AN ABSTRACT OF THE THESIS OF
for the degree of
Benkerroum, Noreddine
Philosophy
in
3, 1992
Title:
Microbiology
Doctor of
presented on
January
Application of Bacteriocins Produced by
Lactic Acid Bacteria in Preserving Dairy Products and Development of
a Selective Medium for Leuconostoc Isolation
Redacted for Privacy
Abstract Approved:
William E. Sandine
Screening lactic acid bacteria for bacteriocin production against
monocytogenes was carried out with 324 strains isolated
from different sources. Four of the five producer strains were
identified as Lactococcus lactis subsp. lactis and the fifth was a
Listeria
Lactobacillus species.
Antimicrobial substance produced by three of
the Lc. lactis strains was nisin.
The fourth (lactococcin LBIIA) was
unique and differed from nisin by sensitivity
to trypsin
and a
chymotrypsin, lack of inhibitory activity against Micrococcus flavus,
and resistance to 16 hours of exposure at pH 10.0. The antimicrobial
substance produced by the Lactobacillus sp. was not protein in
nature; it also was not an organic acid, hydrogen peroxide, or a
bacteriophage.
Nisin was lethal to nine strains of three species of Listeria, including
pathogenic L. monocytogenes.
Effect of nisin on L. monocytogenes in
cottage cheese and yogurt was investigated.
Nisin inhibited this
pathogen in both products.
Fermentation of milk by Streptococcus.
thermophilus and Lactobacillus bulgaricus to produce yogurt was
inhibited by nisin at concentrations higher than 50 IU/ml; less than
50 IU/ml could be used to inhibit growth of L. monocytogenes in
yogurt and cottage cheese.
Use of nisin at 40 IU/ml to extend the shelf-life of Feta cheese
manufactured with starter containing S. thermophilus and L
bulgaricus also was studied. In comparison to control cheese made
without nisin, the nisin-containing cheese actually spoiled more
rapidly, apparently by limiting the growth of L. bulgaricus and
allowing contaminating Gram-negative psychrotrophs to grow with
.
less competion.
Attempts to study bacteriocin production by dairy Leuconostoc (Leu)
strains isolated from natural vegetation was
unavailability
of a
hindered
suitable selective isolation medium.
by
the
Such an
effective medium, was formulated containing 1% glucose, 1% bacto
peptone, 0.5% yeast extract, 0.5% beef extract, 0.25% gelatin, 0.5%
calcium lactate, 0.05% sorbic acid, 75 gg/m1 sodium azide, 0.25%
sodium acetate, 0.1% Tween 80, 15% tomato juice, 30
1.1 g /m1
vancomycin, 0.20 µg /ml tetracyline, 0.05% cysteine hydrochloride
and 1.5% agar.
Over 100 strains of Leu. cultures were easily isolated
from dairy products and vegetables using this medium. Cultures of
Leu. dextranicum and Leu. cremoris produced the same number of
colonies in this medium as in MRS agar.
Application of Bacteriocins Produced by Lactic Acid Bacteria in
preserving Dairy Products and Development of a Selective Medium
for Leuconostoc Isolation
by
Noreddine Benkerroum
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy in Microbiology
Completed January 3, 1992
Commencement June, 1993
APPROVED:
Redacted for Privacy
Professor of Microbiology in charge of major
Redacted for Privacy
He 2
of Depart
t of Microbiology
Redacted for Privacy
Dean of Graduate" School
Date thesis is presented
Typed by,
January 3, 1992
Deborah Norbraten
DEDICATION
To my parents and my family
for their love.
To Mrs. Susan Sandine for her care.
To the Nelsons and the Bonys (France) for their friendship
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to Dr. W. E. Sandine for
his guidance, assistance and fatherly concern.
I wish to also thank Dr. Tantaoui-Elaraki. A, of the Institute of
Agronomy and Veterinary Medicine Hassan II (Morocco) for his
advice, encouragement and friendship,
All the students of the Institute of Agronomy and Veterinary
Medicine Hassan II (Morocco), and the University of Kadi Ayyad
(Marrakech) who helped in carrying out experiments at one time or
another during this research,
All the faculty, Staff, and student body of the Department of
Microbiology who helped in the accomplishment of this work and for
making my stay enjoyable and memorable.
I acknowledge the USAID in their financial support of this
project.
The dedication of Mrs. Deborah Norbraten for typing the
manuscript in such a short time is greatly appreciated.
TABLE OF CONTENTS
PAGE
INTRODUCTION
Literature Cited
1
11
CHAPTER 1: Bacteriocin Production by Lactic
Acid Bacteria
16
Abstract
Introduction
Materials and Methods
17
18
21
31
Results
Discussion
Literature Cited
of Nisin on Growth and
Survival of Listeria monocytogenes
in Cottage Cheese and Yogurt
39
60
CHAPTER 2: Effect
Abstract
Introduction
Materials and Methods
67
Results and Discussion
Literature Cited
68
69
71
78
98
of Nisin on the Keeping
Quality of Feta Cheese
103
CHAPTER 3: Effect
Abstract
Introduction
Materials and Methods
Results
Discussion
Literature Cited
104
105
107
110
112
119
CHAPTER 4: Development and Use of Selective
Medium for Isolation of
Leuconostoc Bacteria from
Vegetables and Dairy Products
Abstract
Introduction
Materials and Methods
Results and Discussion
Literature Cited
BIBLIOGRAPHY
122
123
124
126
130
140
144
LIST OF FIGURES
FIGURES
Figure 1.1:
Figure 1.2:
Figure 1.3:
PAGE
Effect of pH and heat on the antimicrobial
substance produced by Lactococcus lactis
LBII.
53
Growth of Listeria monocytogenes (O.D. at
600 nm) in MRS broth and in Lactococcus
lactis LBII cell-free-supernatant.
54
Dynamics of LBII Inhibitory Substance (IS)
production as a function of time, pH and
growth (O.D.)
Figure 1.4:
Associative growth of Listeria
monocytogenes and Lactococcus lactis LBII in
MRSYG and in reconstituted non-fat dry
milk.
Figure 1.5:
Figure 1.6:
Figure 1.7:
Figure 2.1:
Figure 2.2:
55
56
Associative growth of Listeria
monocytogenes and Lactobacillus sp. Y5 in
MRSYG and in reconstituted non-fat dry
milk.
57
Plasmid patterns of lactococcus lactis LBII
and its derivatives after curing trials with 25
lag/m1 of ethidium bromide.
58
Plasmid patterns of Lactobacillus sp. Y5 and
its derivatives after curing trials at sublethal
temperatures.
59
Effect of pH and added nisin (37 x 102
IU/ml) on growth of Listeria monocytogenes
in Trypticase Soy Broth incubated at 37°C .
92
Effect of pH and added nisin (50 Mimi) on
growth of Listeria monocytogenes ATCC 7644
(log CFU/ml) in reconstituted nonfat dry milk
incubated at 37°C .
93
PAGE
FIGURES
Combined effect of pH and nisin
concentration on growth of Listeria
monocytogenes in Trypticase Soy Broth
incubated at 370C
94
Growth of Listeria monocytogenes 7644 at
4°C and 37°C in the presence and absence of
nisin (2.55 x 103 IU/g) in sterilized cottage
cheese.
95
Figure 2.5:
Effect of nisin on yogurt fermentation.
96
Figure 2.6:
Effect of nisin (50 IU /ml) on Listeria
monocytogenes in yogurt.
97
Figure 2.3:
Figure 2.4:
Figure 3.1:
Figure 4.1:
Effect of nisin on the shelf-life of Feta
cheese.
118
Plasmid profiles of Leuconostoc cremoris 444 and its cured derivative.
139
LIST OF TABLES
PAGE
TABLES
Table 1.1:
Indicator bacteria, their origin and culture
media used for their growth.
Table 1.2:
43
Number and percentage of strains of LAB
antagonizing growth of L. monocytogenes
Table 1.3:
Morphological and physiological tests used
for
bacteriocin
strain
producer
identification.
Table 1.4:
Identification
45
of
bacteriocin-producing
46
strains.
Table 1.5:
Table 1.6:
Table 1.7:
Table 1.8:
44
Effect of various enzymes on the inhibitory
activity of lactic acid bacterial strains against
L. monocytogenes.
47
Stability of inhibitory substances to different
treatments.
48
Stability of LBII and
substances to pH and heat.
Y5
inhibitory
Inhibitory activity of LBII and Y5
Gram positive bacteria and E. coli..
49
against
50
Activity assay of Lc. lactis LBII bacteriocin
after treatment with a -chymotrypsin on L .
monocytogenes and Streptococcus sp.
51
Table 1.10: Plasmid content of the producer strains
(Lactobacillus sp. Y5 and Lactococcus lacits
LBII) and their derivatives after curing
trials.
52
Table 1.9:
TABLES
Table 2.1:
PAGE
Diameter of inhibition zones appearing on
Petri plates seeded with
Listeria
sp. as
caused by nisin (70 ill added to assay well).
Table 2.2:
88
Minimum inhibitory concentration (pH 7.3)
of nisin on Listeria monocytogenes A T C C
7644 determined on Trypticase Soy Agar,
MRS agar at pH 6.8, milk agar and Trypticase
89
Soy Broth.
Table 2.3:
Effect of pH and added nisin (50 IU /ml) on
growth of Listeria
monocytogenes (log
CFU/ml) in reconstituted nonfat dry milk
incubated at 370C .
Table 2.4:
Influence
of
nisin
on
microbial
90
counts
developing on Listeria-selective-isolation
non-sterilized cottage cheese
contaminated with L. monocytogenes ATCC
7644 during incubation at 40C .
medium
Table 3.1:
in
Media and incubation conditions used in the
enumeration of different groups of
microorganisms in Feta cheese.
Table 3.2:
Table 3.3:
Table 4.1:
Table 4.2:
91
Variation
115
of pH during manufacture
and
storage of Feta cheese with and without nisin
added.
116
Chemical analysis of Feta cheese with and
without nisin added.
117
Bacterial and
yeast strains
used
in
the
present study and their origin.
134
Plasmid profiles of cultures used
13 5
TABLES
Table 4.3:
PAGE
Susceptibility
of
cultures
used
to
vancomycin:
Table 4.4:
136
Identification of Leuconostoc strains used in
this study.
Table 4.5:
137
Enumeration of Leu. dextranicum
Leu. cremoris
LuSM.
181
and
JLL8 on MRS agar and on
138
APPLICATION OF BACTERIOCINS PRODUCED BY LACTIC ACID
BACTERIA IN PRESERVING DAIRY PRODUCTS AND DEVELOPMENT OF
A SELECTIVE MEDIUM FOR LEUCONOSTOC ISOLATION
INTRODUCTION
Lactic acid bacteria (LAB) are a heterogeneous group of
microorganisms consisting of rod, coccus and cocco-bacillary shaped
bacteria.
They produce, from fermentable carbohydrates,
lactic acid,
either almost exclusively (homofermentative) or with other
metabolic endproducts (heterofermentatives).
They are also
typically Gram-positive and catalase-negative, though some strains
produce pseudocatalase.
Their natural habitats are milk and dairy
products, herbage, green vegetables, the intestinal tract and vagina
(36, 45).
Taxonomy of LAB has undergone considerable revision in
recent years as microbial classification systems are being based on
more powerful and reliable techniques such as computer assisted
numerical taxonomy and molecular biology.
Traditionally this group
of microorganisms was composed of four genera: Streptococcus,
Leuconostoc, Pediococcus and Lactobacillus. All belonged to the same
family Lactobacillaceae. In the latest completely revised Bergey's
Manual of Systematic Bacteriology (4), these bacteria were placed
into two different families: Lactobacillus remained in the family
Lactobacillaceae while the three other genera were placed in the
family Streptococcaceae. The most important and recent change
however, has been the establishment of a new genus, Lactococcus, to
replace and expand the Lancefield serological group N Streptococcus
genus.
This genus has been known and used by dairy technologists
2
and scientists for almost a century, since the isolation of a pure
culture of Streptococcus lactic in 1909 by Lohnis, as quoted by
Teuber and Geis (45). The new Lactococcus taxon was suggested in
1985 by Schleifer et al. (37) and was validated in 1986 by the
International Union of Microbiological Societies (1).
establishment of this new genus
as well as
Reasons for
detailed information
about its characteristics and those of individual species are provided
by Schleifer et
al.
(37) and Sandine (34).
genera mentioned above, some
In addition to the four
microbiologists are inclined to
consider Bifidobacterium as a fifth genus of LAB since its members
also produce lactic acid from carbohydrates.
They also are Gram-
positive, catalase-negative and have the same natural habitat as
most lactobacilli (5, 24). In fact, Bifidobacterium was for many years
considered as a species of the genus Lactobacillus (5). Weiss and
Rettger (47) even claimed it to be a biovariety of Lb. acidophilus,
which has been strongly objected to by a number of contemporary
investigators (5, 6, 29, 39). Therefore, further strain characterization
was needed to provide data to serve as a basis for clarifying this
matter.
Briggs (6) and Sharp (39) studied 390 and 442 strains,
respectively, for this purpose and they
both
found
extreme
variability among bifidobacterial strains as to their physiological and
then became clear that they were
dealing with a separate group of microorganisms, distinct from
lactobacilli. Therefore, attempts were made to reclassify them into
serological characteristics.
It
another family, but unsuccessfully since the physiological and
serological tests used in these studies proved insufficient to provide
definitive answers. Hence, bifidobacteria continued to belong to the
3
genus Lactobacillus as a single species called Lb. bifidus until the
1970's.
As
molecular
biology
techniques
became
available,
bifidobacteria were placed as a separate genus Bifidobacterium in
the family Actinomycetaceae on the basis of the moles percent of
guanine and cytosine in their DNA and also on the basis of 16S
ribosomal RNA homology. Other species of the genus Streptococcus
(Str.) are also known as LAB, namely Str. faecium and Str. faecalis
(9, 10, 31, 34) of the Enterococcus group and Str. thermophilus (34)
and Str. bovis (23) of the so-called "Other Streptococci" group in the
newly revised Bergey's Manual of Systematic Bacteriology (4).
Previously
those
two
species
were
placed
in
the
Viridans
streptococcal group (28, 34, 40), which no longer exists. Str.
thermophilus has been extensively used in dairy fermentations in
combination with other lactic acid producing bacteria, such as Lc.
lactis subsp. cremoris (previously called Str. cremoris), in Cheddar
cheese manufacture in Australia (9) and with other thermophilic LAB
such as Lb. bulgaricus, Lb. helveticus or Lb. acidophilus in yogurt and
Feta cheese manufacture (34, 30).
Str. faecalis and Str. faecium also
have been used in dairy fermentations (9, 31).
shown to offer
advantages
These strains were
over Lactococcus species
in the
manufacture of some cheeses, especially Cheddar cooked at high
Some of these advantages were: higher
temperature (420C).
resistance to cooking temperature and the salt level normally used,
greater resistance to bacteriophages, and better genetic stability (9).
Other investigations on the use of Str. faecalis and Str. faecium in
Cheddar cheese showed that Str. faecium provided a better quality of
cheese than the regular commercial starter, while Str. faecalis
4
contributed the same or a less desirable quality (31). However, the
use of these two species in cheesemaking was highly controversial
for two reasons: 1) they belong to the Enterococcus group, organisms
from which are used as indicators of fecal contamination in foods
(Jacquet, personal communication); 2) Str. faecalis is I3- haemolytic
and
associated
endocarditis (28).
with
urinary
tract
infections
and
subacute
Also Str. faecium occasionally is associated with
endocarditis and some strains produce biogenic amines resulting in
scombroid disease (34). This contrasts with the fact that LAB are
Generally Recognized as Safe (GRAS).
(28)
these two species are not
contamination.
However, according to Mundt
in reality associated with
fecal
Moreover, 13-haemolysin production by Str. faecalis
and amine production by Str. faecium are plasmid mediated (28), so
cured strains could be used in fermentations.
This would allow non-
haemolytic and non-amine producing variants of both species to be
Str. bovis was also
isolated and used in dairy fermentations.
considered by Jones et al. (23) as a homofermentative lactic acidproducing bacterium. These authors showed that Str. bovis was a
better inoculant in silage than commercial inoculants composed of
homo and heterofermentative LAB. In fact, this species was reported
to be the least fastidious in the genus Streptococcus (20). Therefore,
it grew rapidly, resulting in a swift decrease of pH, with lactic acid as
the major product from carbohydrate breakdown. These are ideal
conditions in silage. Other LAB inoculants were shown to produce
appreciable amounts of ethanol and acetic acid, which are not
desirable in silage (23). To our knowledge, Str. bovis has not been
used in dairy fermentations so far.
5
Although
a
detailed
presentation
on
the
taxonomic
recent
modifications in the genus Streptococcus will not be offered herein, it
seems appropriate to mention some of these changes, especially
those related to organisms used in the research described in this
As mentioned earlier, the most important change recently
thesis.
made was the establishment of the new Lactococcus genus and its
validation by the International Union of Microbiological Societies in
1986 (1).
This
genus was not recognized even
in the latest
completely revised Bergey's Manual of Systematic Bacteriology, (4)
where the previous nomenclature of lactic streptococci was still used.
It
was
mentioned,
however, that
the
of all
"transfer
streptococci (group N) to the genus Lactococcus
is
lactic
in reasonable
agreement with findings from numerical taxonomy".
was probably edited before the new taxon was validated.
The manual
Also, the
lactic streptococci in the revised Bergey's Manual (4) consist of only
two
species: Str lactis and Str.
raffinolactis, while in the new
taxonomy proposed by Schleifer et al. (37) the Lactococcus (Lc.)
genus, replacing the former Streptococcus (group N) genus, consists
of 4 species: Lc. lactis, Lc. garviae, Lc. plantarum and Lc. raffinolactis.
Further changes also were made in the taxonomy of Streptococcus
genus in this new Bergey's Manual. Because of the diversity of
members of this genus and for convenience, Sherman (40) divided
this genus into 4 groups: Pyogenic, Viridans, Lactic and Enterococcus.
This division proved useful for over 50 years. Now, in the new
Bergey's Manual, this genus is divided into 6 categories: Pyogenic,
Oral, Enterococcus, Lactic, Anaerobic and Other Streptococci.
New
species, strictly anaerobic, were included in the Anaerobic group,
6
whereas, species perviously in the Viridans group were moved to the
Other Streptococci group.
From the above discussion two main conclusions may be made:
1).
The definition of LAB provided by Or la-Jensen in 1919 (29)
and quoted by Kandler (24) is still used by workers today:
"Gram-positive, non-sporing, microaerophilic bacteria whose
main fermentation product from carbohydrate is lactate".
definition
is
rather vague.
streptococci,
All
This
including
pathogenic strains and even members of other genera, fall into
this group as so defined. Also, Bifidobacterium species would
be excluded since they are strict anaerobes.
propose the following modification
Therefore we
which
hopefully will
provide well-defined boundaries for this group:
LAB are non-
pathogenic,
forming
Gram-positive,
bacteria
whose
catalase-negative,
main
product
of
non-sporecarbohydrate
fermentation is lactic acid.
2)
In
view of the recent taxonomic
changes, LAB are now
composed of the following genera:
Lactococcus (Lc.), all species included.
Lactobacillus (Lb.), all species included.
Pediococcus (Pd.) all species included.
Leuconostoc (Leu.), all species included.
Bifidobacterium (B.), all species included.
Streptococcus (Str.), only characterized, non-pathogenic strains
of Enterococcus and Other Streptococci.
7
This preoccupation with the taxonomy of LAB is not without
justification.
Indeed LAB have been most useful to humans from the
beginning of mammalian life.
A long time before their existence was
even suspected, ancient civilizations were using them in their food
supplies either for preservation or to enhance the quality of some
foods (38). Cheesemaking practices may be the best and the oldest
example of the use of LAB in foods during early civilization dating
from 6000 to 7000 BC (38). Now that LAB are well known and well
characterized, they are being found to be of genuine benefit to the
well-being of humans.
LAB are used in the fermentation of many foods, including milk,
green olives, cucumbers, cabbage, etc. (8,
12,
16, 45), meats, (2, 41,
42), wine (22, 25) and other products (42, 44). Their primary role in
the fermentation process is the production of lactic acid with a
concomitant pH decrease.
However, they may play other roles,
namely inhibition of undesirable bacteria (e.g. spoilage or pathogenic
bacteria), and enhancement of the texture and the flavor.
In
addition to lactic acid, they also produce a variety of aromatic
substances including acetic acid, ethanol, diacetyl, acetaldehyde, and
dimethyl sulfide (44, 46).
In food preservation they are used to
extend the shelf-life and to control pathogens (17).
by several means.
This is achieved
The most common is the decrease of pH to a level
They also
produce a variety of inhibitory substances, including organic acids,
hydrogen peroxide, diacetyl, and bacteriocins (see Hurst (21) and
Bacteriocin production has received
Daeschel (11) for reviews).
where growth of undesirable bacteria is restricted.
8
considerable attention by dairy researchers in the last decade.
Nisin
bacteriocin known and used for many years in
numerous countries as a food additive to extend the shelf-life or to
was the first
control specific microorganisms such as clostridia
(43), Listeria
monocytogenes (3) and other Gram-positive pathogens (13,
14, 15,
21).
LAB also have been shown to have a positive impact on human
health when consumed as viable cells (probiotic).
The linkage of life
expectancy to yogurt consumption popularized by Metchnikoff (27)
stimulated research in this area.
Relevant literature reviews have
been written with an emphasis on health roles for lactobacilli and
These
bifidobacteria (33, 35, 36) in humans and/or animals.
microorganisms colonize the intestine and are reported to have
different effects.
They produce substances inhibitory to pathogenic
microorganisms, reduce the cholesterol level in the blood serum of
pigs (18, 19, 33), and alleviate lactose intolerance in Vgalactosidase-
They also have been shown to have an
anti-cancer effect (7, 33) and to stimulate the immune system (32,
33, 35). For these reasons consumption of fermented dairy products
has been advocated by some physicians, scientists and non-scientists.
In fact, lactobacilli now are sold in pharmacies and health food stores
under a variety of names and to treat a number of illnesses. Today
this is an intensive research area and more facts demonstrable in
deficient persons (26, 33).
different laboratories are required to substantiate all the health
claims.
9
Since the 1970's, much research has been done on the genetics of
LAB.
These bacteria have been found to harbor plasmids encoding
for important metabolic functions. These plasmids cause genetic
instability in lactic acid producing bacteria but also make possible
genetic manipulation of the organisms.
The aim of the work presented in this thesis was to study the
practical use of LAB to control pathogens (mainly Listeria
monocytogenes) in some dairy products by their production of
bacteriocins as well as to study aroma substances produced by
Leuconostoc.
During the latter study a new selective medium for
Leuconostoc was developed.
Four chapters are presented herein:
Chapter 1:
Bacteriocins produced by lactic acid bacteria
Chapter 2:
Effect of nisin on growth and survival of Listeria
monocytogenes in cottage cheese and yogurt.
Chapter 3:
Effect of nisin on keeping quality of Feta cheese.
Chapter 4:
Development and use of selective medium for
isolation of Leuconostoc bacteria from vegetables
and dairy products.
Research carried out for this thesis was done at three locations.
Work on bacteriocins produced by strains of lactic acid bacteria
isolated from nature (Chapter 1) was done in Rabat, Morocco at the
Institute of Agronomy and Veterinary Medicine, Hassan II; research
on the use of the lactococcal bacteriocin nisin (Chapter 2) to inhibit L.
10
monocytogenes
cheese was done at Oregon State
University and to inhibit this pathogen in yogurt was done in
in cottage
Morocco; studies on the use of nisin in Feta cheese (Chapter 3) were
done at the Federal Dairy Research
Center in Kiel, Germany;
development of the selective Leuconostoc isolation medium (Chapter
4) was done in Morocco as for Chapter 1.
There is great interest today in the use of natural preservatives to
stabilize and protect foods; bacteriocins are the most studied in this
regard. The emphasis for this thesis has been to isolate lactic acid
bacteria which produce bacteriocins and to examine their possible
commercial value. Also, one known bacteriocin (nisin) was used to
inhibit a known pathogen (L.
Efforts
to
isolate
monocytogenes ) in dairy products.
naturally-occurring
bacteriocin-producing
Leuconostoc were thwarted by the unavailability of a useful selective
medium.
Therefore in the latter phases of this research, such a
medium was developed and will find application in future work to
isolate the desired Leuconostoc strains.
11
LITERATURE CITED
Validation of the publication of new names
and new combinations previously effectively published outside
the IJSB. Int. J. System. Bacteriol. 36:354-356.
1.
Anonymous.
2.
Bacus, N. and W. L. Brown.
products.
1986.
1985.
The pediococci: meat
In S. E. Gilliland. (ed.), Bacterial Starter Cultures for
Foods. CRC Press, Boca Raton, Florida.
3.
Benkerroum, N., and W. E. Sandine. 1988. Inhibitory action of
nisin against Listeria monocytogenes. J. Dairy Sci. 71:32373245.
4.
Bergey's Manual of Systematic Bacteriology. 1986 Vol 2., P. H.
A. Sneath, N. S. Mair, M. E. Sharpe and J. G. Holt (eds.). William
and Wilkins, Baltimore, MD.
5.
Bezkorovainy, A. 1989. Classification of bifidobacteria. p. 228. In A. Bezkorovainy and R. Miller-Catchpole (eds.).
Biochemistry and Physiology of Bifidobacteria. CRC Press, Boca
Raton, Florida.
6.
Briggs, M.
Microbiol.
1953.
The classification of lactobacilli.
J. Gen.
9:234-245.
7.
Cole, C. B., and R. Fuller. 1987. The effect of dietary fat and
yogurt on colonic bacterial enzymes (13-glucosidase and [3glucoronidase) associated with colon cancer. Food Microbiol.
4:77-81.
8.
Costilow, R. N., F. M. Coughlin, E. K. Robbins, and W. T. Hsu.
1957.
Sorbic acid as a selective agent in cucumber
fermentations. II. Effect of sorbic acid on the yeast and lactic
Appl. Microbiol.
acid fermentations in brined cucumbers.
5:373-379.
9.
Coventry, M. J.
Aspects of the metabolism of
faecium and Streptococcus cremoris. Ph.D.
1983.
Streptococcus
Thesis, University of Melbourne, Melbourne, Australia.
12
10.
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of lactic acid bacteria in gas-exchanged, brined cucumbers.
Appl. Environ. Microbiol. 40:417-422.
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Delves-Broughton, J. 1990. Nisin and its application as a food
preservative: a review. J. Soc. Dairy Technol. 43:73-75.
14.
Delves-Broughton, J.
1990.
Nisin and its use as a food
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Antimicrobial substances from lactic
acid bacteria for use as food preservatives. Food Technol.
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Eckner, K. F. 1991.
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lactobacilli, pediococci, and leuconostocs: Vegetable products, p.
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17.
Gilliland, S. E.
18.
Gilliland, S. E. and D. K. Walker. 1990. Factors to consider
when selecting a culture of Lactobacillus acidophilus as a
dietary adjunct to produce a hypocholesterolemic effect in
humans. J. Dairy Sci. 73:905-911.
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Beneficial interrelationships between
certain microorganisms and humans: Candidate microorganisms
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21.
Hurst, A. 1983. Nisin and other inhibitory substances from
lactic acid bacteria, p. 327-351. In A. L. Branen, and P. M.
Davidson (eds.). Antimicrobials in Foods. Marcell Dekker, Inc.,
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22.
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Characterization of Leuconostoc oenos
Heatherbell.
isolated from Oregon Wines. Appl. Environ. Microbiol. 50:6806 84 .
23.
Jones, B. A., R. E. Muck, and S. C. Ricke. 1991. Selection and
application of Streptococcus bovis as a silage inoculant. Appl.
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Kandler, 0. 1983.
bacteria.
25.
Carbohydrate metabolism in lactic acid
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Lafon-Lafourcade, S., E. Carre, and P. Riberaeau-Gayon.
1983.
Occurence of lactic acid bacteria during the different stages of
vinification and conservation of wines.
Microbiol.
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Environ.
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Systematic Bacteriology. Vol 2. William and Wilkins, Baltimore,
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3 O.
Fate of Listeria
monocytogenes during the manufacture, ripening and storage
Papageorgiou, D. K., and E. H. Marth.
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Palmer, J. R.
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Master of Science Thesis, Iowa State
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34.
Sandine, W. E.
1988.
New nomenclature of the non-rod-
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3 6.
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Biochimie.
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Roles of Lactobacillus in the intestinal
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1972.
Lactic acid bacteria in food and health: a review with
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certain enteric diseases and their treatment with antibiotics
and lactobacilli.
3 7.
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35:691-702.
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and W. Fischer. 1985. Transfer of Streptococcus lactis and
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Appl. Microbiol.
6:183-195.
Applied Science
3 8.
Scott, R.
1981.
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3 9.
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Gen. Microbiol. 12:107-122.
40.
Sherman, J. M.
1937.
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15
41.
Smith, J. L., and S. A. Palumbo. 1983. Use of starter cultures in
meats. J. Food Protect. 46:997-1006.
42.
Smith, J. L., and S. A. Palumbo. 1981. Microorganisms as food
additives. J. Food Protect. 44:936-955.
43.
Sommers, E. B. and S. L. Taylor.
1987.
Antibotulinal
effectiveness of nisin in pasteurized process cheese spreads. J.
Food Protect.
50:842-848.
44.
Sugihara, T. F. 1985. The lactobacilli and streptococci: Bakery
products, p. 119-125. In S. E. Gilliland, (ed.), Bacterial Starter
Cultures for Food. CRC press. Boca Raton, Florida.
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Teuber, M., and A. Geis.
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Use of Gas-Liquid
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47.
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Appl. Bacteriol.
28:501.
16
Chapter
1
Bacteriocins Produced by Lactic Acid Bacteria
Noreddine Benkerroum*
and
William E. Sandine
Department of Microbiology
Oregon State University
Corvallis, OR 97331-3804
Running Title:
Bacteriocins
*Present address: Department of Food Microbiology, Institute of
Agronomy and Veterinary Medicine, Hassan II, Rabat, Morocco.
17
ABSTRACT
Lactic acid bacteria (LAB), 324 in number and including 101
identified Leuconostoc strains, were screened for bacteriocin
production against Listeria monocytogenes.
Five strains (1.5%) were
found to produce antimicrobial substances other than H202 or organic
acids.
Four of these strains were identified as Lactococcus (Lc.) lactis
Antimicrobial
and the fifth as a Lactobacillus (Lb.) species.
substances produced by Lc. lactis strains were confirmed to be
protein in nature by their sensitivity to proteases and they had a
narrow spectrum of action. Therefore, they were considered to be
bacteriocins. Three of these bacteriocins were nisin; the fourth was
similar to but distinct from nisin as determined by its different
sensitivity to proteases and its spectrum of action.
It has been
named Lactococcin LB11A. The fifth strain produced an inhibitory
substance not inactivated by catalase, not dialysable and with
narrow spectrum of action.
It
was not protein
therefore not considered to be a bacteriocin.
in nature
a
and
18
INTRODUCTION
One of the most important and significant values of LAB to humans is
their beneficial role in health.
They can act directly when consumed
as probiotics (57, 58, 59) or when incorporated in foods such as meat,
vegetables and dairy products, or indirectly by action of the
antimicrobial substances they produce in foods (8, 16, 24, 38, 39).
Several types of antimicrobial substances are produced by various
members of this group of microorganisms. The most well known are
organic acids, hydrogen peroxide, diacetyl and bacteriocins (8, 24,
38).
The latter have received increased research attention during
the last decade, these studies focusing mainly on the search for new
bacteriocins, their characterization, and purification.
An early
definition of bacteriocins was given by Jacob et al. (40) when
referring to colicins (i.e. the first bacteriocins found to be produced
by certain Escherichia coli strains) as proteins that act only on closely
related species.
Subsequently, a more specific definition was offered
by Tagg et al. (65) who stated that for an inhibitory substance to be
considered a bacteriocin it must have the following six
characteristics:
1) a narrow spectrum of action, 2) bactericidal mode
of action, 3) adsorption to specific receptors on cells, 4) production
and immunity are plasmid encoded, 5) lethal biosynthesis, 6) has an
As this definition was quite
restrictive and few "bacteriocins" could meet these requirements, the
same authors later suggested that bacteriocins produced by Gram
essential biological moiety for activity.
positive bacteria should at least be biologically active proteins with a
bactericidal mode of action against sensitive microorganisms.
Now,
19
the most widely accepted definition is that "bacteriocins are proteins
or protein complexes with a bactericidal mode of action directed
against species that are usually closely related to the producer
bacterium" (42).
It
is now known that bacteriocins are produced by numerous genera
of bacteria including those that are Gram-positive, Gram-negative, as
well as sporeformers (55).
At least four genera of LAB, Lactococcus
(32), Leuconostoc (35, 36, 45), Lactobacillus (7, 14, 27, 43) and
Pediococcus (13, 37, 50, 62) have been demonstrated to produce a
variety of bacteriocins, some of which are rather well characterized.
Among these, nisin is the most well known. It is produced by some
strains of Lc. lactis subspecies lactis.
Nisin has been used for many
years in other countries as a food additive to prevent spoilage (16,
20, 21, 24, 39, 54) but only recently has been approved in the United
States (26) for addition to processed cheese spread.
Intensive
research on nisin has been carried out recently, especially to clone
the gene encoding for its production (29, 31, 41, 63) and to
determine its mode of action on sensitive cells (20). The ultimate
goal of bacteriocin research is the legal use of these substances as
natural food preservatives.
Among the food-borne pathogens,
L.
monocytogenes recently has
caused health concerns, especially since this microorganism not only
survives temperature abuse (23) but grows in refrigerated foods at
2-5°C (11, 57). Therefore, intense research efforts have been made
to find means to control the occurrence and survival of this pathogen
20
in foods.
Nisin was reported to be effective in controling Listeria in
cottage cheese (12) and found to control these bacteria in yogurt
(Chapter
3
in this
thesis).
Studies on the sensitivity
of L
.
monocytogenes to bacteriocins other than nisin produced by LAB
have revealed this pathogen is sensitive to bacteriocins produced by
different species of the Lactobacillus (1, 36, 45, 61), Leuconostoc (35,
36, 45), Pediococcus (13, 36, 50, 62), and Lactococcus (15, 36, 45, 62)
genera.
The purpose of the present study was to survey LAB isolated from
raw milk and different dairy products produced in Morocco for the
production of bacteriocins and to characterize the producer bacteria
as well as the inhibitory substance produced.
21
MATERIALS AND METHODS
Bacterial Strains and Media:
Bacteria tested for bacteriocin production were isolated from raw
milk, Lben(1), raw butter(2), bakery yeast and pickle brine.
Except
for the butter, products were serially diluted in 0.85% saline and 0.1-
ml aliquots were surface spread plated on M17 (66), MRS (22) and
Elliker (25) agar media and then incubated for 24 hours at 30 °C .
Butter was melted at 400C and centrifuged at 4000 rpm for 10 min in
a Heraeus Sepatech centrifuge. The aqueous phase was serially
diluted and surface plated as described above. After incubation,
colonies were randomly selected, grown in MRS broth, Gram stained
and tested for catalase production. Gram negative and/or catalase
positive strains were discarded. Those to be studied were grown and
frozen at -200C in sterile 10% reconstituted nonfat dry milk.
Working cultures were transferred to MRS broth and slants of MRS
agar for short time storage. All incubations were at 300C, unless
otherwise indicated.
In addition to the above-described isolates, 101 Leuconostoc spp.
isolated from raw milk and vegetables on a newly developed
Leuconostoc selective medium formulated during the present
research were tested for bacteriocin production.
These strains were
identified as Leuconostoc according to tests recommended by Garvie
(30).
(1) Lben: A traditional Moroccan raw fermented milk, soured
spontaneously and then churned to separate the Lben from the butter( 2)
22
Indicator organisms used in this study (Table 1.1) were maintained
as stab cultures on TSA (Biokar) or MRS agar, at 40C and transferred
weekly.
Prior to use they were propagated in appropriate broth
media.
Detection of Bacteriocinogenic Strains:
Bacteriocin producers were screened against L. monocytogenes using
the spot and the well diffusion methods as described by Spelhaug
and Harlander (62) and Tagg and McGiven, (64), respectively.
In the
spot technique, an overnight culture of the test organism grown in
MRS broth supplemented with 2.5% yeast extract and 2% glucose
(MRSYG) was diluted 10 fold in 10 mM Tris HC1 (pH 7.0) and 2 [t1
aliquots were spotted onto M17 and MRS agar. Inoculated plates
were incubated until growth was evident, then overlaid with 5 ml of
TS soft agar (0.7% agar) seeded with 0.1-m1 of an overnight culture
of L. monocytogenes. Plates were incubated for an additional 18
hours then checked for clear zones around spots of the putative
producers.
In the well diffusion assay, 20 ml of Muller Hinton agar (Biokar), or
MRS soft agar in the case of LAB, were seeded with 0.1 ml of an
overnight culture of the indicator organism, poured in a sterile Petri
Plates were then put into the
dish and allowed to harden.
for 30 min to
Wells, 8mm in diameter, were
punched in the medium using stainless steel tubing and then they
refrigerator
were filled with 60
p. 1
1
hr.
of the test culture supernatant.
The
23
supernatant was prepared by growing the test organisms in MRSYG
broth for 24 hours; one ml of the culture was then transferred to a
1.5-m1 sterile Eppendorf tube and centrifuged in a microfuge (Fisher
Scientific, Model 235C).
At this point, filter sterilization of the
supernatant was avoided since
it has
been shown
that some
bacteriocins are retained on filter surfaces (52).
Strains selected for further study were identified by using the
following tests:
from
glucose
ammonia production from arginine, CO2 production
in
citrate-supplemented
(0.2%)
milk,
dextran
production, growth at different temperatures (10, 43 and 440C), milk
coagulation within
16
hours at 22 °C (fast cultures) and litmus
reduction.
Elimination of Organic Acids, Hydrogen Peroxide and Bacteriophages
as Inhibitory Agents:
The effect of organic acids was ruled out by adjusting the pH of the
test clear supernatant to 6.0 with 10 M NaOH which was then filter
sterilized with a 0.22-0 Millipore filter membrane and tested by the
well diffusion assay for the persistence of the inhibition zone.
To exclude the effect of hydrogen peroxide, catalase (EC1.11.1.6,
Sigma Chemical Co.) was used in two ways:
1.
Incorporation in the overlay agar, for the spot test, to a final
concentration of 68 IU/ml as described by Barefoot and
Klaenhammer (10).
24
2.
Treatment of the cell-free supernatant with
650 'Wm' of
catalase in Tris HC1 buffer (pH 8.0) for 2 hours at 370C.
were;
Controls
Tris HC1 buffer with and without enzyme, and non-
treated cell free supernatant to which has been added the same
quantity of buffer as in the reaction mixture. After treatment,
activity was assayed by the well diffusion method.
Evidence that the inhibitory action was not due to a bacteriophage
was obtained in two ways:
1
A lawn of the indicator strain was streaked with an inoculating
loop, which has been stabbed into
the zone of inhibition
surrounding the well, according to McCormick and Savage (47).
The plate was incubated until growth was evident and then
examined for plaque formation along the streak line.
2.
Phage titer of a 24-hour culture of the test organisms was
determined as described by Terzaghi and Sandine (66).
Mode of Action:
A modification of the cell diffusion assay was used.
Muller Hinton
agar (20 ml) was seeded with 0.1-ml of a sensitive indicator culture,
poured in a sterile Petri dish and allowed to harden. It was incubated
until growth was evident. Then wells were cut and filled with 60 ill
of cell free supernatant.
Incubation of plates was continued and then
they were examined for clearing around the wells at different time
intervals starting from 3 hours. Clearing indicated cell lysis.
25
Another procedure to test for bactericidal action was used.
M17 agar
(20 ml) supplemented with glucose (0.5%) and seeded with 0.1-m1 of
a sensitive indicator culture was incubated as in the first case until
growth was evident. The test culture was then streaked onto this
plate and incubation continued for an additional 24 to 48 hours and
then checked for clearing around the streak line.
In addition to these techniques, and for confirmation, growth of the
indicator strain in neutralized (pH 7) cell-free supernatant (CFS) was
monitored by following the optical density at
incubation.
600 nm during
The CFS was inoculated with 0.1-m1 of an overnight L.
monocytogenes culture and incubated.
Sterile MRS broth (10-ml)
was similarly inoculated with the indicator organism and incubated
to serve as a control. Samples of 1 ml each were aseptically removed
at 0, 2, 4, 7, 9, and 24 hours and their optical densities determined at
600 nm by using a Jenway PCO1 Colorimeter.
Action of Enzymes:
The cell free supernatants of possible bacteriocin producers adjusted
to pH 6 were treated separately with 6 different enzymes. Each
enzyme was obtained from Sigma Chemical Company except trypsin,
which was from Serva: trypsin (EC 3.4.21.4), a-chymotrypsin (EC
3.4.21.1), pronase E (Type XXV), RNase (EC 3.1.27.6), lysozyme (EC
3.2.1.17) and lipase (Type XIII). Trypsin, a-chymotrypsin, pronase
and lipase type XIII were dissolved in phosphate buffer (0.1 M, pH
6).
RNase was in 0.05 M Tris 15 mM NaC1, (pH 8) and lysozyme in 25
26
mM Tris (pH 8). Enzyme solutions were mixed with cell free
supernatants (1:1) to a final concentration of 1 mg/ml. Incubation
was at 37°C in a water bath for 2 hours; then samples were boiled
for 3 min to stop the reactions. This treatment did not terminate the
RNase reaction, which continued during the experiment. Controls
included sterile MRS broth, a 1:1 mixture of culture supernatant with
buffer by without enzyme and buffer with only the enzyme.
After
boiling, each sample was assayed for bacteriocin activity by the well
diffusion method.
Nisin
(Alpin
and
Barrett,
Trowbridge,
England)
to
a
final
concentration of 36 x 102 IU/ml was used along with the CFS for
comparison purposes.
Resistance to chloroform and heat:
Chloroform was thoroughly mixed with the producer culture (1:10)
and left at room temperature (25°C) for 1 hour. The mixture was
then centrifuged at 5000 rpm; chloroform was removed and activity
in the aqueous phase assayed by the well diffusion method.
Aliquots of cell free supernatants also were dispensed in different
test tubes and heated at 60°C for 30 min, 80°C for 30 min, 100°C for
10 min or autoclaved (121°C for 15 min). The samples were cooled
and assayed for activity. Unheated cell free supernatants were used
as positive controls.
27
Testing the effect of heat at various pH levels was accomplished by
using two series of test tubes, each containing 4 ml of clear culture
In each series, the pH of the supernatant was adjusted
to 2, 3, 4, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11 or 12 with concentrated
supernatant.
HC1 or 10 M NaOH. After pH adjustment, one series was boiled for 10
Supernatant from a non-producer strain was treated in the
same way but was not heated and was used as a negative control.
min.
After treatment, activity was assayed against L. monocytogenes.
Spectrum of Action:
The spectrum of activity against different bacteria was determined
by the spot technique (62) using M17 agar as a bottom layer and
MRS soft agar as the top layer when the indicator strains were a LAB
or soft TSA for other indicator microorganisms.
Those used as
indicators are listed in Table 1.1. Producer microorganisms were
also tested against each other and against themselves.
Dynamics of Bacteriocin Production:
Production of bacteriocins was monitored during growth of producer
strains at 300C in MRSYG broth by determination of arbitrary units
(AU) after 2, 4, 5, 7, 10, 24 and 48 hours of incubation. Reciprocal of
the highest dilution showing a definite
inhibition of an indicator
strain in the well diffusion assay was expressed an AU per 60 ml.
The pH of the culture as well as the OD at 600 nm were recorded at
the same intervals.
28
Associative Growth:
The effect of bacteriocin producers on growth of L. monocytogenes in
mixed culture was assessed in MRSYG and in 10% reconstituted
nonfat dry milk. Sterile MRSYG broth (20 ml) and 10% NDM
autoclaved at 121°C for 10 min were simultaneously inoculated with
0.1 ml of an overnight culture of L. monocytogenes and the producer
strain. They were then incubated and numbers of L. monocytogenes
determined on ASL medium (3) modified by omission of moxalactam.
Samples were taken at 0.5, 7, 9, 24 and 48 hours.
Controls included
10% NDM and MRSYG inoculated with L. monocytogenes, as well as
10% NDM and MRSYG inoculated with potential producer organisms
and sterile 10% NDM.
Plasmid Isolation:
A modified small scale lysis procedure described by Anderson and
McKay (6) was used to isolate plasmid DNA. Lysis medium was MRS
supplemented with 20 mM DL threonine (Sigma) for LAB and TSB for
Strains were grown at 300C for 4 to 5 hrs and cells
harvested by centrifugation at 5,000 rpm (Heraeus Sepatech) for 10
E.
coli V517.
min at 250C.
Washed cells were resuspended in 379 ill of TES buffer
(67% sucrose, 50 mM Tris, 1 mM EDTA, pH 8) and transferred to a 1.5
ml Eppendorf tube and then 96 g 1 of lysis buffer (20 mg/ml
After 30 min of
incubation at 370C, 27.6 pi of SDS solution (20% [w/v] in 50 mM Tris,
lysozyme (Sigma) in 0.05M Tris, pH 8.0) was added.
20 mM EDTA, pH 8.0) was added to complete lysis. The DNA was
denatured with 27.6 IA of 3M NaOH, freshly prepared, followed by
29
gentle mixing for 5 min; then the plasmid DNA was renatured by
neutralization with 45.6 µl of 2M Tris (pH 7.0) followed by gentle
mixing for 5 min. Precipitation of chromosomal DNA was achieved
by addition of 71.7 gl of 5 M NaC1 and overnight incubation at 40C .
Samples were then centrifuged
in
a Fisher Scientific microfuge at
Supernatants were collected in
maximum speed for 20 min.
centrifuge tubes and extracted successively with 700 IA of distilled
phenol saturated with 3% NaC1 and 700 g 1 chloroform: isoamyl
alcohol (24:1).
Plasmid DNA was then precipitated in two volumes of
cold ethanol at 40C for 1.5 hours and centrifuged for 15 mins at
Plasmid pellets were dried for 15 min and
maximum speed.
resuspended in 10 p.1 of TAE buffer (40 mM Tris-acetate, 1 mM EDTA,
pH 8).
Five ml of RNase solution (10 mg/ml) was added to plasmid
DNA samples, which were then incubated at 370C for 15 min.
Agarose Gel Electrophoresis:
Electrophoresis was performed
in
a Photodyne apparatus according
to the manufacturer's recommendations.
Agarose concentration was
0.7% (Agarose Type II, Sigma). Gel was run in TAE buffer at 8.0
V/cm on 12 to 15 g1 DNA lysates to which had been added 5 µ1 of
tracking dye.
E.
coli V517 plasmids were used
estimate the molecular weight of unknown plasmids.
as standards to
30
Curing Trials:
Attempts were made to cure putative bacteriocin-producing strains
by two methods: growth at sublethal temperature and exposure to
curing agents. Strains were grown in MRS broth and subcultured
daily for up to 10 days at 43 C using a 1% inoculum. After exposure
to a curing agent, 1 ml of an overnight culture was inoculated into 10
ml of MRS broth containing 25 gg/m1 of ethidium bromide (Sigma)
Test tubes were covered with aluminum foil to
After 3 and 7 days of
protect ethidium bromide from light.
incubation, serial dilutions were made and 0.1-m1 aliquots of
and incubated.
appropriate dilutions
incubated.
were
surface plated
on MRS agar
and
Individual colonies were selected, grown in MRSYG broth
and tested for bacteriocin production by the well diffusion assay.
31
RESULTS
Screening of LAB for Inhibition of L. monocytogenes:
Strains isolated from different plant and food products, 324 in
number, were tested for antibiosis against L. monocytogenes (Table
1.2).
Eighty-seven (26.9%) gave positive results in the well diffusion
assay when the pH of the supernatant was unchanged. However,
most of the inhibition zones were either small (diameter less than 12
mm including the well) or hazy. When the pH of the supernatant
was adjusted to 6, only 4 strains (1.2%) retained their activity.
These
strains plus another one (1.5%) were positive in the spot test with
M17G agar as the bottom layer. When MRS agar was used as a
bottom layer, similar results to those of the well diffusion assay
using non-neutralized supernatant were obtained (i.e., 87 strains
positive).
The five strains found positive
positive in the well
in
the spot test, including the four
diffusion assay with pH of the supernatant
adjusted to 6, were identified by physiological and morphological
tests classically used for LAB identification (Table 1.3).
Four strains
were identified to the species level as Lactococcus lactis and one was
identified to the genus level as a Lactobacillus species (Table 1.4).
Nature of Inhibitor Substances:
Substances produced by the five selected strains were not hydrogen
peroxide, bacteriophages or organic acids. The inhibitory activity
32
was not affected by catalase and no plaques were formed when an
inoculating loop was stabbed in the zones of inhibition and streaked
on a lawn of L. monocytogenes. Also, phage titer determination of
producer strain cell free supernatants were negative. As for organic
acids, despite the fact that one strain, Y5, showed no activity at pH 6,
the substance that it produced was not dialysable (Visking tubing
8000 molecular weight cutoff, Serva), but retained activity at pH 4.6,
suggesting that it was not an organic acid. All substances were
Four culture
insensitive to ribonuclease, lysozyme and lipase.
activities were sensitive to proteases, but the fifth was not tested in
this regard (Table 1.5). Three out of the four tested had the same
sensitivity pattern to enzymes as nisin, while the fourth (LBII)
differed from nisin in that it was sensitive to trypsin and ccchymotrypsin.
Properties of Inhibitory Substances:
Table 1.6 summarizes properties of the inhibitory substances.
They
were retained by a membrane tubing with a molecular weight cutoff
of 8000 when dialysed against polyethylene glycol 8000 (Sigma).
They were stable to the different heat treatments and to chloroform.
As the substances produced by S29, S41 and S60 behaved like nisin,
their resistance to boiling at pH 2 and pH 1.1 was examined. These
results, as well as their range of inhibition against various bacteria,
(data not
shown) indicated that these
substances were nisin.
Therefore strains producing them were discarded.
Further study
was focused on the LBII strain because it was very inhibitory to L.
33
monocytogenes
and the bacteriocin produced was elaborated by a
GRAS (Generally Recognized as Safe) bacterium, Lc. lactis.
Stability of LBII and Y5 Antimicrobial Substances to pH and
Combined pH and Heat:
Both substances were more active at pH values less than 4 (Table
1.7). Y5 activity gradually diminished from pH 2 to pH 5 in both
heated and unheated samples. The non-producer strain used as a
negative control did not show any inhibition zone above pH 3 or
below pH 12.
Small turbid zones could be observed, suggesting that
the large and clear zones produced by Y5 at pH values below 4.6 was
not due to acidity alone and that the low pH was a stimulatory factor.
As for the
LBII inhibitory substance, its activity also decreased as the pH
increased but it was completely lost at pH values above 10 when
samples were not heated and at pH values higher than 8 when they
Heated and unheated samples acted
in
the same way.
were heated at 1000C for 10 min (Figure 1.1). These data indicated
that LBII was behaving like nisin; however, when the pH of LBII
supernatants was adjusted to 11 and held at room temperature for
one hour and then readjusted to
7,
activity was totally recovered.
Nisin activity was irreversibly lost with this treatment, suggesting
that LBII produced a bacteriocin different from nisin.
34
Bactericidal Mode of Action:
bactericidal mode of action was demonstrated by the well
diffusion and streak assays against L.
monocytogenes and
Streptococcus sp. Also, when L. monocytogenes was grown in Lc.
The
lactis LB11 CFS at pH 7.0, the optical density decreased to a very low
level (Figure 1.2).
L.
monocytogenes grew well in spent broth from a
culture of a non-producer strain at pH 7.0 which was used as a
positive control (data not shown). However, since spent broth from
the
producer
strain,
treated
with
protease to
inactivate the
bacteriocin, was not used as a positive control, the best evidentce
presented herein for the bactericidal mode of action remains as
described.
As for Y5, 10-fold concentration of the CFS was necessary
to demonstrate the bactericidal mode of action by the well diffusion
assay, however, it was demonstrated in mixed culture growth with L.
monocytogenes (Figure 1.5).
Spectrum of Action:
Table 1.8 shows the range of activity of LBII and Y5 bacteriocins
LBII and Y5 did not inhibit each
other and, as expected, did not inhibit themselves. LBII was more
against different bacterial strains.
active on Gram positive than on Gram negative bacteria, although it
inhibited E. coli V517 and Salmonella typhimurium to some extent.
Its spectrum of action was similar to nisin, the main difference being
that it did not inhibit Micrococcus flavus which is very sensitive to
nisin and is even used for nisin quantitation in foods, according to
35
Fowler et al. (28).
spectrum of action.
On the other hand, Y5 had a very narrow
The
most sensitive
strains to it were
Streptococcus sp. and Salmonella typhimurium, but it also inhibited
Lactococcus lactis 7962, a nisin producer.
Inhibition zones of Y5 on
sensitive indicators were improved when plates were incubated 48
hours before adding the indicator-seeded top layer or by using MRS
agar as a bottom layer instead of M17G.
Dynamics of LBII Inhibitory Substance (IS) Production:
Figure
1.3
summarizes results
on the
production of LBII IS.
Detection of this substance by the well diffusion assay starts after 5
hours of incubation and reaches its maximum between 10 and 24
hours.
After 7 hours of incubation, 30% of the bacteriocin is
produced when the pH is 4.6 and the cell growth is maximum.
Therefore 70% of activity units are produced during the stationary
phase, indicating that LBIII IS may be a secondary metabolite as is
the case for many known bacteriocins such as Pediocin AcH (13) and
nisin (39).
Associative Growth:
Associative growth of L. monocytogenes with Lc. lactis strains LBII
and Y5 was examined in 10% reconstituted NDM and in MRYG (Figure
1.4 and Figure 1.5, respectively).
higher than
grown alone.
The L. monocytogenes counts were
both media after 24 hours of incubation when
However, when in association with LBII, growth was
108
in
36
reduced about 4 log units after 9 hours of incubation in MRSYG and
in nonfat milk as well. No significant difference (P <0.001) occurred
in reduction of the growth rate of L. monocytogenes when comparing
As for growth of L. monocytogenes
MRSYG and reconsitituted milk.
with strain Y5 (Figure 1.5), a significant decrease in Listeria counts
could be observed only after 24 hours in MRSYG and after 48 hours
The pathogen was killed by Y5 within 48 hours in
in nonfat milk.
MRSYG and within 72 hours in milk.
Such a difference can have two
that MRSYG is a better medium for
production of the inhibitory substance than milk. Glucose and yeast
The first
explanations:
is
extract were shown to have
production (13).
a
stimulatory effect on bacteriocin
The second could be the fact thdt Y5 IS has a
maximum activity at low pH (Table 1.7). In fact, the pH dropped to
about 3.7 in MRSYG after 24 hours while it took 48 hours to reach
that pH in milk (data not shown). The killing of L. monocytogenes in
the mixed culture with Y5 also indicates a bactericidal effect,
however, at this low pH it is difficult to tell whether it is the acidity
or IS activity that plays the major antibiosis role. It is likely that
there is a synergistic action between the acidity and the inhibitory
activity of the substance. This is rather common as shown by Raccah
et al. (51). In fact, lactostrepsins produced by lactococci behave in a
similar way
and
thus
have
Furthermore, Abdel-bar et al.
been
( 1)
named
showed that Lb.
produced a non-proteinoceous antimicrobial
active only at pH 4.0 and below.
"acid
bacteriocins".
bulgaricus
substance which
is
37
LBII Possibly Produces More than One Bacteriocin:
Two observations led us to conclude that LBII produces more than
one bacteriocin: First, after protease treatment, activity was totally
lost (Table 1.9) on L. monocytogenes but only slightly reduced on the
hemolytic Streptococcus sp. Second, after curing, some mutants lost
their activity against L. monocytogenes but still inhibited the
Streptococcus sp.
Strains producing more than one bacteriocin have
been described previously (2).
Plasmid Profile of Producer Strains:
The plasmid profiles of Lc. lactis LBII and Lactobacillus sp.Y5 and
their mutants are shown in Figures. 1.6 and 1.7, respectively.
These
plasmid profiles were consistently observed in repeated trials
Their
estimated molecular weights are shown in Table 1.10. Both wild
type strains harbor plasmids with MW ranging from 1.8 mDa to 30
mDa in the case of Y5, and from 4.9 mDa to 50 mDa in the case of
LBII. The occurrence of multiple plasmids in these organisms is not
unusual.
Curing of Y5 by incubation at sublethal temperatures (430C) gave
mutants lacking the 5.2 mDa plasmid (Figure 1.7, Lines c, d, and e)
without concomitant loss of IS production. LBII, however, gave 10
mutants not producing the IS against L. monocytogenes, among
which 5 were still inhibitory to the Streptococcus sp. After a second
transfer the 5 mutants regained their capability to inhibit L
nionocytogenes. All LBII mutants showed a strikingly different
.
38
plasmid profile from the parental strain, while their morphological
and physiological characteristics remained the same. The mutants
had more plasmids than the wild type (Figure. 1.6); some of the extra
plasmids migrated further than the smallest parental plasmid and
cannot therefore be considered as open forms.
39
DISCUSSION
Results of this study show that antibiosis among LAB is common; 27%
of the 324 isolates were inhibitory to L.
monocytogenes
as
determined by the well diffusion assay. However, the majority of
substances were inactive at pH 6, suggesting that organic acids are
common metabolites used by this group of organisms to antagonize
other microorganisms. Less common, however, is the production of
bacteriocins (32, 36, 62). In this study, 4 strains (1.2% of those
tested) were bacteriocin producers while one is still to be confirmed.
Geis et al. (32) made a survey on 280 lactic streptococci (lactococci)
for bacteriocin production and found 23% producing antimicrobial
substances by direct methods; 6% were confirmed to produce
bacteriocins.
The higher frequency of bacteriocin production found
in their work compared to this work may be explained by the fact
that these workers used four indicator strains related to the test
organism,
which
greatly
increases
the
chances
to
detect
In the present study it was also shown
that the assay technique as well as the media used for screening
have a great impact on the results. Such has been also shown by
Spelhaug and Harlander (62). The well diffusion assay with the pH
of the test supernatant adjusted to 6 is widely used for this purpose,
bacteriocinogenic strains.
but it has two major limitations that lead to false negative results:
diffusion of the substance may be impeded (19),
bacteriocins" would not be detected.
2)
1)
"acid
As for the spot test, media used
as top and bottom layers must be chosen with care. In our hands,
when MRS agar was used as a bottom layer, false positive results
40
were obtained, while M1 7G agar gave better results and would be
appropriate for such studies.
These findings
are
in perfect
agreement with those obtained by Spelhaug and Harlander (62).
Nonetheless, MRS or MRS fortified with glucose and yeast extract was
reported to be suitable medium for bacteriocin production by LAB
(32).
Therefore MRS or MRSYG are recommended for any kind of
study on the production of bacteriocins but the screening.
Among the 5 producers we found, 4 were Lc. lactis and one was a
Lactobacillus species. No Leuconostoc were found to be inhibitory
against L. monocytogenes, although 101 identified Leuconostoc
strains
were
examined.
The
production
of
Leuconostoc seems not to be a common phenomenon.
bacteriocins
by
Few studies, to
our knowledge, have described the production of antimicrobial
substances by this group.
Of the four lactococci found to produce
inhibitory substances, three were producing nisin.
suggest that
bacteriocins against L.
These results
monocytogenes
are
more
frequently produced by Lc. lactis and that strains of this species
mostly produce nisin, but not exclusively.
The Lc. lactis LBII strain appeared to produce two inhibitory
monocytogenes, was
inactivated by trypsin, a -chymotrypsin and pronase, and the second,
inhibitory for Streptococcus sp., was not destroyed by asubstances.
The first, active against L.
The former received the most attention in this study.
Production of more than one inhibitory substance by the same strain
has been reported previously (2). The LBII inhibitor, which was
chymotrypsin.
41
inactivated by the three proteases, fits the definition of a bacteriocin
as given by Klaenhammer (42). Therefore we considered it as a
bacteriocin and propose the name of Lactococcin LBIIA. This
substance shares many properties with nisin but differs from it by
two main characteristics: spectrum of action and sensitivity to
proteases, namely to trypsin and a-chymotrypsin, to which nisin is
resistant.
It needs to be pointed out, however, that there are
conflicting data about sensitivity of nisin to a-chymotrypsin;
some
authors found it sensitive (38, 39) while others demonstrated its
In our case the nisin used (Applin & Barrett) was
resistant; nonetheless, there is a general agreement on resistance of
nisin to trypsin. The production of substances different from nisin
by strains of Lc. lactis has been reported by other workers. Geis et
al. (32) separated the inhibitory substances produced by L. lactis
strains into 3 types on the basis of their inhibitory spectra and
chemical properties. Lactococcin LBIIA would not fit in any of these
types. It is similar to Type VI bacteriocins, but according to these
authors substances of this type are resistant to trypsin which is not
resistance (16).
the case for our substance.
Carminati et al. (15) found 7 strains of Lc.
lactis producing inhibitory substances that differed from nisin in
their sensitivity to proteases. The present investigation involved use
of
fewer proteases and therefore, it
is
difficult to compare
Lactococcin LBIIA to those studied by Carminati et al. (15)
Bacteriocin production was shown to be either plasmid (2, 15, 17, 18,
33, 34, 37, 48, 53, 60) or chromosomally (47, 49) encoded.
Mutation
studies done to relate lactococcin LBII production to plasmids were
42
not conclusive; instead they raised intriguing questions that we could
not answer (Figure 1.6).
Y5 is a Lactobacillus
strain that produces a non-dialyzable inhibitory
substance, not destroyed by catalase, suggesting that it is neither an
organic acid nor hydrogen peroxide.
but it has a bactericidal effect.
It
also is not a bacteriophage
Nonetheless, this substance could not
be considered a bacteriocin or a bacteriocin-like substance since its
protein nature was not demonstrated in this study. Proteases we
used were all active at a neutral pH, where the inhibitory substance
produced by Y5 was inactivated. If we rely on the accuracy of the
spot technique using M17G as a bottom layer for detecting
bacteriocins, this substance would be similar to lactostrepsins (9, 45)
or to MicrogardTM (Wesman Foods, Inc., Beaverton, Oregon).
The
latter, presumably a bacteriocin (5) and produced by
Propionibacterium shermanii, shows similar activity with regard to
pH as reported by Al-Zoreky et al.(4). Further characterization of the
substance produced by Lactobacillus Y5 is being carried out.
43
Table 1.1: Indicator bacteria, their origin and culture media used for
their growth.
Strain
Origin
Listeria monocytogenes
Staphylococcus aureus
Escherichia coli V517
Streptococcus sp.
Micrococcus flavus NCIB8166
Leu. dextranicum 187
Leu. cremoris
Leu. cremoris dM711
Lactococcus lactis ssp. lactis
biovar.diacetylactis BU2
Lc. lactis 7962
Salmonella typhimurium
ATCC
IAVHII
OSU
IAVHII
OSU
OSU
OSU
FDRC
FDRC
OSU
IAVHII
Medium(1)
TS(2)
TS
TS
TS
TS
MRS
MRS
MRS
MRS
MRS
TS
OSU = Oregon State University (Corvallis, USA)
IAVHII = Institut Agronomique et Veterinaire Hassan II Rabat,
Morocco
FDRC = Federal Dairy Research Center (Kiel, Germany)
(1)media were broth in the case of propagation and agar slants for
storage.
(2)TS
Trypticase Soy.
44
Table 1.2: Number and percentage of strains of LAB antagonizing
growth of L. monocytogenes
Well diffusion assay
Spot test
No. of
strains
22
43
83
67
101
8
Source
Supernatant)
N. CFS2
Raw butter
12(54%)
Lben
19(44%)
raw milk
17(40%)
Bakery Yeast
78(42%)
Mix. of Vegetable3 5(5%)
Pickle brine
6(75%)
0
0
1(2.3%)
1(1.2%)
2(2.9%)
2(4.6%)
1(1.2%)
2(2.9%)
0
0
0
0
)Used without pH adjustment
2Neutralized cell-free supernatant (pH 6)
3All Leuconostoc species
M17G agar MRS agar
12(54%)
19(44%)
17(40%)
28(42%)
5(5%)
6(75%)
45
Table 1.3: Morphological and physiological tests used for bacteriocin
producer strain identification.
Strain
Test
Gram reaction
Morphology
Catalase production
Gas from glucose
Gas from citrate
Dextran production
Growth at 10 °C
Growth at 420C
Growth at 440C
Milk coagulation
Litmus reduction
Arginine deamination
LBII
S29
S60
S49
+
+
+
Y5
+
c
c
c
r
c
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
46
Table 1.4: Identification of bacteriocin-producing strains.
Strain
S29
S41
S60
Y5
LB II
Source
Bakery yeast
Raw milk
Bakery yeast
Lben
Lben
Identification
Lactococcus lactis
Lactococcus lactis
Lactococcus lactis
Lactobacillus species
Lactococcus lactis
47
Table 1.5: Effect of various enzymes on the inhibitory activity of
lactic acid bacterial strains against L. monocytogenes.
Strain
Enzyme
Pronase Lipase Lyzosyme Ribonuclease
a-Chym
Trypsin
R
S
R
R
R
R
S
R
R
R
R
R
S
R
R
S
S
S
R
R
S
R
R
R
R
S29
S41
S60
LBII
Nisin
R = resistant
S = sensitive
R
R
R
R
R
48
Table 1.6: Stability of inhibitory substances to different treatments.
Treatment
Heat
1000C for 10 min
600C for 30 min
800C for 30 min
autoclaving (121°C,
15 min)
Chloroform
Dialysis
Filtration
R = Resistant
Ret = Retained
P = Passes
Inhibitory substance
S29
S60
S41
Y5
LBII
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
Ret
Ret
Ret
Ret
Ret
P
P
P
P
P
R
49
Table 1.7: Stability of LBII and Y5 inhibitory substances to pH and
heat.
Diameter of inhibition zones (mm)*
Y5
LB II
pH
Unheated
Samples
Heated
Samples
2
18
16
3
ND
ND
4
17
14.5
4.6
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
ND
ND
17
14.0
13.0
13.0
10.0
15.0
15
14.5
0
0
0
0
14
14
0
0
Unheated
Samples
Heated
Samples
15.00
14.00
13.00
11.00
10.50
15.00
14.00
13.00
11.00
10.00
0
0
0
0
0
0
0
0
0
0
0
0
0
0
*diameter of inhibition zones include the well diameter (8mm)
except when it was zero
ND = not determined
Data are average
duplicate.
values
of two determinations, each
one
in
50
Table 1.8: Inhibitory activity of LBII and Y5 against Gram positive
bacteria and E. coll.
Indicator Strain
Micrococcus flavus
Staphylococcus aureus
E. coli V517
Streptococcus sp.
Lc. diacetylactis BU2
Leu. cremoris 44-4C
Leu. dextranicum 181
LBII
Y5
-H-
+
+
-FH-
+I-
-I-H-
+Ft-
+
AAA-
Lc. lactis Y5
Lc. lactis LBII
Leu. cremoris M711
Lc. diacetylactis F7122
Lc. lactis 7962
L.
-:
+:
A-F:
-H-F:
monocytogenes
No zone of inhibition
Diameter < spot
Diameter > 2 x spot diameter
< 2 x spot diameter
+
-I-H-
_
-H-
-I-H-
-I+
51
Table 1.9: Activity assay of Lc. lactis LBII
bacteriocin after
treatment with oc-chymotrypsin on L. monocytogenes
and Streptococcus sp.
Diameter of Inhibition Zones
Indicator
Treated CFS
L. monocytogenes
0
Streptococcus sp.
12
CFS = cell-free supernatant
CPS
12
15
Buffer
0
0
52
Table 1.10: Plasmid content of the producer strains (Lactobacillus
sp. Y5 and Lactococcus lactis LBII) and their derivatives
after curing trials.
Strain
Production of Inhibitory
Substance)
Plasmid Content
(mDa)
Y5
+
1.8; 3.7; 5.2; 6.8; 8.6; 9.6; 21; 30
Y5(d)2
+
1.8; 3.7; 6.8; 8.6; 9.6; 21; 30
LBII
+
4.9; 5.4; 9.0; 21; 38; 50
LBII(d1)
2.2; 9.0; 27; 30; 43
LBII(d2)
2.2; 3.7; 8.4; 9.0; 27; 30; 43
1+ produces the IS
2All mutants had the same plasmid profiles but only one is shown on
this table.
53
Figure 1.1:
Effect of pH and heat on the antimicrobial substance produced by
Lactococcus lactis LBII.
Samples heated at 100°C for 10 min. before testing.
Right:
*
*
*
*
*
*
Left:
Clockwise from the top:
Cell free supernatant (CFS), pH 10
CFS, pH 8
CFS, pH 6
CFS, pH 4
MRS (sterile)
CFS, pH 11
Non-heated
samples.
*
Clockwise from the top:
Cell free supernatant (CFS), pH
*
*
*
CFS, pH 8
CFS, pH 6
CFS, pH 4
*
*
MRS (sterile)
CFS, pH 11
10
54
0.0
i
0
.
.
i
5
I
i
i
15
10
20
25
Hours
Figure 1.2: Growth of Listeria monocytogenes (0.D. at 600 nm) in
MRS broth and in Lactococcus lactis LBII cell-freesupernatant.
55
1.0
E
.
c
cp
z
c)
co
d
0
CL
0.5 -
0.0 -
,
0
10
.
20
30
40
Hours
Figure 1.3: Dynamics of LBII Inhibitory Substance (IS) production as
a function of time, pH and growth (O.D.)
56
10
--------
6-
o MRSYG: L. m.
D
MRSYG: L. m. +LB11
NDM: L. m.
J
NDM: L. m. +LB11
10
30
20
40
50
Hours
Figure 1.4: Associative growth of Listeria monocytogenes (L.m.) and
Lactococcus lactis LBII in MRSYG and in reconstituted
non-fat dry milk (NDM).
57
40
60
80
Hours
Figure 1.5: Associative growth of Listeria rnonocytogenes (L.m) and
non -fat
Lactobacillus sp Y5 in MRSYG and in reconstituted no
dry milk (NDM).
58
ASCP
lib imp OM
INN
Figure 1.6: Plasmid patterns of lactococcus lactis LBII and its
derivatives after curing trials with 25 µg /m1 of ethidium
bromide.
Lane A: Standard; E. coli V517
Lane B: Parental Strain (Lc. Lactis LBII)
Lane C & D: LBII(dl) and LBII(d2) respectively; LBII
derivatives
chr = chromosomal DNA
59
A 16 CDP
0.c.
Figure 1.7: Plasmid patterns of Lactobacillus sp. Y5 and its
derivatives after curing trials at sublethal temperatures.
Lane A: Standard; E. coli V517
Lane B: Lactobacillus sp. Y5; Parental Strain
Lane C, D & E: Y5 derivatives (Y5d1, Y5d2, Y5d3)
chr = chromosomal DNA
o.f. = open form of plasmid DNA
60
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67
Chapter 2
Effect of Nisin on Growth and Survival of
Listeria monocytogenes in Cottage Cheese and Yogurt*
Noreddine Benkerroumt
and
William E. Sandine
Department of Microbiology
Oregon State University
Corvallis, OR 97331-3804
Running Title: Effect of Nisin on L. monocytogenes
*The part of this chapter dealing with cottage cheese has been
published: Inhibitory action of nisin agains Listeria monocytogenes,
J. Dairy Sci. 71:3237-3245.
tPresent address: Department of Food Microbiology, Institute of
Agronomy and Veterinary Medicine, Hassan II, Rabat, Morocco.
68
ABSTRACT
The sensitivity of nine strains of Listeria to nisin was determined as
well as the minimum inhibitory concentration of nisin necessary to
completely inhibit their growth. All strains tested were variably
sensitive to nisin and different minimal inhibitory concentration
(MIC) values were obtained ranging from 740 to 105 IU/ml in
Trypticase Soy Agar (TSA) and from 1.85 to 3.6 x 103 IU/ml in MRS
agar.
MIC values obtained on TSA were higher than those obtained
on milk agar, MRS agar and Trypticase Soy Broth (TSB).
The
inhibition of L. monocytogenes ATCC 7644 in TSB at different pH
values and different nisin concentrations in nonfat dry milk (NDM)
(50 IU/ml), in sterilized and non-sterilized cottage cheese (37 x 102
and 2.55
x
investigated.
103
IU/g, respectively) and in
yogurt, also was
This bacterium was completely inhibited in TSB (37 x
102 IU/ml) after 24 hours at pH 5 and above and within 24 hours at
pH 4.5 and below. Nisin concentrations ranging from 50 to 200
IU/ml inhibited growth of L. monocytogenes at pH 6.0 and below but
not at pH 6.8.
4.5.
A concentration of 20 IU/ml was inhibitory only at pH
In NDM, 50 IU/ml was inhibitory to the pathogen at pH 6.8 and
below and the activity increased as the pH decreased. In cottage
cheese, and in yogurt as well, no Listeria survivors were found at 24
hours at 370C and 40C, and during storage at 40C, respectively.
Nisin
was shown to inhibit yogurt fermentation only at a concentration
higher than 50 IU/ml.
69
INTRODUCTION
Listeria monocytogenes is a food-borne pathogen, which recently has
been associated with food-borne disease outbreaks (13, 33).
Raw
milk has been reported to be a vehicle for this pathogen (16, 22, 23,
27, 31), which can survive the processing of some dairy products (6,
8, 16, 30) or contaminate the plant equipment and thereafter cause
post-pasteurization contamination.
Although Bradshaw et al. (3) showed L. monocytogenes would not
survive pasteurization, this microorganism has been isolated from
post-pasteurization
contamination or improper pasteurization. However, Doyle et al. (7)
showed that this organism survives HTST pasteurization of milk
pasteurized
milk
(13,
14),
suggesting
(71.70C for 15 s). It has also been shown that L. monocytogenes
survives the manufacture of cottage (6), Camembert (6, 30) and
Cheddar (6, 29) cheese as well as the drying operation to produce
NDM (16). Therefore, Listeria may be present in dairy products as a
result of post-pasteurization contamination or
survival during
manufacture; growth of this bacterium can occur under temperature
abuse but also under refrigeration conditions.
In
this regard,
Rosenow and Marth (28) showed that L. monocytogenes grows well
at 40C in skim, whole, and chocolate milk and in whipping cream and
over a temperature range from 0 to 43°C.
Although many nonspore-forming bacteria are sensitive to nisin,
industrial use of this antibiotic is limited to the prevention of spore
outgrowth in processed cheeses and canned foods (9, 15).
The
70
earliest use was to prevent the late gas defect in Swiss-type cheeses
caused by clostridia (15, 20). It also has been used alone or with
subtilin in canned foods to prevent the outgrowth of bacterial spores.
Recently, Taylor (40) showed that nisin inhibits the outgrowth of
Clostridium botulinum spores or impedes toxin production by this
microorganism. In addition, Ogden (24) and Ogden and Tubb (25)
proposed the use of nisin in brewing to inhibit Lactobacillus and
Pediococcus, the main organisms associated with beer spoilage.
In
the United States, nisin has been placed on the GRAS list and is
approved for use in pasteurized cheese spreads (11). The action of
nisin is pH-dependent; it is more effective in low pH systems (5, 20).
It
is stable at pH 2, where it can resist boiling for 10 min, but it
readily destroyed at pH 11 (18, 20).
is
Campbell and Sniff (4) showed
that 200 IU/ml are enough to inhibit Bacillus coagulans at pH 5.3 but
560 IU/ml failed to inhibit this bacterium at pH 7.2. According to
Henning et al., (17) nisin should be used only in foods where the pH
below 7.0 "to ensure sufficient solubility and stability during
processing and storage."
is
As for the safety of nisin, studies done in USSR, Japan, and England
A joint FAO/WHO committee on
food additives stated in 1968 that 3,300,000 IU/kg of body weight
does not present any undesirable effects, and consequently, they
have established its nontoxicity.
recognize it as a safe food preservative. Recent public health
concerns about Listeria in food products and the fact that it is a
Gram-positive bacterium prompted us to investigate its sensitivity to
nisin.
71
MATERIALS AND METHODS
Cultures:
Nine strains of Listeria spp were tested for their sensitivity to nisin.
The
nine
strains also were used to determine
inhibitory concentration (MIC) of nisin.
the minimum
They included eight strains
isolated from clinical cases and a ninth strain (7644) obtained from
the American Type Collection Culture (ATCC), Rockville, MD 20852.
The clinical isolates were L. monocytogenes 7644K; L. monocytogenes
V7 type la; L. monocytogenes 35152; L. monocytogenes Scott A type
4b; L. monocytogenes 1513, L. ivanovii KC1714 type 5; L. ivanovii
C194 type b and L. seeligerii LA15.
All
strains were grown on slants of Trypticase Soy Agar (TSA)
obtained from Baltimore Biological Laboratory (BBL), Baltimore, MD
and stored at 4 °C.
Before each use they were transferred to 10 ml of
Trypticase Soy Broth [(TSB) (Bacto-Tryptone, Difco Laboratories,
Detroit MI), 15 g; Bacto-soytone (BBL), 5g; NaCl, 5g; and distilled
water, 1000 ml] and incubated 16 hours at 37 °C .
Nisin was purchased from Aplin and Barrett, Ltd., Trowbridge,
Its potency was 37 x 106 IU/g. The stock solution (37 x
103 Wimp was prepared by dissolving 0.1 g of nisin in 80 ml of 0.02
England.
N HC1 solution and holding at room temperature for 2 hours to
The volume was then made up to 100 ml with
0.02 N HC1 and the solution filter sterilized through 0.22-p m
complete dissolution.
Millipore membrane.
This solution was stored at -20 °C .
72
Sensitivity Testing:
A modification of the well assay technique described by Fowler et al.,
(12) was used:
melted TSA was tempered to about 46 °C and
inoculated with 1% overnight culture of Listeria. Twenty milliliters
then were poured in petri dishes and allowed to harden. Wells
(8mm diameter) were then cut into the dishes and filled with 700 of
These cultures were incubated at 370C for
nisin solution (lmg /ml).
24 to 48 hours until inhibition zones were evident. The inhibition
zones were then measured with the aid of a graduated ruler.
Determinations of Minimum Inhibitory Concentration:
Determinations of MIC were done on broth and agar media.
Agar
media used were TSA, MRS (Difco Laboratories, Detroit MI) and milk
The agar incorporation method described by Hogg et al. (19)
was used. An overnight Listeria culture (25111) was dispensed as a
agar.
drop
on TSA, MRS and
concentration of nisin.
24 hours.
milk agar plates containing a given
Inoculated plates were incubated at 370C for
Nisin-free plates (controls) were inoculated in the same
way.
It was dispensed in 10-ml aliquots
and nisin was aseptically added to achieve different concentrations
and the tubes were inoculated with 0.1 ml (ca. 106 cells/ml) of an
Liquid medium used was TSB.
A nisin-free tube was used as a control
and was inoculated similarly. All tubes were incubated at 370C .
After 24 hours of incubation tubes were checked for turbidity.
overnight Listeria culture.
73
Effect of Nisin on L. monocytogenes ATCC 7644:
Varying pH with constant nisin concentration: media used were TSB
and 10% reconstituted NDM.
Three series of test tubes containing 9
ml of TSB each were used. The tubes of each series were adjusted to
different pH values (7.0; 6.5; 5.0; 4.5; 4.0 and 3.5) with 85% lactic
acid. To the first series (test), 1 ml of nisin stock solution (37 x 103
IU/ml) and 0.1 ml of 0.6 x 105 CFU/ml L. monocytogenes ATCC 7644
overnight cultures were added.
The final concentrations of nisin and
microorganism in each tube were then 37 x 102 IU/ml (1 mg = 37 x
103 IU) and 6 x 102 cell/ml.
To the second series, only Listeria w a s
added to the same concentration, i.e. 6 x 102 cell/ml. The third series
was a negative control to test the sterility of the medium; neither
nisin nor the microorganism was added.
Cultures were incubated at
370C. Listeria count was determined by plate count on TSA at 0, 1, 2,
4, 19 and 24 days.
The same experiment was conducted using reconstituted NDM.
However, the final concentration of nisin was 50 IU/ml, the inoculum
of Listeria was about 106 cells/ml and the pH was adjusted to 6.84,
5.5 and 4.5 with 1 M citric acid. Growth of L. monocytogenes w a s
monitored by plate count on TSA at 4, 7, 24 and 48 hours.
Varying pH and nisin concentration:
A series of test tubes containing
9 to 10 ml of TSB each were used. The tubes of each series were
adjusted to different pH values (6.4, 6, 5.5 and 5) with citric acid.
For each pH, a different nisin concentration was used (20, 50, 100,
and 200 IU/ml). Tubes were inoculated with 0.1 ml of an overnight
74
Negative and positive controls were prepared as
described above. Growth of the pathogen was monitored by O.D.
determinations at 520 nm at 2, 4, 6, 24 and 48 hours.
Listeria culture.
Effect of Nisin on Listeria monocytogenes ATCC 7644 in Cottage
Cheese:
This experiment was done on sterilized and nonsterilized cottage
cheese incubated at 37 or 40C. Two trials were conducted.
The first one was two series of three samples containing 250g of
cheese and 50 ml of pasteurized cream (12% milk fat) each. The
samples were thoroughly mixed and sterilized for 20 min at 1210C
Nisin and L. monocytogenes ATCC 7644 overnight cultures were
.
added to one sample of each series to a final concentration of 2.55 x
103 IU/g and 3.5 x 105 cell/g, respectively (test).
To another sample,
the positive control, the culture only was added to the same final
concentration.
The
third sample was a negative control and
remained uninoculated and without nisin addition.
All these samples
were mixed aseptically in a stomacher (Tekmar) for approximately
50 seconds and replaced in sterile 1000-m1 flasks. One series was
incubated at 40C, the other at 370C. They were then sampled for
counting L. monocytogenes on listeria selective isolation agar (LSI) at
0,
1, 2, 5, 9, 14, 24 and 30 days.
LSI (37) consisted of: TSA (Difco),
45g; yeast extract (Difco), 5 g; bromocresol purple (Difco) 0.04 g; and
esculin (Sigma Chemical Co., St. Louis, MO), 5 g; supplemented with
filter sterilized acriflavine hydrochloride (Sigma) and nalidixic acid
(Sigma).
The latter two ingredients were in solution in 0.1 N NaOH to
75
final concentrations of 0.010 and 0.040 mg/ml, respectively.
Filter-
sterilized aqueous solution of polymixin B sulfate (Sigma) to a final
concentration of 16 IU/m1 was also added. When no Listeria was
found in a 1-ml sample by the plate count method, we used the FDA
method for Listeria isolation described by Lovett et al. (22). A 25-m1
sample was enriched in 225 ml of a selective enrichment medium
described by Bannerma and Billie (2) and incubated at 30 °C. At 1
day and 7 days, a 10-g1 loop from the enriched culture was streaked
on LSI agar and incubated for 24 to 48 hours.
In the second trial, three samples were also prepared (test, positive
control, and negative control) in the same way as in the first case
except that they were not sterilized. Nisin concentration in the test
and the initial concentration of Listeria in the test and the positive
control were also the same. The nonsterilized cottage cheese was
incubated at 4 °C and sampled for plate count on LSI agar at 0, 1, 2, 5,
9,
14, 24, and 30 days.
Effect of Nisin on Yogurt Fermentation:
As Lactobacillus
reported
to
be
bulgaricus used in yogurt starter cultures was
sensitive to nisin
(24,
25),
this preliminary
experiment was carried out to determine the concentration of nisin
which can be used in yogurt without affecting its normal processing,
especially acid production.
76
Whole dry milk was
were made as follows:
reconstituted at 17% and pasteurized in water bath at 800C (internal
temperature) for 30 min. Of the pasteurized milk, 100 milliliters
were dispensed in 120-m1 waxed paper cups and inoculated with 2%
Yogurt trials
activated commercial yogurt starter consisting of Lb. bulgaricus and
Streptococcus thermophilus (1:1) (Redi-set, Hansen Laboratories, Inc.
Milwaukee, WI).
The starter culture was activated
according to the manufacturer's recommendations.
sterile NDM
in
Nisin was added
to the cups to a final concentration of 10, 20, 40, 50 and 100 IU/ml.
A sample not containing nisin was used as a control.
were
incubated at 430C until coagulation, (6 to
The containers
7
hrs), then
transferred to the refrigerator for the rest of the experiment.
The pH
and acidity were measured every hour until coagulation then at 1,
13, and 15 days. pH measurements were done with a Crison pH
meter using an ingold combination electrode. The acidity was
measured by titration with N/10 NaOH solution in presence of 1%
phenolphthalein solution.
Effect of Nisin on Listeria monocytogenes in Yogurt:
Two yogurt cups were prepared as described above. Nisin was
added to one of them to a final concentration of 50 IU/ml. The
second was a positive control.
A third cup containing only
pasteurized reconstituted NDM was also used as a second positive
control.
All cups were inoculated with a 0.1 ml (c.a. 105 1U/rap of an
overnight Listeria culture.
Samples were incubated at 43 °C until
those containing the starter coagulated.
They were then held at 40C
77
and sampled for plate count on ASLM (1) at 1, 2, 3, 13 and 15 days.
When no L. monocytogenes was found in a 1-ml sample, enrichment
procedure was followed as described above.
78
RESULTS AND DISCUSSION
Susceptibility Testing:
The well diffusion assay for susceptibility testing showed that
Listeria is inhibited by nisin.
The mean of inhibition zone diameters
for each strain tested is shown in Table 2.1. All strains were
inhibited by nisin, but important strain differences were observed in
the degree of inhibition showing that some Listeria strains are more
sensitive to nisin than others: L. monocytogenes ATCC 7644 and L.
ivanovii KC 1714 type 5 were the most sensitive (18 mm diameter)
while, L. monocytogenes V7 type 1 a was most resistant (10mm
diameter).
Determination of the Minimum Inhibitory Concentration:
The MIC ranges of the nine strains tested on different media are
shown in Table 2.1
These results show again that L. monocytogenes
is the most vulnerable strain to nisin among those tested.
It has the
lowest MIC (740 IU/ml on TSA), followed by L. ivanovii
KC 1714
type 5. The L. monocytogenes V7 type la strain had the highest MIC
(1.18 x 105 IU/ml on TSA).
In general, the MIC ranges obtained
were relativly high and subject to variations, depending on the assay
conditions, mainly pH and composition of the medium.
The pH of
TSA used in this experiment was 7.3, which is not optimum for nisin
action as stated earlier.
Also, MIC ranges obtained on MRS
lactobacilli agar were much lower than those obtained on TSA (Table
2.2).
One explanation for this is the lower pH of MRS (6.8), which is
79
more suitable for nisin action than pH 7.3.
Also, MRS contains some
components which may synergistically inhibit Listeria with nisin
Sodium acetate contained in this
increasing the overall effect.
medium is indeed inhibitory to
gram-negative bacteria.
a number of gram-positive and
Table 2.2 shows also that L. monocytogenes
ATCC 7466 had lower MIC on milk agar and in TSB than on TSA. The
lower MIC in milk agar may be also due to a synergistic action of a
milk inhibitor system (lactenins) and nisin. As for the difference
between TSB and TSA, an explanation would be that nisin has a
better access to the bacterium in the liquid than in the solid state of
the medium. These results suggest that one must be careful when
wanting to minimize nisin addition to products based on MIC
determinations under specific conditions.
Dairy products where nisin
has been mostly used as a preservative or to extend the shelf-life (9,
20) seem to be suitable for nisin action and 10 to 100 IU/ml are
generally used in practice. As regards regulations concerning the use
of nisin as a food preservative, most countries where it is permitted
allow either 12.5 mg/kg (about 5000 IU/g) or do not limit the
amount (10, 20).
Effect of Nisin on Listeria monocytogenes ATCC 7466 at Different pH
Values
monocytogenes at different pH values in absence or
presence of 37 x 102 IU/ml of nisin is shown in Figure 2.1. L
Growth of L.
.
monocytogenes grew well without nisin at pH of 5.5 to 7.0. At pH
5.0, growth was slow but not completely inhibited. After 24 hours,
80
no Listeria were found in a 1-ml sample; however, regrowth (6.3 x
102 cell/mil) was observed by the 19th day at this pH. The inhibitory
action of pH became clear at pH 4.5 and below. The pathogen was
eliminated from the sample after 48 hours at pH 4.5 and after only
24 hours at pH 3.5. No regrowth was observed at pH lower than 5.0.
Similar results were reported by Conner et al., (5) and Sorrel ls et al.,
(38), who showed that L. monocytogenes is completely inhibited at
pH 4.6 and below when lactic acid is used to adjust the pH.
Figure 2.1 shows also the growth of L. monocytogenes ATCC 7466 in
TSB with 37 x 102 Mimi of nisin. No survivors were found after 24
hours in a
1
ml sample at all pH values.
At pH 7 and pH 6.5, an
increase of Listeria count was observed in the first 24 hours to about
104 CFU/ml and then no Listeria were detected in
next 24 hours.
1
ml within the
Similar results were obtained with reconstituted NDM (Figure 2.2,
and Table 2.3). From Figure 2.2 it may be seen that counts of L.
monocytogenes kept increasing at pH 6.8 in the controls (without
nisin), while they decreased steadily in test samples containing 50
IU/ml of nisin.
in
both
At pH 4.5, a decrease in Listeria counts was observed
test and control samples; however, the pathogen was
eliminated from the test sample within 24 hours while in the control,
few cells were still viable even at 48 hours. The same behavior was
observed at pH 5.5 and 5.0 (Table 2.3).
These results suggest that 50
IU/ml of nisin can be effective in the control of L. monocytogenes in
milk and dairy products.
81
In view of these results taking into account that, in practice, high
concentrations of nisin may not always be possible to use, this
experiment was carried out using different combinations of pH and
nisin concentrations. Results are shown in Figure 2.3. At pH 6.8, no
concentration was inhibitory to L. monocytogenes; no significant
difference (P <0.01) between O.D. reached after 48 hours in samples
containing up to 200 IU/ml and the control, was observed.
At pH 6
and 5.5, however, 50, 100 and 200 IU/ml were all effective. A
concentration of 20 IU/ml had no significant (P < 0.01) effect on L.
monocytogenes at pH 6.8, and 5.5. This concentration however was
inhibitory at pH 5. These data show that much less nisin is needed to
control L. monocytogenes in low pH than in high pH systems, either
because nisin is more effective at low pH, as has been shown earlier
17), or because of an additive effect of acidity and nisin action.
However, in contrast to the report of Henning et al., (17), nisin was
(4,
still active at pH 7.0 (Figure 2.1), at least, at high concentration.
According to these authors, nisin is less soluble at pH 7.0, which
impeded its effectiveness. Wei and Hansen (41) also investigated the
effect
of pH on
nisin solubility and
exponentially from pH 2 to 6 and that
pH
8.
Hurst (20),
however,
it
found that it decreases
is almost insoluble around
reported
that
a
high
protein
concentration enhances the solubilization of nisin. According to
Eapen et al., (9) the solubility of nisin in water at pH 7.0 is 75 µg /ml
(the equivalent of 2.8 x 103 IU/ml), which is sufficient to inhibit all
microorganisms susceptible to this substance.
In food preservation,
in general, 10 to 500 IU/g has been recommended (9).
Moreover, it
82
has been shown that an average of 2 to 10 IU/ml was enough to
inhibit a cell concentration of 2 x 105 /ml (18).
Some microorganisms,
however, need a high nisin concentration to be inhibited. In this
regard, Taylor (40) showed that 50 to 200 gg/g (1.9 x 103 to 7.6 x
103 IU/g) were necessary to inhibit C. botulinum in various canned
Also, Mohamed et al., (23) showed that only 32 IU/m1 were
necessary to inhibit L. monocytogenes 4379 at pH 7.4 and 37°C .
foods.
They also showed that the sensitivity of this strain decreases when
the temperature of incubation decreases: 256 IU/m1 are required to
inhibit completely L. monocytogenes 4379 growth at 22°C and pH
7.4. However, this amount is 16-fold reduced at pH 5.5 at the same
temperature. In fact, solubility of nisin in dairy products does not
seem to be a concern, because most have a low pH and they contain
enough proteins to help solubilize nisin.
Furthermore, nisin has
already been used successfully in cheeses, as mentioned earlier.
In
other food systems, however, protein-nisin interactions may occur to
reduce the effectivness of nisin.
Effect of Nisin on Listeria monocytogenes ATCC 7644 in Sterilized and
Nonsterilized Cottage Cheese.
Figure 2.4 shows the growth curves of the pathogen in the positive
control and the test samples of the sterilized cottage cheese at 4 and
37°C. No Listeria could be found in 1 ml of sample after 1 day at 4°C
as well as at 37°C.
Furthermore, the pathogen could not be recovered
by the FDA method for Listeria isolation either after 1 day or after 1
week, which confirms that it was killed by nisin, not only inhibited
83
The same figure shows also that Listeria grows well in
sterilized cottage cheese (positive sample) despite its low pH, which
was in our case 4.9 to 5.0, while the synthetic medium showed that
or injured.
the
pathogen
was
greatly,
although not completely,
inhibited
(regrowth was observed by the 19th day) (Figure 2.1) at pH 5.
This
may be explained by the richness of cottage cheese that provides
Listeria with some growth factors not found in the synthetic medium,
or by the protective effect that cheese proteins may have for the
pathogen as it has been shown with Clostridium botulinum, which
can grow in canned foods at lower pH than in synthetic media.
may be a combination of these two factors as well.
It
Table 2.4 summarizes the effect of nisin on Listeria in nonsterilized
cottage cheese at 40C.
It appears that the cheese starter bacteria are
not inhibited by LSI medium.
However, the latter form larger
colonies and ferment the esculin more slowly when used as a
fermentable carbohydrate in the medium.
Some suspicious colonies
(smaller and turn yellow faster) were selected and Gram stained.
Such colonies were Gram-positive cocci and therefore assumed to be
The pathogen was not frequently found in the
positive control either; one or two colonies, at the most out of 10
were Listeria, sometimes none were Listeria. This suggests that
starter bacteria exhibit an antagonistic effect against Listeria, which
is no surprise; in fact, the inhibitory effect of lactic acid bacteria on
starter bacteria.
pathogens
and
spoilage microorganisms is
now
well
known.
Nonetheless, the inhibition of the pathogen by starter bacteria is not
a guarantee of the safety of this cheese.
Some cells can survive as it
84
was found here and elsewhere (5, 31, 37). However, the addition of
nisin not only inhibits the growth of Listeria, but also kills the
bacterium.
The antibiotic is, therefore, able to protect this product
from Listeria when contaminated either from the raw milk or as a
result of post-pasteurization contamination.
Most important, nisin
conserves its effectiveness at a low temperature for a long time (15),
ensuring protection of the product against Listeria contamination
when it
is
stored under refrigeration.
Furthermore, nisin delays the
growth of other spoilage psychrotrophs that can exist or contaminate
cottage cheese. In effect, in this experiment, nonsterilized cottage
cheese samples without nisin spoiled
1
wk earlier than those
containing the antibiotic (the spoilage was judged by the alteration of
the physical appearance and smell).
Bacterial counts were also lower
in the test than in control samples (Table 2.4).
Effect of Nisin on Yogurt Fermentation:
Figure 2.5 shows the pH decrease and the increase of acidity during
yogurt fermentation
and
with different concentrations
of nisin
It may be seen from
this figure that nisin concentration of 50 IU/ml and below had no
noticeable effect on yogurt fermentation. The pH dropped and the
acidity increased in the same way as in the control. Furthermore,
milk coagulation in all these samples was normal; it occurred from 6
to 7 hours and the curd was firm and without syneresis. However, in
the samples containing 100 IU/ml of nisin, fermentation was greatly
retarded and the curd had an abnormal, viscous body. After 15
compared to a positive control (without nisin).
85
days, the acidity and the pH of these samples were, respectively,
only 0.60% and 4.5 on the average, while in the control it was 1%.
This is due to the fact that nisin is inhibitory to Lb. bulgaricus;
therefore, addition of this substance to yogurt during fermentation to
a certain level would lead to an unbalanced rod-coccus ratio. These
results suggest that if nisin is to be used for yogurt preservation, the
amount added should be 50 IU/ml or less.
Effect of nisin on L. monocytogenes in yogurt:
Figure 2.6 shows the behavior of L. monocytogenes in yogurt in the
presence and absence of nisin during storage at 40C.
Although a
significant decrease in Listeria counts was observed in yogurt
without nisin, the pathogen survived manufacture and
storage at 40C.
15 days of
However, in yogurt containing 50 IU/ml of nisin, no
Listeria was found in
1-ml sample at 24 hours and thereafter.
Also, no survivors were resuscitated by the enrichment procedure.
Survival of pathogens in fermented dairy products, in spite of the
antagonistic effect of lactic acid bacteria used as starter cultures, is
well documented (6, 29, 30, 31, 34, 35). In yogurt and in other dairy
products fermented with the same starter [e.g. Lb. bulgaricus, and
a
Streptococcus thermophilus (ST:LB::1:1)]
and
Feta cheese, L.
such as some cultured milks
monocytogenes was shown to survive
manufacture and storage (26, 34, 35).
Papageorgiou and Marth (26)
showed that L. monocytogenes could survive the manufacture and
more than 90 days of storage at 40C in Feta cheese.
This bacterium
was also shown to survive in cultured milk fermented with STLB and
86
in yogurt from
1
to 12 weeks and
1
to
12 days, respectively (34).
The same authors showed that survival of L. monocytogenes in
yogurt depends on the size of both Listeria and starter culture
inocula, the final pH reached, the temperature and duration of the
fermentation, and Listeria strain. Shaack and Marth (34) showed
that L. monocytogenes survives only between 9 to 15 hours during
the actual fermentation process of yogurt. However, in a typical
yogurt fermentation, which lasts 4 to 6 hours, they showed that this
bacterium was able to grow during fermentation and then survive
during storage at 40C (34). Lammerding and Doyle (21) also could
recover L. monocytogenes from yogurt after 7 days of storage at 40C ,
although the initial inoculum was relatively low (about 32 x
CFU/ml).
102
Survival of L. monocytogenes in yogurt in spite of the low
pH and the antagonistic activity of the starter culture, may be
explained by the protective effect of the milk casein on the pathogen,
as was shown with Salmonella
typhimurium (32).
These data and
those reported elsewhere (6, 10, 26, 35) show that food processors
should not rely on pasteurization and fermentation only to insure full
protection and safety of fermented milk products.
Some food
additives may also be used in addition along with Good Manufacture
Practices.
In our case, nisin proved efficient in controlling growth of
In the latter
case, however, the amount of nisin to be used is crucial and should
be sufficient to inhibit the pathogen without harming Lb. bulgaricus;
L.
monocytogenes in both cottage cheese and yogurt.
otherwise, the product will be defective.
87
This work shows that in addition to the classical use of nisin to
prevent outgrowth of bacterial spores in foods, it can also be used to
prevent growth of some food-borne pathogens of major concern such
as L. monocytogenes. Growth of this bacterium that can occur at a
relatively low pH and under refrigeration can be overcome by nisin
addition.
The sensitivity of Listeria to nisin was shown to be
somewhat strain dependent, which should be taken into account in
determining amounts to be added in order to offer a safety factor.
Also, since nisin has no known toxicity for humans, its use as a food
additive in the United States to inhibit pathogens warrants further
consideration.
88
Table 2.1: Diameter of inhibition zones appearing on Petri plates
seeded with Listeria sp. as caused by nisin (70 Ill added
to assay well).
Inhibition Zone Diameter'
(mm)
Strain
L. monocytogenes 7644K
L. monocytogenes 35152
L. monocytogenes V7 type la
L. monocytogenes Scott A type 4 b
L. ivanovii C194 type b
L. monocytogenes 15313
L. seeligerii LA 15
L. ivanovii KC1714 type 5
L. monocytogenes ATCC 7644
'Mean values of four determinations
2Standard deviation values in parenthesis
12
(.71)2
11
(.41)
(.90)
(.73)
(.16)
(.16)
(1.08)
(1.41)
(1.47)
10
11
1 1
11
12
18
18
89
Table 2.2: Minimum inhibitory concentration (MIC) (pH 7.3) of nisin
on Listeria monocytogenes ATCC 7644 determined on
Trypticase Soy Agar (TSA), MRS agar at pH 6.8, milk agar
and Trypticase Soy Broth (TSB).
MIC (IU /ml)
Strain
Milk
TSA
L. monocytogenes 7644K
L. monocytogenes 35152
L. monocytogenes V7 type la
L. monocytogenes Scott A type 4 b
L. ivanovii C194 type b
L. monocytogenes 15313
L. seeligerii LA 15
L. ivanovii KC1714 type 5
L. monocytogenes ATCC 7644
1.48 x 104
1.48 x 104
1.18 x 105
1.18 x 104
74 x 103
1.48 x 104
1.48 x 104
14.8 x 102
740
MRS
agar
TSB
ND
ND
ND
ND
ND
ND
ND
ND
ND
50
agar
3.7
ND
3.7
ND
3.36 x
ND
1.65 x
ND
37
1.65 x
ND
ND
3.7
ND 3.7
300 1.85
103
103
103
90
Table 2.3: Effect of pH and added nisin (50 IU/ml) on growth of Listeria
monocytogenes (log CFU/m1)1 in reconstituted nonfat dry milk
incubated at 370C .
pH
6,8
Thne(h)
0
5.0
5.5
4.5
+nisin
-nisin
+nisin
-nisin
+nisin
-nisin
+nisin
-nisin
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
4
6.7(0.29)2
7.5(0.53)
6.4(0.59)
6.7(0.56)
5.6(0.82) 6.2(0.16)
4.7(0.37)
7
7.8(0.18)
8.9(0.29)
6.6(0.66)
7.1(0.26)
5.7(0.26) 5.8(0.48)
4.5(0.38) 4.7(0.29)
24
3.8(0.63
9.5(0.41
2.8(0.55)
6.9(0.42)
3.3(0.53) 5.8(0.77)
0.0(0)
2.8(073)
48
1.5(0.50)
9.5(0.29)
1.2(0.30)
8.3(0.33)
2.4(0.43)
0.0(0)
0.8(0.85)
1Values are means of four determinations.
2 Standard deviations in parenthesis.
0.0(0)
5.2(0.70
91
Table 2.4: Influence of nisin on microbial counts developing on
Listeria selective isolation medium in non-sterilized
cottage cheese contaminated with L. monocytogenes ATCC
7644 during incubation at 40C.
DAYS
Treatment
0
1
2
5
9
24
30
.2
.1
.1
3.0
.02
2.0
4.0
3
3
14
(CFU/ml x 107)
Nisin added 1,2
No nisin 1,3
Control 4
.04
.04
.08
.1
.09
.3
.8
.9
.2
.6
.5
4.0
3.0
.06
15 x 105 cell/g of L. monocytogenes added.
2While starter bacteria present in the cottage cheese grew on LSI
medium, L. monocytogenes could not be found on plates of samples
from the cheese containing nisin.
3Colonies were cheese starter bacteria and L. monocytogenes.
4No L. monocytogenes added, so colonies were only cheese starter
bacteria.
92
pH 7.0
pH 6.48
.
pH 5.94 rr
c'
.
4
V
q.--'..
t tr
I
t
,t
- - {I Control
-
N +Nisin
-
I
pH 5.5
.
Cr
I
I
XI
a .. '
_
...--"
I
I
t
d
1
. .°
I
.
.
II
II
a
1
pH 4.5
pH 5.0
pH 4.0
pH 3.5
6
-ff
5
5".
u_
4
0
O 3
0)
0
-.1
2
1
-
1
-
0
A,
t
. .
. .
0
6
12
-
-
1
k
18
0
-
t
t
. ,=7V
1
.
.
6
12
18
0
6 12
18
0
6
12
18
24
Days
Figure 2.1: Effect of pH and added nisin (37 x 102 IU/ml) on growth
of Listeria monocytogenes in Trypticase Soy Broth
incubated at 37°C.
93
10
8
e-- pH 6.8 + Nisin
o pH 6.8
"-E
D
Nisin
4 pH 4.5 + Nisin
6
----o--- pH 4.5 Nisin
u_
0
cm
0 4-
_1
2-
10
20
30
40
50
Hours
Figure 2.2: Effect of pH and added nisin (50 IU/ml) on growth of
Listeria monocytogenes ATCC 7644 (log CFU/ml) in
reconstituted non fat dry milk (NDM) incubated at 37°C.
94
1.0
0.8
E
00c 0.6
(0
Ci 0.4
0
0
Nisin, 1U/m1
o
--o- 20
-o- 50
0.2
--N--
100
-6- 200
0.0
Figure 2.3: Combined effect of pH and nisin concentration on growth
of Listeria monocytogenes in Trypticase Soy Broth
incubated at 37 °C
95
CD
Li_
0
0v--
C)
0
1
0
5
+ nisin/control Oil
a,
20 25
15
10
9
30
Days
Figure 2.4: Growth of Listeria monocytogenes 7644 at 4 and 370C in
the presence and absence of nisin (2.55 x 103 IU/g) in
sterilized cottage cheese.
96
1.0
A
Lactic acid
pH
0.8
011"---
i
I
:0
0
0
X'
0.6 <
U
as
0.4 __I
.
.III'
.' A-
.111°.
,.111
-a
Lactic acid
0.2
0.0
1.0
4
7
C
pH
pH
0.8
,
..a'
--l--11-
Lactic acid
6
Hours
Figure 2.5:
0.2
Lactic acid
4
2
,,
,
....
24 0
4
2
6
Hours
Effect of nisin on yogurt fermentation.
A:
B:
C
D:
Control (no nisin added)
20 IU/ml of added nisin
50 IU/ml of added nisin
100 IU/ml of added nisin
24
0.0
97
0
.
0
10
5
Days
Figure 2.6: Effect of nisin (50 IU/m1) on Listeria
rnonocytogenes in yogurt.
LM = Listeria monocytogenes
SC = Starter Culture
N = Nisin
15
98
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Nisin:
1984.
Food
antibiotic nisin comparative effect on Erysipelothrix and
Listeria. Proc. 4th Int. Symp. Antibiot. Agric., March 27 to 31,
1983 and the University of Nottingham. Blackwell Publ. Ltd.,
London, England.
24.
Ogden, K.
brewing.
1986.
Nisin: a bacteriocin with a potential use in
J. Inst. Brewing.
22:379-383.
25.
Ogden, K., and R. S. Tubb. 1985. Inhibition of beer spoilage
lactic acid bacteria by nisin. J. Inst. Brewing. 91:380-386.
26.
Papageorgiou, D. K., and E. H. Marth.
Fate of Listeria
monocytogenes during the manufacture, ripening and storage
1989.
of Feta cheese. J. Food Protect. 52:82-87.
101
27.
Rodriguez, D., J. F. F. Garayzabal, J. A. V. Boland, and E. R.
Fernandez.
1985.
Isolation de microorganisms du genre
Listeria a partir de lait cru destine a la consommation humaine.
Can. J. Microbiol.
28.
Rosenow, E. E. and E. H. Marth. 1987. Growth of Listeria
monocytogenes in skim milk, whole and chocolate milk and
whipping cream during incubation at 4, 8, 13, 21, and 350C. J.
Food Protect.
29.
31:939-944.
50:452-463.
Ryser, E. T., and E. H. Marth. 1987. Behavior of Listeria
monocytogenes during the manufacture and ripening of
Cheddar cheese. J. Food Protect.
30.
T. and E. H. Marth. 1987. Fate of Listeria
monocytogenes during the manufacture and ripening of
Ryser, E.
Camembert cheese.
31.
50:7-13.
J. Food Protect.
50:372-378.
Ryser, E. T., E. H. Marth, and M. P. Doyle.
1985.
Survival of
Listeria monocytogenes during manufacture and storage of
cottage cheese.
32.
Rubin, H. E.
Salmonella
58:255-206.
33.
J. Food Protect.
48:746-750, 753.
Protective effect of casein towards
typhimurium in acid milk. J. Appl. Bacteriol.
1985.
Schlech, W. F., P. M. Lavinge, R. A. Bortolussi, A. C. Allen, E. V.
Haldone, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E.
S. Nicholls, and C. V. Broome. 1983. Epidemic listeriosis
evidence for transmission by food.
New England J. Med.
308:203-206.
.
34.
Schaack, M. M. and E. H. Marth. 1988. Survival of Listeria
monocytogenes in refrigerated cultured milks and yogurt. J.
Food Protect.
51:848-852.
102
3 5.
Schaak, M. M. and E. H. Marth.
Behavior of Listeria
monocytogenes in skim milk and in yogurt mix during
fermentation by thermophilic lactic acid bacteria. J. Food
Protect.
1988.
51:607-614.
36.
Schultz, G. 1967. Studies on the occurrence of listeria in raw
milk. Montash. Veterinaermed. 22:766-769.
37.
Siragusa, G. R., and M. G. Johnson. 1987. An agar plating
medium for isolation of Listeria monocytogenes inoculated in
yogurt. p. 106 In Abstr. Inst. Food Technol. Mtg., June 16-19.
3 8.
Sorrells, K. M., D. C. Enigl and J. R. Hatfield. 1989. Effect of pH,
acidulant, time, and temperature on the growth and survival of
Listeria monocytogenes. J. Food. Protect. 52:517-573.
39.
Tanaka, N. 1982. Toxin production by Clostridium botuli num
in media at pH lower than 4.6. J. Food Protect. 45:234-239.
40.
Taylor, S. L.
products.
41.
1986.
Nisin as an antibotulinal agent for food
US Pat. No. 4,598,972.
Wei, L., and H. J. Norman.
Some chemical and physical
properties of nisin, a small protein antibiotic produced by
Lactococcus lactis. Appl. Environ. Microbiol. 56:2551-2558.
1990.
103
Chapter 3
Effect of Nisin on the Keeping Quality of Feta Cheese
Noreddine Benkerroum*
and
William E. Sandine
Department of Microbiology
Oregon State University
Corvallis, OR 97331-3804
Running Title: Nisin and Feta Cheese
*Present address: Department of Food Microbiology, Institute of
Agronomy and Veterinary Medicine, Hassan II, Rabat, Morocco.
104
ABSTRACT
Nisin was added to Feta cheese during cheesemaking fermentation to
achieve a final concentration of 40 IU/ml in an attempt to extend
cheese shelf-life. At this concentration, nisin had no adverse effect
on the cheese
starter bacteria.
Neither acid production during
fermentation, nor chemical composition of the final Feta cheese was
affected.
No significant effect (P <0.01) was observed on salt, fat or
ash contents.
Although the total dry matter was
significantly
reduced, its value along with the fat in the dry matter (FDM) still met
Greek regulatory standards.
Shelf-life, however, was reduced to less
than 47 days due
intensive growth of Gram- negative
to an
psychrotrophic bacteria which were not inhibited by nisin.
105
INTRODUCTION
Feta is a soft, white cheese. It originally was a Greek, home-made
cheese characterized by "its smooth, creamy, soluble and sliceable
body and pleasant acidic, salty and mild rancid flavor" when ripened
(9).
For a long time, manufacture of Feta cheese relied on the natural
flora of milk during the fermentation; therefore, its composition and
flavor were not consistent and varied greatly among localities,
climatic conditions and traditions.
Furthermore, defects in body and
flavor were common, as one would expect.
Feta cheese is normally
made from sheep or goat milk or blends of both.
However,
production of these two kinds of milk is not sufficient to yield
significant quantities of cheese.
Therefore, cow milk now is used as
Similar dairy products have been described in Near Eastern
well.
In Egypt, Domiati cheese is very similar to Feta
countries (10).
cheese, the main difference being in the salting procedure.
Feta
cheese is salted in 12% brine, while for Domiati cheese, salt is directly
added to the milk before fermentation to a final concentration of 5 to
15%
(10).
In
1964, Etthymiou
and Mattick (9) developed
a
procedure to manufacture Feta cheese from pasteurized cow milk
The best results were obtained with
a mixed starter culture composed of lactococci and lactobacilli.
Yogurt starter (e.g. Lactobacillus bulgaricus and Streptococcus
thermophilus) has also been used successfully (19).
using selected starter cultures.
The shelf-life of Feta cheese is variable, depending on raw milk
quality
and
the
degree
of
cleanliness
during
manufacture.
106
Papageorgiou and Marth (19) showed that high quality Feta cheese
could be conserved for more than 90 days. In some cases the shelflife of Feta cheese may be less, especially if the curd has been cut
before sufficient acid has been produced (9).
least 25% of
It is well known that at
the world's food supply spoils before it can be
The cost of this, while difficult to accurately estimate, is
nonetheless astronomical, especially in lost income (5). Therefore,
consumed.
intensive research work has been done to extend the shelf-life of
perishable foods.
The most common means used are heat
treatments, refrigeration, use of food additives, bacteriocins or
bacteriocin producer microorganisms and the use of viable lactic acid
bacteria.
Nisin has long been used for this purpose in dairy products,
meats, canned foods, and beverages (4, 6, 8).
In the present study an attempt was made to extend the shelf-life of
Feta cheese made from pasteurized cow milk by addition of nisin.
107
MATERIALS AND METHODS
Starter Culture:
Commercial lyophilized
starter culture (Visbyvac,
yogurt 231)
consisting of Lb. bulgaricus and Str. thermophilus (1:1) was obtained
from Laboratorium Wiesby (Germany).
The starter was regenerated
in sterile reconstituted NDM, which was incubated overnight at 370C .
Nisin:
Nisin was purchased from Applin and Barett, Trowbridge, England.
The stock solution was prepared as described earlier (Chapter
1
of
this thesis).
Manufacture of Feta Cheese:
Feta cheese was manufactured
Marth (19).
trials.
described by Papageorgiou and
Two batches were simultaneously made in each of three
as
In each batch, whole cow milk (101) was pasteurized (750C for
seconds), tempered and placed in stainless steel vats partially
submerged in hot water (35-40 0C) to maintain the milk temperature
16
at 350C.
Starter culture was then added (1% v/v) along with 0.01%
(w/v) of calcium chloride.
When the pH was reduced to 6.4, 2.5 ml of
1/10,000 strength calf rennet was added. To one batch, nisin was
added at the same time as rennet to a final concentration of 40
IU/ml, followed by thorough mixing. No nisin was added to the
108
second batch which served as a control.
The coagulum was cut with
0.63-cm knives 40 to 50 min after rennet addition.
Curds were then
transferred into rectangular perforated stainless steel forms (30 x 22
x 12 cm) and allowed to drain at room temperature (c.a. 220C) for 6
hours.
Forms were turned twice at 2-hour intervals.
After draining,
cheese was cut into six pieces (10 x 11 x 12cm) and placed into 12%
salt brine for 24 hours at room temperature.
The pieces of cheese
were then cut in half (10 x 5.5 x 12 cm) and placed in 6% salt brine.
Cheese was ripened in this brine at room temperature for 4 days.
After ripening, each piece was placed into a sterile metallic can and
covered with 6% brine and stored at 4°C.
Chemical Analysis of Feta Cheese:
Measurement of pH during fermentation and storage of cheese by
using a Corning pH meter equipped with a combination electrode.
Total dry matter (TDM), fat, salt and ash contents were determined
as described in Standard Methods for the Examination of Dairy
Products (22).
Microbiological Analysis of Cheese:
Samples of milk, curd and cheese were serially diluted in a sterile 2%
sodium
citrate
solution and plate counts
were
performed
on
In the case of curd and cheese, to achieve
the first dilution (1:10), 10 grams were weighed and placed into a
sterile stomacher bag containing 90 ml of sterile 2% sodium citrate
appropriate agar media.
109
solution, then blended in a Stomacher for 2 to 4 min.
microorganisms enumerated in
this
study
were:
Groups of
total
coliforms, psychrotrophs and lactic acid bacteria (LAB).
incubation conditions used are summarized in Table 3.1.
aerobes,
Media and
110
RESULTS
Effect of Nisin on the Maufacture of Feta Cheese:
Table 3.2 shows variations in the pH occurring during manufacture
and storage of Feta cheese with and without nisin added. The pH
varied similarly both in control (nisin free) and test (plus 40 IU/ml
It decreased to pH 4.55 and 4.70 at 6 days, then
increased to 4.73 and 5.01, respectively, at 47 days.
of nisin) samples.
Effect of Nisin on the Chemical Compostion of Feta Cheese:
Table 3.3 summarizes results on determinations of Fat in the Dry
Matter (FDM), salt, ash and Total Dry Matter (TDM) content in cheese
after brining in 6% NaC1 (e.g. the first day of storage).
show that
addition
of 40 IU/ml of
These data
nisin during Feta cheese
manufacture had no significant effect (P <0.01) on the fat, NaC1 or ash
contents.
However, TDM was significantly (P <0.01) lower in test
samples than in control.
The average TDM in the control and in the
test samples were 44.95 (± 0.83) and 43.42% (± 0.28), respectively.
Effect of Nisin on the Shelf-Life of Feta Cheese:
During manufacture and storage of Feta cheese, microbiological
analysis consisting of enumerations
psychrotrophs and LAB (Figure 3.1:
of total
a, b,
count, coliforms,
c, and d) were performed.
Cheese appearance was assessed on a regular basis during storage at
111
40C.
Figure 3.1 shows that all groups of microorganisms enumerated
in this study grew well in Feta cheese, regardless of whether or not it
contained nisin.
This
figure
psychrotrophs were not found
also shows that coliforms
and
pasteurized milk but were
detected in the cheese in relatively high numbers, higher in the test
in the
than in the control (Figure 3.1, b, and c). The average coliform counts
at 47 days were 9.8 x 106 CFU/ml in the test and 1.8 x 105 CFU/ml in
The average psychrotroph counts in the test and in the
control were 1.8 x 106 CFU/ml and 6.8 x 104 CFU/ml, respectively.
control.
Total and LAB counts varied in similar ways.
within
5
days
They were maximum
and remained relatively constant during storage
(Figure 3.1: a and d).
The appearance of cheese containing nisin was altered after 40 days
of storage. The surface of the cheese became slimy and soft. This
alteration is typically caused by Pseudomonas bacteria.
112
DISCUSSION
Results of this study showed that addition of 40 IU/ml of nisin to
Feta cheese had no adverse effect on its manufacture. Decrease of
the pH normally observed during lactic fermentation was not
affected.
The increase of the pH after 6 days (Table 3.3) was
observed both in control and test batches; therefore, it could not be
attributed to a nisin effect.
Similar findings were reported by
Etthymiou and Mattick (9), who showed that the pH of Feta cheese
increased from 4.7 to 5.1. 5.3 during one month of storage.
Papageorgiou and Marth (19), however, showed that the pH of Feta
cheese was about 4.3 after brining and this value was maintained
through the whole period of storage (90 days). The increase of the
pH in Feta cheese after 6 days may be due to contamination of the
cheese by microorganisms able to deaminate amino acids, resulting
in release of ammonia.
Such microorganisms are widely distributed
in nature (14). In this regard Pseudomonas was reported to produce
ammonia from arginine (23, 28).
Also, the chemical composition of
Feta cheese was not affected by nisin addition, except for the TDM,
which was lower in the test than in the control. However in both
cases FDM met Greek regulatory standards which provide that it
cannot be lower than 43%. The average value of salt content in the
control and test cheeses (3.1% ± 0.89 and 3.01 ± 0.74, respectively)
were higher than the normal salt content in commercial Feta cheese,
which is about 2.5% (18). However, there is no standard for the
amount of salt.
Greek regulations
describe
only
the
salting
113
procedure; therefore great variations in salt content of Feta cheese
should be expected and are, in fact, tolerated.
As for shelf-life, findings of this study showed that 40 IU/ml of nisin
did not extend the shelf-life;
on the contrary,
it was reduced.
Numbers of coliforms and psychrotrophs were higher
in cheese
containing nisin than in cheese without nisin (Figure 1, b and c). It is
well known that nisin inhibits only Gram-positive bacteria and has
no
effect on Gram-negative bacteria, including coliforms
Pseudomonas (6, 8, 15, 25).
and
Nisin may encourage growth of these
microorganisms by limiting the inhibitory action of the lactic starter
bacteria against them. Lb. bulgaricus has been shown to produce
inhibitory substances against a variety of microorganisms including
Pseudomonas (1, 3), which appear to be responsible for the altered
cheese quality. According to Taylor and Somers (27), the use of
lactobacilli is more efficient to preserve bacon than nisin. Although
nisin has been shown to extend the shelf-life of many foods,
including beverages (6, 8,
17,
18), dairy products (2, 6, 7,
8,
12, 16,
24), canned foods (4, 7, 13, 26) and meat (6, 8, 21, 27), in other cases
it
has been shown to have limited success
(8,
20, 27) or even an
adverse effect, especially in fermented products where the starter
regard we showed earlier
(Chap II, this thesis) that a relatively high concentration of nisin
(more than 50 IU /ml) impedes the yogurt fermentation. Also, in
cultures are sensitive to nisin.
In this
order for nisin to extend efficiently the shelf-life of food products, it
is
necessary to maintain Good Manufacturing Practices because this
bacteriocin will not inhibit the growth of many spoilage bacteria,
114
particularly Pseudomonas, which are common causes of dairy
product deterioration (5).
115
Table 3.1: Media and incubation conditions used in the enumeration of
different groups of microorganisms in Feta cheese.
Group of
Microorganisms
Total count
Medium
Incubation
Psychrotrophs
Plate Count Agar (Difco)
370C for 72 hours
VRBA (Difco)1+1,5% agar (Difco) 320C for 24 hours
Plate Count Agar (Difco)
70C for 10 days
LAB2
MRS (Difco)+1,5% agar (Difco)
Coliforms
1VRBA = Violet Red Bile Agar
2LAB = Lactic Acid Bacteria
300C for 24 hours
116
Table 3.2: Variation of pH during manufacture and storage of Feta
cheese with and without nisin added.
pH
Time
0
0.5
0.7
12
1.703
5.5
7.5
24
5 (days)
6 (days)
11
(days)
47 (days)
Control (without nisin)
Average
SD1
6.65
6.56
6.45
6.41
5.46
5.19
5.24
4.74
4.65
4.55
4.68
4.73
1 Standard deviation (±)
2Rennet addition
3Curd cutting
0.15
0.11
0.04
0.02
0.13
0.08
0.24
0.21
0.16
0.05
0.17
01
Test (+40 IU/ml of nisin)
Average
SD
6.65
6.57
6.49
6.41
6.11
5.79
5.63
4.89
4.79
4.70
4.82
5.05
0.15
0.11
0.06
0.01
0.53
0.54
0.39
0.15
0.03
0.06
0.07
0.05
117
Table 3.3:
added.
Analysis
Chemical analysis of Feta cheese' with and without nisin
Control (nisin. free)
Average
SD2
TDM (%)3
FDM (%)4
44.95
47.07
Salt (%)
Ash (%)
3.1
4.31
Test (+40 IU/ml of nisin)
Average
SD
.83
.60
.89
.47
1 Samples were taken after brining in 6% brine
2Standard deviation (±)
3Total Dry Matter
4Fat in the Dry Matter
43.42
46.63
3.01
4.41
0.28
3.48
0.74
0.40
118
9
7
E
-5.
LL
05
0)
0
_J
3
1
9
,4, -------------------
7
ff
IL
05
0)
0
_i
3
C
10
20
30
40
Days
Figure 3.1:
0
10
20
30
Days
Effect of nisin on the shelf-life of Feta cheese.
A:
Total count
B:
Coliform
C
Psychrotrophs
Lactic acid bacteria
D.
40
119
LITERATURE CITED
1
2.
Abdel Bar, N., N. D. Harris, and R. L. Rill. 1987. Purification and
properties of an antimicrobial substance produced by
Lactobacillus bulgaricus. J. Food Sci. 52:441-415.
Benkerroum, N., and W. E. Sandine. 1988. Inhibitory action of
nisin against Listeria monocytogenes. J. Dairy Sci. 71:32373 2 45.
3.
Blazek, A. B., J. Suscovic and S. Matosic. 1991. Antimicrobial
activity of lactobacilli and streptococci.
W. J. Microbiol.
Biotechnol.
4.
7:533-536.
Campbell, L. L., E. E. Sniff, and R. T. Obrien. 1959. Subtilin and
nisin as additives that lower the heat process requirements of
canned foods.
5.
13:462-464.
Cousin, M. A. 1982. Presence and activity of psychrotrophic
microorganisms in milk and dairy products: A review. J. Food
Protect.
6.
Food Technol.
48:401-411.
Delves-Broughton, J.
1990.
Nisin and its use as food
perservative. Food Technol. 40:100, 102, 104, 106, 108, 111,
112, 117.
7.
Eapen, K. C., R. Sankaran and P. K. Vijayaraghavan.
1983.
present status on the use of nisin in processed foods.
Sci. Technol.
The
J. Food
20:231-240.
8.
Eckner, K. F. 1991. Bacteriocins and food applications.
Vol VI: issue 3:1-5.
9.
Etthymiou, C. C., and J. F. Mattick.
1964.
domestic Feta cheese. J. Dairy Sci. 47:593-598.
Scope.
Developement of
120
10.
Fahmi, A. H. and Sharara, H. A.
Domiati cheese. J. Dairy Res.
1950.
17:312-328.
Studies on Egyptian
11.
Flores. Galaza, R. A., B. A. Glatz, C. J. Bern and L. D. Van Fossen.
1985.
Preservation of high moisture corn by microbial
fermentation. J. Food Protect. 48:401-411.
12.
Fowler, G. G.
au moyen de
1979. La conservation de produits alimentaires
la nisine.
Revue de Fermentations et des
Industries Alimentaires.
13.
34:157-159.
Gibbs, B. M., and A. Hurst. 1964. Limitations of nisin as a
preservative in non. dairy foods. 4th International Symposium
on Food Microbiology, SIK, Goteborg, Sweden. p. 151-164.
14.
Harrigan, W. F. and M. E. McCance. 1976. Laboratory Methods
in Food and Dairy Microbiology. Academic Press. New York.
15.
Hurst, A. 1981. Nisin: A review, p. 85-123. In D. Perlman, and
A. I Laskin (eds.), Advances in Applied Microbiology. Vol 27.
Academic Press. New York.
16.
Kalra, M. S., H. Laxminarayana and A. T. Dudani. 1973. Use of
nisin for extending shelf-life of processed cheese and khoa.
Food Sci. Technol. 19:92-94.
17.
Ogden, K.
brewing.
1985.
Nisin: a bacteriocin with a potential use in
J. Inst. Brew.
92:379-383.
18.
Ogden, K., M. J. Waites, and J. R. M. Hammond. 1988. Nisin and
brewing. J. Inst. Brew. 94:233-238.
19.
Papageorgiou, D. K., and E. H. Marth.
Fate of Listeria
rnonocytogenes during the manufacture, ripening and storage
1989.
of Feta cheese. J. Food Protect. 52:82-87.
121
20.
Rayman, K., N. Malik, and A. Hurst. 1983. Failure of nisin to
inhibit outgrowth of Clostridium botulinurn in a model cured
meat system. Appl. Environ. Microbiol. 46:1450-1452.
21.
Rayma, M. K., B. Aris and A. Hurst. 1981. Nisin: a possible
alternative or adjunct to nitrite in the preservation of meats.
Appl. Environ. Microbiol.
41:375-379
22.
Richardson, G. H. 1985. Standard Methods for the Examinaiton
of Dairy Products. 15th ed. American Health Association,
Washington, D.C:
23.
Rogul, M., and S. R. Carr.
1972.
Variable ammonia production
strains of Pseudomonas pseudomallei
resemblance to bacteriocin production. J. Bacteriol. 112:372among
smooth
3 8 O.
24.
Somers, E. S., and S. L. Taylor.
25.
Stevens, K. A., B. W. Sheldon, N. A. Klapes.
Antibotulinal
effectiveness of nisin in pasteurized process cheese spreads. J.
Food Protect. 50:842-848.
1987.
1991.
treatment for inactivation of Salmonella species
Gram- negative bacteria. Appl. Environ. Microbiol.
Nisin
and other
57:3613-
3 615.
26.
Taylor, S. L.
products.
27.
1986.
Nisin as an antibotulinal agent for food
U.S. Patent 4,597,972.
Taylor, S. L., and E. B. Somers. 1985. Evaluation of the
antibotulinal effectiveness of nisin in bacon. J. Food Protect.
48:949-952.
122
Chapter 4
Development and Use of Selective Medium for Isolation of
Leuconostoc Bacteria from Vegetables and Dairy Products
Noreddine Benkerroum*
and
William E. Sandine
Department of Microbiology
Oregon State University
Corvallis, OR 97331-3804
Running Title: Leuconostoc Selective Media
*Present address: Department of Food Microbiology, Institute of
Agronomy and Veterinary Medicine, Hassan II, Rabat, Morocco.
123
ABSTRACT
A Leuconostoc (Leu.) selective medium (LuSM) for isolation of
bacteria belonging to this genus was developed.
It contained 1.0%
glucose, 1.0% bacto-peptone (Difco), 0.5% yeast extract (BBL), 0.5%
meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05%
sorbic acid, 75 ppm sodium azide (Sigma), 0.25% sodium acetate, 0.1%
(v/v) Tween 80, 15% tomato juice, 30 µg /ml vancomycin (Sigma),
0.20 µg /m1 tetracycline (Serva), 0.05% cysteine hydrochloride and
The medium was successfully used for the
isolation and the enumeration of Leuconostoc bacteria in dairy
products and vegetables. Of 116 strains isolated from fresh raw
milk, curdled milk or various vegetables, 115 were identified as
Leuconostoc. Among them, 89 were successfully identified to the
1.5% agar (Difco).
species level as follows: Leu. cremoris (13.5%), Leu. mesenteroides
subsp. mesenteroides (7.9%), Leu. mesenteroides subsp. dextranicum
(11.2%), Leu. mesenteroides subsp. paramesenteroides (16.9%), Leu.
lactis (10.1%) and Leu. oenos (40.4%).
Comparative enumeration of
two Leuconostoc species, Leu. dextranicum 181 and Leu. cremoris
JLL8, on MRS agar and LuSM showed no significant difference
between counts obtained on both media.
Resistance to vancomycin
was shown to be chromosomally encoded in one strain of Leu.
cremoris.
124
INTRODUCTION
Selective media are among the most useful tools in microbiological
studies, especially in assessing the quality and safety of food
products and to follow the manufacture of fermented foods. For
example, detection and enumeration of some groups, species or even
strains, of microorganisms is essential to assess the hygienic quality
of foods prior to their release for sale or to assess the impact of
specific microorganisms on a technological process.
Leuconostocs are
heterofermentative lactic acid bacteria (LAB) naturally occurring in
milk, grass, herbage, grapes and many vegetables (29).
Members of
this group are widely used in dairy fermentations where they play a
major role in the production of lactic acid and aroma compounds (4,
5, 23, 30). They are also associated with the malolactic fermentation
of wine (15, 18) and other plant products such as pickles, green
olives and sauerkraut (9, 29). Attempts have been made to find
reliable media for their isolation and enumeration and both selective
and differential media have been reported but, unfortunately, none
has proven satisfactory.
Comprehensive reviews on the Leuconostoc
differential and selective media have been given by Garvie (12),
Teuber and Geis (29) and Cogan (5).
Differential media are mostly
based on the ability of leuconostocs to utilize citrate such that they
are recognized by halos developed around colonies growing on media
containing insoluble calcium
citrate (5,
10,
25,
34).
Such
differentation is not accurate since not all leuconostocs utilize citrate;
furthermore,
other
green
plant-associated
bacteria
such
as
Lactobacillus (16) and Lc. Lactis subsp. lactis biovar. diacetylactis
125
also utilize citrate (24, 25).
Other differential media are based on the
inability of leuconostocs to reduce triphenyltetrazolium chloride
(TTC) even in the presence of arginine hydrochloride, thus causing
them to have white colonies.
Arginine hydrolyzing
bacteria,
especially those of the Lactococcus genus, have red or pink colonies
cremoris and some lactobacilli behave in the
same manner. Selective media for leuconostocs were also proposed
from the use of different selective agents such as sodium azide (20),
(4, 3, 32).
Here Lc.
tetracycline (21, 23), and/or growth-promoting agents such as
cysteine hydrochloride and tomato juice (12, 13).
In all these media,
further confirmation is needed and some physiological tests must be
done on isolated colonies, making this procedure laborious and
inappropriate for enumeration.
Chromosomally encoded vancomycin resistance in Leuconostoc
species (8, 22, 27) as well as the sensitivity of Lactococcus
species
(8, 22) and some lactobacilli (27) to this antibiotic led us to consider
its use in a selective medium. The medium (LuSM) in combination
with other ingredients proved successful.
Herein we report on use of
the medium to isolate Leuconostoc species from several natural
sources and food products.
126
MATERIALS AND METHODS
Microorganisms and Their Maintenance:
Microorganisms used in this study are listed in Table 4.1.
They
include bacteria and yeasts. LAB were stored in litmus milk at -20 °C
.
Working cultures of lactococci were propagated in M17G (0.5%
glucose) (28); others were propagated in MRS broth (6) using 1%
inoculum and overnight incubation at 30 °C.
Yeasts were maintained
on slants of nutrient agar (Biokar) at 4 °C and propagated in nutrient
broth (Biokar) using loopful inoculations and overnight incubation at
25°C.
Medium Preparation:
The basal medium contained 1.0% glucose, 1.0% bacto peptone (Difco)
0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin
(Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm sodium azide
Sigma, 0.25% sodium acetate, 0.1% (v/v) Tween 80 and 1.5% of
tomato juice. The tomato juice was prepared as follows: fresh
tomatoes were blended in water (1:1 w/v) using a Moulinex blender.
Tomato juice was then centrifuged at 8000 xg in Servall centrifuge
for 15 min. The clarified tomato juice was used directly without
filtration.
The basal medium was sterilized by autoclaving (121°C for
The final pH of the medium varied from 5.3 to 5.8. No
adjustment of pH was made unless it was below 5.0, whereupon it
15 min).
was adjusted with HC1 to 5.5.
127
Stock solutions of vancomycin (Sigma), tetracycline (Serva) and
cysteine hydrochloride (BDH)
were prepared as follows:
vancomycin
and cysteine hydrochloride were dissolved in water to a final
concentration of 10 mg/ml and
1
mg/ml, respectively.
Tetracycline
solution was prepared according to Maniatis et al. (19) by dissolving
the antibiotic in ethanol to a final concentration of 5 mg/ml. All
these solutions were filter sterilized by using 0.22gm pore diameter
Millipore membranes and aliquots were stored at 200C.
Tetracycline
solution was protected from light by covering the container with
aluminum foil.
For preparation of the completed medium the basal medium was
melted and tempered to about 480C.
cysteine
hydrochloride
solutions
Vancomycin, tetracycline and
final
concentrations of 30µg /ml, 0.211.g/m1 and 0.5 mg/ml, respectively.
were
then
added
to
Action of Vancomycin on LAB:
The following strains were tested for their resistance to vancomycin:
Leu. cremoris 104, Leu. cremoris CAF7, Leu. cremoris 225, Leu.
cremoris 44-4, Leu. cremoris 44-4c, Leu. dextrancum 181, Lc. lactis
biovar. diacetylactis BU2, Lc. lactis biovar. diacetylactis F7/22, Leu.
cremoris M71, Lactobacillus
Vanomycin
was
incorporated
bulgaricus and Lc. cremoris AC1.
in
MRS
agar
(6)
at
various
concentrations (30, 100, 300 and 500 g g/m1), and the agar poured in
sterile Petri dishes and allowed to harden and dry.
An overnight
culture (250) of the microorganism to be tested was dispensed as a
128
drop on the surface of MRS agar containing a given concentration of
Plates were then incubated at 300C and checked for
growth after 2 and 4 days.
vancomycin.
Growth of Microorganisms on LuS.M.:
Microorganisms listed in Table 4.1 were activated in MRS or M17G
broth for LAB and nutrient broth for yeasts. Then a 25111 drop was
dispensed on the surface of the new medium, previously plated and
Plates were then incubated at 300C and examined daily for
growth up to 4 days.
dried.
Isolation and Identification of Microorganisms on the Experimental
Medium:
Microorganisms,
116
number,
in
were
isolated from different
products such as vegetables, milk and curdled milk.
Vegetables were
cut into pieces, soaked with distilled water and microorganisms
isolated from the suspension.
Samples of these products were
serially diluted and surface plated on LuSM and then incubated at
300C for 72 hrs. Colonies were randomly selected from plates
containing uncrowded colonies and purified on MRS agar.
They were
then stored in litmus milk at -200C until needed. For identification of
the
isolates, the
four
following Leuconostoc-identifying
characteristics were tested:
Morphology, catalase production, gas
production from glucose, and arginine hydrolysis. Strains were
identified to species level according to Garvie (11, 12). To test for
129
carbohydrate fermentation, the following sugars were used: lactose,
sucrose, cellobiose, arabinose, galactose, fructose, xylulose, trehalose,
maltose, glucose and esculin.
Quantitative Enumeration:
Enumeration was done on two strains: Leu. cremoris LL8, isolated
and identified in this study, and Leu. dextranicum 181. These strains
were grown separately in MRS broth at 300C for 16 to 18 hours.
Cultures were then serially diluted and plate-counted on LuSM.
Five
replications were done with Leu. cremoris JLL8 and 3 with Leu.
dextranicum 181, each in duplicate. The student test was used for
statistical analysis of the data.
Plasmid Profiles:
The procedure of Anderson and McKay (1), as modified by Wyckoff
et al., (35) was followed.
130
RESULTS AND DISCUSSION
Resistance to Vancomycin:
The results of this study are shown in Table 4.2.
All Lactococcus
strains were sensitive to vancomycin at concentrations lower than 30
lag/ml, while all Leuconostoc strains were resistant to a concentration
higher than 500µg /ml.
Resistance of leuconostocs to vancomycin is
Orberg and Sandine (22) and
(27) found members of this genus to be resistant to
well established (8, 14, 22, 27).
Simpson et al.
more than 2000 gg/m1 of vancomycin.
Noteworthy is that plasmid-
free Leu cremoris 44.4C (Figure 4.1) resists the same concentration of
vancomycin as the parental strain, Leu. cremoris 44.4, which has a
18.5 mDa plasmid (Table 4.3).
This suggests that vancomycin
resistance is a chromosomally encoded trait in leuconostocs.
Among
the 17 strains of Leuconostoc sp. studied by Orberg and Sandine
(22), 3 without plasmids could withstand up to 2000 µg /ml of this
In fact, this phenotype has been used in studies as a suitable
genetic marker to select for engineered leuconostocs (31). It should
drug.
be emphasized however that Leuconostoc is not the only genus of
LAB which is resistant to vancomycin; lactobacilli (8,27) and
pediococci (27) were also shown to be resistant to this antibiotic, L.
bulgaricus could tolerate only up to 100 1.1g/ml.
Nonetheless, unlike
Leuconostoc, this phenotype is not common in lactobacilli.
Eighty
percent of 20 Lactococcus strains tested by Vascovo et al. (33) were
found sensitive.
Simpson et al. (27) showed that homofermentative
lactobacilli in the thermobacterium group were vancomycin-sensitive
131
In contrast, the
sensitivity of lactococci to vancomycin at concentrations less than 10
g/ml is well documented (8, 22, 27).
Simpson et al. (27)
while heterofermentative lactobacilli were resistant.
recommended that 50 la g/ml of vancomycin should be used in
selective media.
Selectivity of the LuSM:
Lactococcus sp., Lb.
completely inhibited.
bulgaricus and the yeasts tested were
all
Although Lb. bulgaricus grew on vancomycin
containing MRS, it did not on the LuSM, suggesting that other
ingredients of the medium were inhibitory for that strain.
Isolation and Identification:
All isolated strains but one were Gram-positive, catalase-negative
gas producing cocci which did not hydrolyse arginine. They were
then regarded as leuconostocs. The exception did not produce gas
and formed cocci assembled in tetrads, typical of pediococci.
Eighty-
nine Leuconostoc strains were identified to the species level (Table
4.4). Results showed that the most common Leuconostoc species
represented was Leu. oenos (36% of the identified isolates), which
may have been due to its stimulation by cysteine hydrochloride (12).
No lactobacilli were found among the isolated strains, despite the fact
that many of them have been reported to be resistant to vancomycin
and that they have the same natural habitat as leuconostocs (8, 12).
The combination of many agents inhibitory to most LAB other than
132
Leuconostoc spp. and the presence of stimulatory agents to some
species of this genus would lead to its outgrowth over the rest of
Sodium azide was reported to be inhibitory to most LAB
LAB.
including
lactobacilli (17) but not leuconostocs (20) or L c .
diacetylactis (Sandine, unpublished data); therefore, this compound
was used by Mayeux et al. (20) as a basis for the formulation of a
Leuconostoc selective medium. Tetracycline was also shown to be
inhibitory to lactococci at low concentrations, less than 10 gg/m1 (21).
No data are available to our knowledge on the effect of this antibiotic
on lactobacilli or pediococci; nonetheless, it has been used in media
selective for leuconostocs (21, 23).
The major contribution of vancomycin in this medium is to achieve a
total inhibition of lactococci and to inhibit some lactobacilli as well,
The latter group of
mainly the homofermentatives (27).
microorganisms is also inhibited by sorbic acid.
Their sensitivity to
sorbate increases at pH values below 6.3 (27). In contrast, sorbic
acid was shown to stimulate growth of leuconostocs (7). It is also
inhibitory to yeasts (2).
Quantitative Determination:
No significant difference (P <0.05) was found between counts on MRS
and LuSM for both strains (Table 4.5). In the case of L e u
dextranicum 181, suitable colony size on the new medium was
.
obtained after 3 to 5 days of incubation, while on MRS 24 hours of
incubation were sufficient. In view of these results, LuSM would be
133
recommended for the isolation and the enumeration of leuconostocs
in mixed starter cultures or in fermented products containing a
heterogeneous flora.
134
Table 4.1: Bacterial and yeast strains used in the present study and
their origin.
Strain
Source
Bacteria
Lc. cremoris AC1
Leu. diacetylactis F7/22
Leu. cremoris 225
Lc. lactis 7962
Lc. lactis LB11
Leu. cremoris 44-4
Leu. cremoris 44-4C*
Leu. cremoris CAF-7
Leu. cremoris 104
Lc. diacetylactis BU2
E. coli V517
Micrococcus flavus
Staphylococcus aureus
Leu. cremoris M71
Lb. bulgaricus
Str. thermophilus
FDRC
FDRC
OSU
OSU
previous study
(26)
(26)
OSU
OSU
FDRC
OSU
OSU
IAV
FDRC
Yogurt starter (Rediset)
Yogurt starter (Rediset)
Yeasts
Saccharomyces cerevisiae
Candida milleri
IAV
IAV
FDRC = Federal Dairy Research Center (Kiel, Germany)
OSU = Oregon State University (USA)
IAV = Institute of Agronomy & Veterinary Medicine (Rabat,
Morocco).
Cured derivative of Leu. cremoris 44-4
135
Table 4.2:
Plasmid profiles of cultures used.
Strain
Molecular weight (Kb)
E. coli V517
56, 7.6, 5.8, 5.3, 4.1, 3.2, 28, 2.1
Leu. cremoris 44-4
18.5
Leu. cremoris 44-4C
None
136
Table 4.3:
Susceptibility of cultures used to vancomycin:
Strain
MIC (µg /m1)
Leu. cremoris 104
>500
Leu. cremoris 225
>500
Leu. cremoris CAF7
>500
Leu. cremoris 44-4
>500
Leu. cremoris 44-4C
>500
Leu. cremoris M71
>500
Leu. dextranicum 181
>500
Lb. bulgaricus
<100
Lc. diacetylactis BU2
<30
Lc. diacetylactis F7/22
<30
Lc. cremoris ACI
<30
137
Table 4.4: Identification of Leuconostoc strains used in this study.
Number
Percent (%)
Leu. cremoris
12
13.5
Leu. dextranicum
10
11.2
Leu. mesenteroides
07
7.9
Leu. paramesentroides
15
16.9
Leu. lactis
09
10.1
Leu. oenos
36
40.4
TOTAL
89
100
SPECIES
138
Table 4.5: Enumeration of Leu. dextranicum 181 and Leu. cremoris
JLL8 on MRS agar and on LuSM.
Leu. cremoris JLL8*
MRS
Leu dextranicum 181**
LuSM
MRS
LuSM
Counts
(CFU/ml)
1.96.108
1.8.108
1.9.108
1.6108
S.D.
1.7.108
1.3.108
2.7.107
3.35.107
*values are means of 5 determination in duplicates
**values are means of 3 determinations in duplicates
S.D. = Standard deviation
139
C
Figure 4.1: Plasmid profiles of Leuconostoc
cremoris 44-4 and its
cured derivative.
Lane A: E. coli V517 (Standard)
Lane B: Leuconostoc cremoris 44-4
Lane C.: Leuconostoc cremoris 44-4C
140
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