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).