATCC
29260).
The basal medium contained the following per liter: proteose peptone no. 3 (Difco), 15 g; K.sub.2
HPO.sub.4, 4 g; KH.sub.2 PO.sub.4, 1 g; NaCl, 5 g; and soluble starch, 1 g. The final pH of the
medium was 7.2. When used for the production of R-type pyocins by P. aeruginosa, glycerol (1%
vol/vol) and monosodium glutamate (8.46 g/liter) were added after autoclaving. When used for the
growth of Neisseria spp., growth factor supplement (1% vol/vol), identical in composition to
IsoVitaleX enrichment (BBL) but lacking glucose; NaHCO.sub.3 (42 mg/liter), and glucose (5 g/liter),
was added after autoclaving. GC agar (Difco) plates containing glucose (5 g/liter) and growth factor
supplement (1% vol/vol) were used where indicated.
Induction and Purification of R-type Pyocin (611 131)
An overnight culture of P. aeruginosa ATCC 29260 was centrifuged (2,100 .times. g for 10 min.) and
resuspended to one-tenth the original volume in a solution containing 0.85% NaCl and 0.1% cysteine
hydrochloride at pH 6.5. A 1% (vol/vol) inoculum was used and the culture incubated on a gyrotory
shaker at 37.degree. C. When the turbidity of the culture reached approximately 150 Klett units,
mitomycin C was added at a final concentration of 1 .mu.g/ml. Incubation was continued until
extensive lysis of the culture occurred, this normally occurring within 3 hours after the addition of
573/1006
mitomycin C. The mitomycin C-induced culture was centrifuged at 2,400 .times. g for 30 minutes to
r