10

2. REVIEW OF LITERATURE

Lactic acid bacteria are group of Gram positive bacteria categorized together

for their morphological, metabolic and physiological characteristics. They produce

lactic acid either through homofermentative of heterofermentative pathway and are

wide spread in nature and also found in human intestine. They are considered

especially as beneficial bacteria. Because they have their ability to break down

proteins, carbohydrates and fats in food and helps in absorption of necessary elements

and nutrients required for the survival of humans and animals. They show a strong

antagonistic activity against many food contaminating microorganisms. Some

lactobacilli produce antimicrobial peptides, bacteriocins which are inhibitory to many

human pathogens. Hence lactobacilli can be considered as a good source of probiotics

in food industry.

2.1 LACTIC ACID BACTERIA

Lactic acid bacteria (LAB), a physiologcally related group of Gram positive

bacteria which are widely used in the production of various fermented products, and

even cosmetics ingredients (Aslim et al., 2005). The presence of these bacteria is

proved to produce desirable and unique flavor in the food stuffs.

2.1.1 ECOLOGY

The lactic acid bacteria are isolated mainly from milk and dairy products.

Beside these products it is also present in industrial effluents, food grains and meat

products, beer,wine, fruits and fruit juices,pickled vegetables and sourdough. They are

also commensal in the intestinal tract and vagina of many homothermic animals

including man (Biswas et al., 1991).

11

2.1.2 CHARACTERIZATION OF LACTOBACILLI

Lactobacillus, an important member of LAB group is a genus of Gram

positive, non-spore forming, non-motile, catalase negative, anaerobic, microaerophilic

or facultatively aerobic bacteria of the family Lactobacillaceae. Morphologically they

are straight or curved rods, ranging from 0.5µm-0.8µm. Some species have been

reported to be bean-shaped with rounded ends usually occurring singly or in short or

long chains. Chain formation is common in late logarithmic phase of growth (Katla et

al., 2003).

2.1.3 ISOLATION AND IDENTIFICATION

Callewaert et al., (1999) have identified species of Lactobacillus from meat

and meat products by a few biochemical characteristics; presence of meso

diaminopimelic acid in the cell wall, the isomers of lactic acid produced, production

of citruline from arginine and fermentation of some carbohydrates. This identification

key was further checked by DNA-DNA hybridization.

Several new genetic approaches have been made for improving the

identification of lactobacilli, such as analysis of plasmid content (Corr et al., 2007)

and total soluble cell protein SDS-PAGE pattern, sequencing of 16S rRNA,

restriction endonuclease analysis, development of species specific probes and M13

DNA finger printing (Gevers et al., 2001).

G + C Content

Kemperman et al., (2003) have reported that determination of carbohydrate

fermentation is not reliable and reproducible. Beside carbohydrate fermentation,

species of lactobacilli could be strongly identified by G + C content. According to

12

G+C content, the genus Lactobacillus is heterogenous, with a wide range 32-51

moles%.

2.1.4 GROWTH AND PHYSIOLOGY

The lactobacilli are very sensitive to physico chemical factors and

environmental conditions. The main factors influencing their growth is pH,

temperature, media composition etc. (Balasubramanyam and Varadaraj 1998). The

optimal growth conditions for lactobacilli could be described as

1. A temperature just below optimum.

2. A pH value of 5.5-6.O

3. A low oxygen content.

4. An adequate growth medium.

5. Absence of toxic factors.

TEMPERATURE

The lactobacilli can be placed in three groups according to the growth

temperature. They grow usually in the range of 30°C-40°C. Homofermentative

species generally grow at 45°C or higher but not at 15°C or 20°C.

Heterofermentative species grow at 25-35°C, and variable growth is observed

at 45°C and 15°C. One of the heterofermentative species L. fructiorans shows good

growth even at 10°C.

13

pH

The acidic properties of lactobacilli are one of their most characteristic

features. It is for this reason that they usually predominate as the final flora in sugar-

containing media under anaerobic condition e.g. vegetables, mashes, milk and cheese .

Lactobacilli are usually unable to grow on alkaline pH except for the intestinal

types which can resist alkaline pH.

MEDIA

The special requirements of the latobacilli are amino acids, peptides, nucleic

acid derivatives, vitamins, minerals, salts, fatty acids or fatty acid esters and

fermentable carbohydrates ( Daba et al., 1991). In addition to this good media must

be slightly acidic (pH 5-6) and should contain reducing substances.

Several media for lactobacilli have been described like tomato juice medium.

Rogosa et al., (1951) described a selective medium designed especially for the

enumeration and isolation of lactabacilli of oral and fecal origin. While one of the best

and most selective medium for lactobacilli was designed by De Man et al., (1960).

2.2 BACTERIOCINS

Many of the antibiotics produced by bacteria are peptides in nature and some

of them have many properties common with the substances referred to as bacteriocin

(Ganz et al., 1985). Although, these bacteriocins share some common properties with

other inhibitory substances produced by bacteria e.g. thionins, defensins and killer

toxins, but they are unique. A number of Gram-positive and Gram-negative organisms

have been reported to be bacteriocinogenic (Fricourt et al., 1994).

In the search for a food biopreservative, investigation on certain antibacterial

proteins (bacteriocins) from lactic acid bacteria has been very popular ( Joerger and

14

Klaenharnmer, 1986). As LAB is already being used in fermentation, the use of

naturally isolated bacteriocin producer is thought to add an extra degree of protection

without posing any special problem (Lindgren and Dobrogosz, 1990), These

bacteriocins have no clinical efficiency like antibiotics, and they pose no threat to the

development of resistance. Bactertocin producing LAB are easily isolated from dairy

products meat, fish and vegetables.

2.2.1 HlSTORICAL PROSPECTIVE

Leroy and Vuyst (2005) provided description of bacteriocins in lactobacilli

after examination of strains for lysogeny. They found that 6% of the strains produced

an inhibitor that was phage unrelated and bactericidal to other Lactobacillaceae. Later,

it was found that twenty five out of 121 L. fermentum strains produced an inhibitory

substance which inhibited the growth of L. acidophilus indicator strains.

TABLE I: MICROBIAL GENERA THAT PRODUCE BACTERIOCIN

Aerornonas Acetobacter Actinobacillus

Agrobactarium Bacillus Bacteroides

Bordetella Brevibacterium Brucella

Carnobacterium Caulobacter Citrobacter

Clostirdiurn Corynebacterium Enterobacter

Enterococcus Erwinia Escherichia

Haemophilus Halobacteria halofexax

Klebsiella Lactobacillus Lactococcus

Leuconostoc Listeria Micrococcus

Morganella Mycobacterium Neisseria

Parecolobacterium Pasteurella Pediococcus

Propionibacterium Proteus Pseudomonas

Salmonella Sarcina Serratia

15

Staphylococcus Streptococcus Shigella

Thermus Vibrio Xanthomonas

Yersinia

Yeast Fungi

Candida Aspergillus

Cryptococcus

Hansenula

Kluveromyces

Pichia

Saccharomyces

Torulopsis

These antagonistic strains are inhibited by the effect of common bacteria present in

urine. Later in 1971 Schroder et al., have discovered that the filtrates of the cultures of

Escherichia coli, called “principle V” strongly inhibited the growth of another strain

of the same species. Since Scroders observations, similar substances were found by

researchers which to be produced by numerous strains of the family

Enterobacteriaceae including Escherichia, Enterobacter, Salmonella, Shigella and

Proteus species. Later on the term was coined as “COLICIN” for this inhibitory

substance, where as the term “bacteriocin” was first used by the successors . They are

called “colicins” because a substance produced by any member of the group may be

active on strains belonging to any other species of the family, including E.coli ( Fisher

et al., 2005).

Although, the nature of the inhibitory substance was not clear previously, but it

was suggested that many of the observed interactions were caused due to substances

that are now classified as bacteriocins (Tagg et al., 1976). This bacteriocin is the

general term and the individual type of bacteriocins are generally named according to

the species of the organisms originally produce it ( Torodov and Dicks, 2005).

16

2.2.2 NOMENCLATURE

Bacteriocins are antibacterial proteins or protein complexes produced by

several Gram-positive and Gram-negative bacteria. Their lethal action is restricted to

only a limited number of related species, and some act only on certain strains of the

same species which produce them (Floris et al., 2003).

The original concept of bacteriocin was colicin based and was defined by Tagg

et al., (1976) as substances possessing:

1. Bactericidal mode of action.

2 A narrow inhibitory spectrum of activity towards closely related species.

3. The presence of an essential, biologically active protein moiety.

4. Attachment to specific cell receptors.

5. Plasmid-borne genetic determinants of bacteriocin production

6. Production by lethal biosynthesis i.e. commitment of the bacterium to produce

bacteriocin will consequently bring about cell lysis.

2.2.3 CLASSIFICATION

Fuller et al.,( 1992) have classified the bacteriocins into two groups depending

on the molecular weights. Bacteriocin of low molecular weight are more susceptible

to trypsin digestion but are less sensitive to heat. The high molecular weight

bacteriocins are trypsin resistant and thermolabile (De Grado et al., 1982).

Richard and Hans ( 1999) have described three classes of antagonistic agents

which have been brought together under the term bacteriocin. One class is represented

by some of the pyocins (aeruginocins) elaborated by strains of Pseudomonas

17

aeruginosa. Strong evidences suggest that these are defective bacteriophage particles,

comprising only the tail elements, which exert their effect on the cytoplasmic

membrane of sensitive cells after binding to specific receptors in the outer membrane.

This resulted in protein synthesis inhibition without cell lysis.

Another class is represented by broad-spectrum megacins produced by

B. megaterium. They cause susceptible cells to leak intracellular contents by attacking

the membrane phospholipids.

Beside these two classes another class includes the classic bacteriocins

D-soluble proteins molecular weight 50,000-100,000 that attack sensitive cells by

more means. Many species produce these type of inhibitors but only colicins is

extensively studied and best understood (Sanni et al., 1999).

Although the criteria suggested by Tagg et al., (1976) were initially used to

characterize proteins of lactic acid bacteria as bacteriocins, it is now clear that these

criteria are universal. Reddy et al., (1984) have reported that most of the proteins

which inhibit the growth of related bacteria without affecting the producing strains are

considered bacteriocins. Proteins which act exclusively via enzymatic activity such as

lysozyme are not classified as bacteriocins.

Klaenhammer (1993) had defined four different classes of bacteriocins.

(1) Lantibiotics, small membrane active peptide (<5 kDa) containing the unusual

amino acids lanthionine, methyl lanthionine and dehydrated residues; e.g.

nisin, lacticin 481, carnocin U149, lactocin S.

(2) Small heat stable, non-lanthionine containing membrane-active peptides (<10

kDa) and moderate (100°C) to high 121°C) heat stability; e.g. pediocin PA-i,

18

lactococcin A, B,M, leucocin A, sakacin A,P, lactacin F. Sub-groups that can

be defined within the class Il bacteriocins are:

a. Listeria-active peptide with a consensus sequence in the N- terminal of

Tyr-Gly-Asri-Gly-Val-Xaa-Cys-.

b. Complexes consisting of two proteinaceous peptides for activity;

lactococci G. M. lactacin F.

c. Thiol-activated peptides requiring reduced cysteine residues for

activity; lactococcin B.

(3) Large, heat-labile proteins (30 kDa); helveticin J, V-1829, acidophilicin A,

lactacins A, and B.

(4) Complex bacteriocins, composed of protein with one or more chemical

moieties (lipid, carbohydrates) required for activity; plantarica 5, leuconocin S,

lactocin 27, pediocin.

The lactobacilli are historically notable for their production of class IV

complex bacteriocins. These proteins are associated with other lipid or carbohydrate

moieties which appear to be required for activity. Jimenez-Diaz et al., (1993) reported

a new bacteriocin produced by L. plantarum that was heterogeneous in its chemical

composition and notably had the widest spectrum of activity of the known

Lactobacillus bacteriocins. To date, the class 3 bacteriocins have been found only in

Lactobacillus and include heat- labile proteins of large molecular mass.

A number of heat stable Class I peptides have been identified and purified

from Lactobacillus species.These include curvacin A, sakacin A, P, lactacin B and F

(Maher and McClean,2006).

Some of the bacteriocinogenic spp. of Lactobacillus reported to date are listed

in table II.

19

Table II BACTERIOCINS PRODUCED BY GRAM-NEGATIVE AND

GRAM-POSITIVE BACTERIAL STRAINS

S.No. Producer Bacteriocin

1 A.aerogenes Aerocins

2 A.rediobacter Agrocin

3 B.cereus Cerecins

4 B.flagilil Fragilicin

5 B.megaterium Megacins

6 B.stearothermophilus Thermocin

7 B.subtilis Subtilin

8 B.thermoleovoeans Thernioleovorin

9 Carnobacterium Carnobacteriocin

10 C.diphtheriae Corycin Diphthericin

11 C.piscicola Pisciococins

12 C.michiganense Cornocin

13 C.bowmum Clostocin Boticin or Butyricin

14 C.perfringens Peringiocins

15 C.welchi Ciostocin or welchicin

Clostridiocin Clostridicins

16 K.pneumoniae Diplococin Paeumocid

17 E. cloacae Cloacins

18 E. coli Colicin

19 Enterococcus faecium Enterocin

20 H. mediterranea Halocins

21 K. pneumonia Kiebicin Pneumococc

22 K. pneumonia Pneumocin

23 Leuconostoc mesenteroides Mesenterocin

20

S.No. Producer Bacteriocin

24 Leuconostoc gelidium Leucocin

25 L. acidophilus Lactacin Acidocin

26 L. amylovorus Amylovorin

27 L. delbrueckii Lacticin

28 L. gasseri Gassericin

29 L. heIveticus Helveticin

30 L. lactis subsp. cremauis Lactococcin A

31 L. lactis subsp. cremoris Diplococcin

32 L. monocytogenes Listeriocin Monocin

33 L.sake Lactocin Sakacin

34 M. morgani Morganocin

35 Micrococcus varians Micrococcin Variacin

36 N. meningititis Meningocin

37 Paracolobactrum Arizonacins arizonae

39 Pasturella pestis Pesticin A-1

40 A.acidilactici Pediocin A-1 Pediocin ACH

41 A.acidilactici Pediocin

42 P.acnes Acnecin

43 P.fluorescens Flavocins

44 A.jensenis Jenseniin G

45 P.pentosaceus Pediocin A

46 A pentosaceus Pediocin

47 A thoenil Propionicin

48 P.aeruginosa Pyocins Aoruginocin

50 P.syringae Syringacin

51 Proteus morganli

52 Salmonella colicin 1 K or B

21

S.No. Producer Bacteriocin

53 Shigella Colicin S1, S2, S3, S4 and S8

54 S. aureus Staphylococci Aureocins

55 S. epidermidis Staphylococcin 1580

56 S. lactis Nisin

59 S. cremoris Diplococcin

60 S. faecalis Enterococcin E-1

61 S.faecium Enterocins EIA and EIB

62 S. mutans Mutacin

63 S. sanguis Strepiocin STN

64 S. viridians Viridin B

65 V. cholera Vibriocins

66 Yersinia pestis Pesticin

2.2.4 NOMENCLATURE

Normally, bacteriocins are named after the genus or species of the strain that

produces them. The suffix “cin” is used to denote bacteriocinogenic activity and is

appended to either the genus or the species name. Many authors have added a suffix

„e‟ to the name of bacteriocins e.g. staphylococcine, listeriocine and corycine etc. For

precise specification of a particular bacteriocin, trivial designation of producing strain

be included within the bacteriocin name e.g. co and cal plasmid are given names such

as K235 which indicates the colicin type K of E. coli strain K235. Furthermore,

colicin immunity is used to subdivide colicin types. Colicins E2 and E3 are both

members of the E group of colicins.

22

2.3 PROPERTIES OF BACTERIOCINS

2.3.1 CHEMICAL COMPOSITION

Bacteriociris are extremely heterogenous group of substances constituting an

active protein moiety alone or in conjugated form i.e. they may be either simple

proteins or proteins linked to lipid and carbohydrates (Tagg et al., 1976). Similarly,

Moniem Abada (2008) had reported that several colicins can exist in two forms,

simple proteins or covalently linked to lipopolysaccharide components of the cell

wall. Colicins of induced cultures are simple proteins, while those uninoculated

cultures are linked to lipopolysaccharide which forms part of the cell wall „0‟ antigen.

However the active chemical group in all cases is protein.

The chemical nature of bacteriocins produced by Gram-positive bacteria are

not very different from those of Gram-negative bacteria. Bacteriocins produced by

lactobacilli have been shown to constitute a heterogenous class of antimicrobial

peptide (Joerger and Klaenhammer 1986).

Bacteriocins of LAB in general are small cationic proteins with high

isoelectric points and amphiphilic characteristics. The bacteriocins of Lactotacillus

spp. are produced as large native complexes containing protein, lipid, carbohydrate

and phosphorus etc. Lactocin 27 is a protein-lipopolysaccharide complex while

lactacin B, a bacteriocin produced by L. acidophilus is a simple protein

(Klaenhammer 1988). Similar findings were reported for bacteriocin of L. fermenti

which is also a lipid and carbohydrate complex. Similarly a bacteriocin produced by

L. acidophilus has also been reported to be associated with a lipid-like material

(Joerger and Klaenhammer, 1986).

23

2.3.2 PHYSICAL PROPERTIES

Bacteriocins are ranges in size from low molecular weight proteins such as

streptococcin A-FF22 with the molecular weight of 8,000 D to complex defective

phage particle with a molecular weight in excess of 106

.

Hancock and Chapple (1999) have concluded that the high molecular weight

forms of the bacteriocins were probably phage related whereas the low molecular

forms were not phage related.

A common characteristics of bacteriocin of Gram-positive species is their

apparent existence in two or more distinct physical forms. The smallest of the

substances to be called bacteriocin include streptococcins A-F22 and 673, and lactocin

LP27. All are having molecular weights in the range of 8,000 - 12,500. Klaenhammer

(1998) reported the molecular weight of lactocin F (L.acidophilus) to be 2,500.

Lactocin S (L. sake) in purified state had a molecular weight less than 13,700.

2.3.3 ANTIGENICITY

On the basis of their high molecular weight and protein composition

bacteriocin could serve as an excellent antigen (Tagg et al.,1976). For example colicin

K is a potent antigen and the antiserum prepared against it agglutinates the bacteria

that produce it. In addition to that, megacin A-216 has also been shown to be

antigenic and can evoke an antibody capable of neutralizing its own killing effect.

One of the characteristics of monocins (a bacteriocin produced by L. monocytogenes)

is its antigenicity (Tagg et al., 1976). Piard et al., (1990) used ELISA to quantify nisin

in foods using antibodies isolated from antisera raised against nisin (conjugated to egg

albumin). Similar studies were also conducted by Pappagiani (2003). But in his study,

albumin-conjugated and non-conjugated pediocin AcH failed to elicit an immune

response in mice or rabbits suggesting that pediocin AcH would not be toxic to

24

humans if used as a food biopreservative. In another experiment Joeger and

Klaenhammer (1986) have obtained anti helveticin J antibodies by injecting purified

helveticin J into rabbits. These antibodies were subsequently used as immunoprobes

to clone the structural gene of this bactoriocin.

Thus, antibodies prepared against purified bacteriocins can be used to localize,

quantify, sequester, and analyse bacteriocins and the corresponding genes that encode

for these proteins ( Hopwood et al.,1985).

2.3.4 GENETIC DETERMINANTS OF BACTERIOCIN

The property of bacteriocin production seems to be a inherited characteristic

determined by bacteriocinogenic gene. In spite of noticeable exceptions, the nature of

the genetic determinants for bacteriocins are plasmid linked, which can be lost either

spontaneously or through the use of curing agents. In plasmid curing the agents most

commonly used are ethidium bromide, acridine orange while there are reports which

show that exposure of the producer strain to elevated temperature enhances the rate of

plasmid loss (Tagg et al., 1976). The irreversible spontaneous loss of

bacteriocinogenicity during serial subcultures or long-term storage has been reported

for strains of L. helveticus.

The bacteriocinogenic factor may determine not only the chemical

composition of bacteriocin but also the regulation of its biosynthesis. This

bacteriocinogenic factor could be transferred to a non-bacteriocinogenic sensitive

strain in mixed culture. The recipient in turn acquires the ability to produce same

bacteriocin and also becomes immune to the action of bacteriocin to which it was

previously sensitive (Ivanovics, 1962), In a very few cases it has been shown that

genetic determinants of bacteriocins are present not on plasmids but on chromosomes

e.g. in L. helveticus, L. plantarum, and L. acidophilus N2 (Hancock et al., 2006).

25

2.3.5 IMMUNITY TO BACTERIOCIN

There are number of different mechanisms by which bacteria protect themselves

from the adverse effect of their own bacteriocins. One of them undergone post-

translational modification after the protein is synthesized as a prepeptide and some

amino acid residues are post-translationally processed to generate the active protein

molecule. This is the case for nisin with the generation of the thio ether amino acids

lanthionine and 3-methyl lanthionine. Other bacteriocins produces a precursor in

many cases splitting the prebacteriocin into an active bacteriocin and an immunity

protein 9 ( Jack et al., 1995).

Bacteriocin immunity is quite different from bacteriocin resistance.

Bacteriocin resistance is due to loss of specific receptors for particular bacteriocin.

The resistant strains are unable to adsorb bacteriocin, but may adsorb to immune cells

in high concentration (Tagg et al,, 1976).

In most cases, colicins are inactive against a cell that contains a related Col

plasmid. The immunity in colicins is conferred by an excreted protein that binds to the

large colicin protein. In case of the colicin cloacin DF13, this protein consists of 3

regions: (1) a receptor-binding region, (2) as RNase, (3) and a region that binds the

immunity protein. The immunity binding segment has a strong negative charge that is

neutralized by the positively charged immunity protein. After binding to the receptor,

the colicin is released. The N-termini remains outside the cell and the RNase segment

enters the cell, leaving the immunity protein on the cell surface. The immunity to

colicin is not absolute and the colicinogenic cells are sensitive to very high

concentration of homologous colicin. This phenomena is called immunity breakdown.

They further described that the formation of a firm complex between a bacteriocin and

its specific immunity protein seems to be the mechanism by which bacteriocinogenic

cells protect themselves from the lethal action of their own product ( Guinance et

al.,2005).

26

In L. acidophilus the bacteriocin producing gene and immunity gene are

reported to be present on a plasmid pLAIO3. The production of acidocirin 8912 and

host immunity might serve as genetic markers in the gene transfer system of these

organisms. Similarly, a plasmid of 110 kb encoding lactacin F production and

immunity was also identified. It was further suggested that the immunity functions of

L. acicdophilus NCK 88 to lactacin F reside within the cell wall.

In L. sake L45 the lactocin S production and its immunity factors reside on an

unstable 50 kb plasmid. Hancock (1997) had identified two regions within L. lactis

subsp. cremoils 9B4 plasmid p984-6. Each were encoding a unique bacteriocin along

with the corresponding genetic determinants.

2.4 FACTORS INFLUENING THE PRODUCTION OF BACTERIOCIN

Various factors influence the yield of bacteriocin, and the conditions for

optimal growth of a bacterium does not necessarily coincide with optimal synthesis

and release of a bacteriocin. Some of the factors affecting the production of

bacteriocin are as follows:

2.4.1 MEDIA

Significant production of bacteriocins have been demonstrated on solid media.

Results obtained by Maher and McClean (2007) had indicated an increase in the yield

of bacteriocins produced by streptococci, after increasing the viscosity of liquid

media. Few year later it was reported that yield of staphylococcin 1580 was 20 times

greater in a semisolid medium than in liquid medium (Tagg et al., 1976). Beside this,

media components have also been reported to affect the yield of bacteriocins, for

example addition of yeast extract to basal trypticase medium enhanced bacteriocin

27

production by various strains of S. mutans. While, addition of manganese was

considered necessary for megacin production and casein hydrolysate for butyricin

7423 production (Tagg et al., 1976).

Parade et al., (2007) compared bacteriocin production by 16 strains of

lactococci in various media. The authors concluded that complex media were essential

for bacteriocin production. while studying the influence of media constituents on

bacteriocin production by Klebsiella pneumoniae it was observed that maximum

production of this bacteriocin is in trypticase soy broth (TSB). This yield was further

stimulated with the addition of 1 % yeast extract to TSB. Biswas et al.,(1991) found

that Tween 80 and Mn allowed optimal biomass and bacteriocin production.

In a similar attempt, Richard and Hans (1999) have studied the factors

affecting the production of two bacteriocins from lactic acid bacteria. Their work

revealed the effect of media components on bacteriocins such as dialysates of

complex media, containing only low molecular weight fractions were effective for

pediocin production but not for the production of lactococcal bacteriocins. They

suggested yeast extract, Tween 80 and pH to have the largest influence on bacteriocin

production.

2.4.2 CULTURAL CONDITION

Several authors have revealed the influence of some cultural conditions e.g.

time of incubation, temperature, aeration and pH on the yield of active bacteriocin.

Generally the yield of bacteriocin is greater at temperature optimal for growth of

producer strain. Production of acidiocin 8912 and mesentericin Y105 was maximum

at 30°C. The production may be suppressed or lost at elevated temperature. Prolonged

incubation of producing strain may also result in substantial loss of bacteriocin

activity (Tagg et al., 1976). The effect of pK is well documented (Joerger and

28

Klaenhammer, 1986) . The degree of adsorption of these peptides on to the cell

surface depends significantly on the pH of the medium. Maximum adsorption

occurring at pH 6.0 to 6.5, but no adsorption was found at pH 2 or below (Piard et

al.,1990).

2.4.3 GROWTH KINEICS

Maximum bacteriocin yield in a culture may occur at different phases of

growth cycle, depending on the type of bacteria. The production of staphylococcin

C55 starts in the log phase reaches a maximum between 24 and 48 hours of growth

and then gradually declines. Whereas, butyricin 7423 is secreted during the late

exponential phase. A bacteriocin from Streptococcus zymogenas was shown to be

maximally produced after 90 minutes of growth with a rapid loss in activity during

late logarithmic growth. Plantaricin A was observed to be maximally accumulated

during the mid-log phase of growth with a decrease in activity thereafter which may

be due to the synthesis of an inactivator or the increasing acid conditions that develop

during the latter stages of growth. A bacteriocin produced by C. perfringens 28

appeared in culture supenatant fluids during late logarithmic growth and ceased to be

produced after stationary growth. Accumulation of helveticin J was detected between

the late log and stationary phases of growth (Joerger and Klaenhammer, 1986).

Mesentericin Y105, was excreted maximally after a lag period of 3 hours, until the

late exponential phase (Odufna and Adeyele, 1985).

2.5 EFFECT OF BACTERIOCINS ON SENSITIVE BACTERIA

Bacteriocin sensitivity depends upon the presence of specific receptors on the

cell surface of the sensitive cells. Thus, the first step of action of the bacteriocin

activity is the adsorption of bacteriocin to specific receptors, which is an energy

independent and a reversible phase. After the specific adsorption step the bacterocin is

29

converted to a new state by an energy-dependent process and finally, specific

interaction between the bacteriocins and target occur. This is resulted in an

irreversible pathological changes. Adsorption of different bacteriocins may have

different biochemical consequences for a cell. For example, colicin E3 specifically

inhibits protein synthesis, whereas colicins El, K and A inhibit DNA, RNA and

protein synthesis as well as the active transport of amino acid (Tina and Legisa, 2006).

Colicins seem to be more potent than antibiotics. Even a single molecule of

colicin is sufficient to kill a sensitive bacterium (a single hit kinetics).

2.5.1 RECEPTORS AND ADSORPTION

The action of colicin is very specific and have a narrow spectrum of action due

to the requirement for specific protein receptors in the outer membranes of bacteria.

Colicin K and T6 share the same receptor since mutant selected for resistance to either

are resistant to the other. The resistant mutants appear to lack the specific receptors for

colicin fixation (Yoneyama and Katsumata, 2006).

One of the bacteriocins of C. perfringens divided in to two groups which

inhibited macromolecular synthesis, while the other was revealed to be involved in the

production of spheroplasts of sensitive cells.

Colicins belonging to different types bind to different receptors immediately

after adsorption to receptors, do not bring about any irreversible change leading to cell

death unless the cytoplasmic membrane is energized. Not all colicins bring about the

same biochemical changes in the sensitive cells; different colicins can kill cells by

affecting different biochemical targets (Zaslof, 2002).

30

Table III : MODE OF ACTION OF SOME BACTERIOCIN

S. No. Colicin Target action specificity

01 Colicin B, lb Damage cell membrane

02 Colicin El, KI Uncoupled energy dependent process by an

unknown effect on cell membrane

03 Colicin E2 Degrades DNA

04 Colicin E3 Cleaves 16 S rRNA

Adsorption is an energy independent process. Cells treated with uncoupling

agents remain susceptible to rescue by trypsin (an agent which dissipates the

energized state). When the uncoupled is also removed, the cells are then able to grow

and form colonies, there by demonstrating that colicin cannot kill cells in the absence

of membrane energy (Zucht et al., 1995).

Some bacteriocinogenic strains have no receptors which would adsorb their

own bacteriocins, and thus the bacteriocins have no effect on their own producer

strain. There are also certain bacteria which lack the adsorption specificity such as

lactocin LP27 (Tagg et al, 1976).

The L. forms known to be devoid of their cell walls and are completely phage

resistant, have shown even higher degree of susceptibilities to colicins than their

parent bacteria. The Colicin may become adsorbed directly to the cytoplasmic

membranes of these L. forms. According to Smarda and Taubeneck (1998) the

specificity of interaction between colicin and bacterial cell cannot be determined by

cell wall receptors, provided the bacteriocin is able to penetrate the cell wall. This

31

suggests that receptors are simply meant to bring the bacteriocins into contact with the

cytoplasmic membrane (Vogelmann and Hertel, 2011).

Bacteriocins of Gram-positive bacteria are reported to have a wider spectrum

of activity as compared to Gram-negative bacteria. This is because of the presence of

specific receptors they have.

Most of the proteins in the outer membrane of E. coli acts as colicin receptors

and are also involved in the transport of various metabolites across the cell. For

example, the colicin E reach a glycoprotein with a molecular weight of 60 KD is also

a receptor for vitamin B. Colicin K receptor is probably involved in the transport of

nucleotides, while colicin K mutants, which lack this colicin receptors are deficient in

the uptake of nucleosides. Certain colicin receptors are necessary for the uptake of

iron by E. coli. The receptor for colicin B is involved in the accumulation of ferric

enterochelin. Similarly colicin M receptor is also receptor for ferrichrome.

A sensitive cell may show adsorption specificity for more than one type of

bacteriocins and consequently is sensitive to more than one types of bacteriocins. Loss

of particular receptors from the surface of sensitive cells renders the cell resistant to

the action of that bacteriocin. The presence of receptors is proved by the action of

antiserum, prepared against the outer surface of sensitive bacteria, which protects the

cells from bacteriocins by blocking the receptors (Wiravan et al., 2005).

2.5.2 MODE OF ACTION

According to Smacchi and Gobbetti (2000) exposure of susceptible bacteria to

colicin results in the death of cells which, however, are not lysed. Thus, the action of

colicin is bactericidal and not bacteriolytic. The kinetics of colicin action seems to

indicate that fixation of a small number of molecules is sufficient to bring about the

death of a bacterium. It was further indicated, that the rate of killing is proportionate

32

to the initial colicin concentration. These facts suggested that a single particle of

colicin are able to kill a bacterium (Minahk et al., 2000).

Studies on the three dimensional structure of colicins indicated that the protein

may be arranged as three independent structural domains, each of which carries a

separate action that leads to the death of susceptible cells (Spano et al., 2002).

The central portion of the molecule binds to specific receptors in the outer

membranes of sensitive bacteria and most are co bind to different receptors (Hardy,

1975), After binding of the colicins to receptors, the protein or a portion of is

translocated across or into the cell membrane of the susceptible cell. The killing action

of the colicin is then carried out by the C- terminal portion of the protein.

Previously it had been reported that the action of colicin on sensitive bacteria

appear to be very similar to the bactericidal action of phage. Furthermore, addition of

colicin to bacteria previously infected with phage blocks the development of phage

(Sitaram and Nagaraj, 1999).

2.5.3 BIOCHEMICAL EFFECTS

There are three major biochemical effects of bacteriocins on the sensitive cells.

These biochemical targets are as follows:

(1) DNA degradation

(2) Inhibition of protein synthesis

(3) Disruption of cytoplasmic membrane

33

2.5.3.1 EFFECT ON DEOXYRIBONUCLEIC ACID

The primary effect of colicin E2 is on DNA. It has been shown that colicin E2

inhibits DNA synthesis within 2 minutes. Lade et al., (2006) had shown that purified

colicin E2 itself an endonuclease that is able to cleave DNA molecules that viewed by

single-strand breaks (SSBs) followed by double-strand breaks (DSBs). It appears that

colicin E2 penetrates the cells to act in a catalytic method as a DNA endonuclease

and the exonuclease in the cell then degrade DNA further. The excessive degradation

of the DNA by bacteriocin leads consequently to the inhibition of RNA and protein

synthesis. Colicin E2 and other colicin not only bring about the DNA degradation but

also inhibit cell division.

Besides colicin, megacin C appears to cause excessive degradation of DNA

and consequently RNA and inhibition of protein synthesis (Verluyten et al., 2004).

2.5.3.2 EFFECT ON RIBOSOMAL RNA

Colicin E2 and cloacin DF13 inhibit protein synthesis by inactivating the

bacterial ribosomes. They cleave the 16S ribosomal RNA molecules and components

of 30S ribosomal subunits, about 50 nuclootides from the 3‟.

2.5.3.3 EFFECT ON CYTOPLASMIC MEMBRANE

Several colicins including El, K and la, and A come into direct contact with

the cell membrane and exert bactericidal action because of their ability to disrupt the

energized state of the cytoptasmic membrane. The high energy state of the membrane

is, therefore, essential for the action of these colicins and appears to be their

biochemical target (Tagg et al.,, 1976). Many other colicins which disrupt the cell

34

membrane are colicins A, B, Ia, lb and SB. These colicins inhibit active transport

process in the sensitive cells and partially disrupts the cytoplasmic membrane.

One of the consequences of the action of colicins on the energized state of the

cell membrane is that the ATP concentration falls low and, thus DNA and RNA

synthesis is inhibited. Megacin A also acts on cytoplasmic membrane. In contrast to

partial disruption by colicins, it completely disrupts the cytoplasmic membrane

(Simon et al.,2002).

2.6 BACTERIOCIN PRODUCTION IN GRAM-NEGATIVE AND GRAM-

POSITIVE BACTERIA

The bacteriocin produced by Gram-negative bacteria have generally proved

easier to isolate and characterise than those produced by Gram positive strains. As

compared to Gram positive bacteria, the bacteriocins of Gram negative bacteria have a

narrow spectrum of activity. The titres of bacteriocins of Gram negative bacteria can

be greatly increased by treatment with inhibitors of DNA synthesis. The most well-

studied bacteriocins are the colicins, which are produced by Escherichia coli and

closely related genera belonging to the Enterobacteriaceae, such as Shigella,

Salmonella and Enterobacter. Bacteriocins produced by Gram-positive bacteria are

generally of low titer which cannot be greatly increased by inducing agents and are

more difficult to purify as compared to Gram-negative bacteria (Tagg et al., 1976).

Bacteriocins produced by Gram-positive bacteria have a wide spectrum of action.

Certain Gram-positive strains produced substances analogous to colicins such as those

produced by some strains of Streptococcus and Staphylococcus (Nakano and Kura

mitsu, 2006).

The followings are the list of some selected species of lactobacilli which

produce wide range of bacteriocin which are very much useful in food and

fermentation industries.

35

L. FERMENTI

Bacteriocin produced by L. fermenti strain 446 have been purified and

characterized. It is a lipopolysaccharide-protein complex (Tagg et al.,1976). The

antagonistic substances produced seemed unaffected by pH or catalase, non-dialysable

and can be precipitated by ammoniurn acetate.

L. HELVETICUS

Upreti and Hinsdill (1973 and 1975) identified and characterized lactocin 27, a

bacteriocin produced by L. helveticus LP27. It exhibited a narrow spectrum of

inhibition, affecting only strains of L. acidophilus and L. helveticus. Lactocin 27 was

isolated from culture supernatant as a protein lipopolysaccharide complex with a

molecular weight in excess of 200,000. However, after purification to homogenicity,

the molecular weight of the glycoprotein was about 12,400. It was extremely heat

stable, retaining its activity even after 1 hour at 100°C (Pascual et al., 2006).

Lactocin 27 adsorbed equally to both sensitive and resistant bacterial cells and

had a bacteriostatic effect on the indicator strain, L. helveticus LS18 (Upreti and

Hinsdill, 1975). It inhibited protein synthesis, but did not effect DNA and RNA

synthesis, neither the ATP levels were affected (Klaenhammer, 1988).

Later, Joerger and Klaenhammer (1986) identified a new bacteriocin,

helveticin J produced by L. helveticus 481. The sensitive indicators of helveticin J

included L. helveticus 1846, and 1244, L. bulgaricus 1373, and L. lactis 970,

Helveticin J was heat sensitive (30 minutes at 1000C) as compared to lactocin 27 and

lactacin B. Pronase, trypsin, pepsin, proteinase K and subtilisin were able to inactivate

helveticin J activity after incubation period of 1 hour at 37°C. This bacteriocin

appeared to be of 37 KDa. Furthermore, L. helveticus 481 was shown to posses a

36

single plasmid of 8 MD. However, pMJ1008 did not encode genetic determinants for

helveticin J production or host cell immunity. The genetic determinant encoding

helveticin J appeared to reside on the chromosome.

Some of the examples of plasmid and chromosomally located bacteriocins of

lactobacilti are listed in table IV.

TABLE IV : BACTERIOCINS OF LACTOBACILLI

S.No. Producers Bacteriocin

1 L. acidophilus

Lactacin F

Lactocidin

Acidocin 8912

Acidophihn

2 L. bavaricus Bavaricin A

3 L. brevis Brevicin

4 L. casei B80 Caseicin 80

5 L. curvatus Curvacin

Curvaticin

6 L. delbrueckii Lacticin A

7 L. gasseri Gassericin A

8 L. helveticus Helveticin J

9 L. plantarum Plantaricin A

Plantacin B

10 L. sake Lactocin S Sakacin A

Joerger and Klaenhammer (1990) had cloned and sequenced the DNA

containing the helveticin J genes from L.h 481. They were successful in transferring

recombinant plasmid pTRKI35 into L. acidophilus NCK 64 via electroporation.

37

L. ACIDOPHILUS

Reinheimer et al., (1995) had first described a bacteriocin type inhibitor

produced by L. acidophilus. This inhibitor was termed lactocidin. It displayed a

broad inhibitory spectrum, was active at neutral pH, insensitive to catalase and non-

volatile and non-dialyzable.

In later years Pascual et al.,(2006) have reported that majority of strains of L.

acidophilus produced bacteriocins. The compound produced by L. acidophilus N2

was responsible for inhibition of, L. bulgaricus, L. helveticus and L. lactis. The crude

bacteriocin had a molecular weight approximately 100,000. It was sensitive to

proteolytic enzymes and heat- stable. It also had its complete activity after 60 minutes

at 100º C. It had a bactericidal effect on the sensitive cells. This bacteriocin was

designated as lactacin B. They further reported that lactacin B adsorbed to both

sensitive and insensitive Lactobacilius species, thus it lacked adsorption specificity.

This indicated that the cell sensitivity or resistance was not solely determined by the

presence or absence of specific cell receptors for lactocin B.

38

TABLE V : CHROMOSOMALLY AND PLASMID ASSOCIATED BACTERIOCIN

PRODUCTION IN LACTOBACILLUS

S.No. Bacteriocin Chromosomally

located Plasmid associated

01 Acidocin 891 2 - pL.A 103

02 Bavaricin A + -

03 Caseicin 80 - -

04 Heiveticin J + -

05 Lactacin B + -

06 Lactacin F + -

07 Lactocin S - pCIMI 50

Plasmid

08 Lactocin 27 + -

09 Plantaricin S + -

10 Sakacin P - +

11 Sakacin A + -

Roger and Montville (1991) have demonstrated the production of lactacin F

from L. acidophilus 11088 (NCK 88), which was more heat resistant and exhibited a

broader spectrum of activity than lactacin B. Production of lactacin F was shown to be

pH-dependent. It was inactivated by proteinase K, subtilin, trypsin and nicin were

unaffected by lyozyme and lipase. The ability to produce lactacin F was conjugally

transferred by 8110 kb plasmid that appears to be an episomal element. Native

lactacin F is associated with a 180 KDa bacteriocin complex, whereas the active agent

is identified as a peptide of approximately 2.5 KDa. Amino acid sequence analysis

39

had identified 25N-terminal amino acids, but they may contain as many as 56 residue

(Turcotte et al., 2004).

Tagg et al., (1976) found that L. acidophilus TK 8912 has produced an

antibacterial substance, designated as acidocin 8912, which was active against strains

of Lactobacillus. It was found stable to heat treatment (120°C for 20 min) and not

affected by pH from 5 to 7. They further presented direct evidence for the

involvement of plasmid ptA 103 in acidocin 8912 production.

Recently, Zapparoli et al., (1998) reported that the inhibitory action of

acidocin 8912 was directed at the cytoplasmic membrane, which might have been due

to the dissipation of the proton motive force and pore formation.

L. SAKE

Schillinger and Lucke (1989) have reported that sakacin A, a bacteriocin

produced by L. sake Lb 706. The inhibitory compound was active against various

LAB and L. monocytogenes. Plasmid profile analysis indicated that a plasmid of about

18 MD may be involved in the formation of bacteriocin and immunity. Sakacin A

possessed some of the characteristics of lactocin S. Both have shown inhibitory

activity against closely related bacteria but not identical strains.

Sakacin P, a small heat stable, ribosomally synthesized peptide produced by

certain strains of L. sake, have shown activity against L. monocytogenes (Minahk et

al., 2000).

In another study conducted by Lindgren and Dobrogosz, 2000, L. sake strain

L45 isolated from naturally fermented dry sausage was shown to produce a

bacteriocin designated as lactocin S. It inhibited the growth of Lactobacillus,

Leuconostoc and Pediococcus. Analyses of these isolates revealed the presence of

40

two plasmids of about 50 kb (pCIM1) and 34 kb (pCIM2). Hybridization experiments

showed that all fragments in pClM2 were found in pClMl. pCIM2 could therefore be a

detected form of pCIM1 (Novick, 1990). However, pCIMI was thought to carry a

second and dominant compatible replicon. It was noted that spontaneous loss of

bacteriocin activity as well as immunity was observed with simultaneous loss or

modification of pCIM1. Whereas loss of pClM2 did not provoke any changes with

respect to these two properties. Their experiments suggested involvement of plasmid

pCIM1 in production of lactocin S and in immunity to this bacteriocin (Minahk et al.,

2000).

L. PLANTARUM

Laemelli (1970) have reported the production of plantaricin B, by L.

plantarum. Similarly, a bacteriocin (plantaricin A) has been demonstrated to be

produced by L.plantarum C1 isolated from a cucumber fermentation. Plantaricin A

was bactercidal towards some species of the four genera of LAB; Lactobacillus,

Pedioccccus, Leuconostoc and Streptococcus. The plantaricin A was rendered inactive

when treated with proteolytic enzymes and had a molecular weight greater than 6000.

L. plantarum C1 is having 2 plasmids of approximately 43 and 65 MDa. Both the

plasmids did not correlate with plantaricin A production or immunity or its

phenotypes.

Recently, Leroy et al., (2006) also indicated the secretion of a small cationic

peptide designated as plantaricin A in L. plantarum C Similarly production of

pediocin Ad-I by L. plantarum WHE92 isolated from cheese has been identified

(Rojers and Montville, 1991).

41

L. GASSEII

Vignolo et al., (1995) investigations showed that fecal L. acidophilus should

be identified as L. gasseii (formerly classfied as group B-1 and B-2 of L. acidophilus.

Yang et al., (1992) detected a heat-stable bacteriocin and its prevalence in L. gassei

strains freshly isolated from infant feces. The agent was inhibitory to L delbrueckii

subsp. bulgaricus JCM 1002. It was not affected by catalase, and not reduced after

heating for 20 min at 120°C, but it was completely destroyed by treatment with

proteinase or trypsin. The bacteriocin from L. gassei (A33 and 39 are termed

gassericin A) have appeared similar in its heat stability to lactacin B and lactacin F

(Klaenhammer, 1980).

L. DELBRUECKII

Biswas et al., (1991) for the first time reported bacteriocins produced by

L.delbrueckii subsp. lactis JCM 1106, JCM 1107 and JCM 1248. The first two were

demonstrated to produce inhibitory agents active against L. delbruckii subsp.

bulgaricus JCM 1002 and MAI yB-62. While L. delbrueckii subsp. lactis JCM 1248

have produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus

NIA yB-62. Heat treatment at 60°C for 10 minutes led to complete loss of activity.

The inhibitory agent, from L. delbrueckii HJCM 1106 and JCM 1107 were called l A

and the bacteriocin from L. delbrueckii JCM 1248 were called lacticin B.

L. BAVARICUS

Andrighetto et al., (1998) reported that bacteriocin producing L. bavaricus

M1401, which exhibited antagonistic effect against closely related species and

inhibited nine out of ten L. monocytogenes. The bavaricin A, activity was inhibited by

proteolytic enzymes and are sensitive to alkaline treatment.

42

L. CURVATUS

The bacteriocin produced by a strain of L. curvatus was shown to be heat

stable and exhibiting a broad spectrum of activity. A plasmid of about 46 Kbp was

thought to be involved in bacteriocin production and immunity to this antibacterial

compound (Vuyst et al., 2004).

. L. SALVARUS

` This bacteriocin salvacin produced by Lactobacillus salvarus was inhibitory

towards L. monocytogenes, Streptococcus mutans, Actinomyces viscosus and

Propionibacterium acnes.

L. AMYLOVORUS

L. amylovorus DCE471 has demonstrated antibacterial effect which was

designated as amylovorin L471. It was a small thermostable and strongly hydrophobic

protein (Vuyst et al., 1996). The production of this bacteriocin was found to be growth

associated as the conditions favoring increase in biomass improve the volumetric

bacteriocin titer (Callewaert et al.,2000).