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Molecular genetic studies of pediocin-like bacteriocin Chapter – 2

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2.1. LACTIC ACID BACTERIA, BACTERIOCINS AND THEIR

APPLICATIONS

2. 1. 1. LACTIC ACID BACTERIA

Lactic Acid Bacteria (LAB) are a group of Gram-positive cocci or rod shaped

bacteria with low G+C content and catalase negative property. They are fastidious,

non-aerobic but aero tolerant, chemo-organotrophic, produce lactic acid through the

fermentation of carbohydrates, and grow only in a complex media (Aly et al. 2006;

Forde et al. 2011). LAB are widely distributed in nature and are commonly found in

foods like dairy products, fermented meats and vegetables, sourdough, silage, plants,

beverages and also in the gastro intestinal tract (GIT) of human and animals (Aly et

al. 2006). Hence, LAB are broadly used as starter or non-starter cultures in the

fermentation of meat, dairy, plant products, etc. (Zhang et al. 2011). The LAB are

considered as a dominant microflora within the GIT contributing to the enhancement

of the immunity and inhibition of the pathogenic bacteria for intestinal integrity

(Vaughan et al. 2005; Forde et al. 2011, Zhang et al. 2011). The major assets of the

LAB include, break down of food and biosynthesis of essential vitamins. They are

involved in the destruction of some of the toxic compounds generated within the body

or ingested in the form of food (Aly et al. 2006). Therefore, the LAB are extensively

studied for benefiting the health of the mankind, and their application in the food

industry as a starter culture or as a food preservative (Klaenhammer, 2000).

The characterization of LAB genomes has been studied for understanding their

biochemical and fermentation pathways. Thus, leading to their application in probiotic

industries or as a cell factory in the development of food-grade additives,

neutraceuticals, vaccine systems, etc. (Hugenholtz et al. 2002; Hanniffy et al. 2004;

Stanton et al. 2005; Mayo et al. 2008). Since LAB are considered as an important

group in the microbiota, they are used extensively as probiotics, which are defined as

“viable microbial dietary supplement that beneficially influences the host through

their effects in the intestinal tract” (Salminen et al. 1998; Roberfroid, 2000).


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Probiotics have many advantages, which include, alleviation of lactose intolerance,

immune enhancement, reduction of colon cancer, anti-cholesterol activity and

reduction of diarrheal symptoms, anti-mutagenic and anti-carcinogenic properties

(Shah, 2007; Van Reneen and Dicks, 2010). As the LAB are considered to enhance

the nutritive qualities of food as well as inhibit the spoilage and pathogenic bacteria,

they are used in many fermented foods (Matilla-Sandholm et al. 1999; Lasagno et al.

2002). The incorporation of such cultures as a biopreservative agent into food models

has additional advantage over inhibition of the pathogenic and spoilage bacteria

(O‟Sullivan et al. 2002). The studies on biosynthesis, mode of action and potency of

bacteriocins may lead to the development of novel drugs or genetically modified

bacteriocins for the treatment of infections or as a food additive for preservation

(Nissen-Meyer et al. 2009).

2. 1. 2. GENERAL FEATURES OF LAB

Till date, approximately 20 different genomes of LAB are available, with an

average genome size of 2-3 Mb coding for about 2000-3000 genes (Makarova and

Koonin, 2007; Mayo et al. 2008). All the genomes display the typical features of the

bacterial chromosome and appear to be conserved among different LAB genomes.

The genetic events like horizontal gene transfer (HGT), gene loss/gain, gene

duplication, gene re-arrangement, mutations, etc., are responsible for the present

genome structure within the LAB species (Mayo et al. 2008; Horvath et al. 2009).

At present, nearly 400 species of LAB are recognized and are generally

classified into four families and seven genera (Zhang et al. 2011), namely

Lactobacillaceae (comprises of the genera Lactobacillus and Pediococcus),

Leuconostocaceae (consists of the genera Oenococcus and Leuconostoc),

Enterococcaceae (include Enterococcus) and Streptococcaceae (consists of the

genera Lactococcus and Streptococcus) (Carr et al. 2002; Salminen et al. 2004).

Table 2. 1. 2. 1. depicts some of the general genomic features of LAB which includes


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variation in the genome length, %GC content, number of plasmids, and proteins. With the

availability of more than 25 LAB genomic maps, the comparative genomic analysis has

helped in the identification of „islands of adaptability‟ which display a key genetic region

involved in adaptation during the process of evolution (Klaenhammer et al. 2002). The

association of insertion elements (IS) elements, bacteriophages and mobile genetic

elements (MGEs) provide a major scope for the HGT among LAB, which display the

significant genetic regions that emphasize the unique and beneficial properties of LAB

(Bolotin et al. 2001; Klaenhammer et al. 2002).

The LAB are of significant importance in dairy industry, because of their

metabolic properties which contribute to the enhancement of the flavour, texture and

nutritional value of the fermented end products (Donkor et al. 2005). Additionally, LAB

protect food products through enhanced production of anti-microbial compounds like

bacteriocins (De Vuyst and Vandamne, 1994), antifungal compounds such as fatty acids

(Corsetti et al. 1998) or phenyllactic acid (Lavermicocca et al. 2000), production of

organic acids, carbon dioxide, ethanol, hydrogen peroxide, diacetyl (De Vuyst and

Vandamne, 1994; Atrih et al. 2001) and antibiotics (Holtzel et al. 2000).


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Table 2. 1. 2. 1: General genomic features of LAB and IS elements

Species-strain Genome

size (Mb)

%GC

content Plasmids

No. of

proteins

GenBank

accession no.

Lactobacillus

Lact. acidophilus NCFM 1.9 34 0 1,864 NC_006814

Lact. brevis ATTC 367 2.3 46 2 2,221 NC_008497

Lact. casei ATTC 334 2.9 46 1 2,776 NC_008526

Lact. plantarum WCFS1 3.3 44 3 3,052 NC_004567

Lact. salivarius subsp.

salivarius UCC118 2.1 32 3 1,834 NC_007929

Leuconostoc

Leuc. citreum KM20 1.7 39 4 1,820 NC_010471

Leuc. mesenteroides subsp.

mesenteroides ATTC8293 2.0 37 1 2,009 NC_008531

Lactococcus

Lac. lactis subsp. cremoris

MG 1363 2.5 35

Plasmid

cured 2,436 NC_009004

Lac. lactic subsp. cremoris

SK11 2.6 35 5 2,509 NC_008527

Lac. lactis subsp. lactis 2.3 35 Plasmid

cured 2,310 NC_002662

Oenococcus

O. oeni PSU-1 1.7 37 0 1,691 NC_008528

O. oeni ATCC BAA-1163 1.7 37 0 1,398 AAUV00000000

Pediococcus

Ped. pentosaceus ATTC

25745 1.8 37 0 1,757 NC_00825

Streptococcus

Strep. thermophilus LMD9 1.8 39 2 1,710 NC_008532

Strep. thermophilus

CNRZ1066 1.7 39 0 1,915 NC_006449

Strep. thermophilus

LMG13811 1.7 39 0 1,890 NC-006448

(Source: Klaenhammer et al. 2002; Makarova and Koonin, 2007; Mayo et al. 2008)


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2. 1. 2. 1. Characteristics of LAB used in food industry

The important genera of some of LAB like Pediococcus, Enterococcus and

Lactobacillus used in the field of food industry was described below.

2. 1. 2. 1. 1. The genus Pediococcus

Pediococcus was first isolated and characterized from plants by Mundt et al.

(1969). They are found to be catalase negative, homofermentative, spherical shaped in

tetrads, non-motile, non-sporulating and facultative anaerobes (Schlegel, 1993; Kumar

et al. 2011). They grow usually in rich nutritional media but differ in their tolerance

level in the utilization to oxygen and carbohydrates, growth at different parameters like

pH, temperature and NaCl concentrations (Papagianni and Anastasiadou, 2009). The

Genus Pediococcus currently includes the Pediococcus acidilactici, Ped. pentosaceus,

Ped. parvulus, Ped. damnosus, Ped. inopinatus, Ped. dextrinius, Ped. halophilus, Ped.

cellicola, Ped. claussenii and Ped. stilesii (Kumar et al. 2011).

Presently, Ped. acidilactici and Ped. pentosaceus are commonly used in the

fermentation of many foods like meat, vegetables, dough, fruit juices and they are also

being used as a commercial probiotic feed (Papagianni and Anastasiadou, 2009).

However, the pediococci have very limited usage or is restricted in the milk

fermentation, due to their inability to ferment lactose (Renye and Somkuti, 2009).

Numerous reports, described about the desirable attributes provided by the

Pediococcus sp. in the manufacture of cheese, where they can be used as an adjuvant

along with a non-starter culture (Caldwell et al. 1996). Besides their generally

regarded as safe (GRAS) status, the Pediococcus sp. also produces bacteriocin,

pediocin PA-1 or pediocin-like bacteriocin which have potential benefits to the food

industry because of its potent anti-listerial activity.


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2. 1. 2. 1. 2. The genus Enterococcus

Enterococci were first described by Thiercelin in 1899. They are Gram-

positive, catalase negative, cocci in shape. Further, they have the ability to grow at 10-

45 oC and can survive even at 65

oC for 30 min (Stiles, 2002). Till date, 28 different

species of enterococci are identified based upon the phylogenetic analysis of 16S

rRNA-DNA sequencing (Bhardwaj et al. 2008). Some of the important species of

enterococci, includes Enterococcus faecium, Ent. faecalis, Ent. durans, Ent.

gallinarum, Ent. hirae, etc., which are being used widely in the improvement of

nutritional value of food products (Granata et al. 2010).

Enterococcus sp. play a beneficial role in the dairy foods, because of their

biochemical properties like lipolysis, proteolysis and improving taste and flavour of

food. Further, they have been used in the development of cheddar cheese, mozzarella

cheese, feta, etc., (Sarantinopoulous et al. 2002; Manolopoulou et al. 2003). Several

species of enterococci are also used as probiotics besides Lactobacillus sp. and

Bifidobacterium sp. (Franz et al. 2003). The enterocins produces by different

Enterococcus sp. are found to display broad spectrum of activity and can act as a

protective starters in different food models (Čanžek Majhenič et al. 2005). The

Enterococcus sp. are also used in food to maintain the normal intestinal microflora,

reduce gastro intestinal disorders, stimulate immune response, etc., (Giraffa, 2003;

Foulquie Moreno et al. 2006, Bharadwaj et al. 2008).

2. 1. 2. 1. 3. The genus Lactobacillus

The lactobacilli are one of the important group of bacteria used in the food

preservation, or as starters in dairy products or in the fermentation of vegetables, fish,

silage, sausages, etc. Further, they are found to be present in a variety of sources like

plants, animals, humans, etc. (Giraffa et al. 2010). They are Gram-positive, rod-

shaped, homo/heterofermentative, aerotolerant and represent the largest group of

Lactobacillaceae. Many species of Lactobacillus like Lact. acidophilus, Lact.


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delbrueckii, Lact. johnsonii, Lact. salivarius, Lact. rhamnosus, Lact. casei, Lact.

paracasei, Lact. fermentum, Lact. plantarum, Lact. helvaticus, etc., are presently used

as starters or non-starters in dairy and vegetable fermentation and are found to play an

important role in the health of humans by improving nutrition (Pot, 2008; Pot and

Tsakalidou, 2009).

Most of the Lactobacillus sp. are used as starters because of their proteolytic

activity, production of aroma compounds and bacteriocins, which extend their

biotechnological properties (Leroy and De Vuyst, 2004). A variety of dairy products

are available in market which include pasteurized milk, ice creams, fermented milks,

cheese, where the lactobacilli are used extensively (Tamime et al. 2005; Grattepanche

et al. 2008).

Hence, the genera of Pediococcus, Enterococcus and Lactobacillus are found

to have wider application in the field of food industry.

2. 1. 3. BACTERIOCINS

Several of the bacteria have developed the weapons to kill the other competing

bacteria for their survival in the long term struggle for niche and nutrients. A classic

example is the increase in the antibiotic resistance mechanisms in these bacteria (Moll

et al. 1999). The discovery of the anti-microbial peptide‟s which diminishes the

spread of resistance in the target species has inspired the researchers to study further

the bacteriocin‟s mode of action (Cotter et al. 2005). The awareness for the health,

high quality foods by the consumers has led to the development and utilization of

these naturally produced anti-microbial compounds instead of the chemical

preservatives, which are found to have several side effects on the health of the

humans (Settanni and Corsetti, 2008). The anti-microbial peptides (AMP‟s) produced

by the Gram-positive and Gram-negative bacteria have shown potential activity

against bacteria that are resistant to antibiotics (Cole et al. 1997; Nes et al. 2002).



Such ribosomally synthesized AMP produced by one bacterium and inhibiting the

growth of other bacteria, either in the same species (narrow spectrum) or across

genera (broad spectrum) are called as Bacteriocins (Tagg et al. 1976; Moll et al.

1999; Cotter et al. 2005). Gratia in 1925 discovered the bacteriocin and the term

“bacteriocin” was coined in 1953 to define colicin produced by Escherichia coli

(Garneau et al. 2002; Settanni and Corsetti 2008). The production of such bacteriocin

has been found in numerous species of LAB, which are given “Generally regarded as

Safe” (GRAS) status (Adams, 1999).

The bacteriocins due to their proteinaceous nature have attracted the

researchers for their potential application in the food industry as a food preservative

(Jack et al. 1995). The use of bacteriocin as a preservative was first reported in 1951

and has benefited the mankind from the health point of view (Hirsch et al. 1951). The

LAB which have an essential role in fermented foods also produce bacteriocins and

play a defining role in the microbial safety and preservation of food products (Caplice

and Fitzgerald, 1999). This has promoted the utilization of LAB in the long term

fermentation of foods, like meat, sea foods, vegetables, dairy products, etc., (Ennahar

et al. 1998; Morgan et al. 1999; Benech et al. 2002, Garriga et al. 2002; Nilsson et al.

2004; Grande et al. 2005; Sobrino-Lopez and Belloso, 2006; Gálvez et al. 2007) and

non-fermented foods, like vaccum packaged meat products (Vold et al. 2000;

Castellano et al. 2004). In general, studies have focused on food safety using

bacteriocins as preservative, characterization of bacteriocin and in situ application of

the bacteriocin in a food model system (Settanni and Corsetti, 2008).

2. 1. 4. CLASSIFICATION OF BACTERIOCINS

The LAB producing the AMPs are of great interest because of their food grade

quality and industrial importance (Nissen-Meyer et al. 2009). The bacteriocins

produced by LAB are widespread in nature and play an important role as a non-toxin

and natural food preservative (Cotter et al. 2005; Foulquie-Moreno et al. 2006). Most



of the bacteriocins of LAB are cationic, hydrophobic or amphiphilic and composed of

20 to 60 aminoacids residues (Nes and Holo, 2000). Though bacteriocins are

generally classified into three major classes based on their biochemical and genetic

properties (Nes et al. 1996; Eijsink et al. 2002), Cotter et al. (2005) has revised this

classification into class I and II.

Class I are the Lantibiotics (lanthionine-containing antibiotic) that are small

(<5 kDa) peptides (19-38 aminoacids in length) containing the unusual aminoacids

lanthionine (Lan), α-methyl lanthionine (MeLan), dehydroalanine and

dehydrobutyrine. These residues form covalent bridges between aminoacids, resulting

in the formation of internal ring and give the lantibiotics their characteristic structural

features. This class has been divided into two subclasses based upon their chemical

structure and properties (Guder et al. 2000; Chen and Hoover, 2003; Aly et al. 2006).

Type A lantibiotics are elongated peptides, positively charged and their mode of

action is through the formation of pores on the cell membrane of bacteria (eg. nisin

from Lactococcus lactis) (Cotter et al. 2005). Type B lantibiotics are globular small

peptides, negatively charged or carry no net charge and their activity is related to the

inhibition of specific enzymes (eg. mersacidin produced from B. subtilis, Cinnamycin

from Streptomyces cinnamoeus) (Altena et al. 2000; Chen and Hoover, 2003).

Class II includes unmodified bacteriocins, that are small (<10 kDa), non-

lanthionine peptides which are subdivided into four subclasses, namely- Class IIa, IIb,

IIc and IId. The multiple alignment of the class II bacteriocins are represented in Fig.

2. 1. 4. 1 where they are classified based upon the variation in the C-terminal region

of the bacteriocins (Nissen-Meyer et al. 2009).

Class IIa includes pediocin-like peptides having an N- terminal consensus

sequence –Tyr-Gly-Asn-Gly-Val-Xaa-Cys, that are hydrophilic and are thought to

facilitate the non-specific binding to the target surface. This group has been



extensively studied because of their anti-listerial activity (Ennahar et al. 2000; Chen

and Hoover, 2003; Diep et al. 2007) The C-terminal regions are located after the

hinge region and are less conserved and are thought to determine the non-listerial

anti-microbial spectrum. (Eg: Pediocin PA-1/AcH from Ped. acidilactici, Sakacin A

and P from Lactococcus sake).

Class IIb is a two peptide bacteriocin that requires the combined activity of

both the peptides for their action through the dissipation of the membrane potential,

leakage of ions, etc. and thus resulting in the target cell depletion (Nissen-Meyer et al.

2009). At least fifteen different class IIb bacteriocins have been isolated and

characterized, which have the conserved GxxxG motif (Chen and Hoover, 2003;

Nissen-meyer et al. 2009). (Eg: Lactococcin G and M from Lactococcus lactis,

Plantaricin S or EF from Lact. plantarum).

Class IIc is a circular bacteriocin or non-pediocin like or one peptide

bacteriocin and includes sec-dependent secreted bacteriocins (Drider et al. 2006). The

N- and C- terminal ends of these bacteriocins are covalently bonded resulting in the

formation of cyclic structure (Cotter et al. 2005). They are cationic, hydrophobic, and

range from 3.5 to 7.2 kDa proteins. They attach to the target cell membrane, disrupt

the proton motive force (PMF) and results in cell death (Chen and Hoover, 2003;

Nissen-Meyer et al. 2009). (Eg: Acidocin B from Lact. acidophilus, Divergin A from

Clostridium divergens, Enterocin P from Ent. faecium).

Class IId is the linear non-pediocin-like one peptide bacteriocins with no

similarity to the pediocin-like bacteriocins (Chen and Hoover, 2003; Cotter et al.

2005; Iwatani et al. 2011). (Eg: Enterocin B or L50A or L50B or Q from Ent.

faecium, Lacticin Q or Z from Lactococcus lactis QU5 and QU14).



Fig 2. 1. 4. 1 Multiple sequence alignment of class II pediocin-like bacteriocins

(Source: Nissen-Meyer et al. 2009)

Class III peptides are bacteriolysins or large (>30 kDa), heat labile, lytic

proteins which are often murein hydrolases. This class has not been well characterized

and are of lesser interest among the food scientists. They have a domain type of

structure, where each domain has specific functions for translocation, receptor binding

and lethal activity (Klaenhammer et al. 1993; Biswas et al. 1991; Cotter et al. 2005;

De Vuyst and Leroy, 2007). Their mechanism of action is different from the other

bacteriocins as they function through the lysis of sensitive cells by catalysing cell-wall

hydrolysis. Further, the N-terminal region shows homology to the endopeptidases and

the C-terminal represents the target recognition site (Eg: Helveticin J or V-1829 from

Lact. heleveticus) (Lai et al. 2002; Chen and Hoover, 2003; Johnsen et al. 2004).

Class IV- These include bacteriocins that form large complexes with other

chemical moieties, lipids or carbohydrates to exhibit anti-microbial activity. These

bacteriocins are not well characterized, and no bacteriocin of this group have yet been

convincingly demonstrated (Cotter et al. 2005).



2. 1. 5. GENE CLUSTERS, ORGANIZATION AND MODE OF ACTION OF

LAB BACTERIOCINS

The genes encoding the bacteriocin are usually organized in operon clusters

(McAuliffe et al. 2001). The unmodified bacteriocins such as plantaricins,

carnobacteriocins, sakacins are stimulated by specific peptides and located on the same

gene cluster. The bacteriocins like subtilin are located on the chromosome and some

bacteriocins like mersacidin, pediocin PA-1 are located in an operon on the plasmid or

on transposons as in the case of nisin and lacticin 481 (Chen and Hoover, 2003).

The lantibiotic biosynthesis operon generally consists of specific genes. The

prepeptide structural gene (lanA) was found to be responsible for the modification

reactions involved in the formation of lanthionine and methyl lanthionine (lanB). The

processing of these proteases is responsible for removal of the leader peptide (lanP).

The lanT encodes a membrane associated ABC transporter that transfers the

lantibiotic across the membrane. Finally, the two proteins LanK and R encodes two

components, a regulatory proteins that transmit an extracellular signal thereby leading

to the lantibiotic production (Chen and Hoover, 2003; Aly et al. 2006).

The class II bacteriocins are often arranged in one or few operons and are

usually plasmid encoded except enterocin A, divercin V41, sakacin P,

carnobacteriocins B2 and BM1 which are chromosomally encoded (Drider et al. 2006;

Aly et al. 2006, Nissen-Meyer et al. 2009). Basically four genes are required for the

production of class IIa bacteriocins. They are

a. The structural gene which encodes a pre-bacteriocin.

b. The immunity gene that protects the bacteriocin producer from its own bacteriocin.

c. The gene that encodes an ABC (ATP-binding cassette) transporter necessary for

secretion and lastly,

d. The gene that encodes an accessory protein with unknown function.



The mode of action of the class I and class II bacteriocins produced by the

LAB is depicted in the Figure 2. 1. 5. 1. The class I lantibiotics (eg. nisin), has been

shown to have a dual action. The nisin molecule can bind to the lipid II and prevent

the cell wall synthesis, leading to the cell death. Additionally, they use the lipid II as a

docking molecule to initiate the membrane insertion and pore formation, leading to

the leakage of ions and result in rapid cell death. The class II peptides are attached to

the cell membrane with their amphiphilic helical structure and get inserted into the

membrane, leading to depolarization and cell death (Cotter et al. 2005).

Fig. 2. 1. 5. 1 Mode of action of bacteriocins produced by LAB

(Source: Cotter et al. 2005)



2. 2. PEDIOCIN PA-1 LIKE BACTERIOCINS AND THEIR

DISTRIBUTION AMONG LAB

2. 2. 1. STRUCTURE AND MODE OF ACTION OF PEDIOCIN PA-1 LIKE

BACTERIOCIN

The pediocin PA-1 bacteriocin is one of the most extensively studied class IIa

bacteriocin belonging to the “pediocin family” (Nes et al. 1996; Rodriguez et al.

2002). The members from this group show a strong anti-listerial activity with 40-60%

sequence similarity (Fimland et al. 2005). Studies on the structure and function of the

class IIa bacteriocin have shown that the pediocin molecule contains two major

structural regions, a highly conserved N-terminal with a consensus motif -YGNGV-

and a less conserved C-terminal region (Drider et al. 2006; Papagianni and

Anastasiadou, 2009). The structure of the pediocin-like or the class IIa bacteriocin

helps in understanding the mode of action of these molecules. The action of pediocin

PA-1 like molecule on the target cells involves three major steps:

a. The binding of the pediocin molecule to the cytoplasmic membrane of the

sensitive cells,

b. Insertion of the molecule into the membrane and

c. Formation of the pores on the target cell membrane resulting in the cell death that

may occur with or without cell lysis (Montville and Chen, 1998; Rodriguez et al.

2002; Cotter et al. 2005).

The basic structure of the anti-microbial peptides (AMPs) of class IIa

bacteriocins has the N-terminal region that forms a three-stranded antiparallel β-sheet

like structure supported by the disulphide bridge (Wang et al. 1999; Fimland et al.

2005). A hairpin-like structure is formed in the C-terminal half region along with the N-

terminal β-sheet region, leading to the formation of a flexible hinge that allows the two

domains to move relative to each other (Uteng et al. 2003; Fimland et al. 2005). The in

vitro site-directed mutagenesis studies have revealed that the N-terminal β-sheet domain

was responsible for the binding of pediocin-like AMPs to the target cell surface by

electrostatic interactions (Chen et al. 1997; Kazazic et al. 2002, Fimland et al. 2005).



The more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the

hydrophobic part, whereas the hinge regions enables the C-terminal hairpin domain in

penetrating deeper into the hydrophobic part of the target cell membrane (Fig. 2. 2. 1.

1). Miller et al. (1998) reported that the N-terminal end of pediocin PA-1 molecule

binds to the C-terminal end of maltose-binding protein, resulting in the membrane

leakage.

Figure 2. 2. 1. 1: A schematic representation of the structure and orientation of the

pediocin-like AMPs

(Source: Nissen-Meyer et al. 2009).

The proton motive force (PMF) which is required for the cells metabolic

processes helps in understanding the mode of action of such bacteriocin molecules.

After the pore formation, the PMF is increased, which is a result of the

electrochemical gradient of protons across the bacterial cytoplasmic membrane and is

composed of the membrane potential (Δ ) and the pH gradient (ΔpH) (Rodriguez et

al. 2002). Christensen and Hutkins, (1992) provided the first evidence for the

involvement of PMF in the mode of action of pediocin PA-1 molecule. A

concentration-dependent dissipation of the PMF of the sensitive target cells was

caused by the pediocin molecule, thereby leading to the increase in the membrane

permeability of the target cells to the protons and thus resulting in death of the target

cell (Christensen and Hutkins, 1992; Rodriguez et al. 2002).



2. 2. 2. BIOSYNTHESIS AND ORGANIZATION OF THE PEDIOCIN PA-1

LIKE BACTERIOCIN MOLECULE

The genetic determinants for the biosynthesis of the pediocin PA-1 or pediocin-

like are encoded in plasmid of the LAB strains (Rodriguez et al. 2002; Pappagianni and

Anastasiadou, 2009). The pediocin operon comprises of four genes pedA, pedB, pedC

and pedD which are localized on a 3.5 Kb sized DNA fragment (Fig. 2. 2. 2. 1). Each

gene is preceded by a ribosome binding site (RBS) and they are organized in a single

operon (Marugg et al. 1992). The pediocin PA-1 operon produces two transcripts, the

most abundant major transcript comprises of the pedABC and has an approximate size of

1.2 Kb and the second transcript corresponds to the pedABCD of 3.5 Kb size (Venema et

al. 1995), and each gene is preceded by a RBS.

Figure 2. 2. 2. 1: A representation of the pediocin PA-1 operon from Pediococcus

acidilactici H (P- promotor, R- Ribosome binding site, T- Transcriptional terminator)

(Source: Rodriguez et al. 2002).

The four genes of pediocin operon are described below,

papA/pedA (Structural gene): The first gene of pediocin PA-1 operon encodes a

62 amino acid precursor called as pre-pediocin PA-1. The leader peptide sequence of the

pre-pediocin is removed with the secretion and results in the formation of the mature

pediocin, which comprises of 44 amino acids (Rodriguez et al. 2002).

papB/pedB (Immunity gene): The pedB gene comprises of 112 amino acids

involved in the immunity of the producing cells. Kim et al. (2005) studied the crystal

structure of the native PedB protein which consists of one molecule in an asymmetric

unit. Out of 112 amino acids, the residues from 7-93 are visible in the crystal

structure, which reveals that the PedB protein forms a compact globular domain of



four helices (α1, α2, α3 and α4). The α1 and α3 run in the same direction and the α2

and α4 in the opposite direction (Fig. 2. 2. 2. 2). The molecule is maintained in a

network of hydrophobic interactions between the chains. Moreover, the last residues

from 94-112 are comprised in the C-terminal region and are considered to be

important for immunity (Kim et al. 2005).

Figure 2. 2. 2. 2. A diagrammatic representation of the PedB protein structure

(Source: Kim et al. 2005).

papC/pedC: This gene encodes 174 aminoacids that are considered to be

essential for secretion, belonging to the group of “accessory proteins” or “membrane

fusion proteins (MFP)” involved in ATP-binding cassettes (ABC) transporters

(Rodriguez et al. 2002).

papD/pedD: The last gene of the ped operon, pedD is known to be poorly

transcribed. The pediocin activity can be enhanced by placing the pedD behind a

strong promoter. It is presumed that the increase in the pedD production leads to an

efficient maturation/translocation of pediocin (Vanema et al. 1995). The N-terminal

domain of pedD is located in the cytoplasm, and the processing involves cleavage of

the leader sequence behind the glycine doublet at the processing site (Henderson et al.

1992).



2. 2. 3. DISTRIBUTION OF PEDIOCIN PA-1 LIKE BACTERIOCIN

AMONG LAB GENOMES

Pediocin PA-1 is produced by a large number of LAB strains isolated from

different sources. These include dairy products (Bhowmik and Marth, 1990),

fermentation of vegetables (Bennik et al. 1997; Knorr, 1998; Halami et al. 2005),

meat (Luchansky et al. 1992; Mattila-Sandholm et al. 1993), forage crops (Cai et al.

1999), sausages (Anastasisdou et al. 2008a), human breast milk (Osmanagaoglou et

al. 2011) etc. Several studies were carried out to demonstrate their mode of action

(Jack et al. 1995; Ennahar et al. 2000) and for their possible utilization in

biopreservation (Schoeman et al. 1999). In the recent past, it is reported that class IIa

bacteriocins are not only synthesized in cross-species but also in cross-genera (Halami

et al. 2005). The different pediocin and pediocin PA-1 like bacteriocin producers from

various LAB species are enlisted in Table 2. 2. 3. 1.

Initially, pediocin PA-1 was reported in Ped. acidilactici strain PAC 1.0

(Gonzalez and Kunka, 1987) and Ped. acidilactici H (Bhunia et al. 1987; Bhunia et al.

1991) and later it was reported in Lact. plantarum WHE 92 (Ennahar et al. 1996),

Ped. parvulus ATO34 and ATO77 (Bennik et al. 1997). Plantaricin 423 from Lact.

plantarum 423 has few changes in amino acid and reported to have 99% homology

with pediocin PA-1 (Van Reenen et al. 1998) and as coagulin in Bacillus coagulans I4

with only one amino acid substitution at 41 position (Le Merrec et al. 2000). Miller et

al. (2005) characterized the genetic organization in Ped. pentosaceus S34. Similarly, a

derivative of pediocin PA-1 was reported in Ped. pentosaceus ST44AM of 6.5 KDa

bacteriocin isolated from marula (Todorov and Dicks, 2009). Recently, two strains of

Lact. plantarum isolated from pork products showed high homology to the pediocin

PA-1 of 3.5 and 10 KDa molecular weight proteins (Todorov et al. 2010a). Mlalazi et

al. (2011) detected the pediocin PA-1/AcH structural gene in the native non-starter

cultures of Lact. casei, Lact. paracasei and Lact. rhamnosus isolated from retail

cheddar cheese.



The intergeneric microorganisms are defined as those bacteria which are

evolved by transfer of the genetic material from organisms of different genera

belonging to the same familiy. Similarly, the interspecific microorganisms can be

defined as the bacteria evolved from the transfer of genetic material from organisms

belonging to different species of same genera. The ability of the LAB to different

environmental conditions for their survival leads to the genetic modifications,

rearrangements, gene transfer either gene loss or gain. Thus, their is a high possibility

of HGT responsible for the presence of such kind on interspecific and intergeneric

pediocin (Le Marrec et al. 2000).



Table 2. 2. 3. 1: Distribution of pediocin PA-1 like bacteriocin in different species of LAB and their properties

Bateriocin Producer organisms Source Molecular size

of plasmid (Kb)

Protein

weight (kDa) Properties of pediocin

Pediocin AcH Ped. acidilactici H, E, F, M Fermented

vegetable and

meat

8.9 Kb

(pSMB74)

4.6 Temperature and pH (2.5-9.0) stable ,

degraded by proteases (trypsin, papain, α-

chymotrypsin, proteinase K), bactericidal,

bacteriolytic

Pediocin PA-1 Ped. acidilactici PAC1.0

NRRL-5627

- 9.4 Kb

(pRSQ11)

4.6 Temperature and pH (2.0-10.0) stable ,

degraded by proteases (papain, α-

chymotrypsin), bactericidal, bacteriolytic

Pediocin JD Ped. acidilactici JD-123 Commercial

culture

- - Heat stable, degraded by trypsin,

bactericidal

Pediocin AcM Ped. acidilactici M Sausage - 4.6 Heat stable, pH (1-12) stable, degraded by

trypsin

Pediocin SA-1 Ped. acidilactici NRRL

B5627

- - 3.66 Temperature stable, degraded by only

proteinase K and resistant to trypsin, α-

chymotrypsin, papain, pepsin, bactericidal

Pediocin PA-1 Ped.pentosaceus S34 buffalo milk 8.9 Kb - -

Pediocin ST18 Ped. pentosaceus ST18 Boza Belogratchik - - Temperature and pH (2-12) stable, degraded

by proteases, bacteriostatic

Pediocin ST44AM Ped. pentosaceus ST44AM Marula - 6.5 Temperature and pH stable (2-12), degraded

by many proteases, bactericidal



Pediocin PA-1 Ped. parvulus ATO77,

ATO34

Minimally processed

vegetables

plasmid encoded - -do-

Pediocin PA-1/AcH Lact. plantarum WHE92 Munster cheese 11 Kb 4.6 -do-

Coagulin/Pediocin

PA-1/AcH like

Bacillus coagulans I4 Cow dung 14 Kb 4.6 -do-

Plantaricin 423/

pediocin PA-1 like

Lact. plantarum 423 Sorghum beer Plasmid encoded - -do-

Pediocin PA-1 Lact. plantarum ST202Ch,

216Ch

Beloura, Chourico - 3.5 and 10.0 Temperature and pH (2-12) stable, degraded

by proteases (trypsin, papain, pepsin, pronase)

Pediocin PA-1 Ent. faecium ST5Ha Smoked salmon Chromosome

encoded

Temperature and pH stable, degraded by

proteases, anti-bacterial, anti-viral

Pediocin PA-1 Lact. casei Retail cheddar

cheese

- - Heat stable, inactivated by only proteinase

K, anti-bacterial

Pediocin PA-1 Lact. paracasei Retail cheddar

cheese

- - -do-

Pediocin PA-1 Lact. rhamnosus Retail cheddar

cheese

- - -do-

Source: Rodrequiz et al. (2002); Todorov and Dicks (2005b); Papagianni and Anastidastou (2009); Kumar et al. (2011)



2. 3. IDENTIFICATION OF LACTIC ACID BACTERIA

The identification and characterization of LAB is very important, because of

several advantages afforded by them for the improvement of health and nutritional

status in humans (Karna et al. 2007). Basically, phenotypic and genotypic methods

are used in the characterization of several LAB strains.

2. 3. 1. PHENOTYPIC METHODS

The phenotypic identification of LAB basically involves the morphology,

physiology and biochemical properties, which help in preliminary characterization of

LAB (Badis et al. 2004). Based upon the phenotypic characters, they are classified

into six major groups, which include, facultative hetero-fermentative rods, obligate

hetero-fermentative rods, tetrad-forming homo-fermentative cocci, homo-

fermentative cocci, hetero-fermentative cocci and an unidentified group (Banwo et al.

2012). One of the major problems associated with the characterization of LAB by

these methods is due to, their complex nutritional requirement and their growth and

adaptability to several environmental conditions (Vandamme et al. 1996; Aquilanti et

al. 2007). In addition, results obtained can be inappropriate due to their low

reproducibility and discriminatory power (De Angelis et al. 2001). However, the

several dominant microflora were isolated from the fermented vegetable products of

Eastern Himalayas by using phenotypic methods (Tamang et al. 2005). Further,

preliminary differentiation of the selected LAB isolates can be clustered on the basis

of their cell morphology, acid production from various carbohydrates, tolerance to

different salt concentration, pH and temperatures.

2. 3. 2. MOLECULAR METHODS

Understanding the strengths and weaknesses of the phenotypic methods used

in the identification of LAB, has created a need to develop, several molecular typing

tools to detect the LAB strains at their sub-species level. The major advantage of

these molecular typing techniques is its reproducibility, discrimination, interpretation,

fast generation of data, ease of use etc., (Farber, 1996; Mohania et al. 2008). The

following section focuses on the detection, identification and typing of LAB species

and strains from different sources.



2. 3. 2. 1. Species specific and genus specific PCR detection

There is a tremendous influence of polymerase chain reaction (PCR) on the

research in diverse fields of biological sciences. An organism can be identified

directly through PCR assays using genus specific or species specific primers in a short

span of time than the conventional cultural tests. Singh and Ramesh, (2008) detected

several Pediococcus, Leuconostoc, Lactobacillus and Enterococcus species in a

fermented cucumber sample by using genus specific primers. This method helps in

understanding the microbial dynamics, in terms of dominance and co-existence of

microflora during the process of fermentation. Several reports have been published

with species-specific and strain specific primers to demonstrate the presence of a

bacterial group and/or species for identification (Giraffa and Neviani, 2000; Todorov

et al. 2010b). An overall, PCR-based methodologies provide rapid detection of a

bacteria and also helps in understanding the diversity in a mixed population of

bacteria that are present in an environmental source (McCartney, 2002).

2. 3. 2. 2. 16S rRNA gene sequencing

The 16S rRNA gene sequencing is widely used to determine the taxonomical

status and phylogenetic relationships among bacteria. The fact that 16S rRNA gene

has highly conserved consensus sequence they are presently used for the identification

of an unknown bacteria. The available online databases like, Basic Local Alignment

Search Tool (BLAST) helps in the identification of the bacteria based on the sequence

data of the variable regions (Pozo-Bayon et al. 2009). Based upon this, several

universal primers have been designed for specific identification of culture in a family

of bacteria (Heilig et al. 2002). A comparative 16S rRNA gene sequence analysis

helps in the identification of different species of LAB (Holzapfel et al. 2001).

Sukumar and Ghosh, (2010) identified several species of Pediococcus isolated from

an Indian fermented food by 16S rDNA sequencing. However, the major drawback of

this method is that, it cannot be used to differentiate the different species of highly

related organisms (Pozo-Bayon et al. 2009).



2. 3. 2. 3. Hybridization of nucleic acids

The interpretation of the data generated by the restriction enzyme analysis

(REA) of the chromosomal DNA is quite moderate. Hence, for higher resolution of

microorganism detection, the nucleic acid hybridization (DNA- DNA; DNA- rRNA)

is performed. The probes of 16S-23S rRNA regions of a bacterial genome are widely

used as a powerful molecular technique in the identification at species level (Rodas et

al. 2003). Hence, the use of probes followed by Southern hybridization gives more

easily interpretable data with high reproducibility (Mohania et al. 2008). In this

context, Ennahar et al. (1996) have characterized the pediocin PA-1 producing

bacteriocin from Lact. plantarum WHE 92 isolated from cheese by hybridization

method. This method can also be used effectively in the identification of a bacteria

present in a mixed population by colony hybridization (Pozo-Bayon et al. 2009).

2. 3. 2. 4. Pulse field gel electrophoresis (PFGE)

PFGE employs the separation of large DNA fragments with a rare-cutting

restriction enzyme, which is then subjected to electrophoretic separation with

increasing pulse timings (Holzapfel et al. 2001). Although, this method is more time-

consuming and requires special expensive electrophoretic equipment, the data

generated by this method is more discriminatory and superior than ribotyping.

Tynkkynen et al. (1999) has shown that this method of typing is more discriminatory

in the closely related Lact. casei and Lact. rhamnosus strains. This technique is also

used in studying the diversity of Oenococcus oeni and Lact. plantarum strains during

malolactic fermentation (Hernandez et al. 2007).

2. 3. 2. 5. Random amplified polymorphic DNA (RAPD) fingerprinting

This technique is basically a PCR-based typing technique, which uses short

arbitrary primers of about 10-15 bp, that anneal to the DNA template randomly at

multiple sites (Mohania et al. 2008). The PCR reaction is performed at low-stringency

annealing conditions leading to the amplification of different fragments sized

amplicons. The main advantage of this method is that, it is fast, more reproducible,



highly discriminatory, easy to perform and inexpensive (Pozo-Bayon et al. 2009).

This method has been used in the identification and discriminating several LAB

species of Pediococcus, Lactobacillus, Enterococcus, Oenococcus etc. (Nigatu et al.

1998; Mora et al. 2000b; Moschetti et al. 2001; Schillinger et al. 2003).

2. 3. 2. 6. Restriction analysis of amplified rDNA (ARDRA)

ARDRA is otherwise called as restriction fragment length polymorphism

(RFLP). This method involves the use of restriction enzymes (AluI, HaeIII, FokI) to

digest the amplified 16S rDNA amplicons. This method has low discriminatory power

when compared to the ribotyping, PFGE, RAPD analysis (Mohania et al. 2008).

However, several species of Lactobacillus, Enterococcus, Pediococcus, Weissella,

Oenococcus, etc., has been successfully differentiated by this method (Aquilanti et al.

2007; Pozo-Bayon et al. 2009).

2. 3. 2. 7. Amplified fragment length polymorphism (AFLP)

AFLP combines the power of RFLP, wherein PCR based method is followed

by ligating the primer-recognition sequences (adaptors) to the digested DNA. These

adaptors act as primer binding sites for PCR amplification. AFLP is a newly

developed technique and has proven to be useful in differentiation of LAB from

various fermented food products at their intraspecies level (Ben Amor et al. 2007). A

high resolution by AFLP analysis was observed for different strains of Lact.

plantarum (Van Hoorde et al. 2008).

2. 3. 2. 8. Enterobacterial repetitive Intergenic consensus (ERIC) PCR and

Repetitive extragenic palindromic (REP) PCR

The ERIC and REP sequences are dispersed throughout a bacterial genome

and helps in the rapid detection of a bacterial strain with good discriminatory power

(Versalovic et al. 1991). Ventura and Zink, (2002), characterized Lact. johnsonni

isolates at their strain level by ERIC and REP PCR. The data generated by these



methods is relatively fast, more discriminatory than REA and RFLP. The (GTG)5

REP PCR produces high-quality fingerprinting for several of LAB isolates (Van

Hoorde et al. 2008). The REP PCR was successful in the detection and typing of

several LAB strains of Lactobacillus, Pediococcus, Leuconostoc, and Enterococcus,

isolated from fermented food samples like dahi, idli batter and salt-fermented

cucumber (Singh and Ramesh, 2009). Moreover, the diversity among Lact. plantarum

strains isolated during the fermentation of carrot and beet root was achieved by using

Rep-PCR fingerprinting (Kingston et al. 2010).

2. 3. 2. 9. Multiple Locus Sequence Typing (MLST)

MLST has emerged as a powerful typing tool used in studying the ecology,

epidemiology, evolution, diversity, leading to the evaluation of intra-species genetic

relatedness among a group of bacteria (Maiden et al. 1998; Urwin and Maiden, 2003).

This method involves the sequencing and comparing the internal regions of several

housekeeping genes, leading to the differentiation among strains, because of its

unique allele combination (Maiden et al. 1998). Though MLST method has its own

advantages with respect to discrimination and reproducibility, it is also associated

with sequencing of atleast 6-8 housekeeping genes, laborious, time consuming and

cost effective. This MLST method was found to be a powerful tool in differentiating

the strains of Lact. plantarum when compared to the ribotyping, RFLP and 16S-23S

rDNA hybridization (de las Rivas et al. 2006). More recently, MLST was used

successfully in the differentiation of two closely related species of Pediococcus i.e

Ped. parvulus and Ped. damnosus, where detection by conventional PCR and real-

time PCR was not accomplished (Calmin et al. 2008).

All the above mentioned molecular methods are very useful in characterization

of LAB upto their sub-species/strains level. Based upon the requirement in the

characterization and identification of LAB, these molecular typing methods enlisted

in Table 2. 3. 2. 1, can be employed for the LAB.



Table 2. 3. 2. 1. Comparison of molecular typing techniques used for LAB identification

Molecular

typing

technique

Reproducibility Discriminatory

power

Ease of

interpretation

Ease of

performance Disadvantages

Genus and

species PCR

Good No

discriminatory

power within a

set of primers

used

moderate Excellent Occurrence of false

positive results

16S rRNA

gene

sequencing

Excellent Excellent Excellent Excellent Requires costly

equipment and

reagents

Hybridization

of nucleic

acids

Good Good Good Good More time

consuming, requires

costly restriction

enzymes and

reagents

PFGE Excellent Excellent Good Moderate More time

consuming,

laborious, requires

costly equipments,

restriction enzymes

RAPD Excellent Excellent Moderate Excellent Standardization of

protocol is required

RFLP Excellent Moderate to

excellent

Moderate Moderate Requires restriction

enzymes

AFLP Excellent Excellent Excellent Excellent Requires costly

equipments,

restriction enzymes

and reagents

MLST Excellent Excellent Excellent Excellent Requires costly

equipments and

reagents, time

consuming,

laborious.

(Source: Aquilanti et al. 2007; Ben Amor et al. 2007; Pozo-Bayon et al. 2009)

2. 3. 3. MOLECULAR TECHNIQUES USED IN THE DETECTION OF

PEDIOCIN-LIKE BACTERIOCIN AND THE PRODUCING BACTERIA

Several molecular methods have been developed to identify the pediocin

producers in Ped. acidilactici, to study their relationship between different species at

the genetic level by DNA-DNA homology (Dellaglio et al. 1981; Garvie, 1986), for

taxonomical level identification, sequencing of 16S rRNA gene was performed that

would reveal their evolutionary status (Collins et al. 1990). Several PCR methods

were used to detect the pediocin gene in different organisms of LAB (Bennik et al.



1997; Rodriguez et al. 1997; Mora et al. 1998; Halami et al. 2005). The typing

techniques such as, PFGE (Simpson et al. 2002), RAPD PCR (Welsh and

MacClelland, 1990; Kurzak et al. 1998; Nigatu et al. 1998; Mora et al. 2000a; Mora et

al. 2000b) were used in discriminating and differentiating the pediococcal strains.

Moreover, this methodology was also employed in characterizing the Lactobacillus

strains and proved to be a useful method for typing the distinctive strains of same

species (Johansson et al. 1995) or different species of same genera of Pediococcus

and Lactobacillus (Van Reneen and Dicks, 1996; Nigatu et al. 2001).

In addition, pediocin PA-1 structural gene was characterized by several

biochemical and molecular techniques like PCR, DNA sequencing and Southern/dot-

blot hybridization (Bhunia et al. 1994; Rodriguez et al. 1997). Further, monoclonal

anti-body based immunoassay for pediocin was performed by Bhunia (1994).

Martinez et al. (1999) have employed immunochemical techniques to detect the

accurate specificity and sensitivity of a pediocin PA-1 bacteriocin in a food system.

The bacteriocin gene stability and production were studied by using genetic

engineering techniques like heterologous expression (Miller et al. 1998). Further, the

polyclonal anti-peptide antibodies specific to pediocin PA-1 has been designed

(Martinez et al. 2000a, Martinez et al. 2000b). Transcriptional analysis was developed

by using reverse transcriptase PCR for the detection of pediocin in Ped. acidilactici

UVA1 and real time PCR for analyzing the pediocin PA-1/AcH in Pediococcus

strains isolated from human faeces (Mathys et al. 2007). A metagenomic approach

was adapted to detect class IIa bacteriocin encoding genes looking for valuable

probiotic and bacteriocinogenic microbiota, contributing to ecological studies

(Weickowicz et al. 2010). Table 2. 3. 3. 1, enlists about the different methods used in

the identification of pediocin PA-1 like bacteriocin and the producing LAB.



Table 2. 3. 3. 1. Identification of pediocin PA-1 and the producing bacteria

Bacteriocin

producer

Technique used

References Pediocin PA-1 like

bacteriocin Producing organism

Ped. acidilactici

PAC1.0

Sequencing of the plasmid

encoding the bacteriocin -

Bhunia et al.

(1994)

Lact. plantarum

WHE92

Aminoacid sequencing,

mass spectrometry analysis DNA-DNA hybridization

Ennahar et al.

(1996)

Ped. parvulus

ATO34 and ATO77 Dot-blot hybridization

Carbohydrate fermentation

(using API CH strips)

Bennik et al.

(1997)

Ped. acidilactici

PAC1.0,

Lact. plantarum

WHE92,

Ped. parvulus

ATO34 and ATO77

PCR-based site directed

mutagenesis of pedB gene -

Mora et al.

(2000c)

Ped. pentosaceus

S34

Southern hybridization of

pediocin gene as probe

Carbohydrate fermentation

pattern (using API 50CH

system)

Miller et al.

(2005)

Lact. plantarum 423 Southern hybridization and

Northern blot analysis -

Van Reenen et

al. (2003)

Ped. acidilactici M33

PCR with pediocin specific

primer and sequencing the

obtained product

- Millette et al.

(2007)

Ped. acidilactici

UVA1

Sequencing of pedA, pedB,

and pedC genes. Real-time

PCR for pedA gene

16S rDNA gene sequencing

and carbohydrate

fermentation pattern (API

50 CHL system)

Mathys et al.

(2007)

Ped. acidilactici HA-

111-2 and HA-5692-

3

Sequencing of the complete

operon -

Albano et al.

(2007)

Ped. pentosaceus

ST44AM

PCR targeting the pediocin

PA-1/AcH gene and

sequencing the obtained

product

RAPD-PCR and 16S rDNA

sequencing

Lact. plantarum

ST202ch and

ST216ch

Amplification and

sequencing of structural

gene

Species-specific primer for

Lact. plantarum

Todorov et al.

(2010a)

Ent. faecium ST5a

PCR targeting the pediocin

PA-1/AcH gene and

sequencing the obtained

product

RAPD-PCR and 16S rDNA

sequencing

Todorov et al.

(2010b)

Ped. pentosaceus

OZF

PCR and sequence analysis

of the complete pediocin

operon

16S rDNA sequencing and

carbohydrate fermentation

pattern (API 50CH system)

Osmanagaoglou

et al. (2011)

Lact. casei, Lact.

paracasei, Lact.

rhamnosus

PCR and sequencing of the

pediocin genes

16S rDNA sequencing,

PFGE, carbohydrate

fermentation (API 50CH)

Mlalazi et al.

(2011)



2. 3. 4. GENETICALLY MODIFIED BACTERIA PRODUCING PEDIOCIN

LIKE BACTERIOCIN

The development of genetically modified organisms or heterologous

production of bacteriocins in other bacteria has led to improvement in their

biosynthesis, mode of action, and potency (Fimland and Nissen-Meyer, 2007). As the

pediocin production is a plasmid linked phenotype, several studies were performed in

heterologous system. The Pediocin PA-1 has been cloned and expressed in several

bacteria like Escherisia coli, Lactococcus lactis, Lactobacillus sakei, Streptococcus

thermophilus, Enterococcus faecalis, Ped. acidilactici, Bifidobacterium longum,

Saccharomyces cerevisiae and in methylotrophic yeast Pichia pastoris. Table 2. 3. 4.

1, summarizes the cloned and expressed pediocin for its application in food industry.

Further, the development of new and improved bacteriocins requires a detailed

understanding of the mechanism, involved in its activity against the pathogens.

Moreover, a number of natural and genetically modified bacteriocins play a major

role in the preservation of food and animal feed and will also be important for the

treatment of infections in future (Fimland and Nissen-Meyer, 2007).

Table 2. 3. 4. 1: Pediocin production in a heterologous system

Pediocin Producing organism Vector Expression host

Pediocin PA-1 Ped. acidilactici PAC 1.0 pSRQ11 and pVA891 E. coli

Pediocin AcH Ped. acidilactici H Shuttle vector pHP59 E. coli and Ped. acidilactici

Pediocin PA-1 Ped. acidilactici PAC 1.0 - Lac. lactis

Pediocin Ped. acidilactici pMC117 Lac. lactis subsp. MM210

Pediocin Ped. acidilactici PAC 1.0 Shuttle vector pST Strep. thermophilus, E.coli,

Lac. lactis, Ent. faecalis

Pediocin PA-1 Ped. acidilactici PAC1.0 yT&A, Yeast

expression vector

Saccharomyces cerevisiae

Y294

Pediocin PA-1 Ped. acidilactici PAC 1.0 Yeast expression vector Pichia pastoris KM71H

Pediocin Ped. acidilactici pPC418 Lac. lactis ssp. lactis, Strep.

thermophilus, Ent. faecalis

Rec-pediocin

PA-1

Ped. acidilactici pPSAB (E. coli);

pPSAB1 (B. longum)

Bifidobacterium longum MG1

6XHis-Xpress-

PedA

Ped. acidilactici K7 pTZ57R/T subcloned

in pRSET-A

E. coli BL21 (DE3)

Pediocin PA-1 Ped. acidilactici 347 Lactococcin A

secretory apparatus

Lactococcus sp.

Source: Halami and Chandrashekar, (2007); Arques et al. (2008); Papagianni and Anastidastou (2009);

Kumar et al. (2011)



2. 4. HORIZONTAL GENE TRANSFER AND MOBILE GENETIC

ELEMENTS

2. 4. 1. HORIZONTAL GENE TRANSFER (HGT) IN LAB

The transfer of genetic material between cells is known as lateral or horizontal

gene transfer (HGT) and is considered as an important phenomenon in the field of

Evolution, Ecology, Biotechnology and Medicine (Jain et al. 1999; Ochman et al.

2000; Frost et al. 2005; Fraser et al. 2007; Keeling and Palmer, 2008). The HGT in

any genome deserves a detailed study because of its diversity and societal

implications (Zaneveld et al. 2008). Based on the complete genome sequence data of

LAB, it was reported that the genomes of LAB consists of a core genome which

encodes all house-keeping genes responsible for maintaining the species identity and

an auxiliary genome encoding accessory functions associated with transportation of

non-essential nutrients, bacteriophages and mobile elements, responsible for cell

surface modifications, etc., (Rasmussen et al. 2008; Družina et al. 2009). The

accessory genes contain different G+C content when compared to the core genome, as

they are obtained from other species of bacteria (Hacker and Kaper, 2000; Malachowa

and DeLeo 2010). The HGT usually occurs mainly through three processes,

conjugation, transformation and transduction (Družina et al. 2009; Malachowa and

DeLeo 2010; Van Reneen and Dicks 2010). Additionally, the transfer of genes is

facilitated by the mobile genetic elements (MGEs) that encode the proteins important

in the movement of DNA within or between the genomes (Družina et al. 2009). The

comparative genome analysis revealed that the LAB are masters in adaptation, and

have the ability to adapt to diverse environmental conditions (Morelli et al. 2004; Van

Reenen and Dicks, 2010).

The MGEs were first discovered in the maize genome in the late 1940s by

McClintock (McClintock, 1950). The MGE are known to play a major role in the

adaptation process and transfer of the genetic material like DNA among and within

the bacterial species (Malachowa and DeLeo, 2010). The MGEs consist of insertion



sequences (IS), transposons (Tn), plasmids, pathogenicity islands, integrons,

bacteriophages, gene cassettes, and chromosome cassettes (Fig. 2. 4. 1. 1). Such

segments facilitate the movement of the DNA between or within the genomes

(Družina et al. 2009; Siefert, 2009). The major function of MGEs in LAB include

metabolism of carbohydrates, amino acids and citrate, production of bacteriocins and

exopolysaccharides, resistance to antibiotics and heavy metals, phage resistance,

hydrolysis of proteins and DNA restriction modification systems (O‟Driscoll et al.

2006, Družina et al. 2009).

Plasmids: They are self-replicating DNA molecules, that act as MGE. They

are extrachromosomal replicons and carry an origin of replication as well as encode

some proteins involved in plasmid replication and maintenance (Frost et al. 2005).

McKay et al. (1972) first reported the presence of plasmids in Lactococcus sp. Most

of the LAB harbour different number of plasmids, around 1-16 in a single strain, and

are found in enterococci, pediococci, lactococci, leuconostoc and lactobacilli species

(Mathur and Singh, 2005). The plasmids of LAB are usually associated with lactose

fermentation, production of bacteriocins, conjugative transfer, citrate permease

activity, proteolytic activity, etc. (Družina et al. 2009). Such characters are

horizontally transferred for competitive advantage (Siezen et al. 2005).

Insertion sequences (ISs): They are the simplest type of MGEs that encode

transposase, and are important for movement of IS within or between bacterial

genomes. Mahillon and Chandler, (1998) first defined the IS elements as segments of

DNA, more than 2.5 Kb and capable of inserting at multiple sites in a target molecule.

The presence of several IS elements facilitate recombination events in the bacterial

genomes by means of site-specific recombination at the transposition site. The IS

transposition may occur at higher rates in certain bacteria under various stressful

environment conditions (Ohtsubo et al. 2005; Twiss et al. 2005). Several

Lactobacillus sp. are found to contain IS elements like ISL2, ISL3, IS1223, ISLpl1,



IS1163, etc. (Nicoloff and Bringel, 2003). The comparison of DNA sequences

revealed that the possible means of integration of IS elements by HGT was mostly

through conjugation process (Guédon et al. 1998).

DNA transposons (Tn): These elements have the capability to get integrated

or move or rearrange the chromosomal DNA in a cell or may get transferred from one

cell to other by means of plasmids, phages or integrative conjugative elements (Frost

et al. 2005). The conjugative transposons or integrative and conjugative elements

(ICEs) are wide spread in LAB and are self-transmissible MGEs and spread to new

hosts by conjugation. These ICEs are also found to promote mobilization of genomic

islands (Burrus et al. 2002). They are large transposable elements and have been

found to confer resistance to several antibiotics like tetracycline, erythromycin,

chloromphenicol and kanamycin (Gevers et al. 2003). Such transposons are also

found to be associated with nisin production and sucrose fermentation (Mathur and

Singh, 2005).

Integrons: This is a unique class of MGEs which were identified in the late

1980s and are associated with antibiotic resistance genes (Stokes and Hall, 1989).

These are immobile elements consisting of a nonmobile int gene for a tyrosine

recombinase, a cognate attI site for recombination and a strong promoter (Fluit and

Schmitz, 2004). The site specific recombination module of integrons are usually

associated with one or more mobile elements such as plasmids or transposons or gene

cassettes and are responsible for transfer between different genera of bacteria

(Toussaint and Merline, 2002; Fluit and Schmitz, 2004; Mazel, 2006). The Fig. 2. 4.

1. 1 describes the mechanism of HGT by various MGE involved in the transfer of

genes or a set of genes.



Fig. 2. 4. 1. 1. Schematic representation of inter and intracellular mechanisms of gene

transfer revealing the HGT phenomenon. (Major MGEs like Plasmids, Transposons, Insertion elements, Integrons are represented (Source: Zaneveld et

al. 2008).

2. 4. 2. CURRENT ACCOMPLISHED APPROACHES OF HGT

Over the last three decades several reports have been published on the in vitro

transfer of various plasmids, Tn, IS and other MGEs. The HGT has been identified in

several of the bacterial genomes, by two methods: Phylogenetic and Compositional

method (Zaneveld et al. 2008). The phylogenetic methods can describe the

distribution of or relationships between the genes in a multiple taxa and thus indicates

the HGT of a gene (Syvanen, 1994; Page and Charleston, 1997; Zaneveld et al. 2008).

The BLAST hits, gene presence/absence within the species phylogenies, ratios of

evolutionary distances are the common phylogenetic techniques used to reach the

approximation of a horizontally transferred gene (Zaneveld et al. 2008). In contrast,

the compositional methods examine sequence characteristics which include G+C

content, codon adaptation index, amino acid usage, relative synonymous codon and

dinucleotide usage (Hooper and Berg, 2002). The major disadvantage of this method

is that the sequences of similar compositional characteristics cannot be detected. Such

computerized approaches have helped many researchers to uncover the sequence

features to exploit and categorize the transferred genes (Zaneveld et al. 2008).



2. 4. 3. MGES INVOLVED IN THE TRANSFER OF BACTERIOCIN

ENCODED GENES OF LAB

The wide distribution of bacteriocin producing gene among LAB is basically

because of the presence of conjugative transposons or plasmids. Table 2. 4. 3. 1

enlists the MGEs involved in the transfer of bacteriocin under in vitro and in vivo

conditions among LAB. Coakley et al. (1997) reported the conjugal transfer of

lantibiotic “lacticin 3147” on a 60 Kb conjugative plasmid into L. lactis DPC3147, a

commercial starter culture. Such plasmid transfer was facilitated by the presence of

transposons on the plasmids harbouring the bacteriocins. Similarly, bacteriocin

encoded plasmid transfer was reported in pheromone induced plasmid transfer

systems in Ent. faecalis in a 56 Kb pAD1 plasmid. The conjugative transfer was due

to the presence of oriT and repA genes adjacent to the bacteriocin encoded genes

(Francia and Clewell, 2002). Interspecies transfer of pMG1, a conjugative 65.1 Kb

plasmid from Ent. faecium to Ent. faecalis strains and vice versa was reported in broth

mating experiments (Ike et al. 1998). Several of the gene clusters of lantibiotics like

nisin, lacticin 3147 and lacticin 481 are located on transposons or composite

transposons and are found to be transferred among LAB. The bacteriocin pediocin

PA-1 produced from Ped. acidilactici was found in other LAB that are reported to

encode mobilizable elements in the plasmids encoding the bacteriocin (Van Reenen

and Dicks, 2010). However, the in vitro transfer of pediocin PA-1 from the native

producer to other genera of LAB was not reported till date.



Table 2. 4. 3. 1. List of MGEs involved in the bacteriocin transfer

Bacteriocin Bacteriocin

producer

Recipient

strain

Plasmid size

(in Kb) MGE involved Mode of transfer Reference

Enterocin 226

NWC

Ent. faecium 226

NWC

Ent. faecalis

JH2-2 5. 2 Kb

The cell-to-cell contact led to the transfer

of conjugative plasmid pEF226.

In vitro conjugal

transfer Salzano et al. (1992)

Nisin Lactococcus

lactis

Ent. faecalis

JH2-2

Association of conjugative transposons

in the biosynthesis of nisin and sucrose

fermentation

Tn5276 and

Tn5307

Dougherty et al.

(1998)

Enterocin

1071A &1071B Ent. faecium

Ent. faecalis

OGX1 &

FA2-2

50 The cell-to-cell contact led to the transfer

of plasmid pEF1071

Conjugal transfer to

OGX1 Balla et al. (2000)

Lacticin 481 L. lactis - - Association of transposons-like structure

adjacent to lantibiotic operon

Insertion element

IS1675 Dufour et al. (2000)

Lacticin L. lactis

Commercial

Lactococcal

strains

60.2 oriT linked and association of mobilized

ML3/712sex factor element Conjugation Hickey et al. (2001)

Ruminococcin Ruminococcus

gnavus - - Presence of Insertion element ISRgn1

A transposon

responsible for

transfer of

bacteriocin

Gomez et al. (2002)

Bacteriocin 43 Ent. faecium

Ent. faecalis

FA2-2, JH2S-

S, OG1-10

6.2 (pDT1)

Bacteriocin producing plasmid also

harbours mobilization region (mob

genes)

Conjugative

mobilizable plasmid

Todokoro et al.

(2006)

Molecular genetic studies of pediocin-like bacteriocin Chapter 2


2. 5. APPLICATION OF BACTERIOCIN AND BACTERIOCIN

PRODUCERS

2. 5. 1. FOOD-BORNE PATHOGENS AND LISTERIOSIS

Over the last few decades there has been incidence of food-borne diseases

globally and an increase in the mortality and morbidity rates worldwide due to food

processing (Scott, 2003). Most of the developing and industrialized countries

experience an increase in the food-borne illnesses caused by several food-borne

pathogens like Listeria, Salmonella, Staphylococcus, Aeromonas, Escheria coli,

Clostridium, Shigella, etc. (Scott, 2003; WHO, 2007). In the past decade, several

sporadic and outbreak cases of food-borne listeriosis in human beings was observed

with consumption of foods of dairy and animal origin (Nayak et al. 2011). Though,

the rate of infection is relatively rare by L. monocytogenes, it is considered to have the

highest fatality and hospitalization rate of about 20 – 50% and 90%, respectively,

when compared to other food-borne pathogens (Zhang et al. 2004; Mook et al. 2011).

Listeria monocytogenes causing Listeriosis can grow in a wide variety of

environmental sources like soil, water, effluents, processing plant sources, red meat,

poultry, sea foods, dairy products, ready-to-eat (RTE) foods, etc. (Brouwer et al.

2010, Pinto et al. 2010). Listeriosis is mainly affected in three major group of persons,

which include, immuno-compromised elderly people, pregnant women and the

unborn or newly born infants (CDC, 2009; Mook et al. 2011). A survey of L.

monocytogenes presence in different food systems was detected in pastries (10%),

salami (20.5%), cheese (27%), mayonnaise based salads (34.1), smoked salmon

samples (34.1%), raw milk (2-38%), white cheese (3-4%), semi-hard cheese (3-64%)

(Mahmoodi et al. 2010; Pinto et al. 2010; Kasalica et al. 2011).

To improve the microbial safety of the processed or fermented or vaccum-

packed or RTE foods, several good manufacturing practices are followed, which

include, chemicals like sodium chlorite, sodium lactate, ozone, chlorine, etc., and

irradiation processes like UV-C irradiation, gamma-irradiation, electron-irradiation

(Garcia et al. 2003; Kim et al. 2004; Han et al. 2004; Chun et al. 2010). However,



these processes change the texture, flavour, colour, nutritional components and cause

serious health related problems to the consumers (Ko et al. 2005; Chun et al. 2010).

Hence, the inhibition of food-borne pathogens (mainly Listeria), and to improve the

shelf-life of RTE and dairy products, the class IIa anti-microbial bacteriocins

produced by LAB are presently used (Papagianni and Anastasiadou, 2009; Yadav and

Prakash, 2012).

2. 5. 2. APPLICATION OF BACTERIOCIN IN DIFFERENT FOOD SYSTEM

LAB plays an important role in the preservation of fermented foods, due to

their attributes such as, adaptation to various ecological niche, producing anti-

microbial compounds, and contributing to the improvement of flavour, texture, taste

and nutritional value (McKay and Baldwin, 1990; Leroy and De Vuyst, 2004; Giraffa

et al. 2010). Hence, LAB can be used in biopreservation, which refers to the extension

of shelf-life and improvement of the safety of foods using micro-organisms and/or

their metabolites (Ross et al. 2002) (Table 2. 5. 2. 1). Because of the GRAS status

assigned to LAB, more importance is given to the AMPs in the practical application

as a food preservative (Montville and Chen, 1998; Giraffa et al. 2010). The increasing

awareness among consumers on health risks by the artificial chemical preservatives

has led to the development of bacteriocins as a biopreservative agent (Settani and

Corsetti, 2008). The attractive characteristic features of bacteriocins to be considered

as a good biopreservative agent are their proteinaceous nature, non-toxicity, thermo

and pH stability, broad anti-microbial activity. The application of LAB and/or

bacteriocins in food are diverse (Ananou et al. 2007), which include-

a) Inoculation of food with LAB which act as starter culture or as protective culture,

where in situ bacteriocin production is observed.

b) Use of food which was previously fermented by the bacteriocin-producing strains

(NisaplinTM

, MicrogardTM

, AltaTM

2341) as an ingredient in food processing.

c) Addition of either purified or partially purified bacteriocins as additives in a food

system to improve its shelf-life.



Table 2. 5. 2. 1: Application of bacteriocins and bacteriocin producing LAB in different food system

Bacteriocin Producing strain Target pathogens Application

Nisin,

Enterocin,

Pediocin-like

bacteriocin

Lac. lactis,

Ent. faecium,

Ped. acidilactici, Lact. plantarum

Inhibits the growth of Listeria

monocytogenes, Clostridium sp.

Improves the flavour, texture, taste in the

fermentation of dairy foods when used as a starter

culture

Sakacin 674

Pediocin PA-1

Lact. sake Lb674,

Ped. acidilactici H

Controlling Listeria monocytogenes growth and

also Gram positive pathogenic bacteria in cooked

meat during extended refrigerated storage

Used as a biopreservative agent in meat products

Nisin Z, Lac. lactis Inhibits the growth of Clostridium botulinum

spores

Improves the growth and shelf-life of brined

shrimp (Pandalus borealis) and hence can be

used in the biopreservation of fish products

Nisin Lac. lactis ssp. lactis IFO12007 Inhibits the growth of Bacillus spores Improving the fermentation and safety of Miso, a

fermented soyabean paste

Mundticin

Pediocin PA-1

Ent. mundtii AT06

Ped. acidilactici H

Inhibits the growth of Listeria

monocytogenes

The shelf-life of minimally processed food from

mungbean sprouts on refrigeration was also

found to get improved

Enterocin Ent. faecium J96 Salmonella pullorum Prevents the gastro intestinal infection in young

broiler chickens

Bovamine™ Lact. acidophilus E. coli O157:H7 Prevents colon infections in cattle



Enterocin Ent. faecium EK13 Salmonella dusseldorf SA13 Reducing infections in Japanese quails (poultry)

Enterocin EJ97 Ent. faecalis EJ79 Prevents the growth of endospore forming

bacteria, Bacillus macroides/Bacillus

maroccanus in Zucchini puree.

Enetrocin EJ97 has the potential to preserve food

spoiled by Bacillus macroides/Bacillus

maroccanu when used in pure form

Enterocin AS-48 Ent. faecium Inhibits the growth of Listeria

monocytogenes, Bacillus cereus and

Staphylococcus aureus, Bacillus coagulans,

Clostridium botulinum and also

Alicyclobacillus acidoterrestris

Enhances of the stability of bacteriocin for a

period of 120 days under refrigeration conditions

in fruit and vegetable juices; also used to improve

the efficiency of canned foods

Pediocin Ped. acidilactici Enterococcus, Listeria monocytogenes,

Klebsiella, Pseudomonas, Shigella

Restricts human gastric infections

Enterocin Ent. faecium E. coli, Clostridia sp. Prevents gastric infection in rabbit

ABP-118 Lact. salivarius UCC118 Listeria monocytogenes Prevents gastric infection (mouse model)

Enterocin AS-48 Ent. faecium Protects of canned foods from spoilage by

Bacillus coagulans, Clostridium botulinum

Improving the efficiency of canned foods

Source: De Vuyst and Leroy (2007); Galvez et al. (2007); Settani and Corsetti (2008)



2. 5. 3. APPLICATION OF LAB IN SOYMILK FERMENTATION

Soya beans (Glycine max) and soymilk have been used in several developing

countries as an important nutritional component and the use of soymilk in the

preparation of yogurt-like products is widely accepted (Karleskind et al. 1991). The

soy-based foods provide beneficial effects to the health of the consumers due to their

hypolipidemic, anti-cholesterolemic, anti-atherogenic effects, reduction of

allergenicity and alleviation of lactose intolerence (Messina et al. 1994; Lopez-Lazaro

et al. 2002, Ewe et al. 2010). Further, the incorporation of probiotic bacteria as dietary

adjuncts has increased the consumption of several foods in the Europe, Asia and

United States (Nagata et al. 1998; Kristo et al. 2003).

Soy products are rich in isoflavones, dietary fibre, oligosaccharides, proteins,

trace minerals and vitamins, which has a tremendous influence on the hosts health

(Slavin et al. 1999). The isoflavones occur in two forms, glucosides and aglycones.

The aglycone isomer binds to the estrogen receptor sites and mimics the function of

estradiol and it is found to reduce the incidence of osteoporosis, menopausal

symptoms, mortality from cardiovascular disease and cancer (Rekha and

Vijayalakshmi, 2008). Many soy products have limited use by the consumers because

of their undesirable off-flavours (Kanada et al. 1976; Pinthong et al. 1980; Favaro

Trindade et al. 2001).

The consumption of soymilk and their products is beneficial to the ecosystem

of the GIT of the humans, resulting in an increase in the population of probiotics and

reducing the unwanted bacteria. In addition, the presence of isoflavones and saponin

is the major advantage of soymilk and these does not exist in other dairy products

(Cheng et al. 2005). The nutritional composition of soymilk was compared with the

skim milk, cow milk and buffalo milk and is represented in Table 2. 5. 3. 1.



Table 2. 5. 3. 1: Nutrient composition of different milk types

Constituents

Milk Type

Soymilk Skimmed cow’s milk Cow

milk Buffalo milk

Calories 133 90 150 101

Calories from fat 34 0 70 -

Total fat (g) 4 0 8 6.71

Saturated fat 1 0 5 4.2

Cholesterol (mg) 0 <5 35 8

Sodium (mg) 104 125 125 40

Carbohydrates (g) 13 13 12 4.9

Fibers (g) 3 0 0 -

Sugars (Lactose) (g) 0 12 12 4.9

Proteins (g) 9 8 8.2 4.5

Vitamin A (%) 24 10 6 13

Vitamin C (%) 0 4 4 -

Iron (%) 9 0 0 20

Calcium (%) 19 30 30 19.5

Acidity (%) 0.24 - 0.21 -

(Source: Hajirostonloo, 2009; www.soyconnection.com, www.buffalomilk.co.uk; http://dspace.dial.pipex.com).

2. 5. 4. IMPROVEMENT OF NUTRITIONAL VALUE, SHELF-LIFE AND

FERMENTATION OF SOYMILK BY LAB

The fermentation of soymilk by LAB was found to reduce beany flavours and

anti-nutritional factors like phytic acid with good acceptance of the soy end products

(Buono et al. 1990, Wang et al. 2006). The beany flavour in the soy products are due

to the formation of aldehydes such as hexanal and pentanal mainly from the

hydroperoxidation of polyunsaturated fatty acids catalyzed by lipooxygenases (Rekha

and Vijayalakshmi, 2008). The fermentation processes dealing with soymilk yogurt

production, enabled the researchers to characterize the product completely, including

the sensory evaluation and calculation of the residual amounts of phytates and α-

galactosidase (Favaro Trindade et al. 2001; Raghavendra et al. 2011). The major

carbohydrates present in soymilk are sucrose, raffinose and stachyose and no lactose,

http://www.soyconnection.com/

http://www.buffalomilk.co.uk/



which is abundant in bovine milk (Sharma et al. 2009). The presence of these

galactosides (raffinose and stachyose) has led to controversial opinions among

researchers for safe consumption. The α-galactosidase enzyme which is required for

the breakdown of galactoside are absent in the human gut. Moreover, the undigested

galactoside leads to the production of carbon dioxide, hydrogen and methane in the

intestine and results in flatulence and abdominal pain (Favaro Trindade et al. 2001,;

Ewe et al. 2010). However, most of the LAB utilize these oligosaccharides as energy

source and reduce the beany flavour in product, thus reducing the gas production in

the human intestine (Cheng et al. 2005).

The most commonly found food-borne pathogen in the food industry is the

Listeria monocytogenes. The Listeria has the ability to grow in foods with low pH,

storage temperatures from 1 to 45 oC (Kouakou et al. 2010). Lui and Lin (2008)

described about the crucial concern of L. monocytogenes contamination in soymilk

products and could not successfully prevent its growth. Hence, controlling the growth

of Listeria in any of the food system became necessary. Several of the LAB are found

to destroy the Listeria with their antagonistic effects and are widely used in

fermentation of dairy, food and meat processing industries (Kouakou et al. 2010).

Hence, in this regard the inhibition of Listeria can be obtained by the growth of LAB

producing pediocin like bacteriocin in soymilk.

The preparation of soy yogurt like products using LAB such as Lactobacillus,

Streptococcus, Enterococcus and Bifidobacterium was found to result in good texture,

taste and essential attributes for the product acceptability by the consumers (Donkor et

al. 2005). Presently, the probiotic bacteria like Bifidobacterium, Lactobacillus and

Streptococcus sp. are widely used in the fermentation of dairy and soymilk (Tamime

et al. 2005). Božanić et al. (2008a and 2008b) observed the fermentation of soymilk

by Lact. casei, Lact. acidophilus, which lasted for 12-17 h. The fermentation of

soymilk by Bifido. animalis subsp. lactis Bb12 was observed within 4 h of incubation



at 42 oC (Božanić et al. 2011). However, the LAB like Strep. thermophilus, Lact.

delbrueckii subsp. bulgaricus, Lact. rhamnosus, Lact. jonsoni are commonly used in

the fermentation of soymilk (Donkor et al. 2007; Farnworth et al. 2007). Thus, these

organisms are used as commercial starter cultures which are found to improve the

flavour, texture, aroma of the end product (Rattanachaikunsopon and Phumkhachorn,

2010). Hence, the bacteria which has the ability to survive in soymilk, produce

pediocin like bacteriocin, ferment the soymilk efficiently, inhibit Listeria, extend the

shelf-life of the end product with desirable sensory properties is required.

Enterococcus sp. has been used in several of the dairy products as adjuvant

starters, as a protective culture in fermented foods or as a probiotic strain (Giraffa,

1995; Franz et al. 1999, Gardiner et al. 1999). Several species of Enterococcus has the

ability to withstand the adverse environmental conditions such as high salt

concentrations, extreme pH and temperatures and hence play an important role in

fermented food products (Giraffa, 2003). The bacteriocins of Enterococcus sp. have

been used as a food additive during fermentation of several of the food products like

soymilk (Laukova and Czikkova 1999); meat products (Aymerich et al. 2000); cheese

(Farias et al. 1994) etc. Enterococci play an important role in dairy products because

of their specific biochemical properties like proteolysis, lipolysis and hence

contributing in the production of flavour and taste to the end product (Foulquié

Moreno et al. 2003). Most of the class IIa bacteriocins are isolated from fermented

meat and dairy products. However, limited literature is available from vegetable

sources, where Listeria is found to be major contaminant (Halami et al. 2005; Singh

and Ramesh, 2008). Hence the use of pediocin PA-1 like bacteriocin produced by Ent.

faecium has several advantages in the dairy industry.

The present review of literature described about the biotechnological

properties of LAB in a food system. Hence considering the advantages of LAB, the

characterization of the pediocin PA-1 like bacteriocin and the producing organisms

are described in the upcoming chapters.

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