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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).
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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
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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
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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).
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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).
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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.
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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)
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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).
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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).
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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
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
20 S. Manjulata Devi
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).
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
21 S. Manjulata Devi
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.
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
22 S. Manjulata Devi
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).
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
23 S. Manjulata Devi
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
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
24 S. Manjulata Devi
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)
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
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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.
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
26 S. Manjulata Devi
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).
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
27 S. Manjulata Devi
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,
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
28 S. Manjulata Devi
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
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
29 S. Manjulata Devi
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.
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
30 S. Manjulata Devi
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.
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
31 S. Manjulata Devi
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.
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
32 S. Manjulata Devi
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)
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
33 S. Manjulata Devi
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)
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
34 S. Manjulata Devi
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
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
35 S. Manjulata Devi
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,
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
36 S. Manjulata Devi
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.
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
37 S. Manjulata Devi
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).
Molecular genetic studies of pediocin-like bacteriocin Chapter – 2
38 S. Manjulata Devi
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.
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39 S. Manjulata Devi
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)
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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,
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
41 S. Manjulata Devi
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.
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
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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
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
43 S. Manjulata Devi
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)
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
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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.
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
45 S. Manjulata Devi
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,
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
46 S. Manjulata Devi
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
Molecular genetic studies of pediocin-like bacteriocin Chapter 2
47 S. Manjulata Devi
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.