4

2. REVIEW OF LITERATURE

Fermentation is process where microbes oxidise complex organic compounds like

carbohydrate into simple substances where organic acids act as electron acceptor.

The microbes digest the food substrate with its enzymes, increase the flavour, aroma, and

texture, make the food edible, enrich the food with vitamins, essential amino acids and above

all preserve food naturally. Fermented foods are produced across the world using various

techniques, raw materials and more. However, they fall in four categories namely alcoholic,

lactic acid, acetic acid and alkaline fermentation (Soni and Sandhu, 1990). Alcoholic

fermentation involves the production of ethanol as the end while lactic acid and acetic acid

fermentation produce respective acids as end products. The alkaline fermentation are not well

known but widely consumed in South east and African countries. In alkaline fermented foods,

the proteins are broken into amino acids and peptides releasing ammonia during fermentation

resulting in alkaline pH of the food which is achieved spontaneously by mixing bacteria,

especially Bacillus subtilus. A typical example for alkaline fermented food is Japanese natto

(Wang and Fung, 1996). Among these, lactic acid fermentation is widely usedfor preservation

of foods. This process is acheived by lactic acid bacteria which includes Lactobacillus,

Lactococcus, Pediococcus, Enterococcus, Leuconostoc, Weissella, Aerococcus,

Carnobacterium, Oenococcus, Sporolactobacillus, Teragenococcus, and Vagococcus.

Because of their presence in food and contribution of healthy microflora for animals, they are

‘generally recognised as safe’ (GRAS) (Donohue, 2004). LAB also known as “Lactics” are

often associated with plants (Corsetti et al., 2007; Chen et al., 2010), animals (Audisio et al.,

2011), meat (Pringsulaka et al., 2012), fermented products and often used as starter culture in

manufacture of various dairy products like cheese, curd and yogurt.

2.1 Approaches to Identify Lab

The bacteria were identified by their phenotypic characters like morphology, mode of

glucose fermentation, growth at various temperatures, pH, salt concentrations, carbohydrate

fermentation pattern, cell wall protein or whole cell protein analysis. However, these methods

are not accurate as the phenotypic characters depend on the environmental conditions which

are not reproducible. Thus, additional genotypic characterisations of isolates are also essential

to identify the organisms. Certain organisms that exhibit closely related phenotypic

characteristic feature may be well differentiated using certain techniques like RAPD, PFGE,

RFLP, DGGE and TGGE.

5

2.1.1. Randomly Amplified Polymorphic DNA (RAPD) PCR

The RAPD-PCR technique is widely used technique to discriminate closely related

organisms in which the polymorphic DNA was amplified using arbitrary primers. One

advantage of RAPD is that it does not require any knowledge of DNA sequence and amplified

product. This method is now been widely used to differentiate closely related organisms in

large number of isolates (Van Reenen and Dicks, 1996; Rossetti and Giraffa, 2005;

Albesharat et al., 2011; Bourouni et al., 2012). If there is any mutation in primer binding site,

the pattern of RAPD-PCR will be changed resulting in non-reproducibility hence it has to be

performed under controlled conditions.

2.1.2. Pulse Field Gel Electrophoresis (PFGE)

This is another fingerprinting technique which is used to distinguish closely related

organisms where DNA was subjected to electrophoresis under constant voltage with a change

in directions of 60 degree at regular interval. The DNA of higher molecular weight will

slowly realign with their charge when the field directions are changed that results in effective

separation. Unlike RAPD, this technique requires specialised instrument where standard

electrophoresis was run under constant voltage in three directions at regular interval and this

technique is laborious as it takes longer time to separate large sized DNA. Certain studies

have used this technique to identify the distinct isolates of same species (da Cunha et al.,

2012; Gonzalez-Arenzana et al., 2012). This technique gives variation that is more accurate in

the sub-typing of the organisms.

2.1.3. Restriction Fragment Length Polymorphism

In this technique, the variation in homologous DNA was explored by digesting DNA

with restriction enzymes which was separated according to their size by electrophoresis.

However, the process is cumbersome as it is time consuming and requires large amount of

DNA hence terminal restriction fragment length polymorphisms (T-RFLP) was developed. In

this method, a segment of DNA was amplified using fluorescent labelled primers following

with restriction digestion with enzymes and electrophoresis in polyacrylamide gel

electrophoresis. As the size is determined using fluorescent detector, only terminal fragments

are detected while other fragments were ignored hence this technique was used for detection

of LAB from a mixed community (Nieminen et al., 2011; Bokulich and Mills, 2012).

2.1.4. Temperature and Denaturing Gradient Gel Electrophoresis (TGGE And DGGE)

The TGGE is a method that separate molecules like nucleic acid by their melting

behaviour while DGGE separates the molecule with the aid of denaturing agents. Unlike other

methods, this is more sensitive since DNA with single nucleotide variation can be separated

6

very efficiently. A gene was amplified by PCR methods which give single band in normal

agarose gel electrophoresis will exhibit variation in DGGE because of the variations in

nucleotide sequence. This method was widely used to detect LAB from various sources like

cheese (Gori et al., 2012), milk products (Bao et al., 2012) and more. The survival rate of

LAB in intestine which was isolated from silage had been compared using DGGE (Han et al.,

2012). DGGE was widely used method as it is simple and inexpensive that does not require

specialised apparatus while TGGE required dedicated apparatus hence not widely used.

2.1.5. Phylogenetic Analysis

The 16S rRNA, a well conserved universal marker gene is the most commonly

exploited target for identification of organisms. The gene has constrained function established

in early stage of evolution which are relatively unaffected by environmental factors hence

used as a vital tool in the the study of evolutionary relationship of organism and identification.

However, it has limitations as it is well conserved with limited resolution that makes difficult

to differentiate very close organisms like Lactobacillus plantarum and Lact. pentosus. As the

copy number varies with bacterial species, it makes additional difficulty to represent certain

bacterial strains (Mohania et al., 2008). Amlified restriction digestion of ribosomal DNA is

RFLP of 16S rRNA gene product where 4 mer restriction digestion enzymes like AluI, HaeIII

are used to digest the gene product (Rodas et al., 2003). Similarly intergenic space region

(ITR) has also been used to determine the phylogenetic relationship of organisms by

comparing with prevailing sequences (Zavaleta et al., 1996). However, these could not

differentiate strains having high degree of relatedness but could differentiate species. Since it

was very difficult to identify closely related organisms, nested multiplex PCR was used to

identify them with other genes like recA (Torriani et al., 2001). Recently, various methods

like FTIR (Samelis et al., 2011), MALDI-TOF MS (Duskova et al., 2012) were introduced to

identify and cluster LAB. As whole cellular components were evaluated with FTIR and

MALDI-TOF MS, it gives complete information of phenotypic and genotypic properties. The

FTIR was used to differentiate between pathogenic and non-pathogenic Listeria and

Staphylococcus (Lamprell et al., 2006; Rebuffo-Scheer et al., 2008) while MALDI-TOF MS

was used to discriminate food borne bacteria (Mazzeo et al., 2006).

2.2. Lactic Acid Bacteria in Fermentation

Fermented foods are from plant or animal source which is processed by fungi or

bacteria that thrive over on the surface of the source. LAB are isolated from various

fermented products kimchi, doenjang, dongchimi (Lim and Im, 2009), kallappam, koozh,

morkuzhambu (Kumar et al., 2010), appam batter, vegetable pickle (Jamuna and Jeevaratnam,

7

2004a). Many new species like Leuc. kimchii (Kim et al., 2000), Leuc. inhae (Kim et al.,

2003), Weissella koreensis (Lee et al., 2002), Lact. kimchii (Yoon et al., 2000) are explored

from kimchi hence fermented food has diverse group of organisms. They are widely

distributed in nature and have a strong capability to survive in any environmental conditions

(Liu et al., 2011b).

Fermented foods can be classified as cereal based, vegetable based, vegetable/fruits

based, fish based, meat based of which cereal based fermented food are widely popular in

Asian countries and are extensively studied. Lactobacillus is the predominate organism in

theis type of fermented foods (Table 2.1) that modifies the organoleptic properties of the

fermented foods (Rathore et al., 2012). The organic acid produced by them has a role in

preservation and gives a special taste to the food. The diacetyl compounds produced during

metabolism gives a unique flavour to the food (Liu et al., 2011b). During the process of

fermentation LAB produce vitamins like riboflavin, thiamine, folic acid, etc., and enhance the

digestibility (Ghosh and Chattopadhyay, 2011) and increase the free amino acid content in the

food (Ding et al., 2009) theremy increasing its nutritional value.

In addition to fermented foods, lactic acid bacteria also play a vital role in

fermentation of medicine which was evidenced by isolation of Lact. acidophilus Kanjika,

fermented rice which is used as food and also as medicine (Reddy et al., 2007). Ayurvedic

medicine kutajarista a traditional fermented decoction of Holarrhena antidysentrica is widely

used for the treatment of various diseases like indigestion, amoebic dysentery, diarrhoea,

piles, intestinal parasite infestation and problems, fever, (Sekar and Mariappan, 2008) and

Lact. plantarum isolated from this decoction ameliorates the cellular damages caused by

Aeromonas veronii (Kumar et al., 2011a). Thus, fermentation not only enhances the nutritive

and but also the medicinal value of food making them as functional foods.

The functional foods are processed food fortified with health benefit components like

vitamins, flavones and a well-known example is addition of iodine to table salt. Fermentation

plays a major role in formulation of functional foods. Soymilk fermented with probiotic

strains increase free amino acid contents, vitamin B6, γ-aminobutyric acid, isoflavone (Li

et al., 2012). The antioxidant activity of the LAB fermented soymilk was also higher than that

of unfermented soymilk (Wang et al., 2006) which was because of changes in conjugation of

flavone and soyasaponins in soymilk (Hubert et al., 2008). In cereals fermented with

Lact. rhamnosus and Saccharomyces cerevisiae, the total phenolic content and antioxidant

activity was increased (Dordevic et al., 2010). Not only in fermented milk and cereals but

fermented fruits also have significant increase in antioxidant property which also inhibits

8

intestinal glucose and sugar uptake enzymes (Wu et al., 2011) produced during fermentation.

Ankolekar et al., (2011) has observed a significant increase with antioxidant activity,

-glucosidase and angiotensin converting enzyme inhibitors in the fermented apple juice.

LAB possesses many enzymes like polyphenol oxidase, which modify the phenolic

content in the food thereby increasing the functionality. By successive cleavage, gallatonines

were converted into gallic acid, while flavanol glycosides like kaempferol and quercetin were

converted into aglucones and bioactive polyphenol (Duckstein et al., 2012; Santos et al.,

2012) which has higher antioxidant and antimicrobial activity. Recently dihydrodaidzein

racemase has been identified in Lactococcus which is involved in conversion of daidzein, a

phytoestrogen, into equol which has beneficial effects in human (Shimada et al., 2012).

Certain other enzymes responsible for metabolism of phenolic content were discussed by

Rodriguez et al., (2009). Further characterisation of these enzymes will help in improvement

of food quality and in development of functional foods.

2.3. Lactic acid bacteria and its metabolites

Lactic acid bacteria are known to produce several metabolites that are beneficial for

humans and sometime detrimental. One of the well-known end products is lactic acid which is

used as preservative. Besides lactic acid, they also produce variety of compounds like

diacetyl, acetoine, butanediol, flavone, organic acids and various volatile components which

depend on the sources of fermentation. During fermentation process, they metabolise certain

flavanol glycoside in the plant materials into 4-hydroxybenzoic acid, gallic acid (Duckstein

et al., 2012) that exhibit antimicrobial activity (Broberg et al., 2007) and antioxidant activity

(Duckstein et al., 2012). Ganzle et al., (2009) reviewed metabolic pathways of various

carbohydrate and their end products that exhibit antifungal activity. Some organisms like

Lact. buchneri, Lact. reuteri, and Ped. pentosaceus produce propionate and propanediol that

are industrially useful and exhibit antimicrobial activity.

9

Figure 2.1. Metabolism of plant components by LAB. Adopted from Duckstein et al.,

(2012) with permission copyright © 2012.

10

Table 2.1: Certain cereal based fermented foods from Asian countries

Food Cereal Organism Reference

Appam,

Kallappam

Rice Lactobacillus plantarum (Jamuna and Jeevaratnam,

2004a),

(Kumar et al., 2010)

Boza barley,

oats, millet,

maize,

wheat or

rice

Lactobacillus plantarum

Enterococcus faecium

Leuconostoc lactis

Leuconostoc mesenteroides

subsp. dextranicum

Pediococcus pentosaceus

Lactobacillus fermentum

Lactobacillus paracasei

Lactobacillus pentosus

(Todorov and Dicks,

2005a)

(Todorov and Dicks,

2005b)

(Todorov, 2010)

(von Mollendorff et al.,

2006)

(Petrova and Petrov, 2011)

Brem Maize Pediococcus pentosaceus

Enterococcus faecium

Lactobacillus curvatus

Weissella confuse

Weissella paramesenteroids

(Sujaya et al., 2001)

Burong Isda Rice Lactobacillus plantarum (Olympia et al., 1995)

Chili Bo Maize Lactobacillus plantarum

Lactobacillus fermentum

Lactobacillus farcimini

Pediococcus acidilactici

Enterococcus faecalis

Weissella confusa

(Leisner et al., 1999)

Dosa,

Idly,

Dhokla

Rice Lactobacillus plantarum

Pediococcus pentosaceus

Lactococcus lactis

Enterococcus faecium

Leuconostoc mesenteroides

(Iyer et al., 2011)

(Sawale and Lele, 2010)

(Vijayendra et al., 2010)

Injera Wheat,

Barley,

Corn, rice

Leuconostoc mesenteroides,

Streptococcus faecalis

Pediococcus spp.

Lactobacillus spp.

(Gashe, 1985)

(Nigatu et al., 1998)

Kanjika Rice Lactobacillus acidophilus (Reddy et al., 2007)

Khanomjeen Rice Lactobacillus plantarum (Oupathumpanont et al.,

2009)

Koozh Millet Weissella paramesenteroides

Lactobacillus plantarum

Lactobacillus fermentum

(Kumar et al., 2010)

Miso Rice/Wheat Enterococcus durans

Enterococcus faecium

Lactococcus spp.

(Onda et al., 2002)

Puto Rice Leuconostoc spp.

Enterococcus faecium

(Kelly et al., 1995)

(Shibata et al., 2007)

Sourdough Wheat Many Lactobacillus spp. (Gobbetti, 1998)

11

2.3.1. Antimicrobials

2.3.1.1. Organic Acids

When the carbohydrates are fermented, they produce wide variety of organic acids that

will reduce the pH of the environment resulting in reduction of unfavourable organisms. Two

major acids, namely lactic acid and acetic acid are produced by LAB that exhibit

antimicrobial activity at low pH than at neutral pH among which acetic acid is more effective

as they control the growth of yeast, molds and Bacilli (Reis et al., 2012). The dissociated

lactic acid in cytoplasm causes acidification resulting in failure of proton motive force. A

synergistic effect of acetic acid and lactic acid was observed at reduced concentration when

compared to individual concentration. In addition to antimicrobial activity, acetic acid also

contributes for the aroma (Reis et al., 2012). Propionic acid though produced in low

concentration is known to interact with cell membrane and neutralise the electrochemical

proton gradient but not as like lactic acid which also inhibit amino acid uptake (Reis et al.,

2012). Certain Lactobacillus spp., and Pediococcus are known to produce 2-pyrrolidone-5-

carboxylic acid that exert antimicrobial activity against spoilage bacteria (Yang et al., 1997)

whose mode of action may also similar to that of lactic acid (Ouwehand and Vesterlund,

2004) but was not as efficient. Additional to these organic acids, they produce benzoic acid

(Niku-Paavalo et al., 1999), 5-hydroxyl ferulic acid (Ou and Kwok, 2004; Knockaert et al.,

2012), 3-phenyllactic acid (Rodriguez et al., 2012), 3-hydroxydecanoic acid (Broberg et al.,

2007), hydroxyl fatty acid (Sjogren et al., 2003) and more that exhibited antimicrobial

activity. Reuterin, chemically β-hydroxypropionaldehyde produced by Lact. reuteri is a non-

peptide broad spectrum antimicrobial compound synthesised from glycerol exhibit

antimicrobial active against prokaryotes and eukaryotes (Chung et al., 1989). Reuterin as

analog of D-ribose, inhibit B1 subunit of ribonucleotide reductase and thioredoxin suggesting

its inhibition of sulfhydryl enzymes (El-Ziney et al., 2000). Some of the widely studied

organic acids that exhibited antimicrobial activity are given in Table 2.2.

2.3.1.2. Bacteriocins

Bacteriocins are diverse group of modified or unmodified antimicrobial peptide

extracellularly secreated by bacteria which are protected by dedicated immune system (Rea

et al., 2011). Since the discovery in 1925 by Gratia, search of bacteriocins from various

organisms has been intensified resulting in purification and characterisation. Though many

bacteriocins were reported from various organisms, the peptide antimicrobials from LAB

were considered to be innocuous as they are naturally associated with human and animals and

can safely be employed in food preservation and medicine (Rea et al., 2011) as they are active

12

against diverse group of microbes including fungi (Adebayo and Aderiye, 2010). Gram

positive bacteria has gained specific regulatory mechanisms for synthesis and secreation of

bacteriocins sparing the producer strain. Bacteriocin gene operon which was usually

associated with plasmid (Daeschel and Klaenhammer, 1985; Mathys et al., 2007) encodes

specific transport system for bacteriocin, an immunity protein and the prepeptide of

bacteriocin which was secreted extracellularly after processing.

Table 2.2: Some organic acids produced by LAB that exhibit antimicrobial activity

Compound Structure

Lactic acid

Acetic acid

Propionic acid

2-pyrrolidone-5-carboxylic acid

Benzoic acid

5-Hydroxy ferulic acid

3-phenyllactic acid

Reuterin

13

Figure 2.2. Synthesis and secreation of bacteriocin by Gram positive bacteria Adopted

from Ennahar et al., (2000) Copyright© 2000

14

2.3.1.2.1 Detection of Bacteriocin

The concentration of bacteriocin are expressed as arbitrary units (AU) which is

defined as reciprocal of highest dilution exhibiting minimal inhibition. Parente et al., (1995)

have detailed various ways of screening bacteriocin activity among which agar well diffusion

is widely used because of its reliability. While screening for bacteriocins, it is essential to

eliminate the inhibition by non bacteriocin agents like bacteriophage, organic acids, H2O2 and

non-ribosomally synthesised antimicrobials like mevalonolactone and cyclic dipeptides.

Evaluation of bacteriocin preparation at the pH of 6 eliminates the effect of acids and

treatment with protease should lead to loss of activity (Moraes et al., 2010).

2.3.1.2.2. Classification of Bacteriocin

The classification of bacteriocin produced by LAB is much complicated because of

heterogeneity hence various classification schemes has been proposed. However,

Klaenhammer, (1993) classification of bacteriocin into four groups has formed a basis of all

prevailing classification. According to Klaenhammer,

Class I bacteriocins or lantibiotics are small peptides (<5 kDa) that has unusual amino

acids like lanthionine, β-methyl lanthionine (lantibiotics).

Class II bacteriocins are unmodified heat stable membrane active peptides whose

molecular weight are less than 10 kDa. These are further classified into three classes

Class IIa bacteriocin that has a conserved YGNGV sequence in N-terminal region

exhibiting antilisterial activity.

Class IIb bacteriocins are two peptide bacteriocins

Class IIc bacteriocins are thiol activated peptide whose activity was lost if disulphide

bridges are broken

Class III bacteriocins are unmodified heat labile proteins whose molecular weight are

greater than 30 kDa. These are associated with enzymatic activity.

Class IV bacteriocins are complex proteins that are associated with lipids or

carbohydrate moieties.

This classification was further refined by Cotter et al., (2005) as Class I lantibiotics

and Class II peptides which are further classified as IIa: pediocin like bacteriocins, IIb: two

peptide bacteriocins, IIc: cyclic peptides and IId: non-pediocin unmodified bacteriocins and

Class III bacteriolysin which are lytic peptides. Heng et al., (2007) and Nissen-Meyer et al.,

(2009) have further refined the classification which has sub-classified Class II bacteriocins

based on amino acid sequence and moved cyclic peptides to Class IV.

15

2.3.1.2.2.1. Class I: Post Translationally Modified Bacteriocins

The bacteriocins of this class undergo post translation modification to get lantibiotics.

This group of bacteriocins are well characterised as Nisin of this group is used as food

preservative. Because of additional post translational modifications, this group was further

classified into three groups by Rea et al., (2011)

Class Ia: Lantibiotics

The lanthionine containing antibiotics are generally less than 5 kDa with 19-30 amino

acids that undergo post translation modification to produce unusual amino acids. Lantibiotics

are further classified into type A linear lantibiotics which includes Nisin and subtilin while

type B globular bacteriocin that include mercsacidin, cinnamycin, mutacin II and lacticin 481.

The mode of action of type A lantibiotics is by membrane pore formation resulting in

dissipation of membrane potential causing cell death while type B acts as inhibitor of cell wall

synthesising enzymes.

Class Ib: Labyrinthopeptins

Labyrinthopeptins are globular hydrophobic peptide which was recently identified

from Actinomadura namibiensis (Meindl et al., 2010). This bacteriocin was distinguished by

the presence of labionins derived after modification from S-X-X-S-X-X-X-C motif by

LabKC, a bifunctional protein which posses Ser/Thr kinase and lanthionine cyclase. This

bacteriocin has a notable activity against herpes simplex virus.

Figure 2.3. Crystal structure of Labyrinthopeptins with labionins Adopted from

Meindl et al., (2010) copyright © 2010.

16

Class Ic: Sactibiotics

A circular bacteriocin, subtilosin A produced by Bacillus subtilis (Kawulka et al.,

2004) that has unusual sulphur cross linkage between Cys and -carbon of Phe and Thr was

not considered to be circular bacteriocin hence Martin-Visscher et al., (2009) has proposed to

place it in a unique class. Hence it was included in Class I bacteriocin as it was post

translationally modified to produce such linkage. Another two peptide bacteriocin thuricin CD

produced by Bacillus thuringiensis 6431was also identified to have such cysteine to -carbon

linkage (Rea et al., 2010) which does not show any sequence similarity with subtilosin A. The

genetic sequence of thuricin operon consists of two regions, one coding for bacteriocin and

another overlapping ORF of trnC and trnD that code for radical S-adenosyl methionine (SAM

superfamily protein) (Rea et al., 2010). The unusual cysteine to -carbon linkage was

produced by two [4Fe-4S] clusters of radical SAM enzyme where one involved in cleavage

reaction while another is required for generation of thioester linkage (Fluhe et al., 2012).

Figure 2.4 Structure of subtilosin A that shows unique thioester linkage Adapted with

permission from Kawulka et al., (2004) Copyright © 2004

17

2.3.1.2.2.2. Class II: Unmodified Bacteriocins

The class II bacteriocins are essentially unmodified bacteriocin consisting standard

amino acids with molecular weight <10 kDa. According to Heng et al., (2007), it is further

subdivided into Class IIa: Pediocin like bacteriocin, Class IIb: two peptide bacteriocin,

Class IIc: other unmodified bacteriocins

Class IIa: Pediocin Like Bacteriocin

A large collection of Class II bacteriocins produced by Gram positive bacteria belong

to this sub-class. It has a potential application as food preservative and possible biomedical

application as it is effective against Listeria, Staphylococcus aureus, Bacillus cereus and

Clostridium perfringens. Pediocin PA-1 is the best characterised bactericion of this class and

is the only antilisterial bacteriocin from this class used as preservative as a constituent in

AltaTM

2341. This group of bacteriocin was characterised by the conserved amino acid motif

YGNGV at N-terminal end and presence of disulphide bridge. The bacteriocin exhibits

bactericidal mode of action where the conserved region binds with IIC and IID components of

mannose permease phosphotransferase system and insertion of bacteriocin inside the

membrane forming pore that caused membrane permeabilization.

Figure 2.5. Sequence alignment of Class IIa bacteriocin sequence collected from

BACTIBASE showing conserved region

18

Figure 2.6. Mode of action of of Class II bacteriocin adopted with permission from

Ennahar et al., (2000) copyright © 2000

19

Class IIb: Two Peptide Bacteriocin

The bacteriocins of this class include plantaricin EF which has two peptides and

requires both the peptides in equal concentration to exert its activity. Till date, only 16

bacteriocins were reported since the discovery of lactococcin G (Nissen-Meyer et al., 2009).

The mode of action of this bacteriocin has revealed the permeability of target cells leading to

cell death (Nissen-Meyer et al., 2009). The gene coding for this bacteriocin are found adjacent

to each other with immunity protein and ABC transporter. Although conserved regions were

not detailed much in this class, a common GXXXG motif was essential for antimicrobial

activity (Rea et al., 2011). The peptides are active only with their counterpart though 60-70%

similarity was observed. However, the activity was observed when lactococcin G was

combined with its complimentary peptide of enterocin which exhibited nearly 88% homology

(Nissen-Meyer et al., 2009).

Figure 2.7. 3D structure of plantaricin J

Cass IIc: One Peptide Non-Pediocin Like Bacteriocin

The unmodified non-pediocin like one peptide cationic, hydrophobic bacteriocin

which does not have similarity with Class IIa bacteriocins are placed in this group. It includes

lactococcin A, enterocin B, cornobacteriocin B, acidocin 1B, mesenterocin 52B and more.

Lactococcin A was the first of this type isolated from Lactococcus lactis which also increases

permeability of membrane. This bacteriocin also binds to mannose phosphotransferase

permease embedded in the target cells and inserted inside the membrane resulting in pore

formation. Several enterococcal bacteriocins like enterocin EJ97, LA50, LB50, Q belong to

this group (Heng et al., 2007).

20

2.3.1.2.2.3. Class III: Large Bacteriocins

These bacteriocins are heat labile large proteins of molecular weight more than 10 kDa

with exception of propionisin SM1 which is heat stable. This group is further subdivided into

two groups as IIIa: bacteriolysin and IIIb: non-lytic antimicrobial proteins. Bacteriolysins are

plasmid mediated glycyl-glycine endopeptidase that hydrolyse pentaglycine cross bridges in

peptidoglycans of Gram positive bacteria resulting in cell lysis. One well studied bacteriocin

of this group is lysostaphin, a 27 kDa bacteriocin produced by Staphylococcus simulans that

exhibit antimicrobial activity against pathogenic organisms like Staphylococcus aureus and

Staphylococcus epidermidis (Heng et al., 2007). Unlike this, dysgalacticin and streptococcin

A-M57 does not kill the target organism by lysis mechanisms. Dysgalacticin produced by

Streptococcus dysgalactiae subsp. equisimilis blocked the glucose uptake targeting

PEP-dependent glucose and mannose transporter and also exhibited bactericidal effect by

increasing the membrane permeability by leaking intracellular potassium ions on

Streptococcus pyogenes (Swe et al., 2009). The structural gene of dysgalacticin was located in

plasmid and the strain lost its activity when the plasmid was cured. In addition to this, the

strain also became resistant for exogenous dysgalacticin which showed that the producer

strain had immunity protein dsyI which acted at membrane level to prevent its effect on target

cells (Swe et al., 2010).

Figure 2.8. 3D structure of lysostaphin a class IIIa bacteriocin Rendered from Protein

database PDB 1QWY.

21

2.3.1.2.2.4. Class IV: Cyclic Peptide Bacteriocins

These bacteriocins were classified as separate class because of its unique feature

(Heng et al., 2007). As like other bacteriocins, these are also ribosomally synthesised which

undergo post translational modification to form a covalent amide linkage between N and C

terminal last amino acids of mature peptides. Because of this nature, these bacteriocins are

resistant towards various proteases and are stable across wide temperature. Ever since the

discovery of enterocin AS48 (Samyn et al., 1994), the study on circular bacteriocins had been

increased that lead to the discovery of gassericin A (Kawai et al., 1998), carnocyclin A

(Martin-Visscher et al., 2008), lactocyclicin Q (Sawa et al., 2009), leucocyclicin Q (Masuda

et al., 2011). Based upon the amino acid composition this bacteriocin is further subdivided

into group IVa which is cationic peptides that include enterocin AS-48, circularin A,

uberolysin, lactocyclicin Q, carnocyclin A and garvicin ML which has high pI value

of nearly 10. Group IVb is anionic peptides that included gassericin A and butyrivibriocin

AR10.

Figure 2.9. Protein sequences of Class IV cyclic peptide showing two different subclasses

and 3D structure of Enterocin AS-48 and Carnocyclin A Adapted with permission from

van Belkum et al., (2011). Copyright © 2011

22

2.3.1.2.3. Applications of Bacteriocin

The bacteriocins produced by LAB have several striking features that made attractive

for using them in food preservation and medical purpose. They are considered as safe and

easily digested by intestinal enzymes thus do not alter the intestinal microbiota. As they exert

synergistic effect with other antimicrobials, exerting wide spectrum of inhibition against food

spoilage and pathogenic bacteria, it can effectively be used as food preservative (Jamuna

et al., 2005). Though many bacteriocin preparations were studied, only Nisin and Pediocin are

commercially available for direct use in food because of regulations in laws across many

countries. As it is cationic and hydrophobic, they build electrostatic interactions with negative

charges of bacteria and make pore on the membrane. In addition, they exhibit spermicidal and

antitumor activity (Silkin et al., 2008) (Villarante et al., 2011). Hence, the topical application

of bacteriocin in the field of medicine for treatment against various pathogens in human and

cattle are focused. Mersacidin produced by Bacillus inhibited cell wall synthesis of

methicillin-resistant Staphylococcus aureus in mice while nisin inhibited the growth of

Staphylococcus aureus in respiratory tract of rats, Clostridium tyrobutyricum, C. difficile

(Dicks et al., 2011). Nisin produced by Lactococcus lactis AMB1 and IB-367 has given

successful results against Helicobacter pylori in phase I clinical trials. Likewise, subtilosin, a

class Ic bacteriocin, isolated from Bacillus amyloliquefaciens has proved to exhibit

antimicrobial activity against Gardnerella vaginalis, bacterial vaginosis associated bacteria.

This bacteriocin was active only against pathogens sparing native vaginal Lactobacillus with

negligible cytotoxicity and exhibited spermicidal activity (Sutyak et al., 2008a; Sutyak et al.,

2008b) hence this bacteriocin can safely be used in personal care products for treatment of

bacterial vaginosis. In addition, as this bacteriocin also exhibit spermicidal activity on higher

animal models like horse, it can be used in animal models as spermicidals. This has another

advantage than nisin which also exhibited spermicidal activity (Aranha et al., 2004) as it does

not affect the native vaginal isolates while the fate of vaginal isolates upon usage of nisin is

unknown. Dicks et al., (2011) had detailed about the application of bacteriocin in treatment

against various pathogenic bacteria associated with respiratory diseases, neuroparalytic

diseases, oral and dental diseases, gastric diseases. Certain lantibiotics like duramycin also

play a vital role as anti-inflammatory agents by sequestering phosphatidylethanolamine or

inhibit angiotensin converting enzymes. However, further use of bacteriocins as medicines

need thorough investigations as they may also cause some immune responses.

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2.3.2. Exopolysaccharides

Exopolysaccharides (EPS) are metabolite of bacteria which are simple or complex in

structure and composition for protection of cells against stress like metals, phage attacks,

desiccation, for facilitation of adhesion onto substratum, for biofilm formation and more

(Farnworth et al., 2007). Depending on the composition and structure of EPS produced by the

bacteria, the texture of food is modified hence it has essential value in food industry. Among

the EPS producing bacteria, study of EPS produced by LAB has increased because their

GRAS status. Based on the composition, EPS was classified into homo-polysaccharide that

has single type of sugar unit and hetero-polysaccharide that has more than one type of sugar

unit (Jolly et al., 2002).

2.3.2.1. Homo-polysaccharides

Most of the homo-polysaccharides are synthesised from sucrose with the help of

glycosyltransferase family. Some commonly studied homo-polysaccharides are dextran,

levan, alteranan, reuteran which are synthesised by respective glycosyltransferase enzymes

namely dextransucrase, levansucrase, alteransucrase and reuteransucrase. Among these homo-

polysaccharides, dextran that has glucose with (16) glycosidic linkage as main chain and

varied degree of (12), (13) and (14) branching is widely studied. It is used for

matrix preparation of chromatographic columns, to restore blood volume, though it has side

effects in medical application (Patel et al., 2012).

2.3.2.2. Hetero-polysaccharides

It is made of several repeated units of two or more monosaccharide units or its

derivatives like N-acetyl-D-glucosamine. Synthesis of hetero-polysaccharide involves gene

cluster that include region for enzyme regulation, chain length determination,

glycosyltransferase, for export of synthesised EPS (Ruas-Madiedo et al., 2012).

2.3.2.3. Application of Exopolysaccharides

The EPS is not digested by intestinal enzymes and because of its high viscosity; it

increases the bulk faecal transit. They are digested by colonic bacteria especially

Bifidobacterium longum and produces short chain fatty acids which has many beneficial

properties in host (Hongpattarakere et al., 2012). It is also observed to alleviate

immunomodulatory property (Liu et al., 2011a), posses antitumor, antiviral activity and used

to eliminate heavy metal (Kumar et al., 2007) for lowering blood glucose level in borderline

type II diabetes (Farnworth et al., 2007). Thus, intense research in structural and functional

characterisation of EPS will enhance applicability in the field medicine.

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2.4. Application of Lactic Acid Bacteria

LAB have wide application as probiotic organisms for its multifactorial benefits to

human being and other animals. Probiotics are defined as “Live microorganisms which when

administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). In

early 20th

century, Metchnikoff proposed the role of gut flora in human health which was

termed as probiotics by Werner Kollath in 1953 (http://en.wikipedia.org/wiki/Probiotic).

Many organisms possessing probiotic property from various sources were reported, among

them Lact. rhamnosus GG (Lebeer et al., 2010) and Bifidobacterium (Schell et al., 2002)

strain have exhibited an effective probiotic property. World health organization (FAO/WHO,

2002) and Indian Council of Medical Research (ICMR-DBT, 2011) has suggested certain

properties which probiotic organisms should posses. According to them, the probiotic

organisms must be tolerant to acid and bile, secrete antimicrobial substances and bile

hydrolase, should produce β-galactosidase and adhere to intestinal epithelial cells. Probiotic

Lact. plantarum AS1 isolated from kallappam (Kumar et al., 2010) binds with

adenocarcinoma cells via carbohydrates and proteins (Kumar et al., 2011b) and also reduced

the development of DMH-induced cancer by antioxidant dependent mechanism (Kumar et al.,

2012b). Bhakta et al., (2010) has identified probiotic Pediococcus exhibited resistance to

heavy metals like arsenic. Several studies have reported the ability of LAB to remove metals

and certain toxic compounds from environment (Haskard et al., 2001; Halttunen et al., 2007b)

(Zinedine et al., 2005). However, removal is influenced by pH, where maximum removal

occurs at pH 4-6 while below pH 3 there is a sharp decrease in adsorption (Halttunen et al.,

2007a; Bhakta et al., 2012). Unlike other cationic metals like cadmium, lead which can bind

on the negative charges on the surface of cell membrane, the cell surface charge of LAB have

to be modified for removal of arsenic (Halttunen et al., 2007a). In addition to heavy metal

resistance, LAB is also known to degrade phytate, anti-nutritional factor (Anastasio et al.,

2010). Thus, LAB exhibiting probiotic properties with removal of heavy metals and toxic

substances may exhibit promising medical application as probiotic supplement. These

potential values of LAB have prompted to devise this study of isolation and characterisation

of LAB from south Indian fermented food source Idly batter. The antimicrobials substances

and exopolysaccharide produced by potent isolates having beneficial properties like heavy

metal resistance were characterised.