BACTERIOCIN PRODUCTION AND CHARACTERIZATION FROM

LACTIC ACID BACTERIA FOR STRAIN DEVELOPMENT BY

PHYSICO-CHEMICAL METHODS

Dedicated
i

TO
Someone
Who always encouraged me for the achievement of this
Goal of my life without whom it would be impossible
For me to play the chess of life

TABLE OF CONTENT

INTRODUCTION

1.1) Inhibitory Characteristics Of Bacteria Applied In Food Formation

Or Fermentation.......................................................................................................
ii

1.2) Applications Of Bacteriocin.............................................................................

1.2.1 Food conservation........................................................................................

1.3)

Bacterial Impedance.....................................................................................

2. LITERATURE REVIEW

Literature Review

22

MATERIALS AND METHODS

3.1) Isolation Of Bacteriocin Producing Bacterial Strain........................

22

3.1.1 Selection of strain..............................................................................

22

3.1.2 Procurement of samples.............................................................

22

3.1.3 Sterilization of glass ware.........................................................

22

3.1.4 Media Preparation..................................................................

22

3.1.5 Sample preparation and dilutions...............................................

23

3.1.6 Inoculation and Incubation.....................................................

23

3.2

24

Bacteriocin Production................................

3.2.1 Preparation of bacterial inoculum (test organism)................................

24

3.3) Antimicrobial Analysis By Well Plate Method.......

24

3.4) Optimization Of Growth Conditions..........................

24

(a)

Sensitivity of bacteriocin to pH.......................................................

24

(b)

Sensitivity of bacteriocin to heat..................................................

25

(c)

Sensitivity of bacteriocin to enzymes...........................................

25

3.5) Effect of NaCl on bacteriocin production.................

25

3.6) Partial purification..............................................

25

3.6.1 Partial purification by ammonium sulphate precipitation..................

25

3.7)

26

Purification by gel filtration.....................

a)

Swelling of gel.........................................................

26

b)

Packing the column............................................

26

c)

Equilibration of the column..................................

26

iii

d)

Application of sample..........................................

26

e)

Elution .........................................................

26

3.8) Purification by ion exchange chromatoghraphy .............

27

3.9 Application of sample and elution........................

27

3.10

SDS-Polyacrylamide Gel Electrophoraresis..............

27

3.11) Strain Development By Using Physico-chemical Mutagenic

Agents............................................................

28

3.12) Statistical Anaysis............................................

28

4.

28

RESULTS..........................................................................

4.1 Production Of Bacteriocin ....................................

28

4.2

Characterization Of Bacteriocin.....................

29

a) Effect of temperature ................................................

29

b) Effect of pH ......................................................

29

c) Effecet of enzymes............................................

30

d) Effect of NaCl.......................................................

30

4.3)

32

Partial Purification..................................

a) Precipitation of protein by gel filtration pattern............

33

b) Ion exchange chromatography................................

33

4.4 Strain Development by Using Physico-chemical Mutagenic

Agents...........................................................................................

34

5. DISCUSSION....................................................................

39

REFERENCES.....................................................................

42

LIST OF TABLES
Tables

Page

Table (4.2.a) Effect of temperature on the activity of bacteriocins.................

29

Table 4.2 b. Effect of pH on bacteriocin activity.........................................

30

iv

Table 4.2 c. Effect of proteinase-K, chlroform and trypsin on bacteriocin activity against
Straphforiase............................................................................
30
Table 4.2 d. Effect of different concentrations of NaCl on bacteriocin production by Lactic acid
bacteria against Strapforease..................................................
31
Table 4.2. e. Grading of antimicrobial activity in symbolic and digitalized form along with their
interpretation for the activity..................................................
31
FIG.4.3.a. Gel filtration pattern of Ammonium Sulphate precipitated residues of bacteriocin by lactic
acid bacteria on Sephadex G-100..................................
32
Table 4.3 Antimicrobial activity of different samples from different steps of purification samples of
Lactic acid bacterial species...........................................................
33
Table 4.4. Details of dose and time of UV exposure for physical mutagenesis.......

35

Table 4.5. D etails of dose of Nitrosoguanidine for chemical mutagenesis..............

35

Table 4.6. Comparison of the amount of Vitamin B12 produced by parental and different physically
mutant strains at 30C........................................................
36
Table 4.7. Comparison of the amount of Vitamin B12 produced by parental and different physically
mutant strains at 32C.................................................
36
Table 4.8. Comparison of the amount of Vitamin B12 produced by parental and chemically induced
mutant strains at 30C.................................................
37
Table 4.9. Comparison of the amount of Vitamin B12 produced by parental and chemically induced
mutant strains at 32C................................................
37

LIST OF ABBREVIATIONS
LAB
Bact

Lactic acid bacteria

Bacteriocins
v

EMS

Ethyl methane sulfonate

MMS

Methyl methane sulphonate

MNNG

N-methyl-N -nitro-N-nitrosoguanidine

IS

Insertion sequence

MRS

De-man ragosa sharp

SW

Soy whey

Normal

SDS

Sodium dodesyl sulphate

UV

Ultravoilet

W/V

Weight by Volume

v/v

Volume by volume

oC

Degree celcius

Microgram

Microliter

g/l

Gram per liter

ml

Mililiter

Gram

mg

Miligram

rpm

Rotations per minute

mm

Milimol

nm

Nanometer

SUMMARY
Over the last two decades, a variety of bacteriocins, produced by bacteria that kill or inhibit
the growth of other bacteria, have been identified and characterized biochemically and genetically.

vi

Lactic acid species were isolated from indigenious dairy source (whey) and purified using different
microbiological techniques. The pure culture was maintained in selective broth and used for
bacteriocin production. The produced bacteriocin was concentrated and characterized heating up to
100C was found to have less effect while more heating minimized the bacteriocin activity. Similarly
at low pH, activity of bacteriocin reduced while at higher (6.8), near to neutral, it showed higher
antimicrobial activity. On treating with choloroform, bacteriocin activity ceased while NaCl
concentration, up to 4% had no effect bacteriocin production.
The selected extract was partially purified by ammonium sulphate at 80% saturation level and
then further purified by using gel filtration chromatography using Sephadex-100. Most of the extract
exhibited anti-bacterial activity against bacterial strain i.e. Strpforease. On the basis of these results,
it is suggested that LAB is a source far anti-bacterial peptides and in future, may be used for
industrial extraction and isolation of anti-bacterial compounds which may found place in food
industry as alternative of antibiotics.

vii

viii

CHAPTER NO.1

1. INTRODUCTION
There has been scientific awareness of a valuable demand to overcome damaging
microorganisms in our experiment. The big goal for penicillin by Alexander Fleming in 1929
has opened the door in order to utilize therapeutic antibiotics by the medical and veterinary
communities to compete for typical diseases spreading by microbes. These therapeutic
antibiotics have been precluded in order to make their appliances in food and the use of
antagonistic elements with conservative or antimicrobial attributes has become a trade
mark approach to conserve and protect food. Addition of antimicrobial compounds to
refined artifacts such as in foods and drinks has become a traditional approach in the
process of food conservation (ARQUES et al., 2010).
Lactic acid is a very valuable artifact of industrial significance. Several techniques
using traditional and modern biotechnological approaches have been used for
improvements in the production of lactic acid. The lactic acid bacteria have been altered to
increase the lactic acid production. The progress in biotechnology and identification of
industrial utilization of lactic acid has led to the struggles being attracted on use of
biotechnological weapons to alter lactic acid bacteria (LAB) and other entities for lactic acid
production. The early struggles in LAB genetic alterations were considered closely to
update LAB with increased features for food grade applications, using accustomed
techniques. The casual mutations were also used by insertion sequence (IS) elements. The
LAB conducted to genetic developments has been used in dairy industry for flavour
enhancement, resistance to bacteriophages, addition of nutritional components and stability
and structure of end products. The controlled gene expression systems for industrial grampositive bacteria with low G + C content have already been described. Anyhow, with the
acceptance of poly lactide as a biodegradable polymer, struggles were directed to low the
cost of lactic acid production by genetically modifying the organism, by using various easily
available agro-industrial residues and by process alterations to remove the lactic acid
produced during the time of fermentation (SINGH et al., 2006).
Lactic acid bacteria (LAB) are a taxonomically diverse group of Gram-positive
bacteria that share the property of converting fermentable carbohydrates initially to lactic
acid bacteria and hence acidify the medium in which they grow. Becouse of being aero
tolerant anaerobes, the LAB family occupies a broad range of natural ecological niches.
They are present on plant surfaces, among the resident microflora of the gastrointestinal
1

tract of vertibrates, as well as in sewage, milk and soil as well (CHEN et al., 2005;
MUSIKASNG et al., 2009). LAB consisting of a group of Gram-positive bacteria, including
Lactococcus, Lactobacillus, Leuconostoc, Straptococcus, and Pediococcus as well as the
more peripheral Aerococuus, Carnobacterium, Teragenococuus, Vagococcus, Entrococcus
and Weisella; these belong to the order Lactobacillales (WOOD AND HOLZAPEL, 1995).
The LAB is feasibly the only second to yeast in significance for their services to mankind.
They are being used in all over the world in order to create free from harm, stockable food
stuffs for mallinea (ZHU et al., 2009). These foods consisted of fermented milk artifacts
(such as chees, yogurt and kefir), beverages (malolactic fermentations in wines),
vegetables (sauerkraut, silage,kimchi) (GEIS, 2003). Therefore, LAB shows a valuable part
in the food industry as a result of their fermentative capablities. Over the past decade,
interest in the study of LAB has increased dramatically. This shows not only the growing
industrial value of these bacteria for a broad range of fermentation processes but also the
emergence of their uses as probiotics, i.e., starins to which nutritional and human or
animal health benefitial properties are attributed (ALI, 2010).
Lactic acid bacteria are being increasingly used in order to produce different
extensions of fermented foods with better shelf-life, sour and nutritional properties.
(BERNARDEAU et al., 2006; STEINKAUS et al., 1983). The use of certain strains of lactic
acid bacteria, called probiotics, has given health benefits by stoping disease symptoms.
Specially, Lactic acid bacteria are being cultivated in (micro) anaerobic food conditions and
have been (historically) grouped as non-respiring, facultative anaerobes (JUNGH, 2006;
PARVEZ et al., 2006).
Naturally, lactic acid bacteria (LAB) present in food fermentation environments and
mostly used as starter cultures for fermenting different types of food. The metabolic
products of lactic acid fermentation have important roles in formation of characteristic
aroma/flavour and texture of several foods. In recent years, great improvements have been
gained in genetic engineering that made consumer-oriented alterations in starter systems
possible.

Although,

LAB

consist

of

twelve

genera,

six

of

them

(Lactococcus, Lactobacillus, Leuconostoc, Enterococcus,Streptococcus and Bifidobacteriu

m) are greatly used as starter cultures in the production of commercial food (OZER et al.,
2007).
Significantly, most of the cogent progress in the research of bacteriocin has come
from explorations of the colicins, the anticedent bacteriocins produced by different members
belonging to the family of Enterobacteriaceae, and this concluded for remarkable
2

knowledge of the genetic basis, domain structure and killing action of these molecules.
Altho
ugh, there is now a tremendous amount of research activity that focused on the bacteriocinlike actions of Gram-positive bacteria, typically LAB. A big number of LAB are food-grade
organisms that at present broadly being used in the food industry but now offer the deep
anticipation of food preservative uses to stop bacterial pathogens and spoilage
microorganisms. It was Pasteur with his college Joubert who, first consistantly write down
an estimation of opposing interferences between bacteria. In encapsulating their
conclusions that common bacteria (perhapes Escherichia coli) could conflict with the
growth of co-inoculated anthrax bacilli, either in urine (used as a culture medium) or in
experimentally infected animals. After all, in most cases the considerations were of an
analytic rather than empirical type, there is no knowledge in order to separate and
distinguish the any restrictor chemical substance (PUGSLEY, 1984).
The first fine affirmation of the nature of an antibiotic factor produced by E. coli was
given by Gratia, who showed that

the strain V (virulent in experimental infections),

produced in liquid media, dialyzable and heat-resistant substance (later referred to as

colicin V) that stops the growth of E. coli even with high dilution. Later a chain of different
colicins were discovered over a period of time. Jacob created bacteriocins in 1953.
Bacteriocins were paticularly assigned as protein inhibitors of colicin type, in other words
these are molecules recognized by lethality after amalgamation, killing actions, and
assimilationn with specific receptors on the surface of bacteriocin sensitive cells. Acoording
to Klaenhammer, there are two main qualities that make a difference of the majority of
bacteriocins from antibiotics: (1) bacteriocins formed ribosomally, while antibiotics are not
and (2) bacteriocins posses almost confined killing range, while most antibiotics have broad
killing ranges. The family of bacteriocin consists of a number of proteins that are different in
many respects, and could be grouped into two main types, those produced by Gramnegative and Gram-positive bacteria (HIRAK et al., 2010).
The proteinacious assets (bacteriocins) of Gram-positive bacteria come in progress
with the clearity that a number of inquires in the field of bacteriocins was centered on those
of Gram-negative bacteria but conclude an enhancement in research focusis on
bacteriocins of Gram-positive lactic acid bacteria. It reveales that much of the interest in
these substances is an absolute reaction to the practical uses of these factors, either for the
preservation of foods or the avoidance and treatment of bacterial infections (TAGG et al.,
1976).
3

1.1)

Inhibitory Characteristics Of Bacteria Applied In Food Formation or

Fermentation

Agitation (fermentation) processes depend on natural or altered environments to select

against spoilage and pathogenic microorganisms and to enhance the growth of adorable
microorganisms, either those naturally occurring or willfully added. Numerous fermentations
progress via a sequence of microbial species, where the adorable species usually
overweigh and convey the characteristic identity associated to the particular food or
beverage. Examples of disruptive activities that can bock the fermentation process and
compass the product are phage infection of dairy starters, growth of lactobacilli in wines
and growth of staphylococci in fermented meats. The use of bacteriocins as fermentation
benefit has been studied and presents an exclusive feature, (the desirable microorganisms
used in fermentation can also be the source of bacteriocins (NAQHMOUCHI et al., 2008).
Few Lactococcus lactis (lactic acid bacteria) strains form lactic acid and Nisin (an
antibacterial peptide from sugars by fermentation). Its cells and culture solution have
bacteriostatic and antibacterial effects to microorganisms. Recently, these strains have
gained much interest for its quality to develope food preservation. Nisin is an antibacterial
peptide having molecular weight of approximately 3.5 kDa consisting of 34 amino acids and
having lanthionine, -methyllanthionine, dehydroaranine and dehydrobutyrin in a molecule
(GILL et al., 2003). Nisin A, Nisin Z and Nisin Q have been announced as natural amino
acid alternate substances. It has been known that the antibacterial spectrum is broad and
the antibacterial effect is showed in not only Gram-positive bacteria but as well as in Gramnegative bacteria. One of the first utilization of bacteriocins was using nisin-producing
starter cultures in cheese formation. Several inspections came into light that did not support
their further advancement and uses. Nisin-producing starters were effective in controlling
Clostridia spoilage; however, it was analized that cheese made with these starters was not
as high a quality as compared to cheese made with regular starters. Nisin producing starter
cultures were also sensitive to phage attack (HURST, 1981).
A second entrance was using nisin contrary starters with good assets. The nisin
contrary starters were analyzed having some usefull differences in terms of starter
performance and durability when comparisions were made to the regular parent starter
cultures (LIPINSKA, 1977).

Pediococcus acidilactici that produced a strain, pediocin A1 as a lactic starter for the
production of anhydrous agitated sausage was investigated. The objective was to estimate
the ability of this strain to stop delibratelly introduced Listeria monocytogenes. It was
ceased that the lactic fermentation by itself was enough to control L. monocytogenes if
sufficient acid was produced. Anyhow, pediocin production was exhibited to give a
safeguard against L. monocytogenes if acid production was not enough. The use of
bacteriocins and bacteriocinogenic LAB to charge microbial populations in food and
beverage fermentations has been displayed with different assets. Assertive problems that
may arise are the appearance of bacteriocin-resistant populations of spoilage LAB,
increased likelihood of phage infection because of the dependence on one or two starters
instead of a heterogeneous mixture of LAB, and inactivation of inhibitory properties by
interaction with product components (FOEGEDING et al., 1992).

1.2) Applications Of Bacteriocins

1.2.1 Food conservation
The ustilization of lactic acid bacteria in food production is feasibly one of the ancient
models of biotechnology. It is most likely that fermented milk has been exhausted since
man started milking animals, conceivably far back as 11,000 years before. Over the years,
many fermented foods have been evolved, each with its own microbiological flora involved
in production. These fermented foods have been emerged without microbiological
accomplishment; only closely recently have we been able to comprehend the biological
process underlyingi food fermentation by lactic acid and other bacteria (RICHARD, 1990).
There are some important properties of bacteriocins that made them acceptable to
preserve food: (1) They are mainly identified as protected substances, (2) are not active
and dangerous on eukaryotic cells, (3) digestive proteases become them calm, having
some consequence on the gut microbiota, (4) are usually pH and heat-resistant, (5) they
have a almost wide antimicrobial spectrum, against several pathogenic and spoilage
bacteria caused by food, (6) they exhibit bactericidal activity, mainly acting on the bacterial
cytoplasmic membrane: no cross resistance with antibiotics, and (7) their genetic orginators
are mostly encoded by plasmid, giving genetic guidance.
The collection of studies carried out recently clearly show that the ustilization of
bacteriocins to preserve food can provide numerous advantages (THOMAS et al., 2000):
(a), an longer shelf life of foods, (b) give additional care during temperature abuse
conditions, (c) decrease the risk for transmission of foodborne pathogens via the food
5

chain, (d) improve the economic calamity due to food spoilage, (e) decrease the uses of
chemical preservatives, (f) allow the utilization of less severe firm teatments without making
trade off with food protection: bitterly to preserve of food nutrients and vitamins, as well as
other assets of foods, (g), allow the advertisment of novel foods (less acidic, with a low
contents of salt, and with a higher contents of water), and (h) they can help to fulfill the
demands of industries and consumers. In this way, directions of the food industry in Europe,
such as the demand to remove the use of artificial elements, the needs for least-processed
and fresher foods, as well as for ready-to-eat food and nutraceuticals could be pleased, by
use of bacteriocins. Although nisin is only bacteriocin in the United States approved as a
direct food additive, there is a big agreement of interest in other bacteriocins that have
similar assets and show wide span activity for prevention or restriction. Bacteriocins
assembled by fermentation could be absolved and added to foods as classic chemicals to
stop food pathogens and spoilage organisms only after gaining approval as a direct food
additive by the FDA. Bacteriocins have several characteristics that make them ideal as food
preservatives. Several bacteriocins can stand against with high temperature used in food
processing and can remain functional over a broad pH range. Bacteriocins can be digested
by many enzymes in the human gastrointestinal tract just like other proteins in the diet and
not become a problem for beneficial gut microflora. Bacteriocins are not dangorous,
odorless, colorless, and tasteless. Finally, bacteriocins are considered by consumers to be
more natural than chemical preservatives. The efficicy of using bacteriocins as food
preservatives will need to be determined for each food system (ROBERTSON et al., 2004).

1.3)

Bacterial Impedance

Peptide medicines such as bacitracin and gramicidin that are formed by bacteria by multienzyme complexes or sequential enzyme reactions have not till gained a number of uses in
order to treat infectious diseases. Anyhow, current examinations of mersacidin and
epidermin, formed by ribosomes are peptides belonging to the class of the lantibiotic, have
recommended that they may be as productive as few presently used therapeutic elements
for the treatment of staphylococcal infections in mice (LIMBERT et al., 1991) and acne in
humans (UNGERMANN et al., 1991), correspondingely. In view of Pasteur, it shows that
there has been a small subgroup of microbiologists who have made progress in
bacteriotherapy and microbial impedance for the treatment and to stop infectious diseases.
The uncovering and progress in penicillin, giving the onset of the history of medicines for
possible applications of antagonistic microorganisms to safe the human or animal host from
6

infection. Anyhow, one important debarment was the successful utilization of the relatively
avirulent 502A strain of Staphylococcus aureus in order to stop serious staphylococcal
diseases in neonates and in the treatment of furunculous. Currently, there has been
enhanced care for prescribing of medicines and resultant enhanced progress for antibiotic
resistance, the pharmaceutical industry may not be able to arrange beneficial new
medicines effectively. This resposbility is now being transmitted into a recovery of interest in
the opinion into the original microflora of bacteriocin-producing bacterial strains of credible
low virulence that are able of meddling with establishment and infection by more species of
pathogens (ALY et al., 1982).
Biological command has gained a lot of consideration over the last decades, as a
substitute to the use of chemical bacteriocides. It has come from increasing public care
over the use of chemicals in the enviornmant in general, as well as a reduction in the
availability of providing broadlyy used effective substances in particular.
The research work was planned with the following main objectives:

Isolation and purification of bacteriocin producing bacterial strains.

Strain development by using physico-chemical mutagenic agents.

To optimize the conditions for bactericin production.

CHAPTER NO. 2

2. REVIEW OF LITERATURE
Few species of LAB (Lactococcus, Lactobacillus, Pediococcus, Leuconostoc) are used for
the production of fermented foods. These can be small proteins, with molecular weights of a
few thousand Daltons, or complex structures having subunits in excess of 106 Da, with
associated carbohydrate or lipid moieties. The varity of bacteriocins is matched for
conditions for activity, way of action, and genetic basis (chromosomal or plasmid).
Bacteriocin producing strains have used mechanisms of immunity to the inhibitory action of
their own bacteriocins, and these immunity genes are usually linked to production genes.
Few strains of Lactococcus lactis produced an important bacteriocin (Nisin) and have been
used as a food preservative. The most distinguished pretein produced by LAB is nisin. It
has been used to inhibit spore-forming organisms in cheese, canned foods, bakery
products and pasteurized milk. It is primarily effective on Gram Positive pathogens. Nisin is
a pentacyclic peptide containing three unusual amino acids. It is inactivated by
chymotrypsin, but resistant to treatment with pronase, trypsin, and heat (100C) under
acidic conditions (ZAJDEL et al., 1985).
The fatal and mutagenic results of various mutagenic compounds for 3 strains of
Streptococcus lactis were examined. Fatality investigations confirmed that S.lactis was
comparably acute to ultraviolet radiations, MMS and MNNG, and, with a few amount, to
EMS. A casual derivative Lac, having a 37-Md plasmid, was to some extent more opposite
in action as well as with little variable than the normal type after treatment with ultra violet
radiation.Thus 3 strains were efficiently mutated by using EMS for the genetic marker
assayed (Rif), an incriment in the frequency of mutatiom was also noted after the
treatments with MMS and MNNG (LAUTIER et al., 1987).

Some bacteriocin-like substance synthsized by lactococci and lactobacilli come

forward possesing congested inhibitory span, the majority have greatly alive rather than the
colicins. These subtances have a trend being active toward a broad extend or area of
Gram-positive bacteria, and a little has described to stop Gram-negative bacteria.
Bacteriocin-like agents

possessing actions towards delicate bacteria can sometimes

amplified by testing them at particular pH values or in the existence of chemical agents that
reduce the strength of cell wall (STEVENS et al., 1991).
Anyhow the specific immunity of bacteriocin synthesizing Gram positive cells to
their similar bacteriocin is weak, genes which encode membrane linked molecules have
been found in a few Gram-positive bacteria. In the case of nisin it was examined that the
presence of the bacteriocin structural gene and the immunity gene is central for assertion of
privilege. Privilege to lactococcin A was revealed to concern at the membrane level through
a method by restricting door to an accepted receptor molecule, prevents its inclusion, or it
becalm the proteins of LAB (VAN BELKUM et al., 1991).
Chemical mutagenesis with EMS was appiedd to improve strains of Lactobacillus
delbrueckii that are resistant for enhanced amounts of lactic acid while constantly forming
the acid. The comparison of three mutants (DP2, DP3 and DP4) was made with normal
L.delbrueckii through agitations with various amonts of glucose. All three mutants generated
greater amounts of lactic acid than the normal type. With pH 6.0 stirred tank group
fermentations, mutant DP3 in 12% glucose, 1% yeast extract or mineral salt medium
generated lactic acid at the ratio that was more than 2-times quicker than the normal.
Mutant DP3 also generated 76 g/l lactic acid as compared to 57 g/l for the normal type. The
mutants DP2 and DP3 exhibited faster specific growth ratios, big lactic acid amounts,
beared great amounts of lactic acid, and formed 13% lactic acid in 13% glucose, 3% yeast
extract/mineral salt medium which needed an extra 10% glucose when the residual glucose
concentration decreased to 4%. Mutant DP3 was resistant for 1.5 years. The strain
development procedure was very advantageous; mutants having enhanced lactic acid
producing capacity achieved by the methods being used (DEMIRCI et al., 1992).
It clears that colicins showed two main types of killing activity. Few make channels in
the membrane of cytoplasm, while others show nuclease action having access to an acute
cell. Anyhow, the low-molecular weight bacteriocins of Gram-positive bacteria exemplify
being active for membrane (BRUNO AND MONTVILLE, 1993; CHIKINDAS et al., 1993).
The lantibiotic subgroup of bacteriocins aims to vary from other classes in the voltage
dependence for their membrane insertion. Poration complexes was designed being made
9

between more than two species of sensitive peptides or proteins following the ion leakage,
loss of proton energy, and finally led to cell death (GARCERA et al., 1993).
The majority of the polypeptides (bacteriocins) from lactic acid bacteria are cationic,
hydrophobic, or amphiphilic molecules having twenty to sixty amino acid residue.
Bacteriocins were grouped into three main types consisting of polypeptides from other
Gram-positive bacteria (KLAENHAMMER, 1993; NES et al., 1996). Lantibiotics (from
lanthionine having antibiotic) are small having molecular weight less than five KDa these
are

peptides

possesing

different

amino

acids

lanthionine,

P-methyllanthionine,

dehydroalanine, and dehydrobutyrine. These bacteriocins were put into class I

bacteriocins. Class I was further redivided into type A and type B lantibiotics with respect to
their chemical structure and actions against microorganisms. Type A lantibiotics are
extended peptides having a clear positive charge that apply their action via the composition
of openings in the membranes of bacteria. Type B lantibiotics possesing round peptides
and have a negative, or no clear charge, and their mode of action was connected in order
to inhibit the exact enzymes.

While class II, the largest group of bacteriocins having

molecular weight less than ten KDa, temperature resistant and non-lanthionine possesing
peptides were put in this system of classification. These were further classified into three
subtypes. Class II-a having peptide similar to that of pediocin with an N terminal consensus
sequence -Tyr-Gly-Asn-Gly-Val-Xaa-Cys. This subtype possesed a big attraction because
of their opposite action for Listeria. Class IIb having bacteriocins needed two various
proteins for their action, and class IIc having the bide peptides of the class, involving secdependent disguised proteins. The class III bacteriocins were not yet defined betterly. This
class with molecular weight greater than thirty kDa are teperature-labile proteins that gained
much attention for scientists of food. A fourth type consisting of complicated proteins or
polypeptides which need carbohydrate for their action had also been recommended through
Klaen Hammer. Anyhow, bacteriocins in this class had not been distinguished significantly
and had been recommended that its definition needs extra explanatory knowledge
(JIMENEZ-DIAZ et al., 1993).
LAB had been reported having actions against mutagens as well as with agents of
cancer in vitro and in vivo. One procedure for this result includes a physical binding of the
mutagenic compounds to the bacteria. The rule of the analysis was to examine the binding
ability of 8 human intestinal or lactic acid bacterial strains of mutagenic heterocyclic amines.
Binding of the mutagenic compounds such as Trp-P-2, PhIP, IQ and MeIQx by the strains of
bacteria was analyzed with HPLC. There were only few differences in the binding abilities of
10

the analyzed strains but the mutagenic compounds were bound with absolutly various
qualities. Trp-P-2 was totally bound and this was not to be of a reversible type. The binding
of PhIP with 50% access was usefull as it is a great mutagen in the western diet. While IQ
and MeIQx were not very well bound. Although pH revealed of being useful for binding.
Binding having relations for decrease in mutagenicity noted after the treatment of
heterocyclic amines to the strains of bacteria. The results showed that mutagens for cooked
food can be found in bacteria from microflora in vitro (ORRHAGEA et al., 1994).
The antimutagenic results of uninoculated milk and milks cultured with starins of
Bifidobacterium or Lactobacillus for the mutagenicity induced by two direct mutagens, 4nitroquinoline N-oxide and 2-nitrofluorene, and three dietary indirect mutagens, aflatoxin
B1, benzo(a)pyrene and quercetin, were examined by applying in vitro Salmonella
typhimurium test. Every model of this milk and control milk had a valuable antimutagenic
influence with a different level for the mutagen being used. Uninoculated milk showed a big
stopping influence rather than cultured milks for dietary indirect mutagens (CASSANDA et
al., 1994).
The effect of controlled pH (5.06.5) and primary dissolved oxygen level (090% air
saturation) on nisin Z production in a yeast extract/Tween 80-supplemented whey permeate
(SWP) was examined within batch fermentations with Lactococcus lactis subsp. lactis
UL719 strain. The total activity corresponding to the sum of solveble and not cell free
functions, as measured through a critical dilution method, was more than 50% lower at pH
5.0 than in the range 5.56.5, even though the specific manufacture decreased as pH
increase. A maximum nisin Z activity of 8200 AU/ml (4100IU/ml) was observed in the
supernatant after 8h of culture for pH in different ranges. Prolonging the culture beyond 12h
decreased this activity at pH 6.0 and 6.5 but not at pH 5.5 or 5.0. A subsequent increase in
cell-bound activity was possibly due to adsorption of soluble bacteriocin to the cell wall.
Aeration amplified cell-bound and total activity to maximum values of 32800 and 41000
AU/ml (16400 and 20500IU/ml), in that order, with an first level of 60% air saturation after
24h of incubation at pH 6.0 (AMIALI et al., 1998).
Two strains of Lactobacilli and four Pediococci generating bacteriocin-like product of
metabolic action secluded from sucuk were analyzed with agar spot tests and as well as
with well diffusion assays for their preventing actions toward sixteen Listeria strains (CON
et al., 2001).
LAB performs against Gram-positive microorganisms by concealing bact. These are
generally grouped into lantibiotics and non-lantibiotics. In the past decade a lot of
11

pertinacious toxins have been set apart and distinguished and others chaseing the
production of peptide proteins had been unseen. Bacteriocins delibrately possessing not
deep stoping span, and are mainly active against less related bacteria. Currently many are
appreciated about peptide proteins of LAB and controlling their production was controlled
(VINCENT et al., 2002).
Strains of lactic acid bacteria to be used for biomass production with the use of
whey-based medium inriched with a salt of ammonium and with reduced levels of yeast
extract (0.25 g/L) was isolated and characterized. Five strains of LAB were cut
off from naturly soured milk after enrichment in whey-based medium. One bacterial
separate, designated MNM2, exhibited a remarkable capability to use whey lactose and
grant a high biomass yield on lactose. That was recognized by the name of Lactobacillus
casei through the 16S rDNA sequence. A kinetic study of cell growth, lactose consumption,
and irritable acidity production of this bacterial strain was performed in a bioreactor. The
biomass yield on lactose, the percentage of lactose utilization, and the highest increase in
cell mass obtained in the bioreactor were 0.165 g of biomass/g of lactose, 100%, and 2.0
g/L, respectively, which were 1.44,1.11, and 2.35 times higher than those present in flask
cultures. The results propose that it is possible to make LAB biomass from a whey-based
medium supplemented with least amounts of yeast extract (MONDRAGON et al., 2006).
Seventeen LAB was far aparted using MRS agar medium from Jeotgal, a Korean
fermented food, purchased at the Jukdo market of Pohang. To categorize the
strains isolated, they were tested by exploring their cell morphologies, gram-staining,
catalase activity, arginine hydrolase activity, D-L lactate form and carbohydrate
fermentation. According to the phenotypic characteristics, three strains were identified as
Lactobacillus spp.,

ten

were

Enterococcus spp.

(or

Streptococcus spp.,

or

Pediococcus spp.) and the rest were Leuconostoc spp. (or Weissella spp.). Five strains
involving, 17 were chosen by preliminary bacteriocin activity test. Four bacterial strains
which repressed both indicator microorganisms were known by 16S rRNA sequencing. The
results

are

as

follows;

Leuconostoc

mesenteroides

(HK

4),

Leuconostoc

mesenteroides (HK 5), Leuconostoc mesenteroides(HK 11), Streptococcus salivarius(HK

8). In order to analysis LAB which were showing a high survival rate in gut, three strains
inhibiting both indicator microorganisms in synthetic gastric acid and bile juice -all except
HK8 had been investigated. The three strains mentioned above grow in extreme low acid
conditions (Sung et al., 2006).

12

The production of nisin, by Lactococcus lactis subsp. lactis was connected with the
actual formulation of lactic acid during agitation enriched with whey. As an end result of the
decreased concentration and huge separation on expense of lactic acid, bringing back
lactic acid as an artifact may not be careful, but its expulsion from the agitation broth was
useful because the addition of lactic acid stops nisin formation. In this way, lactic acid
removal

was

attained

by

biological

sources.

combined

culture

of L.

lactis and Saccharomyces cerevisiae was recognized to encourage the generation of nisin
through the in situ utilization of lactic acid by the starin of yeast, which was able of using
lactic acid as a source carbon. The S. cerevisiae in the combined culture did not go up
against nisin generating bacteria as the yeast does not use lactose, the big carbohydrate in
whey for bacterial development and nisin formation. The results displayedd that lactic acid
made by the microbes was completely used by the yeast and the pH of different culture
could be maintained at around 6.0. Production of nisin by the combined culture system
reached to150.3 mg/L, which was 0.85 times greater than that by a pure culture of L. lactis
(CHUANBIN et al., 2006).
Cultural people of the Himalayan areas of different countries ustilized a range of
domestic fermented products of milk formed from milk of cow as well as from milk of others.
These were not as well-known as other cultural foods. The quantity of LAB ranked
from 107 to 108 cfu/g in these Himalayan milk products. The entire one hundred and twenty
set apart of LAB were cut offed from fiftyeight portions of cultural milk products
aggregated from various

regions

of

different

countries.

Based

on

phenotypic

characterization including API sugar test, the superior lactic acid bacteria were recognized
as

Lactobacillus

bifermentans, Lactobacillus

paracasei

subsp.

pseudoplantarum, Lactobacillus hilgardii, paracasei subsp. Paracasei and many such other
strains. LAB produced a broad span of catalysts and exhibited their big actions. They
displayed antagonistic atributes towards targeted Gram-negative bacteria. Few strains of
LAB displayed a big content having no affinity to water. Commending these strains may
have benefitial sticky future (DEWAN AND JYOTI, 2007).
It was revealed that the two genes, glpQ and pde genes were concerned with
moderate resistance of Enterococcus faecalis JH2-2 to DvnV41, a class IIa peptide protein
formed through divergensV41. The listerial orthologs of these two genes might be lmo
0052 and lmo1292 genes. Here, the function of these genes having resistance to DvnV41
and MesY105 was examined. L. monocytogenes EGDe was not functioned in the above
genes through similar recombination. Listerial mutant strain EGDSC02 (non-functioned in
13

the putative glpQ gene), was litterly resistant to DvnV41. This strain EGDSC01 remained as
normal strain, also acute to DvnV41, but was influenced in growth conditions has been
reported (CALVEZ et al., 2007).
The influence of basic buffer salts on the development of S. thermophilus ST110,
medium pH, and addition of the antipediococcal bacteriocin thermophilin 110 were
examined in whey immersed media over a time period of 24 hours. In a medium having no
buffer,

thermophilin

110

generation

at

37C

paralleled

the

development

of S.

thermophilus ST110 and accessed at a high rate after 8 to10 hours. Affixing of basic buffer
salts reduced the fall in medium pH and resultantly an enhanced biomass and big
productions of thermophilin 110 was evaluated. The best end results were obtained with the
addition of 1% (w/v) MES to the medium, that decreased the pH fall to 1.8 units after 10
hours of development and resultantly an increased cell mass in thermophilin 110 production
was obtained. The outcomes displayed that whey enriched with permeate may be
appropriate for generating big amounts of thermophilin 110 which have the demand for
taking controll over dying pediococci in industrial wine and beer agitations (SOMUTI et al.,
2007).
Bacteriocin generated by Bacillus mycoides far aparted from whey showed strong
inhibitory action towards complex microbes (Listeria monocytognes and Leuconostoc
mesenteroides) produced by food. Fractionally, antibacterial substance was made clear via
salt saturation method. Fractionally cleared bacteriocin could tolerate temperature up to 100
C, functioned at broader pH rank (4 to 11) and was highly active for tripsin. This substance
with opposing functions displayed bactericidal actions towards indicators of peptide protein.
Henceon the basis of above attributes, the antibacterial substance generated by B.
mycoides was the most effective bacteriocin and can be applied to preserve or protect food
was reported (SHARMA AND GAUTAM, 2008).
The composition of whey relyes on the methodolgy of cheese maker, but the main
parts are proteins, lactose and minerals. Few whey components possesing attributes which
made them usefull economically as rigins of taste enhancers, fat alternatives, food binders,
and currently, as functional constituents having biological actions (RECIO et al., 2008).
The result of antimicrobial peptides: divergicin M35 and nisin A on Listeria
monocytogenes LSD 530 potassium (K+) channels: ATP-sensitive (KATP), calciumactivated (BKCa), and depolarization-activated (Kv) types. Enhancement in K+ efflux and
inhibition of cellular growth were observed after adding K+ channel activators pinacidil,
NS1619, and cromakalim to divergicin M35. enhance in K+ efflux from log-phase cells was
14

about 18 1.1, 11 0.63, and nmol mg1 of cell dry weight (CDW) for pinacidil and
NS1619, in that order, over the efflux obtained with divergicin M35 alone. Increases in
K+efflux obtained by adding the same K+ channel activators to nisin A fit a totally different
profile. Divergicin M35 activates K+ channels, mainly of the Kv and BKCa types and to a
lesser degree the KATPtype, causing K+ efflux and thus cell death (NAQHMOUCHI et al.,
2008).
The bacteria used for microbial appliances aids in order to control the natural
microbial flora of host. The capacity of such bacteria to unasked species is because of
increased via the production of effective toxicants against microbes. The abundantly
occuring of these are bacteriocins having a huge and working various family of antimicrobial
presents in all dominant origins of Bacteria. Current analysis exhibits that these
proteinaceous toxicants hit an important role in starting antagonistic actions between the
strains of bactria and closely related species. The effective use of bacteriocin as microbes
and safe elements has currently gained enhanced attraction. Current struggles including
the applications of such strains, with an important center on stemming microbial therapies
for mankinds, bovine animals, and forming were reported (GILLOR et al., 2008).
The significance of new bio-conservation actions and their appliances to make
secure seafood feature and protection mostly in the circumstance of increasing need for
essentially handled aquatic artifacts of food. The big troubles linked with dying and disease
causing agents found in brand-new and refined see foods, particularly ready-to-eat aquaticfood conducted for temporary storage, and the biological approaches that can be applied in
order to low their growth. Which was chased with a survey of present study about the
preventing bacteriocins generating LAB far-aparted from see food artifacts or that was
being assessed for insuring defense on aquatic food artifacts as well as the characteristics
of their bactriocins. Many procedures for maintenance of see food artifacts, like safed
cultures and their current and expected appliances in order to conserve artifacts of food
were also analyzed (CALO-MATA et al., 2008).
The abundance to conserve food systems was concluded by the techniques that are
amalgamated, the basic properties of the artifacts of food and the marked microbes.
Currently, the novel peptide protein such as nisin, enterocins A and B and sakacin K were
used for cooked and anhydrous better grandstander stick with Listeria monocytogenes,
Salmonella enterica and Staphylococcus aureus and endured to a high pressure
medication of 600 MPa. Ante pressurization nisin made meaningful decrease in counts of L.
monocytogenes and S. aureus, typically in anhydrous better grandstander stick. After to
15

pressrizing, Salmonella and L. monocytogenes were not discovered in 25 g of both cooked

and anhydrous grandstander stick and continued at this stage during the complete arsenal.
S. aureus levels, conversely, only reduced below the aspial border (1 log CFU/g) in the
nisin amount. After this, when arsenal was accomplished at a censorious heat, the capacity
of S. aureus to form was dependant on peptide protein used and the type of meat byproduct. In this way, at the last of storage, while S. aureus counts were <1 log CFU/g in all
anhydrous grandstander stick groups, only nisin could decrease its growth in cooked stick
(JOFRE et al., 2008).
Formulation of a novel protenacious toxins (Nisin) from three genetically modified
strains, (LAC338, LAC339 and LAC340) with freedom and regulating

genes of nisin

formation on plasmids in the Lactococcus lactis LL27 nisin producer, were checked below
pH controll and pH-uncontroll batch fermentations. Maximization analysis showed that
extracts of fructose and yeast supplied high functions of nisin. The above modified strains
made 25%, 44%, and 45% much nisin, than mormal or natural L. lactis LL27 after twelve
hours in process of early development. Thus, acute decrease in the production of nisin
were analized at the last level of fermentation with two strains namely LAC339 and LL27 as
compared to other two strains (LAC340 and LAC338) to which the big level of nisin may be
continued for a long time (SIMSEK et al., 2009).
Lactic acid is commonly utilized in many interprises. Nonel appliances of lactic acid
for the production of ecological polymers have developed the need for it. Lactic acid can be
achieved from cheese whey and many others through microbial fermentation by lactic acid
bacteria (LAB). Unmixed sugar and cheese whey can be fermented in a direct way through
LAB. LAB can alter formal biomass to lactic acid. Lactic acid productivity of 6.34 and
4.87 g/lh

and

yields

of

0.98 g/g

lactose

and

0.97 g/g

glucose

have

gotten

from cheese whey and wheat starch, correspondingly, utilizing cell-recycle duplicated batch
fermentation

through

Lactobacillus

sp.

RKY2.

LAB

such

as Lactobacillus

pentosus, Lactobacillus brevis and Lactococcus lactis are able to convert glucose to lactic
acid through homolactic fermentation and also to convert in many others by heterolactic
fermentation (JEBO AND FENGJIE, 2010).
Lactobacillus bulgaricus florishing on whey was condensed via an easy heat
anhydarting procedure in the varying temperature of 34 to 54C and its ability for the
fermention of lactic acid of whey was assesstted. Condensing of cells in whey solution in
the analyzed temperature rank did not alter importantly their survival rate (8186%),
magnifying a safe result of whey as both growth and condensing medium. The kinetics of
16

fermentation of whey and combinition of whey or molasses with the ustilization of

condensed culture were equivalent to those of non-condesed cells, and only decreased pH
had an evaluated influence on the fermentation capacity of the condensed cells.
Furthermore, condensed L. bulgaricus, in both forms either free or dynamic on casein
coagulates, was applied as starter to produce unsalted hard-type cheese. The solid
influence of the amount of starter culture and the immobilization procedure, the assessment
of microbial counts, and the sensory attributes of the cheeses were assessed during
ripening time at different temperatures (KATECHAKI et al., 2010).
For nisin production, the effect of reduced or negative value Soy Whey (SW) as a
substitute

and

easily

available

fermentation

substrate

to

culture

Lactococcus

lactis subsp. Lactis was studied. In the beginning, a micro titer plate assay using a
Bioscreen C Microbiology Plate Reader was applied in order to maximize the conditions for
culture. Many tests were analyzed in order to try to maximize the production of nisin from
SW, by using various procedures for SW sterilization. In resultant flask based experiments,
condensed bacterial mass and production of nisin gained from SW was 2.16 g/L and
620 mg/L, respectively, as compared to 2.17 g/L and 671 mg/L from a medium, that was de
Man-Rogosa-Sharpe broth (MRS broth). Ultrasonication of soybean flake slurries (10%
solid content) in water before to produce SW was resulted in 2% enhancement in biomass
production and 1% reduction in nisin. Nutrient nourished with SW resulted in 4% and
8%increariment in cell and nisin production, orderly. This study displayed the influence in
order to utilize a low value waste in aquas form from the processing of soybean to make a
high-value fermentation by- product (MITRA et al., 2010).
Geobacillus stearothermophilus was a thermophilic bacterium mainly responsible for
the Xat-sour spoilage of low-acid canned food with high water activity. Control of vegetative
cells and spores of G. stearothermophilus strains ECT 48 and CECT 49 through enterocin
EJ97 formed via enterococcus faecalis EJ97 was described. Both strains were highly
sensitive to EJ97 in a culture medium. In samples rom canned foods inoculated with a
cocktail of vegetative cells or endospores of the two strains and stored at 45 C or 30 days,
viable cell counts were reduced fewer detection levels. The time course of microbial
inactivation depended on the food sample and bacteriocin amount. Dormant endospores
were opposed to EJ97 short-time treatments (5 min), but endospores activated to develop
by heat became bacteriocin sensitive. The simultaneous application of enterocin EJ97 and
heat treatments (90 and 5 C) on dormant endospores had an improved antimicrobial effect
that depended both on the bacteriocin concentrations and temperature. Results from this
17

study strengthen the potential of enterocin EJ97 for biopreservation against G.

stearothermophilus in canned vegetable foods and drinks (VIEDMA et al., 2010).
Whey is an end-product of cheese manufacture that is generally regarded as an
extra one. Anyhow, it was a combinition of proteins possesing vital properties with respect
to food and other aspects. In order to get these usefull proteins, whey fractionation was
devised using three main methods; namely chromatographic (e.g., ion-exchange and
hydrophobic adsorption), membrane (e.g., traditional pressure-driven and electroseparation), or combined methods. Currently, new good techniques are being used such as
aqueous two-phase separation and magnetic fishing (EI-SAYED AND HOWARD, 2011).
Enterococci are unique lactic acid bacteria, and also natural inhabitants of mankind
and intestinal tracts of animals. They may have entrance in products of food during
processing via direct or indirect spoilage and are mostly found in processed products of
food, like cheese, sausages, olives, etc. In these days, they are examined to
produce bacteriocin (enterocins), which inhibits the growth of various microbes produced by
food,

like Staphylococcus

aureus,

Listeria

monocytogenes,

Escherichia

coli,

Pseudomonas spp., Bacillus spp. and Clostridium spp. Enterocins relates to class I, class
IIa, class IIc, and class III of bacteriocins. Enterocins can be applied in various products of
food to increase their shelf life due their resistance for temperature and dislay actions with a
brad range of pH. Enterocins were much good as well as protective to be used in the systen
of

food

as

they

are

faecium and Entero.coccus

"generally

recognized

faecalis were

the

as

most

safe"
dominant

(GRAS). Enterococcus
bacteriocin-producing

species of Enterococcus in the by-products of food (JAVED et al., 2011).

Few species of Leuconostoc were much important for fermented dairy products, as
they participate in the organoleptic characteristics of butter and cream, and also contribute
to the formation of openings in some soft, semi-hard (Edam and Gouda cheeses), many
artisanal

or

in

blue-veined

cheeses,

such

as

Roquefort.

In

this

study,

14 Leuconostoc strains from cheese and cheese-related products were characterized by

genotypic and phenotypic methods, and their technological performance assessed for their
potential use as dairy adjunct starters. Phenotypic characterization allowed these strains to
be divided to genus level, and genotypic studies (RAPD-PCR and 16S rRNA gene
sequencing) identified them to species/subspecies level. Five Leuconostoc strains grew
well and acidified milk, and most of them grew even at 8 C. They showed moderate
resistance to heat treatments (30 min at 63 C) and grew well in the presence of 3% and
18

4% NaCl, and were significantly inhibited at pH 5. All strains showed resistance against
the bacteriophages tested. Commonly, the antibacterial properties observed were slight and
due to acid production, with the exception of Leuconostoc citreum MB1, which strongly
inhibited Listeria monocytogenes ATCC 15313 by the production of a bacteriocin-like
compound. All Leuconostoc strains examined were susceptible to gentamicin, tetracycline,
erythromycin and ampicillin. Fewer strains also showed technological and antimicrobial
characteristics, thus potentially appropriate as adjunct starters in fermented dairy products.
This study highlights that adventitious lactic acid bacteria can be a great source of novel
strains with interesting characters that could be used for fermented dairy (CARDAMONE et
al., 2011).

CHAPTER NO. 3

19

3. MATERIALS AND METHODS

The research work was performed in protein molecular biology laboratory,
The details of the materials and methods employed during the research are given below.

Isolation of Bacteriocin producing bacterial strain

Bacteriocin production

Antibacterial assay by well plate method

Strain development by using physico-chemical mutagenic agents.

Optimization of growth conditions or characterization for bacteriocin production

3.1) Isolation Of Bacteriocin Producing Bacterial Strain:

3.1.1 Selection of strain
Lactic acid bacterial species were selected as the target strain for the production of
bacteriocin.

3.1.2 Procurement of samples

A sample of whey was collected from local market and used as a source for the isolation
and purification of lactic acid bacterial species. The sample was stored in sterilized bottles
at 4C.

3.1.3 Sterilization of glass ware

The glass ware required during the study was washed with detergent and sterilized in hot
air oven at 150C for 30 minutes as described by (HARRIGAN AND MECANCE, 1976).

3.1.4 Media Preparation

Following media were used in the study:
Nutrient agar
Nutrient broth

20

All the media were prepared according to their standard compositions (WOOD AND
HOLZAPFEL, 1998). The pH of the medium was maintained by using 0.1 N NaOH and 0.1
N HCL.

Compositions

Compositions of the media used were as fellowing:

a)

Nutrient agar
A general purpose medium used for the culture of non-fastidious organisms contains:

Pepton

5.0 g/L

Sodium choloride

8.0 g/L

Beef Extract

3.0 g/L

Agar No.2

12.0 g/L

The pH of the medium was adjusted at 7.4 and autoclaved at 121c for 15 minutes.

b)

Nutrient broth

Pepton

5 g/L

Sodium choloride

5.0 g/L

Yeast extract

2.0 g/L

Lab-Lemco powder

1.0 g/L

3.1.5 Sample preparation and dilutions

Whey was diluted separately in a ratio of 1:10 in normal saline solution as 10 -1 dilutions and
then further serial dilution was done.

3.1.6 Inoculation and Incubation

Petri plates (10), 5 containing Nutrient agar and 5 containing Nutrient broth were
prepared. Each dilution (1 mL) was poured separately on plates containing solid agar
medium and then spread with a sterile rod. The Petri plates were incubated at 37C for 48
hours.

3.2

Bacteriocin Production

1% of bacterial culture was dissolved in selective broth (M17) in a conical flask. The flask
was put in shaker at 37C and 120 rpm for 48 hours. The mixture was subjected to
21

centrifugation at 10,000 rpm (4C) for 20 minutes. The residue was discarded and cell free
supernatant was concentrated up to 200 mL in a rotary evaporator at 25C by evaporating
the water and then stored in a flask at 4C (GUERRA AND PASTRANA, 2002).

3.2.1 Preparation of bacterial inoculum (test organism)

Nutrrient broth (Oxide) was mixed at a concentration of 1.3 g/100 mL in distilled water and
autoclaved at 121C for 15 minutes. A loop full from pure culture of bacterial strain was
mixed in the medium after cooling and the flask was placed in shaker at 37C for 24 hours.
Inocula for all the bacterial strains were prepared in this manner and stored at 4 C.

3.3) Antimicrobial Analysis By Well Plate Method

Nutrient agar was prepared by adding 2.8 g/100 mL of distilled water and autoclaved at
121C for 15 minutes in autoclave machine. Before transferring this medium in sterilized
Petri plates, 10 L inoculums of Straphforiase as target organism were added to it while it
was liquid and quite cool. Mixed them and poured into plates. Then a small well was made
with the help of a micropipette large tip and spatula and a small drop of nutrient agar was
used to seal the well. After this, small filter paper (whatman paper) discs were laid flat on
growth medium and 100 mL of extract (supernatant) and control of rephampicin was put on
each disc. The Petri plates were then incubated at 37C for 48 hours for growth of bacteria.
The extract having antimicrobial activity, inhibited the bacterial growth, clear zones of
inhibition was formed (HAUNG et al., 2005).

3.4) Optimization Of Growth Conditions

(a)

Sensitivity of bacteriocin to pH

In order to check the sensitivity of bacteriocin to pH mother culture was prepared by adding
2.6 g of nutrient broth in 200 mL of distilled water and autoclaved at 121C for 15 minutes.
Then after mantaining the pH from 3 to 11 in separate test tubes, placed in shaker at 37C
for 24 hours. Then after centrifugation, culture supernatant was used to check the
antimicrobial activity by well plate method (KAREN et al., 2004).

(b)

Sensitivity of bacteriocin to heat

22

Sensitivity of bacteriocin to heat was checked by changing the pH of culture supernatants

by 0.1 N NaOH and 0.1 N HCl at 6.0. While control with Ph of 6.8 was used. The residual
activity was checked by well plate method (TAGG et al., 1976).

(c)

Sensitivity of bacteriocin to enzymes

Sencitivity of bacteriocin to enzymes was checked when culture supernateant was treated
with proteinase k, trypsin, and chloroform. 1mL of crude extract was treated with 1mg
proteinase k, 0.5 mg trypsin and 1 mL of chloroform separately, incubated at 37C for 2
hours and then residual activity was checked by well plate method (Scolari et al., 1993).

3.5) Effect of NaCl on bacteriocin production

The effect of NaCl was checked by growing the bacteriocin producing strain in agar
medium with 2%, 4%, and 6% of NaCl. Then indicator strain was mixed with agar
containing the bacteriocin producing colonies followed by over night incubation. Then
bacteriocin activity was assayed by well plate method (Larsen et al.,1993).

3.6)Partial purification
3.6.1 Partial purification by ammonium sulphate precipitation
The partial purification of bacteriocin was done by ammonium sulphate precipitation.
The crude extract was precipitated with (NH 4)2SO4 at 80% saturation level, calculated from
saturation table. i.e, that is 56.1 g (NH 4)2SO4 was put into 100 mL of crude extract to gain
80% saturation level of initial saturation level (HUYNH et al., 2001). The crude extract was
stirred with periodical addition of measured quantity of ammonium sulphate into it. The
precipitated crude extract was then centrifuged at 10,000 rpm and 4C for 10 minutes to
separate residues and supernatants from crude precipitated extracts; the supernatants
were stored at 4C in 100 ml sterilized bottles while residues were resuspended in minimum
quantity of phosphate buffer (pH 8).

3.7)

Purification by gel filtration

The dialysed sample was purified by gel filtration using sephadex G-200.
The steps involved in gel filtration were as follow:

a)

Swelling of gel
23

Sephadex G-200, 2 g was put into 200 ml of distilled water and kept at room
temperature for over night growth for swelling (Jackob, 1975).

b)

Packing the column

The column was washed with detergent and distilled water and fixed vertically on a

stable stand after drying it. The gel slurry was poured into the column of 4 cm width and 21
cm length. It was left undisturbed until the distinguished layers of water and gel were
appeared.

c)

Equilibration of the column

After packing the column, it was equilibrated with the 10 mM Tris-HCl buffer of pH 8,

after the equilibration, the input buffer pH was equal to output buffer pH.

d)

Application of sample
The outlet of the column was opened to remove excessive buffer from it. The

sample showed antimicrobial activity, was applied on the gel and put the reservoir on the
column.

e)

Elution
The elution of sample was performed by 10 Mm Tris-HCl buffer of pH 8. All the

possible fractions, each with a volume of 5ml, of each eluted sample were collected at
elution rate of 1 drop per 25 seconds. The absorbance of the fraction was recorded at 280
nm as graph was plotted. The fractions with maximum protein contents were pooled out
and appied for antimicrobial assay.

3.8)Purification by ion exchange chromatoghraphy

The fractions obtained from the gel filtration were assayed to check the antimicrobial
activity. The samples showing the activity was further purified by anion exchange
chromatography. Sigma DEAE Cellulose was used as resin in anion exchange
chromatography. Equilibarte it with the 10 Mm phosphate buffer of pH 7 until the pH of input
buffer become equal to output buffer.

3.9

Application of sample and elution

Now column is ready for applying sample. The output of the column was opened to remove
excessive buffer from it. The sample showed antimicrobial activity, was applied on it. The
24

sample was allowed to elute and the fractions of 0.5 mL was collected until the entire
sample eluted. Here the elution was done by the gradient of 10 mM phosphate buffer and
0.1-1.0 M sodium chloride solution of pH 7.
All samples of crude extract and partially purified extracts were then subjected to
antimicrobial assay.

3.10

SDS-Polyacrylamide Gel Electrophoraresis

The characterization of the bacteriocin (peptides/proteins) was done by SDS-PAGE

(Laemmli, 1970). The SDS-PAGE was carried out for crude and purified protein samples
possessing antimicrobial activity. The experiment was carried out on Mini-PROTEIN 3 mini
vertical electrophoresis apparatus (BioRad). Twelve percented gel was used. The samples
were prepared by mixing the protein 2:1 ratio with SDS-sample buffer of sigma. The
samples were heated in order to boile in water bath for 5 minutes and loaded after quick
spin. Tris-HCl, glycine, SDS buffer (sigma) was used as running buffer. The processed
samples (30 L) were loaded along with 5 L of molecular weight Fermentas PAGE Ruler
Prestained Ladder. The gel was run until tracking dye (bromophenol blue) front vereached
the bottom. Running conditions for the gel were same as that of native PAGE. At the
completion of the run the gel was developed by staining with Coomassie Brilliant Blue G250 dye followed by destaining. The gel was documented on SynGene gel documentation
system.

3.11) Strain Development By Using Physico-chemical Mutagenic Agents

Isolates of lactic acid producing strains for different sources were exposed to UV radiation
and Nitrosoguanidine for the improvements of strains for bacteriocin production. For
anaerobic fermentation (Mc. Intosh Mark III) anaerobic jar and for aerobic fermentation
orbital shaking incubator was used. The production of vitamin B 12 was measured by the
growth of lactic acid bacterial species in response to vitamin B 12 produced through
spectrophotometric method.
3.12) Statistical Anaysis
All results (data) were obtained in triplicate and their mean values and standard deviations
were calculated as described by (STEEL et al., 1997).

25

CHAPTER NO.4

4.RESULTS
The present study was conducted for the production, characterization and strain
development of bacteriocin from lactic acid bacteria
In first phase; lactic acid bacterial species was isolated from indigenous source like whey,
while in second phase; bacteriocin was produced, purified and characterized.

4.1 Production Of Bacteriocin

Lactic acid bacteria occur naturally in several raw materials like milk, whey, meat and flour
used to produce food (RODRIGUEZ et al., 2002). These are important because they
produce

antimicrobial

compounds

(bacteriocins). These

compounds

have

grown

substantially due to their potential usefulness as natural substitute for chemical food
preservatives to enhance the shelf life of food (CLAVELEND et al., 2001).

26

The target bacteria were used to produce bacteriocin and then was purified and
characterized.

4.2 Characterization Of Bacteriocin

a) Effect of temperature
Bacteriocin is probably stimulated by environmental conditions like temperature and pH.
The rate of thermal inactivation of the bacteriocins was determined by heating crude
samples of bacteriocin at various temperatures (4C, 37C, 65C, 100C, 121C) for 15
minutes. The bacteriocin was stable at 37C, 4C and 65C but partially stable at 100C and
retained its activity. But bacteriocin completely lost its activity when heated at 121C. In
colony count, at 65C less colonies of target organism (Stapforiase) appeared ranging from
39-35 indicating that bacteriocin is active at this temperature and inhibit the growth of target
organism. At 100C, 230-260 colonies appeared indicating that bacteriocin is partially active
at this temperature but it showed complete inactivation at 121C because colonies
appeared were greater than 300 showing no inhibition against Strapforiase (Table 4.2 a).

Table 4.2 a.

Effect of temperature on the activity of bacteriocins

Temperature treatments

Sample

(15 minutes each)

37C (control)

4C

65C

100C

121C

b) Effect of pH
Activity of bacteriocin was determined at different pH values; sterile cell free supernatants
were adjusted with 1mol.NaOH/HCl to different pH values (3, 4, 5, 6, 7, 8, 9, 10, 11) (Table
4.2 b).

27

Table 4.2 b.

Effect of pH on bacteriocin activity

pH Values

Sample

3.0

6.8 (control)

6.0

c) Effecet of enzymes
Antimicrobial activity of crude extract of bacterocin was determined by treating the
sample with proteinase k, trypsin and chloroform. The samples treated with them showed
no activity against Strapforease when disc diffusion method was performed as shown in
table c. This indicated that bacteriocin is protein in nature and digested by these
proteolytic enzymes (Table 4.1 c).

Table 4.2 c.

Effect of proteinase-K, chlroform and trypsin on bacteriocin activity against

Straphforiase

Enzyme treatment

Sample

No enzyme (control)

Proteinnase-K

Chloroform

Trypsin

d) Effect of NaCl
The research led to the environmental conditions that maximized bacteriocin activity,
which can be expressed as polynomial function of NaCl. The isolated culture was treated
with different concentrations of (0%, 2%, 4%, and 6%) of NaCl to check the ba cteriocin
production (Table 4.2 d).
Table 4.2 d.

Effect of different concentrations of NaCl on bacteriocin production by Lactic acid

bacteria against Strapforease

28

Table 4.2. e. Grading

NaCl Concentration

Sample

0%

2%

4%

6%

of antimicrobial activity in symbolic and digitalized form along with their

interpretation for the activity

4.3)

Sr.#

Mathematical sign

Zone size (mm)

Intrpretation

No or poor antimicrobial activity

1-15

Activity present

++

16-20

Strong activity

+++

21-35

Very strong activity

Partial Purification

The extract was subjected to protein purification of the peptides responsible for
antimicrobial activity. Partial purification of peptides exhibiting antimicrobial activity was
performed by ammonium sulphate precipitation, gel filtration and anion exchange (FIG 4.3
a)

a) Precipitation of protein by gel filtration pattern

The samples were applied to Sephadex G-100 columns as fractions were collected.
Absorbance of each fraction was noted at 280 in ELISA machine (FIG 4.3.a).

29

FIG.4.3.a.

Gel filtration pattern of Ammonium Sulphate precipitated residues of bacteriocin by

lactic acid bacteria on Sephadex G-100.

b) Ion exchange chromatography
The pooled fraction having the strong antimicrobial activity was subjected to anion
exchange chromatography.Then absorbances were noted at 595nm and graph is plotted,
different peaks were subjected to antimicrobial assay (FIG 4.3.b).

30

FIG.4.3.b.

Fractionation of gel filtration on Sigma DEAE Cellulose anion exchanger column

equilibrated with 10 mM of phosphate buffer (ph 7). The column was washed with
phosphate buffer to remove unabsorbed fractions, and then eluted with NaCl gradient (0 1.0 M) in the same buffer to desorb the absorbed fractions which contained antimicrobial
activity.
Table 4.3

Antimicrobial activity of different samples from different steps of purification

samples of Lactic acid bacterial species

Extract

Straphoriase

Crude extract

(NH4)2SO4

++

Supernatant

Gel filtration

++

Ion exchange

++

4.4 Strain Development By Using Physico-chemical Mutagenic Agents

Vitamin B12 has been produced in the laboratory at 30 oC and 32oC temperature by the
mutant strains of Lactic acid bacterial species through fermentation technique. During the
production when the vitamin B12 reaches certain limit i.e., 18 mg/liter, the further production
of vitamin B12 is stopped as simultaneous gradual production of propionic acid also takes
place in the process which inhibits the activity of vitamin B 12 production, as the growth of the
organism is inhibited by propionic acid. Treating conditions for the UV irradiation, such as
the dose and time for each set was properly selected. All sets (set A-5 tubes and set B-5
tubes) were irradiated with a dose of 300 erg/mm2 for 30 sec, 60 sec, 90 sec, 120 sec and
150 sec respectively (Table 4.4).
In the similar way for the chemical mutagenesis, the another culture sets (Set A-5
tubes, Set-B5 tubes) were treated with inducing agent Nitrosoguanidine with different
concentrations (60 mg, 70 mg, 80 mg, 90 mg and 100 mg/lit) for 30 min, then centrifugation
method was adopted for separating the treated cells (Table 4.5). The parental type and
above 20 sets of mutant strains (10 physical mutants and 10 chemical mutants) were
cultivated in a liquid production medium anaerobic and aerobic conditions at 30 oC and 32oC
respectively as discussed in material and methods. After 24 hours the growth was observed
31

in all sets (flask) in the form of turbidity which indicates growth of the organism and
production of vitamin B12. Investigations have shown that during the production of vitamin
B12 propionic acid also formed in the production media and gradually increases in amount,
when the amount of propionic acid in the production system exceeds certain limits (i.e., 18
mg/lit), the growth of the organism was inhibited and stops further production of vitamin B 12.
So parental strain and No-1 and No-2 flasks of the physically mutant strains of set A and B
could not produce vitamin B12 after reaching 18 and 19 mg/lit.
Table 4.4.

Details of dose and time of UV exposure for physical mutagenesis

Number of test tube

Table 4.5.

For the fermentation at

For the fermentation at

30C Set - A (Sec)

32C Set - B (Sec)

30

30

60

60

90

90

120

120

150

150

Total

5 Tubes

5 Tubes

Details of dose of Nitrosoguanidine for chemical mutagenesis

Number of test tube

Table 4.6.

For the fermentation at

For the fermentation at

30C Set - A (mg)

32C Set- B (mg)

60

60

70

70

80

80

90

90

100

100

Total

5 Tubes

5 Tubes

Comparison of the amount of Vitamin B 12 produced by parental and different

physically mutant strains at 30C

32

Dose of Physical
Sr #

Strains

Mutagen (UV

Time of exposure

Yield of vitB12

(Sec)

(mg/liter) at 30C

light) (erg/mm2)
1

Parental

19

Set A mutant

300

30

19

Set B mutant

300

60

20

Set C mutant

300

90

21

Set D mutant

300

120

22

Set E mutant

300

150

22

Table 4.7.

Comparison of the amount of Vitamin B 12 produced by parental and different

physically mutant strains at 32C

Sr #

Strains

Dose of physical

Time of Exposure

mutagen (UV

(Sec)

Light) (erg/mm2)

Yield of vitB12
(mg/liter) at 30C

Parental

18

Set-A mutant

300

30

18

Set-B mutant

300

60

18

Set-C mutant

300

90

18

Set-D mutant

300

120

20

Set-E mutant

300

150

21

Table 4.8.

Comparison of the amount of Vitamin B12 produced by parental and chemically

induced mutant strains at 30C

33

Sr #

Strains

Chemical

Conc. of

Yield of

mutagen

chemical

Time

vitB12

used in

mutagen

(min)

(mg/liter)

mutagenesis

(mg/liter)

at 30C

Parental

19

SET-A1

Nitrosoguanidine

60

30

19

SET-B1

Nitrosoguanidine

70

30

20

SET-C1

Nitrosoguanidine

80

30

21

SET-D1

Nitrosoguanidine

90

30

22

SET-E1

Nitrosoguanidine

100

30

22

Table 4.9.

Comparison of the amount of Vitamin B12 produced by parental and chemically

induced mutant strains at 32C

Conc. of
Sr #

1
2
3
4
5
6

Strains

Parental
SET-A1
SET-B1
SET-C1
SET-D1
SET-E1

chemical

Chemical mutagen used in

mutagen

mutagenesis

(mg/liter)

Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine

60
70
80
90
100

Yield of
Time
(min)

30
30
30
30
30

vitB12
(mg/liter)
at 30C
18
18
18
18
20
21

The above results regarding to mutation infer the ability for the mutant culture strains in the
production of vitamin B12. It is observed from studies that the selected UV radiation (dose
and duration), selected quantity of chemical mutagen nitrosoguanidine were improving the
resistance in the organisms against the propionic acid. Further the fermentation parameter
temperature also influenced the yield, as high yield was observed at 30 oC. Based on this it
can be concluded that the conditional mutagenesis can improve the vitaminB 12 production
capacity in the organism, modification of temperature in fermentation process may also be

34

beneficial. Hence this technique can be used in strain improvement program and also in the
production of vitamin B12 at industrial level.

CHAPTER NO. 5

5.DISCUSSION
Lactic acid bacteria occur naturally in several raw materials like milk, whey, meat and flour
used to produce food. These are important because they produce antimicrobial compounds
(bacteriocins). These compounds have grown substantially due to their potential usefulness
as natural substitute for chemical food conservatives to enhance the shelf life of food
(CLAVELEND et al., 2001).
The target bacteria were used to produce bacteriocin and then was purified and
characterized.
Crude sample of bacteriocin without any heat treatment showed maximum inhibition
or activation revealed its proteinacious nature that denatures at certain temperatures.
This bacteriocin is markedly less thermolabile than the bacteriocides T1-I
bacteriocin, as discussed by (MOSSIE et al., 1979). According to their results, 3% fraction
of B.fragilis bacteriocin is stable after heating at 121C.
As it was stable at low temperatures, the microorganism could act as a potential
barrier to inhibit the growth of psychotropic or mesotropic spoilage and food born
pathogens, such as Lactobacillus ssp., L. monocytogenes, S. aureus, B. cereus and Cl.
Perfringens, frequently found in foods stored under refrigeration (Table 4.2 a).
When this pH treated sample assayed against Strapforease it showed negative
results in acidic pH i.e. 3.0, but showed maximum inhibition at ph 6.0. It was also active at
pH 6.8 indicated that activity increased towards basic pH (Table 4.2 b).
The results indicated that bacteriocin is protein in nature and digested by these
proteolytic enzymes. (MORENO et al., 2000) also described that bacteriocin produced by
35

lactic acid bacterial species showed sensitivity to proteases similar to that of nisin.
However, several factors can have an effect on antimicrobial activity including the
interection between bacteriocin and constituents from the cell or the growth medium.
Purity and concentration of enzymes and the technique used to test for the sensitivity. The
inactivation of antimicrobial activity by proteases suggested that the substances evaluated
in this study could be antimicrobial peptides or bactriocin.
They described that loss of the antimicrobial activity after treatment with enzymes
indicated that sensitivity of the active compounds. All bacteriocins including nisin were
fully or partially inactivated by Proteinase-K, Trypsin and Chloroform (Table 4.2 c).
The isolated culture was treated with different concentrations of (0%, 2%, 4%, and
6%) of NaCl to check the ba cteriocin production. The culture showed maximum production
in the presence of NaCl but stable at 2% and 4% NaCl. The culture showed complete
inactivation when 6% NaCl was added. (UGEN et al., 1999) showed that the production of
lacticin was higher in the presence of NaCl than in its absence (Table 4.2 d).
The proteins were precipited at 80% saturation level of ammonium sulphate followed by gel
filtration. (ENNAHAR et al., 2000) underwent their targeted protein at the 80% saturation
level. (HUANG et al., 2005) precipitated an antibacterial protein at saturation level of 70% of
ammonium sulphate. Similarly, other workers performed precipitation at 60% and 65%
respectively for the partial purification of desired protein (FIG 4.3 a).
In present investigation also the production of vitamin B 12 and growth of parental
strain was arrested and the same type of performance of certain mutant strains was also
noted, particularly in strains which were exposed for less duration for irradiation. This
situation indicates that the mutant strains, which were irradiated for 30 sec, 60 sec did not
developed tolerance towards propionic acid, so they ceased their activity after reaching 18
mg/l and 19mg/l, perhaps the less irradiation time was not helping in the development of
resistant character in the organism towards propionic acid (Table 4.6, 4.7). In contrast to
this the sets which were exposed for longer duration, i.e., 90, 120 and 150 sec were
showing their ability to produced more quantity of vitamin B 12 as higher production of
vitamin B12 was recorded in their flasks. This condition indicates that these organisms have
developed tolerating ability towards propionic acid. It suggests that when the organisms
were exposed to UV radiation for longer time, i.e., 120 sec and 150 sec, they acquired the
resistance to propionic acid and as a result they produce more vitamin B 12 (Table 4.6, 4.7).
Regarding the chemical mutant strains and their vitamin B 12 producing ability the
results show the following findings: The lower concentrations of chemical mutagen
36

Nitrosoguanidine has not shown any positive effect in developing the tolerance in the
organism towards the propionic acid, but higher concentrations (i.e., 90 mg and 100 mg of
nitrosoguanidine/lit) have shown positive effect in developing tolerance in the organism.
Hence, more quantity of vitamin B12 was produced by the mutants which were treated with
high concentration of mutagen when compared with the parental strain and also mutants
which were treated with lower concentrations (Table 4.8, 5.9). This condition also suggests
that higher concentration of nitrosoguanidine is developing tolerating ability towards
propionic acid in the organism. Further the influence of temperature is also noted in the
fermentation process on the vitamin B 12 production ability of parental, physical and
chemically treated mutant strains. High activity of the organism i.e. production of vitamin B 12
was recorded at 30oC temperature and low yield was recorded at 32 oC temperature (Table
4.8, 4.9).
From tables regarding to mutation for strain improvements in chapter no. 4, it
became clear that the growth inhibition which is caused by higher concentration of propionic
acid under anaerobic conditions can be minimized when temperature was decreased.
Bacteriocins are one of most excellent microbial protection systems. While we are
yet in the premier levels of investigation of their historical or developmental associations
and eco-friendly performance, it is very clear from their affluence and variety that they are
the microbial weapons of choice. Categorizing why they are such an advantageous family
of toxicants will need an important assurance to future research. Further more, we need
more cultured eco-friendly models (both empirical and theoretical) to aid in our developing
sense of various actions of the toxicants in starting microbial changes and continuing
microbial diversity. The impact of such investigations is not entirely pedantic. The effect of
bacteriocins to serve as alternatives to classical antibiotics in treating bacterial infections is
realistic, and uses of bacteriocins in food conservation is back fired.

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