Production of Bacteriocin from isolates Natural Lactic acid
Production, Purification,
Stability and Efficacy of
Bacteriocin from Isolates of
Natural Lactic Acid
Fermentation of Vegetables
By
Uma B. Shinde
MITCON Biopharma, Pune.
Under the Guidance of
Miss Priya Bhande
MITCON Biopharma, Pune.
Page 1
Production of Bacteriocin from isolates Natural Lactic acid
CERTIFICATE
This is to certify that Miss. Uma Bhagwat Shinde student of Industrial
Biotechnology has successfully completed her project work entitled
“Production, Purification, Stability and Efficacy of Bacteriocin from
Isolates of Natural Lactic Acid Fermentation of Vegetables”
during 19Dec 2011 to 19 feb 2012 at MITCON biopharma centre, Pune
Miss Priya Bhande
(project guide)
Page 2
Production of Bacteriocin from isolates Natural Lactic acid
ACKNOWLEDGEMENT
It gives me immense pleasure to express my deep sincere gratitude to my guide
Miss. Priya Bhande for her suggestions, guidance, encouragement and support
throughout the period of project.
I am grateful to our head of department Mr. Kulkarni sir, for giving me opportunity
to join this esteemed institute and extending all laboratory facilities.
I am grateful to Miss Angha and Miss Neha for the positive support in my whole
project work and the guidance for handling the instruments and in technical work
and for all help, understanding me and my problems, supporting me in all possible
way throughout my work.
I am thankful to our lab assistants Mr. Amit and Mr. Sandeep for the co-operation
and providing all necessary things in all possible way.
I wish to extend my thanks to my family, especially my parents, my elder brother
and my elder sister without whose love and support I could not be here.
Last but not least my final thanks to my all lovely room mates my all friends in
MITCON, and specially my friend Aditi Ambekar. They understood mi and tried to
help mi in all possible way they could. Thank you very much for being by my side,
believing in me and putting up with me in all my good and bad times.
Page 3
Production of Bacteriocin from isolates Natural Lactic acid
Student Introduction
Name of the Student: Shinde Uma Bhagwat
Registration No.: BTN -33/2008
Name of the Training Program: Hands on Training
Name of the Training Organization: MITCON Institute
Duration of the Training: Dec to Feb
Name of the College: K. K. Wagh College of Agricultural Biotechnology, Nashik.
Page 4
Production of Bacteriocin from isolates Natural Lactic acid
Index
Sr No.
Title
Page No
1
Introduction & objectives
9 to 17
2
Review of the Literature
18 to 25
3
Aim and Scope of the 26
Work
4
Material and Methods
27 to 37
5
Result and Discussion
38 to 61
6
summary
62
7
Reference
63
Page 5
Production of Bacteriocin from isolates Natural Lactic acid
List of Tables
Sr. no
Table no
Page no
1
Media composition
29
2
Composition of crystal
30
voilet
3
Composition of gram
30
iodine
4
Composition of saffrain
31
5
Composition of Nutrient
32
agar
6
Preparation of acetone
33
7
Preparation of sodium
34
phosphate buffer
8
Inhibition zone of
40
antimicrobial activity
9
Observation of
48
biochemical test on
bacteriocin
10
Observation of sugar test
49
on bacteriocin
11
Zone of inhibition of
52 to 53
crude bacteriocin and
precipitated
12
Zone of inhibition of heat
55
stability
13
Zone of inhibition of
57
effect of pH
Page 6
Production of Bacteriocin from isolates Natural Lactic acid
List of Figures
Sr.no
Figures
Page no
1
Tomato,cucumber
37
inoculated Minimal media
2
Garlic inoculated Minimal 38
media
3
Growth
of
bacteriocin 39
(tomato) on MRS media
4
Growth
of
bacteriocin 40
(garlic ) on MRS media
5
Growth
of
bacteriocin 41
(cucumber)
on
MRS
media
6
Pure culture of cucumber
41
7
Pure culture of garlic and 42
tomato
8
Gram
staining
of 43
bacteriocin(cucumber)
9
Gram
staining
of 44
bacteriocin(tomato)
10
Inhibition
zone
of 46
antimicrobial activity
11
Inhibition
zone
of 47
antimicrobial activity
12
Citrate test utilization
49
13
Urease test
50
14
Indole test
50
15
Methyl red test
51
16
Zone
of
inhibition
of 53
C.B& precipitated
Page 7
Production of Bacteriocin from isolates Natural Lactic acid
17
Zone of inhibition of heat 55
stability
18
Zone of inhibition of heat 56
stability
19
Zone of inhibition of pH
59
Page 8
Production of Bacteriocin from isolates Natural Lactic acid
List of Abbreviations
1
G.S
Gram staining
2
GPB
Gram phosphate broth
3
E.coli
Escherichia coil
4
MRT
Methly red test
5
MRS
De Mann Rogose Sharpe
6
LAB
Lactic acid bacteria
7
Nacl
Sodium chloride
8
TSS
Triple sugar test
9
N.A.
Nutrient agar
10
Y.E
Yeast extract
11
PBS
Phosphate buffered saline
Page 9
Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 1
Introduction
Natural fermentation is one of the oldest means of preservation. Lactic acid bacteria
(LAB) subject the vegetables to a fermentation process. The vegetables when
fermented, it develops a pleasant sour taste and it is rich in vitamin and minerals.
Fermented vegetables are important for healthy nutrition.
In spite of the introduction of modern technologies and safety concepts, the
reported number of food borne illnesses and intoxications are on the rise. Many of
the ready-to-eat and novel food products represent new food systems with respect to
health and spoilage risks.
In the production of food, it is crucial to take proper measures for ensuring
its safety and stability during the shelf life. Food preservation is carried out to
maintain the quality of raw material and physicochemical properties as well as
functional quality of the product. Whilst providing safe and stable products.
Preservation of vegetables by lactic acid fermentation is an ancient practice
involving lactic acid bacteria (LAB), which predominantly product lactic acid
besides certain compound such as bacteriocin, which has antimicrobial activity
against other group of microorganisms. The antimicrobial activity of bacteriocin
produced by LAB has been detected in foods such as dairy products, meat, barley,
sourdough, fermented vegetables, etc. therefore, the strains of lactic acid bacteria
have also potential to act as a biopreservative or natural food preservative.
Preservation aims at either to eliminate or reduce the outgrowth potential of
spoilage and pathogenic organisms in foods. Lesser the severity of preservative
treatment, lower is the product damage, meeting both the food industry as well as
consumer interests for high quality products with improved organoleptic and
nutritional quality while maintaining microbial safety.
Page 10
Production of Bacteriocin from isolates Natural Lactic acid
The extensive use of antibiotics in food preservation has increased the risk of
development of resistance in human and animals. Consequently, there has been an
interest with the use of natural antimicrobials like bacteriocins. The exploration of
naturally occurring antimicrobials in food preservation receives increasing attention
due to consumer’s awareness of natural products. So these naturally synthesized
bacteriocins present high potential to be applied in hurdle technology.
Lactic acid bacteria have long been used as commercial vegetables,
fermented beverages for their contribution to flavor and aroma development and
spoilage retardation (Gilliland, 1986, Kleerebezen and Huqennotz, 2003).
The use of the LAB is a natural method for extending the shelf life of food.
The use of bacteriocin ro the microorganisms that produce them is attractive to the
food industry in the face of increasing consumer demand for natural products and
the growing concern about food-borne disease. It has Also necessitated the need to
exploit the biologically derived antimicrobial substances produces by LAB in
natural or inoculated fermentation the bacteriocin produced by the strains isolated
from natural fermented vegetables has neither been characterized nor checked for its
efficiency in various food products.
The use of bacteriocin is among the new approach, which may further
contribute to reduce risks of food borne disease, outbreaks and increased food
quality. Bacteriocins have attracted as potential substitutes for antibiotics to cure or
prevent bacterial infections. Research work accomplished over the last few decades
has focused on the detection, characterization and purification of bacteriocins.
The term Bacteriocin was first coined by Jacob et al. in 1953.bacteriocins are
naturally synthesized microbial substance produces by gram positive and gram
negative bacteria. Besides other antimicrobial compound, bacteriocins of lactic acid
bacteria gained worldwide attention.
The bacteriocin produced inhibited food spoilage and pathogenic bacteria
such
as
Salmonella
typhi,
Shigella
dysenteriae,
Klebsiella
pneumonia,
Page 11
Production of Bacteriocin from isolates Natural Lactic acid
Pseuadomonas Fluroscence, E-coli(Escherichia coli), Candida and Bacillus Cerus
which are recalcitrant to traditional food preservatives method.
Lactic acid bacterial strains are known to produce various types of
bacteriocins, which have bactericidal effects against gram positive bacteria including
food borne pathogens.
Organic vegetables are becoming increasingly popular among modern
consumers, LAB isolated from the same type of vegetables or products in which
they will be subsequently used as biocontrol agents, may have the greatest success
chance in controlling pathogenic bacteria (Vescovo, et al., 1996 Breidt and Fleming,
1997). This may be applied interest as biopreservative of minimally processed
organic vegetables. They also find application in various industries like brewing,
pharmaceuticals and healthcare. Nisin, a bacteriocin produced Lactobacillus
acidophilus is the only commercially used bacteriocin, which is generally recognized
as safe.
The present goal was to investigate LAB with potent antimicrobial activity
within the microbial ecosystem and finally to select interesting strains that have the
role of biopreservation
Bacteriocins:
Natural antimicrobials as novel means of food preservation.
Bacteriocins of LAB are used as the novel method of natural food preservation.
An ideal natural food preservative should fulfill a number of criteria, such as
a) Acceptable low toxicity
b) Stability to processing and storage.
c) Efficacy at low concentration.
d) No deleterious effect on the food and
e) Economic viability, while most bacteriocins fulfill these criteria.
Page 12
Production of Bacteriocin from isolates Natural Lactic acid
The bacteriocins produced by LAB offer several desirable properties
that make them suitable for food preservation:
i. Are generally regarded as safe substances,
ii. Are not active and non toxic on eukaryotic cells,
iii.
Become inactivated by digestive proteases, having little influence on
the gut microbiota.
iv. Are usually pH and heat tolerant,
v. They have a relatively broad antimicrobial spectrum against many food
borne pathogenic and spoilage bacteria.
vi. They show a bactericidal mode of action, usually acting on the bacterial
cytoplasmic membrane.
These bacteriocins are not like therapeutic antibiotics that can
potentially illicit allergic reactions in humans (Cleveland et al., 2001) and
also bacteriocins are proteinecious, ribosomaly synthesized where as
antibiotics are not. There are several large categories of bacteriocins which
are only phenomenologically related. Both gram positive and gram negative
bacteria produce Bacteriocins. These include the colicins, the microcins, and
the bacteriocins from Archaea. The bacteriocins from E. coli are called
colicins (formerly called ‘colicins,’ meaning ‘coli killers’). They are the
longest studied bacteriocins. Colicins produced by E. coli, are often proteins
larger than 20kDa that inhibit the closely related genera. In contrast, gram
positive bacteria produce bacteriocins smaller that 6 kDa. Bacteriocins of
LAB are generally regarded as safe. Most of the bacteriocins from LAB are
cationic, hydrophobic (or) amphophilic molecules composed of 20 to 60
aminoacids residues (Nesand holo, 2000).
Methods of Classification:
Methods of classification include:
Page 13
Production of Bacteriocin from isolates Natural Lactic acid
i.
Method of killing (pore forming, dnase, nuclease, murein production
inhibition, etc),
ii.
Genetics (large plasmids, small plasmids, chromosomla),
iii.
Molecular weight and chemistry (large protein, polypeptide, with/without
sugar moiety, containing atypical amino acids like lanthinonine) and
iv.Method of production (ribosomal, post ribosomal modifications, nonribosomal).
Bacteriocins are classified into four general classes. They are Class I,
Class IIa/b/c and Class III, Class IV.
Class I bacteriocin (lantibiotic):
Lantibiotics are a family of membrane active peptids that conatin the
unusual thio-ether amino acid (lanthionine) as well as other modified amino
acid, such as dehydrated serine and lanthionine. They are small composed of
one or two peptides of opproximately 3kDa. Their distinguishing feature is
the presence of post- translationally modified residues.the best example in
this group is nisinproduce by lactococcus lactic subsp. Lactis. Class I is
being further subdivide into Ia and Ib. classIa bacteriocin, which include
nisin, consist of cationic and hydrophobic peptidies that form pores in the
target membranes and have a flexible structure compared to the more rigid
ones of classIb. ClassIb bacteriocins, which are globular in nature, have no
net negative charge.
Nisin:
Nisin is the star of bacteriocins. It is the only bacteriocin allowed as a
food additive (E234, 1983), recognized as safe (FAO/WHO, 1968) and
accepted by the American Food and Drug Administration (1988). Its
structure has been solved, revealing modified amino acids and lanthionine
rings, and its chemical and enzymatic syntheses have been successful.
Killing activities of nisin include formation of membrane pores, blockage of
cell wall biosynthesis, release of cell wall hydrolases, and prevention of
spore outgrowth. The structural gene ( nisA, nisZ, nis Q, and nsuA), and the
Page 14
Production of Bacteriocin from isolates Natural Lactic acid
genes (nisBTCIPRKFEG) require for its synthesis, regulation, and slefprotection have been cloned, sequenced, and functionally characterized. The
regulation includes auto induction by nisin. The nisin inducible promoters
have been used for protein production and for bioassays to quantify nisin in
food. Properties of nisn have been improved by genetic engineering.
Class II bacteriocin:
The class II bacteriocin is small, heat-stable, bioactive protins. These
are generally unmodified peptides of <5 kDa. They are also further subdivide
into classIIa and classIIb. The action of class IIa bacteriocin seems to involve
disruption of mannose transport into target cells. ClassIIb bacteriocin form
pores in the membranes of target cells and disrupt the proton gradient of
target cells. Class IIa includes pediocin-like bacteriocin having antilisterial
activity. Other bacteriocins can be grouped together as Class IIc. These have
a wide range of effects on membrane permaeability, cell wall formation and
pheromone actions of target cells.
Class III bacteriocins:
Large, heat-labile protein bacteriocins of large molecular weight
include Helviticin-J, Lactacins A and B which are generally > 30kDa like
Enterolysin produced by Enterococcus faecium.
Class IV bacteriocins:
This group is consisting of complex bacteriocins that require
carbohydrate or lipid moieties for activity.
Bacteriocins isolated from different Lactobacillus species
Substance
Producing species
Acidolin
L. acidophilus
Lactacin B
L. acidophilus
Lactacin F
L. acidophilus
Page 15
Production of Bacteriocin from isolates Natural Lactic acid
Factors affecting Bacteriocin production:
Bacteriocins are economically important, so it has to be produced in
large amounts and preferably by growing the strains in media containing
food grade ingredients (Halami et al, 2005; Yang and Ray, 1994). Production
of bacteriocin in a simple medium can be increased by growing the cells at
an optimum pH and supplementing with nutrients specific for a species or
strain. Growing cells under optimum environment for 16 hours facilitates
high bacteriocin production.
Productions of the bacteriocins:
Class I and class II a bacteriocins are usually very stable acidic pH.
For example Rodriguez et al (2002) found that pediocin-PA-was perfectly
stable after 2ld of storage at 15c at 4to6; However half of the activity was
lost at pH7 Larsen and others (1993) found that Bavaricin A was very stable
at pH 2 to 9.7. But storage of Bavaricin A at pH 12.5 for 4 hours resulted in
the complete loss of activity. In addition, bacteriocins from these two classes
are heat stable at acidic pH. As PH increases the heat stability decreases. In
general, bacteriocins are usually sensitive to proteolyytic enzymes, such as
tyrpsin, due to their Proteinaceous nature.
Extraction of bacteriocin:
Extraction of bacteriocin in a large, pure, and concentrated from
(Bibek et al, 1992) is important to study its physical, chemical and
antimicrobial properties and to apply in food systems. Muriana and
Luchansky (1993) have summarized the method to concentrate the proteins
from the culture filterate it involves vaccum concentration, solvent
Page 16
Production of Bacteriocin from isolates Natural Lactic acid
precipitation, acid precipitation. Ammonium sulphate precipitation is the
most common and effective method for the extraction of the concentrated
protein. Yang et al (1992) have also reported a cell adsorption method, which
allowed bacteriocin in the broth to be absorbed to the producer cells at an
optimum pH.
Identification of LAB:
Classical
and
molecular
techniques
were
involved
in
the
identification of bacteriocinogenic LAB. Traditionally, LAB have been
identified and grouped by conventional techniques such as morphological,
physiological and biochemical tests (Bergey’s manual 1986’s edition and
Sharpe, 1979).
Page 17
Production of Bacteriocin from isolates Natural Lactic acid
Objective
The aim of the work was to evaluate the potential use of Bacteriocin
produced by Organisms isolated after Natural Lactic Acid fermentation of
vegetables.
1. The production, identification, purification of Bacteriocin.
2. Checking the stability and efficiency of bacteriocin produced.
Page 18
Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 2
Review of Literature
The LAB comprise a clade of Gram positive, catalase negative, acid
tolerant, rod shape that are associated by their common metabolic and
physiological characteristics. They are widely distributed in nature. They are
isolated fr4om fermented vegetables and lactic products produce lactic acid.
This trait has historically linked LAB with food fermentations as
acidification inhibits the growth of spoilage agents. Proteinaceous
bacteriocins are produced by several LAB strains and provide an additional
hurdle for spoilage and pathogenic microorganisms. The industrial
importance of the LAB is further evidenced by their generally regarded as
safe (GRAS) status, due to their ubiquitous appearance in food and their
contribution to the healthy microflora of human mucosal surfaces.
LAB can be divided into two groups based upon the products produced from
the fermentation of glucose they are.
Homofermenters :
The fermentation of one mole of glucose yields two mole of lactic
acid, generation a net of 2 ATP per mole of glucose metabolized
C6H12O6
2CH3CHOHCOOH
Page 19
Production of Bacteriocin from isolates Natural Lactic acid
Glucose
lactic acid
Heterofermenters:
The fermentation of 1 mole of glucose yields 1 mole each of lactic
acid, ethanol and carbon dioxide. One mole of ATP is generated per mole of
glucose.
C6H12O6
CH3CHOHCOOH + C2H2OH + CO2
Glucose
Lactic acid
Ethyl Alcohol
Classification of Lactic acid bacteria based in type of fermentation
Homofermenters
Facultative
Obligate heterofermenters
homofermenters
Lactobacillus acidophilus
Lactobacillus
Lactobacillus cellobiosus
coryniformis
Lactobacillus lactis
Lactobacillus curvatus
Lactobacillus confusus
Lactobacillus delbrueckii
Lactobacillus plantarum
Lactobacillus coprophillus
Lactobacillus leichmannii
Lactobacillus sake
Lactobacillus
fermentatum
Lactobacillus bovis
----
Lactobacillus sanfrancisco
Lactobacilli are gram positive and vary in morphology from
long that can be either hetero-or homofermentative. They are widespread and
can be isolated from many vegetables sources. Lactobacilli are more tolerant
to acid than the other genera of lactic acid bacteria and this property makes
them important in the final phases of many food fermentations when other
organisms are inhibited by the low pH.
Probiotic application of LAB
The comcept of probiotics was in use in the early 1900s, response
however, the term was coined in 1965 by Lilly and Stillwell. Probiotic is a
Page 20
Production of Bacteriocin from isolates Natural Lactic acid
preparation of live microorganisms, which when applies to man or animal,
beneficially affects the host by improving the properties of the indigenous
microbiota (Havenaar et al, 1992).
LABs are regarded as a major group of probiotic bacteria (Collins
at al., 1998; Schrenmeir and de Vrese, 2001). The probiotic concept has been
defined by Fuller (1989) to mean “ a live microbial feed supplement which
beneficially affects the host animal by improving its intestinal microbial
balance”, Salminen et al, (1999) proposed the probiotics are microbial cell
preparations or components of microbial cells that have a beneficial effect on
the health and well-being of the host. Several lactobacilli, lactococci and
bifidobacteria are held to be health-benegiting bacteria (Rolfe, 2000; Tuohy
et al., 2003), but little is known about the probiotic mechanisms of gut micro
biota (Gibson and Fuller, 2000).
LAB constitute an integral part of the healthy gastrointestinal (GI)
micro ecology and are involved in the host metabolism (Fernandes et al.,
1987). Fermentation has been specified as a mechanism of Probiotics
(Gibson and Fuller, 2000; Metchnikoff, 1908). LAB along with other gut
micro biota ferment various substrates like lactose, biogenic amines and
allergic compounds into short-chain fatty acids and other organic acids and
gases (Gibson and Fuller, 2000; Jay, 2000).
Reaction of organisms on the pathogens.
1. Salmonella typhi:
A rod-shaped flagellated, facultative anaerobic, Gram-negative
bacterium, and a member of the genus Salmonella.
The given salmonella typhi was inoculated in sterile saline. This 1 ml
saline was spread on Nutrient agar media and kept for 5 min in laminar air
flow.The obtained inhibition forming organisms were streaked on the media,
and kept in incubator at 37ºc for 24 hours.
2. Shigella dysenteriae:
Page 21
Production of Bacteriocin from isolates Natural Lactic acid
Shigella dysenteriae is a species of the rod-shaped bacterial genus
Shigella.[1][page needed] Shigella can cause shigellosis (bacillary dysentery).
Shigellae are Gram-negative, non-spore-forming, facultatively anaerobic,
non-motile bacteria.[2]
This strain also spread alike salmonella, and organism forming
inhibition zone was streaked and kept for incubation.
3. Klebsiella pneumoniae:
Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated,
lactose fermenting, facultative anaerobic, rod shaped bacterium found in the
normal flora of the mouth, skin, and intestines.[
Same procedure as like salmonella and shigella is done and kept for
incubation.
4. Pseudomonas Fluroscence:
Pseudomonas fluorescens is a common Gram-negative, rod-shaped
bacterium.[1] It belongs to the Pseudomonas genus; 16S rRNA analysis has
placed P. fluorescens in the P. fluorescens group within the genus,[2] to
which it lends its name.
The inhibiting organism is streaked on plate on which suspension of
pseudomonas is spread,and incubated overnight.
5. E-coli: ( Escherichia coli)
Escherichia coli (commonly abbreviated E. coli) is a Gram-negative,
rod-shaped bacterium that is commonly found in the lower intestine of
warm-blooded organisms (endotherms). Most E. coli strains are harmless,
but some serotypes can cause serious food poisoning in humans, and are
occasionally responsible for product recalls.[2][3] The harmless strains are part
of the normal flora of the gut, and can benefit their hosts by producing
vitamin K2,[4] and by preventing the establishment of pathogenic bacteria
within the intestine.[5
The pathogenic strain of E-coli was spread on nutrient agar and the
inhibition forming organism was streaked and incubated overnight.
6. Candida:
Candida albicans is a diploid fungus that grows both as yeast and
filamentous cells and a causal agent of opportunistic oral and genital
Page 22
Production of Bacteriocin from isolates Natural Lactic acid
infections in humans.[3][4] Systemic fungal infections (fungemias) including
those by C. albicans have emerged as important causes of morbidity and
mortality in immunocompromised patients (e.g., AIDS, cancer
chemotherapy, organ or bone marrow transplantation). C. albicans biofilms
may form on the surface of implantable medical devices. In addition,
hospital-acquired infections by C. albicans have become a cause of major
health concerns.
Candida albicans is a diploid fungus that grows both as yeast and
filamentous cells and a causal agent of opportunistic oral and genital
infections in humans.[3][4] Systemic fungal infections (fungemias) including
those by C. albicans have emerged as important causes of morbidity and
mortality in immunocompromised patients (e.g., AIDS, cancer
chemotherapy, organ or bone marrow transplantation). C. albicans biofilms
may form on the surface of implantable medical devices. In addition,
hospital-acquired infections by C. albicans have become a cause of major
health concerns.
The given strain of Candida was spread on nutrient agar and streaked
with the same organism streaked in all above plates. And incubated at 37º c
for 24 hours.
Biochemical tests:
Principle:
According to the physical characters, the biochemical tests are done.
When we come to know the gram’s nature, motility and colony structure, we
can conclude the biochemical tests referring to the Bergey’s manual.
The presence of the particular enzyme in a micro organism can be
tested by incorporating a specific substrate in a medium, (if necessary), and
then detecting the products formed or even checking the disappearance of the
substrate from the medium.
These biochemical tests employ various media (having different
substrate) which when inoculated with a particular species of bacteria will
follow a specific metabolic pathway to hydrolyze the substrate available to
them. Some of the routine biochemical tests used for determining metabolic
activities of bacteria can be broadly classified as




Utilization of carbohydrates and acids
Utilization nitrogenous compounds
Decomposition of large molecules
Miscellaneous tests
Page 23
Production of Bacteriocin from isolates Natural Lactic acid
According to the physical characters, as the bacteria are gram
negative, the biochemical tests for the obtained bacteria are mentioned
below:
a) Carbohydrate fermentation (Sugar utilization) test Principle:
Sugars are metabolized through different metabolic pathways
(Depending type of species and aerobic and anaerobic environment) to form
various acids like pyruvate, lactase, succinate, formate, acetate ect. These
acids so formed may further break down to gases (formic hydrogenlyase will
split formic acid to H2 and CO2 ).
Due to acid formation, the PH of the medium is lowered and phenol
red indicator is being faint pink to colorless. Gas formation is demonstrated
by the use of Derham’s tube (a small tube inverted in the sugar solution.)
which collect gas.
b) Methyl Red testPrinciple:
Only mixed acid fomenters ( e.g. Escherichia coli) produces
sufficient quantity of acids during initial phase of incubation (PH below than
4.4) which can be detected by methyl red indicator. This is because the fact
the medium glucose phosphate broth is strongly buffered, hence minute
quantities of acids if produced, will not permit the PH of the medium to drop
down. Moreover, methyl red is a PH indicator having ranges between 6.2
(yellow) to 4.4 (red), so the PH at which Methyl red detects acid is
considerably lower than the PH for other indicators used in bacteriological
medium.
c) Indole Production Test:
Principle:
Indole, a benzyl pyrrole, is one of the metabolic degradation products
of the amino acid tryptophan. Organisms that possess the enzyme
tryptophanase are capable of hydrolyzing and deaminating tryptophan with
the production of indole, pyruvate and ammonia.
Indole so produced react with the aldehyde group of a weakly acid
alcoholic solution of Þ-dimethylaminobenzaldehyde (Kovac’s reagent)in
Page 24
Production of Bacteriocin from isolates Natural Lactic acid
presence of heat to form pink colored rose-indole complex. The reaction can
also occur without heat, if the reagent is prepared with HCL.
Indole is a substance which reduces surface tension and hence it is
concentrated in the surface layer of the medium. Moreover, because indole is
soluble in organic compounds. It is recommended that chloroform or xylene
be added prior to adding Ehrlich’s reagent. This serves two purposes, firstly
it extracts indole from whole of the medium and secondly it forms a separate
layer above the medium. As a result, reagent reacts with the indole extracted
in the xylene and forms a pink color. Organic solvents like chloroform, ether,
and light petroleum can be used instead of xylene. This step is not necessary
with kovac’s reagent because the amyl alcohol is used for the diluent is
capable of extracting sufficient indole from the aqueous medium to produce
a positive reaction.
d) Vogous Proskauer (VP) testPrinciple:
In presence of alkali and air (vigorous shaking) acetoin is oxidized to
diacetyl which reacts with guanidine nucleus of arginine present in proteins
present in proteins of peptone to produce pink color. At times a pinch of
creatine is added to provide an additional source of guanidine nucleus and
thus accelerate pink color formation. Test is made sensitive by adding αnaphthol,which serves as catalyst.
e)Citrate utilization test:
Principle:
The test determines the ability of the bacteria to use citrate as sole of
carbon and energy. This ability depends on the presence of a citrate permease
that facilitates transport of citrate into the bacterium. Once inside the cell,
citrate is converted to pyruvate and CO2 . Citrate agar slant contain sodium
citrate as the sole source of carbon, ammonium phosphate as a sole source of
nitrogen, and bromothymol blue as a PH indicator [PH 6 (yellow)-PH 7.6
(blue)]. This test is done on slant since o2 is necessary for citrate utilization.
When bacteria oxidise citrate, they remove it from the medium and liberate
CO2. This CO2 combines with sodium (Supplied by sodium citrate and
water to form sodium carbonate – an alkaline product. Similarly,bacteria
that utilize citrate can also extract nitrogen from the ammonium salt, with the
production of ammonia, which is convertwd to ammonium hydroxide
Page 25
Production of Bacteriocin from isolates Natural Lactic acid
(NH4OH). These alkaline products raise PH, and turn pH indicator to a blue
color and represents a positive citrate test
f)Urea Hydrolysis Test (Urease Test):
Principle:
A strongly buffered medium in which urea is only nitrogen source is
used for the test. Urease is an enzyme possessed by many species of micro
organisms that can hydrolyze urea.
The ammonia so produced reacts in solution to form ammonium
carbonate, resulting in alkalinization and an increase in pH of medium. This
is indicated by change in color of the indicator phenol red (pH 6.8-8.4 yellow
to purple red).
Due to high buffering capacity of the medium, only those organisms
possessing vigours urease activity (proteus vulgaris) can given test positive.
f) Catalase test :
Principle:
Catalase is an enzyme that splits up hydrogen peroxide into oxygen
and water. Chemically catalase is a hemoprotein, similar in structure to
hemoglobin.
Catalase is present, often in high concentrations in the majority of
aerobic organisms but is absent from most obligate anaerobes. Thus when
H2O2 is added externally in the medium,catalase activity results in the
production of molecular gaseous oxygen. Catalase activity can be tested
either by slide test or tube test.
g) Oxidase test:
Principle:
Gordon and McLeod (1) introduced oxidase test for identifying
Gonococci based upon the ability of certain bacteria to produce indophenol
blue from the oxidation of dimethyl-p-phenylenediamine and α-naphthol.
Gaby and Hadley (2) introduced amore sensitive method by using N,Ndimethyl-p-phenylenediamine oxalate where all Staphylococci were oxidase
negative.
In presence of the enzyme cytochrome oxidase (gram-negative bacteria) the
N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol react to
indophenol blue
Page 26
Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 3
Aims and Scope of Study

Production of the Bacteriocin from Natural Lactic Acid fermented
vegetables.

Purification, Biochemical, Stability of the Bacteriocin.
Page 27
Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 4
Materials and Methods
Fermented Vegetables
Vegetables like a Cucumber, Tomato, and Garlic were washed,
peeled and grated/sliced. The sliced of Cucumber, Tomato, Garlic were
inoculated in minimal salt media and Micro flora were isolated from there
samples.
Composition of Minimal Salt 1000ml

30g Na2HPo4

15g NH5Po4

5g NH4Cl

25g NaCl

15g CaCl2
I was taken 3 Bottle in 1 flask 100 ml minimal salt media.
In each flask I inoculate the in flask and keep at room temperature.
All this procedure I had done luminal air flow.
Pathogenic Bacterial Cultures.
I was taken Standard Bacterial Culture 7 standard Bacterial Culture
were taken to sub – culturing of standard bacterial culture. By using N.A
o Standard Bacterial Culture

Salmonella typhi

Shigella dysenteriae

Klebsiella pneumonia

Pseuadomonas Fluroscence,

E-coli(Escherichia coli),
Page 28
Production of Bacteriocin from isolates Natural Lactic acid

Candida

Bacillus Cerus
I were take 7 test tubes and prepare N.A
o Composition of Nutrient Agar 100ml

Peptone – 1gm

Yeast extra – 1gm

NaCl – 05gm

Agar – 2gm
Standard bacterial cultures were procured from MITCON Institute
Pune, were used in bacteriocin screening procedure and all cultures were sub
- culture and were used.
Autoclave the test tubes and N.A than inoculate in test tubes and
prepare the slant streaked the standard bacterial culture and keep it for
incubation at 370c in incubator.
Isolation and Identification of Bacteriocin producing Bacteria
The Bacteriocin procedure from naturally fermented Cucumber,
Tomato and Garlic were isolated by pour plate method technique. By using
MRS Agar (de Mann Rogosa and Sharpe).
Bacteriological Media
The media used for the work were MRS (de Mann Rogosa and
Sharpe) broth and agar were purchased from Himedia, India.
Page 29
Production of Bacteriocin from isolates Natural Lactic acid

Media Composition
MRS (de Mann Rogosa and Sharpe) Medium
Table 1

Ingredient
Amount(g/l)
Protease Peptone
10.00
Yeast extract
5.00
Beef extract
10.00
Dextrose
20.00
Polysorbate 80
1.00
Ammonium Citrate
2.00
Sodium Acetate
5.00
Magnesium Sulphate
0.10
Manganese Sulphate
0.05
Dipotassium Phosphate
2.00
MRS Agar Medium
MRS Agar Medium was prepare by adding 2gm in 100ml to the broth
and boiled to dissolve the agar.
The requisite amount of the medium were taken is Conical flask,
plugged with cotton and autoclaved.
Pour the plate and from Cucumber 1ml, Tomato 1ml and Garlic 1ml
sample were taken were spreading, streaking method were done and
inoculated for 24 – 48 hr at 320c.
Page 30
Production of Bacteriocin from isolates Natural Lactic acid
After incubation typical colonies were isolated and pure culture is
formed in N.A by streaking method.
The isolated were differentiated on their Morphological and
Biochemical Test, staining is done to identify the M.O.
The native isolate of LAB were subjected to species level
identification by various microbial, biochemical test, according as described
in Bergey’s manual of Bacteriology.
Study of physical properties
The study of physical character includes colony characters and gram
staining i.e. Morphology of the organism
The gram staining of the organism is done as well as the characters of
the formed colonies are noted down.
o Microbiological tests(Morphological Tests)

Gram Staining
 Material
Table 2:

Crystal Voilet
Ingredients
Amount g/100ml
Crystal Voilet
2.0
Ethyl Alcohol
20.0
Ammonium Oxalate
0.8
D/W
80.0ml
Crystal voilet and ammonium oxalate were dissolved separately in
ethyl alcohol and d/w respectively and the two solution were then mixed.
The prepared stain was filtered and stored in a clean, dry glass stoppered
bottle.
Page 31
Production of Bacteriocin from isolates Natural Lactic acid
Table 3:

Grams Iodine (Mordant)
Ingredients
Amount g/100ml
Iodine
1.0g
Potassium Iodine
3.0
Table 4

Saffranin ( Counter Stain)
Ingredients
Amount g/100ml
Saffranin
2.5g
Ethyl Alcohol
100ml
Methods (Hucker’s Modification)
 Prepare a heat fixed smear of the culture
 Cover the smear with crystal voilet stain for 1min
 Add gram’s iodine to wash off crystal voilet stain and cover it with
iodine till smear turns coffee brown in color (Approx. 1min)
 Rinse the slide in the running water.
 Add decolorizing solution drop wise at the upper end of slide held in
inclined position, till voilet color fails to come out from the smear, for
normal smear 10 – 15 sec are enough.
 Rinse the smell with water
 Counterstained with Saffranin for 45 – 60min
 Rinse with the tap water, drain, blot, air dry and examine under electro
microscope
The morphology of cell is record.
Page 32
Production of Bacteriocin from isolates Natural Lactic acid
For the identification of the bacteria some common tests are done for
identification of the specific bacteria. Physical characters studied first
accordingly the chemical tests are done.
 Screening of Isolates for Antimicrobial Activity
Antimicrobial activity of the bacterial isolates against all the
pathogenic microorganism was determined by well diffusion method (13 16) under the aerobic conditions.
Material
Table 5:
Nutrient agar media composition
Ingredients
Amount gm/l
Peptone
1 gm
Yest extract
0.3 gm
Nacl
0.5 gm
Agar
2 gm
Method
N.A. medium was prepare & the requisite amount of the medium
wear taken in conical flask plugged with cotton and autoclaved.
Then the bacterial culture wear grown in MRS broth and dilution
approx.
The N.A. pour in plate& make media solidify then the lawn of pathogen
is formed with the help of spreader.
There wear seven pathogen are used.
a. Salmonella typhi
b. Shigella dysenteriae
Page 33
Production of Bacteriocin from isolates Natural Lactic acid
c. Klebsiella pneumonia
d. Pseuadomonas Fluroscence,
e. E-coli(Escherichia coli),
f. Candida
g. Bacillus Cerus
Before preparing lawn prepare the saline and inoculate the pathogen
saline and lawn with the help of spreader and form well and isolated stain
were placed into each well. After all this procedure plates were kept in
incubator for incubation at 370c for 24 hr.
The antimicrobial activity was determined by measuring the diameter
of the inhibition zone around the well.
Partial purification of Bacteriocin
Isolated strain which were isolated from vegetables by the screening
of isolates for antimicrobial activity which showing widest zone of inhibition
against the target microorganisms having the maximum antimicrobial zone
where used for partial purification of Bacteriocin.
The maximum antimicrobial zone was grown in MRS broth at 370c
for 24hrs.
The isolated strain were inoculated in MRS broth and used.
After incubation, the broth was centrifuged at 5000x g for 10min. I
was taken 4 centrifuge test tubes for centrifugation. After centrifugation, the
cells were separated out and only superannuated was used as a crude
bacteriocin.
Material
Table 5:

Preparation of Acetone
Ingredient
Ml
Page 34
Production of Bacteriocin from isolates Natural Lactic acid
Acetone
80ml
Distilled Water
20ml
The acetones were added to the supernatant. I were taken 4
test tubes in equal amount of Acetone were added in supernatant.
After that it was kept undisturbed at 40c overnight.
Next day it removes from freezer. Precipitates formed were
collected by centrifugation at 5000 *g for 5min and again centrifuge
at 5000*g for 5min.
Preparation of Sodium Phosphate buffer
Table 7:
Ingredient
Ml/Gm
Sodium Phosphate
O.335 gm
Distilled Water
100 ml
PH
6
And after preparation of sodium phosphate buffer with
PH=6.0
The comparison with crude bacteriocin and precipitate bacteriocin.
Again i preparing the NA. 5 pathogen were used against the strain.5
pathogen
There wear five pathogen are used.
a. Salmonella typhi
b. Klebsiella pneumonia
Page 35
Production of Bacteriocin from isolates Natural Lactic acid
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus substils
N.A wear taken in conical falsk & plugged with cotton & autoclave ,
petri plate wear also autoclave.
Then pour in petri plate then keep for solidification after that I wear
prepar lawn of pathogen with help of spreader. after that wells wear formed.
then by 200 µl micropippte the crude bacteriocin & precipitation were
incoculated in well & it keep for incubation at 37ºc at incubator.
Next day i wear observe the inhibition zone of different fraction was
recorded in comparision with the crude bacteriocin.
Characterization of bacteriocin
Heat stability
Protocol
Taken 4 test tube in each test tube 5 ml of sample. Two test tube is
heated with 68ºc for 10 to 20 mins respectively & two remaining test tube
wear heated at 121ºc for 15 mins.heat treated with bacteriocin samples wear
assy by antimicrobial activity.
Material
Nutrient agar, pathogen, bacteriocin.
Method
Prepare the nutrient agar then autoclave the N.A. & Petri plate then
pour the N.A. in Petri plate make media solidify ,then prepare a lawn of
pathogen
There wear five pathogen are used.
Page 36
Production of Bacteriocin from isolates Natural Lactic acid
a. Salmonella typhi
b. Klebsiella pneumonia
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus Cerus
Then form well, inoculated the sample in well & keep it for
incubation in incubator at 37ºc for 24 hrs.
Effect of PH
Protocol
Taken 8 test tube in each test tube purified bacteriocin wear taken in
each test tube with different PH 4,5,6,8.activity checked by antimicrobial
activity.
Material
PH meter, N.A.,bacteriocin,pathogen.
Method
Prepare the nutrient agar then autoclave the N.A. & Petri plate
then pour the N.A. in Petri plate make media solidify ,then prepare a
lawn of pathogen
There wear five pathogen are used.
a. Salmonella typhi
b. Klebsiella pneumonia
Page 37
Production of Bacteriocin from isolates Natural Lactic acid
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus Cerus
Then form well, inoculated the sample in well & keep it for
incubation in incubator at 37ºc for 24 hrs.
Page 38
Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 4
RESULT & DISCSUSSION
SCREENING OF BACTRRIOCIN ISOLATES FROM
VARIOUS SAMPLE
Samples like cucumber, tomato, garlic were collected from pune
market and they were subjected for the presence of bacteriocin were isolated.
MRS medium was used as the selective medium to isolate the LAB
.the isolate were screen by overlaying the freshly grown indicator strain of
lactobacillus few of the isolates showed bacteriocin against the standard
bacterial cultural which shown by the prominent zone of inhibition with (fig)
different in zone diameter. the cultural were pricked and inoculated into
MRS broth to reconfirm and purify the isolates.
The inoculated colonies were once again pour plated in N.A & make
the colonies purified.
The bacteriocingenic isolate were obtain from the cucumber, garlic,
tomato.
Apart from cucumber samples tomato samples, tomato sample had
bacteriocin strains. i did not get bacteriocin from garlic. only two strains
were slected for further analysis.
Page 39
Production of Bacteriocin from isolates Natural Lactic acid
Fig 1:Incoculated in minimal media
Page 40
Production of Bacteriocin from isolates Natural Lactic acid
Fig 2: Incoculated in minimal media
Fig 3:growth of bacteriocin(tomato) on MRS media
Page 41
Production of Bacteriocin from isolates Natural Lactic acid
Fig 4: Growth of bacteriocin(garlic) on MRS broth
Page 42
Production of Bacteriocin from isolates Natural Lactic acid
Fig 5: Growth of bacteriocin(cucumber) on MRS broth
Fig 6: Pure culture of cumcumber
Page 43
Production of Bacteriocin from isolates Natural Lactic acid
Fig 7:Pure culture of garlic and tomato
Fig 8: Pure culure of tomato
Page 44
Production of Bacteriocin from isolates Natural Lactic acid
Fig 9: Gram staining of bactreiocin (cumcumber) Lactobacillus acidophillus
Page 45
Production of Bacteriocin from isolates Natural Lactic acid
Fig 10: Gram staining of bactreiocin (tomato) Bacillus spp
Page 46
Production of Bacteriocin from isolates Natural Lactic acid
Charaterization of bacteriocin LAB
The
isolate
were
subjected
to
microbiological
biochemical
physiological examination based on” Bergey’s manul” of classification.
Microbiological characterization
The morphological examination of the isolate were identified through
the gram staining, which show the isolate were gram positive,rod shaped of
cucumber and tomato gram postive short rod shaped & by observing the
electron microscopic showed they are
Lactobicillus acidophlis from
cucumber & Bacillus spp from tomato.morphological examination shows
that the isolate were LAB.the result are shown in fig.
Antimicrobial activity of the purified isolates various pathogenic
strian
The neutralize cluture filterate checked for the antimicrobial activity
against various strains like lactobacillus acidophillus inhibitory action
against various strains with different rates are tabulated in table.
The seven pathogen strains wear used
a.
Salmonella typhi
b.
Shigella dysenteriae
a. Klebsiella pneumonia
b. Pseuadomonas Fluroscence,
c. E-coli(Escherichia coli),
d. Candida
e. Bacillus subtilus
They show the antimicrobial activity against this strain like
Lactobacillus spp from cucumber & bacillus spp from tomato
the result are present in the table.
Page 47
Production of Bacteriocin from isolates Natural Lactic acid
The LAB isolate from the vegetables products by Marjon
H.J. Bennik et al 1997 also showed broad range of inhibitory
action.
Table 1: inhibition zone of antimicribal activity
Sr.no
Specis
Lactobacillus
Bacillus
acidophilus
spp dimeter
dimeter
of of zone
zone
1
E.coli(Escherichia
1 cm
0.6cm
0.2 cm
-
coli
2
Shigella
dysenteriae
3
Candida
o.6 cm
0.4
4
Salmonella typhi
0.8 cm
0.6 cm
5
Bacillus subtilus
0.5 cm
-
6
Klebsiella
0.6 cm
0.5 cm
pneumonia
Fig 10: inhibition zone of antimicriobal activity
Page 48
Production of Bacteriocin from isolates Natural Lactic acid
Fig 11: inhibition zone of antimicriobal activity
Page 49
Production of Bacteriocin from isolates Natural Lactic acid
Biochemical chacterization:
Based on biochemical tests, all the isolates were identified as
belonging to lactic acid bacteria (LAB) group, which was identified. the
isolating
lactobacillus
acidophilus,
Bacillus
spp
giving
maximum
antimicrobial activity was gram positive, long rod shaped, negative for
catalase and oxidase is positive, having circular and white colonies on the
MRS media. the strain was also positive test for glucose, sucrose,
maltose,indole test, methyl red test, citrate, urease, oxdiase and there is no
change for mannitol, rhammnose, lactose, vogos proskrous test.
: Observations for the biochemical tests on inhibition forming
bacteria.:
Table 2
Sr. No.
Test name
Observations on bacteriocin Observations
cucumber
bacteriocin tomato
on
1.
Indole test
No change occured
2.
Methyl red test
Ring of red color on the Ring of red colour on the
surface
surface
3.
Vogos proskrous test
No color change
Ring of orange color
No color change
Page 50
Production of Bacteriocin from isolates Natural Lactic acid
4.
Citrate test
Blue color
Blue color
5.
Urease test
Color changed
Pink color
6.
Oxidase test
Paper showed no color change Paper showed voilet color
7.
Catalase test
No effervesces of O2
Bubbles are observed
Fig 12:Cirtrate test
Page 51
Production of Bacteriocin from isolates Natural Lactic acid
Fig: urease test
Fig 13: indole test
Page 52
Production of Bacteriocin from isolates Natural Lactic acid
Fig 14: Methly red test
Carbohydrate fermentation
Carbohydrate fermentation is carried out to identify the isolate is
herofermenters or homofermenters.the culture were incoculated into the
MRS broth durhams tube (to detect the gas production)the culture utilize the
glucose and produced acid and gas the indicates that hetrofermenters the
result shown in table.
Sugar utilization test
The sugar utilization test was carried out to test the ability of the
isolate to utilize various sugar. the isolate to utilize various sugar Like
lactose, sucrose, maltose, raffinose , arabinose individually and allow to
grow are shown in table.
Page 53
Production of Bacteriocin from isolates Natural Lactic acid
Observations for the sugar (Utilization of carbohydrate)
Table 3
Sr No.
Test name
(sugars used)
Observations on organism Observation on organism
lactobacillus acidophillus
inhibiting bacillus spp
1.
Glucose
Faint pink color
Faint orange
2.
Sucrose
No change
Orange color
3.
Maltose
Red orange color
No change
4.
Mannitol
Orange color
Orange color
5.
Rhamnose
No change
No change
6.
Lactose
No change
No change
Partial purification of bacteriocin
An increase in antimicrobial activity after partial purification of crude
bacteriocin by ammonium sulphate precipitation took place (fig).the fraction
with the highest bacteriocin activity precipited with 10 to 20% by
ammonium sulphate.
The antimicrobial activity increase from 0.8 to 1.4 cm the precipated
bacteriocin shows more antimicrobial activity.
Observation of zone of inhibition
Table 4:
Sr.no
Species
Crude bactreiocin
Precipated
Page 54
Production of Bacteriocin from isolates Natural Lactic acid
1
Salomenella typhi
0.6 cm
0.4 cm
2
Bacillus substils
0.4 cm
0.6 cm
3
E.coli
0.5
0.7
Fig 15: zone of inhibition of crude bacteriocin and precipated
Fig 16: Zone of inhibition of crude bacteriocin and precipated
Page 55
Production of Bacteriocin from isolates Natural Lactic acid
Fig 17 :Observation of zone of inhibition of crude bacteriocin and precipitates.
Characterization of bacteriocin
Heat stability
Paritially purified bacteriocin was found to be stable at 68ºc for upto
10 mins at 100ºc it could not show antimicrobial activity at 100ºc temp.
Page 56
Production of Bacteriocin from isolates Natural Lactic acid
However, after incubation for 15 mins at 121ºc they complete loss of
activity took place. the compared to the earlier report on bacteriocin, residual
activity was lower in our study than report of earlier.
Furthermore tolerance of bacteriocin to heat is known to depends on
the stages of purification, PH, presence of culture medium other protective
components, etc. the antimicrobial activity in our finding too.
Observation table
inhibition zone diameter
Table 5: Heat stability on different temp
Sr.no
temperature Min
E.coli
B.substils
1
68
10
0.3 cm
0.4 cm
2
121
15
0.3 cm
0.2 cm
Fig 17: Heat stability on different temp
Page 57
Production of Bacteriocin from isolates Natural Lactic acid
Fig 18: Heat stability on different temp.
Effect of PH
The partially purified bacteriocin shows maximum activity against
the target microorganism at PH 4 to 5. But the activity of the bacteriocin
gradually continuosly decreased. at PH 8 the antimicrobial activity was
drastically reduced to more than 2.5 time. Thus , the bacteriocin was found
active over a wide PH range with the highest activity at low PH range of 4 to
5 earlier. Earlier, the bacteriocin produced by a newely
isolated
Lactobacillus acidophilus active the PH range at 4 to 6 another bacteriocin
produced by bacillus spp was active over. zone of inhibition shown in table.
Page 58
Production of Bacteriocin from isolates Natural Lactic acid
Table 6 : effect of pH and zone of inhibtion
Sr.no
PH
E.coli
Salo
monella
1
4
o.4 cm
0.2 cm
2
6
0.3 cm
-
3
8
-
0.2 cm
Page 59
Production of Bacteriocin from isolates Natural Lactic acid
Fig 19: effect of pH and zone of inhibtion
Page 60
Production of Bacteriocin from isolates Natural Lactic acid
Fig 20: effect of pH and zone of inhibtion
Page 61
Production of Bacteriocin from isolates Natural Lactic acid
Conclusion
The study revealed that bacteriocin from Lactobacillus sp. Isolate
from natural lactic acid fermentation of vegetables possesses a wide
spectrum of inhibitory activity against Escherichia coli , bacillus substlis,
candida, shigilla, salomenella & klebcilla
Therefore, it has a potential for application as a biopreservation in
different food products as such or in combination with other preservation
methods. Since lactic acid fermentation is employed mostly for development
of products, especially for flavour and taste of the fermented products, the
production of bacteriocin in such products assumes more significance as
biopreservative apart from imparting probiotic effect to the product.
Page 62
Production of Bacteriocin from isolates Natural Lactic acid
CHAPTER 6
Summary
LAB were isolated from fermented product as vegetables. Lactic
acid bacteria (LAB) have been used for centuries in the preparation,
processing and preservation of food & beverages.
LAB have long been employed in fermentation as a preservation
techniques because of the ability of LAB to inhibit the growth of undesired
bacteria due to the production of antimicrobial substance such as bacteriocin.
The industrial importance of the LAB is further evidence by their generally
regard as safe (GRAS) status, due to their ubiquitous appearance in food and
their contribution to the healthy microflora of human mucosal surface
.though many gram positive and gram negative bacteria were able to produce
bacteriocin, the bacteriocin produced by the LAB is widely used because of
it’s GRAS status
.
From various vegetables produce, the bacteriocin LAB were isolated
bacteriocin were subjected to microbial, biochemical and characterization of
bacteriocin. They were tentatively identified as Lactobacillus acidophilus
and bacillus spp they produce the bacteriocin. Their wide range of the
antimicriobial property indicates that it is a wide spectrum bacteriocin.
The bacteriocin is a particular importance to the food industry and
also to other pathogens E Coli, Shigella, Candida, Salmonella, Klebsieva,
pseudomonas and Bacillus Substis. They are inhibitory towards both Gram
negative and Gram positive organisms, so they are said to be broad spectrum
bacteri-ocin.
The bacteriocin is treated with heat at 680C for 10min and 1210c for
15min, the results indicate that all the bacteriocins were 680C stable even at
autoclaving temperature. Hence it can persist and exhibit its inhibitory action
in the cooked foods. Therefore, it can be used as an effective preservative.
Page 63
Production of Bacteriocin from isolates Natural Lactic acid
C HAPTER 7
Reference
1.R Ananthnarayn and c k Panikar’s textbook of microbiology sixth
edition.
2.Experimental Microbiology by J patel Volume 1 and 2
3.Farkas-Himsley H (1980). "Bacteriocins--are they broad-spectrum
antibiotics?". J. Antimicrob. Chemother. 6 (4): 424–
4. doi:10.1093/jac/6.4.424. PMID 7430010.
5. Cotter PD, Hill C, Ross RP (2006). "What's in a name? Class distinction
for bacteriocins". Nature Reviews
Microbiology 4 (2). doi:10.1038/nrmicro1273-c2. is author reply to
comment on article :Cotter PD, Hill C, Ross RP (2005). "Bacteriocins:
developing innate immunity for food". Nature Reviews
Microbiology 3 (?): 777–
88.doi:10.1038/nrmicro1273. PMID 16205711.
6. HENG, C. K. N., WESCOMBE, P. A., BURTON, J. P., JACK, R. W.,& TAGG,
J. R. (2007). The diversity of bacteriocins in Gram-positive bacteria. In:
Bacteriocins: Ecology
7. Z. Yildirim, M.G. Johnson, Detection and characterization
of a bacteriocin produced by Lactococcus lactis subsp. cremoris
Lett. Appl. Microbiol. 26 (1998)
297–304.
8. R. Bromberg, I. Moreno, C.L. Zaganini, R.R. Delboni, J. De
Oliveira, Isolation of bacteriocin producing lactic acid bacteria
from meat and meat products and its spectrum of inhibitory
activity, Braz. J. Microbiol. 35 (2004) 137–144.
9.Characterization of the heat stable bacteriocin produced by lactic acid
bacteria isolated from dairy products and veg Reg no 06PY11, department
of microbiology.
Page 64