CHAPTER 2

REVIEW OF LITERATURE
2.1. Lactic acid bacteria
Lactic acid bacteria (LAB) are group of Gram-positive bacteria that are devoid of cytochromes
and preferring anaerobic conditions, fastidious, acid-tolerant and strictly fermentative. They are
usually non-motile and non-sporulating bacteria that produce lactic acid. This bacterial group
contains both rods (Lactobacilli and Carnobacteria) and cocci (Streptococci). Different species
of lactic acid bacteria (such as Streptococcus, Leuconostoc, Pediococcus, Aerococcus,
Enterococcus, Vagococcus, Lactobacillus, Carnobacterium) have adapted to grow under widely
different environmental conditions. They are found in the gastrointestinal tract of various
animals, dairy products, seafood products, soil and on some plant surfaces (Ring & Gatesoupe,
1998). Although lactic acid bacteria are not dominant in the normal intestinal microbiota, several
trials have been undertaken to induce an artificial dominance of lactic acid bacteria (Verschuere
et al., 2000). Based on their carbohydrate metabolism LAB are divided into two distinct groups.
The homo-fermentative group utilizes the Embden-Meyerhof-Parnas (glycolytic) pathway to
transform a carbon source chiefly into lactic acid. Hetero-fermentative bacteria produce
equimolar amounts of lactate, CO2, ethanol or acetate from glucose exploiting phosphoketolase
pathway. Homo-fermentative group consist of Lactococcus, Pediococcus, Enterococcus,
Streptococcus. Hetero-fermentative group include Leuconostoc, Weisella (Vasiljevik & Shah,
2008).

2.2. Probiotics
The word probiotics originates from the Greek word for life, and is currently used to name
bacteria associated with beneficial effects for humans and animals. According to WHO
5

guidelines probiotics defined as live organisms which when administered in adequate amounts
confer a health benefit on the host. Probiotics bacteria were first studied by Elie Metchnikoff, a
Noble laureate of 1908 in the field of medicine. He theorized that proteolytic microbes in the
colon produce toxic substances responsible for the aging process and proposed that consumption
of fermented milk would coat the colon with LABs, decreasing intestinal pH, suppressing
proteolytic bacteria and thus leading to slowing of the aging process (Gordon, 2008).
Metchnikoff and his followers ingested milk fermented with Bulgarian Bacillus and reported
health benefits (Vaughan, 1965). To qualify as a probiotic, certain criteria need to be met by a
bacterium: a bacterial strain must be identified completely, be harmless for ingestion, adhere to
mucosal membrane, able to colonize the gut epithelium, stable when stored, must survive the
acid and bile salt concentration persisting in upper GI tract (Verna & Lucak, 2010). Researchers
have studied and used probiotics in a variety of medical conditions. Bowe & Logan, (2011)
discussed the possibility of probiotics to cure acne vulgaris although there was no suitable trial
conducted up to now. Rerksuppaphol & Rerksuppaphol, (2010) tested Lactobacillus acidophilus
and Bifidobacterium bifidum against acute diarrhea in infants and children aged 2 months to 7
years. Probiotics shortened duration of diarrhea (34.1 and 34.8 h as against 58 h with placebo)
and also reduced the number of stools (7.3 and 8 vs 15.9 with placebo). Probiotics are also
helpful in preventing intestinal barrier dysfunction in acute pancreatitis (Lutgendorff et al.,
2009). Probiotic pre-treatment diminished acute pancreatitis induced increase in E. coli passage
(Probiotics 57.4 vs. placebo 223), Cr-EDTA flux (16.7 vs. 32.1 cm/s 10 -6), apoptosis, lipid
peroxidation (0.42 vs. 1.62 pmol MDA/mg protein). Ouwehand et al., (2009) reported efficacy
of their strain L. acidophilus NCFM in the alleviation of allergic rhinitis. Probiotic strain
Lactobacillus fermentum VR1-033PCC diminished atopic dermatitis in fifty-six children aged 6-

18 months and reduced the cases by 54% as compared to only 30% in placebo group. Probiotic
bacteria were also studied for their beneficial effect on autoimmune Encephalomyelitis (Lavasani
et al., 2010), childhood constipation (Bekkali et al., 2007), hypertension (Huey-Shilye et al.,
2009) and found them to be effective.

2.3. Antimicrobial peptide Bacteriocin produced by lactic acid bacteria

Bacteriocins are biologically active protein molecules with a bactericidal mode of action (Tagg
et al., 1976). Bacteriocin may serve as anti-competitors enabling the invasion of strain into an
established microbial community (Margaret & John, 2002). Bacteriocins of Gram positive
bacteria are diverse and their production is not necessarily the lethal event as in the case of Gram
negative bacteria. Some Gram positive bacteria have evolved a bacteriocin specific transport
system whereas others employ sec-dependent export pathway (Margaret & John, 2002). Among
Gram positive bacteria the lactic acid bacteria (LAB) are particularly prolific in bacteriocin
production. Based upon the mass, structure and characteristics, bacteriocins have been divided
into three (Klaenhammer, 1988): Class I bacteriocins are otherwise known as lantibiotics since
they contain post-translationally modified amino acid such as lanthionine and methyllanthionine (Guder et al., 2000). It is further divided into A and B subgroups based on
structure and mode of inhibition (Jung & Sahl, 1991). Type A inhibits bacterial species by
depolarizing the cytoplasmic membrane. They usually range from 21 to 38 amino acids and
larger than type B lantibiotics. Nisin is a type A lantibiotic who is best studied and commercially
exploited bacteriocin. Type B lantibiotics inhibit bacteria by suppressing their enzymes e.g.
mersacidin, it interferes with cell wall biosynthesis (Brotz et al., 1995).

Class II LAB bacteriocins are small (30-60 amino acids), heat stable, non-lanthionine containing
peptides (Jung & Sahl, 1991). They have three subgroups a, b and c. Class IIa members share
conserved amino-terminal sequence (YGNGVXaac) and inhibitory activity towards food borne
pathogen Listeria. Example includes Pediocin ACH, Sakacin A, Leucocin A. Class IIb
bacteriocins inhibit target cells by forming pore in the membrane. Example include Lacticin F
and Lactococcin G. Class IIc bacteriocins are sec-dependent e.g. Acidocin B (Leer et al., 1995).
Class III consist of large, heat-labile bacteriocin such as Helveticins J and V (Joerger &
Klaenhammer, 1986; Vaughan et al., 1992). An additional class of bacteriocin is known recently
that require lipid or carbohydrate moiety for their activity e.g. Leuconocin S (Bruno &
Montiville, 1993) and Lactocin (Uprati & Hindsdill, 1975).

2.3.1. Bacteriocin produced by Streptococcus sp.

Most Streptococcus strains are reported to be pathogenic. Subsequently, bacteriocins isolated
from Streptococcus are mostly pathogenic Streptococci (Nes et al., 2007). Also, those
bacteriocins that have been characterized were originated from few species i.e., Streptococcus
salivarius, S. pyogenes, S. macedonicus, S. mutans, S. bovis, S. uberis, S. thermophilus, S. rattus,
S. phocae (Table 1). Most Streptococci bacteriocins were characterized to be lantibiotics among
that cationic type A-lantibiotics are prevalent (Nes et al., 1997). S. salivarius bacteriocin was
first among Streptococci lantibiotic characterized that effectively inhibited human pathogen
Streptococcus pyogenes (Ross et al., 1993). Mutacins are peptide bacteriocins produced by
Streptococcus mutans. Atleast three different lantibiotics namely mutacin I, II and III were
reported from some S. mutans isolates. Mutacin II showed structural resemblance to the lacticin
481 group of type A lantibiotics (Krull et al., 2000). S. mutans UA140 was reported to produce a

two peptide class II bacteriocin (mutacin IV). Nisin is most prominent lantibiotic bacteriocin that
was reported from Lactococcus lactis. Recently, nisin U produced by Streptococcus uberis
revealed 78% identity to Nisin A, subsequently considered to be a nisin variant (Wirawan et al.,
2006). Whitford et al., (2001) described a lantibiotic bovicin 255 produced by a Streptococcus
bovis isolated from cow rumen, whereas a class II non-lantibiotic, bovicin HJ50 was reported
from similar source by Xiao and co-workers (Xiao et al., 2004). In another study, two different
peptides bacteriocins named BHT-A and BHT-B were reported from Streptococcus rattus
(Hyink et al., 2005). During S. pyogenes screening 10% of the strains were found to produce
bacteriocins. Consequently, a lantibiotic Streptin was produced to homogeneity that showed a
molecular mass of 2.42 kDa (Wescombe & Tagg, 2003). Streptococcal bacteriocins were
reported from fermented foods. Macedocin is a lantibiotic from S. macedonicus, isolated from
artisan cheese (Georgalaki et al., 2002). Thermophilin 13, a two-peptide class IIb bacteriocin
produced by S. thermophilus, isolated from yoghurt (Marciset et al., 1997). An anti-listerial
bacteriocin phocaecin PI80 was produced by S. phocae PI80, isolated from the gut of Penaeus
indicus (Satish & Arul, 2009).

2.3.2. Bacteriocins produced by Enterococcus faecium

Enterococcus faecium is a Gram-positive, homo-fermentative, lactic acid bacteria that is natural
inhabitant of the gastrointestinal tract. Nevertheless, they are also found in fermented foods and
are frequently isolated from starter cultures and cheese producers (Galvez et al., 1998). E.
faecium bacteriocins have gained attentions in recent years as they could be isolated easily from
several fermented foods and because many of them are active against food-borne pathogens such
as Listeria monocytogenes (Giraffa, 1995). E. faecium T136 was isolated from Spanish dry

fermented sausages that produced enterocin A and B. They were active against a wide range of
Gram-positive bacteria, including Listeria and Staphylococci. N-terminal amino acid sequencing
revealed the similarity of enterocin A with pediocin family of bacteriocins whereas enterocin B
showed strong homology to carnobacteriocin A (Casaus et al., 1997). Enterocin P inhibited most
of food-borne Gram-positive pathogenic bacteria, such as L. monocytogenes, S. aureus,
Clostridium perfringens and C. botulinum. It withstood high temperature treatment (121C for
15 min) as well as wide exposure to pH (2.0 11.0), freeze thawing, lyophilization and longterm storage at 4 and 20C (Cintas et al., 1997). Enterocin L50, initially referred to as pediocin
L50 is a plasmid encoded broad-spectrum bacteriocin produced by E. faecium L50. It showed
similarity to small group of cytolytic peptides secreted by certain Staphylococci (Cintas et al.,
1998). Enterocin A, enterocin B and enterocin P like bacteriocins were reported from E. faecium
JCM 5804. They inhibited the growth of Clostridium spp., L. monocytogenes, and vancomycin
resistant Enterococcus (Park et al., 2003). Elotmani et al., (2002), Characterized anti-L.
monocytogenes bacteriocin from E. faecium isolated from Raib, a Morrocon tradition fermented
milk. In a similar manner, another research group reported anti-L. monocytogenes bacteriocinlike inhibitory substance from E. faecium UQ31 (Alvarado et al., 2005). Biochemical and
genetic characterization of enterocin A was performed by Aymerich et al., (1996) and reported
that enterocin A leader sequence contain 18 amino acid residues. This belongs to the doubleglycine leaders which are found among most other small non-lantibiotics bacteriocins, some
lantibiotics and colicin V. Similarly, genetic characterization of enterocin I from E. faecium 6T1a
was performed and it was reported that enterocin I does not belongs to the pediocin family of
bacteriocins (Floriano et al., 1998). E. faecium EK13 produces enterocin A and possess probiotic
properties and proved as a candidate for rabbit probiotics (Laukova et al., 2006).

10

2.3.3. Bacteriocins produced by Lactobacillus plantarum

Lactobacillus plantarum are isolated from various sources but more prominently from fermented
food products. Todorov et al., (2007), isolated L. plantarum AMA-K from naturally fermented
milk and characterized its bacteriocin AMA-K. Bacteriocin production is stimulated by the
presence of Listeria innocua. L. plantarum AMA-K grows in milk and produces only 800 AU/ml
after 24 h. Similarly, bacteriocin ST8KF produced by a kefir isolate L. plantarum ST8KF was
characterized by Powell et al., (2007). Bac ST8KF was heat resistant (121C for 20 min), and
showed bacteriostatic mode of activity. Leal et al., (1998), studied bacteriocin production and
competitiveness of L. plantarum LPC010 in olive juice broth. Todorov et al., (2011), for the first
time isolated bacteriocinogenic L. plantarum ST16Pa from papaya (Carica papaya). It was
active against Pseudomonas, Streptococcus, Staphylococcus, Listeria spp. In another report, L.
plantarum NCIM 2084 was attempted to grow in a simple glucose broth to produce bacteriocin.
Its bacteriocin Planatricin LP84 exhibited a bactericidal and lytic effect against Bacillus cereus
F4810 and Escherichia coli D21 (Suma et al., 1998). A bacteriocin produced by L. plantarum
ATCC8014 showed inhibition of Staphylococcus aureus, E. coli, L. innocuo, P. aeruginosa. Its
apparent molecular weight based on SDS-PAGE analysis was 122 kDa (Lash et al., 2005). Hata
et al., (2010) characterized Plantaricin ASM1 from L. plantarum A-1. It showed stability in a
wide pH range, heat and finally concluded as potential food bio-preservative. Xie et al., (2011)
reported an anti-listerial Pediocin LB-B1 produced by L. plantarum LB-B1, isolated from
Koumiss, a fermented dairy product from China. The gene cluster encoding Pediocin LB-B1
showed 99.8% homology with the operon encoding Pediocin PA-1, suggesting that the two
bacteriocins are identical.

11

2.4. Probiotics in aquaculture

Bacteria are found in every small corner of the aquatic environment. Aquatic animal egg is the
first stage of their life-cycle that could be exposed to bacteria. Therefore, a relatively dense,
nonpathogenic, and diverse adherent microbiota present on the eggs would probably be an
effective barrier against the formation of a colony by pathogens on fish eggs. In addition, the
establishment of a normal gut microbiota may be regarded as complementary to the
establishment of the digestive system, and under normal conditions it serves as a barrier against
invading pathogens (Farzanfar, 2006). Larvae may ingest substantial amounts of bacteria. It is
obvious that the egg microbiota will affect the primary colonization of the larvae (Verschuere et
al., 2000). Kennedy et al., (1998) used probiotic bacteria in the culture of marine fish larvae.
They identified and used probionts for the culture of common snook, red drum, spotted sea trout
and striped mullet. They then observed that the application of probiotic bacteria to larval fish
tanks (from egg through transformation) increased survival, size uniformity, and growth rate.
The periodic addition of bacteria to the tanks altered the microbial communities of both tanks
and fish. In addition, they noticed that the fish eggs incubated with probiotic bacteria were less
likely to develop bacterial overgrowth and die than those incubated without probiotic bacteria.
Carnevali et al., (2004) isolated Lactobacillus fructivorans (AS17B) from sea bream (Sparus
aurata) gut, and then administered it during sea bream development using Brachinons plicatilis
and/or Artemia salina and dry feed as vectors. At the end of the experiments, they found a
significantly decreased larvae and fry mortality in their treated groups. Previously, Gildberg et
al., (1997) had analyzed the effect of a probiotic of lactic acid bacteria in the feed of Atlantic cod
fry (Gadus morha) on growth and survival rates. In their study, a dry feed containing lactic acid
bacteria (Carnobacterium divergens) that had been isolated from adult intestines was given to

12

cod fry. After 3 weeks of feeding the fry, they were exposed to a virulent strain of Vibrio
anguillarum. The number of death was recorded during a further 3 weeks of feeding with feed
supplemented with lactic acid bacteria. A certain improvement in disease resistance was
obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in
surviving fish given feed supplemented with lactic acid bacteria. Lara-Flores et al., (2003) used
two probiotic bacteria and the yeast, Saccharomyces cerevisiae as growth promoters in Nile
tilapia (Oreochromis niloticus) fry. The results of this study indicated that the fry subjected to
diets with a probiotic supplement exhibited greater growth than those fed with the control diet. In
addition, they suggested that the yeast is an appropriate growth-stimulating additive in tilapia
cultivation. Gopalakannan & Arul, (2011) isolated Enterococcus faecium MC13 from the gut of
grey mullet Mugil cephalus and studied their protective effect on Cyprinus carpio after
challenging with pathogen Aeromonas hydrophila. In their study, fish group treated with
probiotic showed reduced mortality (22%) and also fish were healthy but untreated fish group
resulted in 100% mortality.

2.5. Indian traditional fermented foods as source of lactic acid bacteria

Traditional fermented foods are popular products since early history that have formed an integral
part of the diet and it can be prepared in the household or in cottage industry using relatively
simple techniques and equipment (Aidoo et al., 2006). Fermentation was evolved as a
preservation technique during lean periods and prevention technique to counter spoilage of food
products. It is one of the oldest and most economical methods for producing and preserving
foods. In addition to preservation, fermented foods can also have the added benefits of enhancing
flavor, increased digestibility, improving nutritional and pharmacological values (Jeyaram et al.,

13

2009). Lactic acid bacteria perform an essential role in the preservation and production of
wholesome fermented foods. Homo-fermentative and the hetero-fermentative lactic acid bacteria
are generally fastidious on artificial media but they grow readily in most food substrates and
lower the pH rapidly to a point where other competing organisms are no longer able to grow.
Leuconostocs and lactic Streptococci generally lower the pH to 4.0-4.5 and some of the
Lactobacilli and Pediococci to about 3.5 (Steinkraus, 1983). Lactic acid bacteria (LAB)
comprise large part of probiotic microflora. There are many LAB strains that have obtained
generally regarded as safe (GRAS) status and used commonly in commercial food products for
human consumption. Probiotics are mono or mixed cultures of live microorganisms that might
beneficially affect the host by improving the characteristics of indigenous microflora (Holzapfel
et al., 1998). Lactic acid bacterial genera consist of Lactobacillus, Lactococcus, Enterococcus,
Streptococcus, Pediococcus, Leuconostoc, Wesiella etc.
India is traditionally rich in fermented foods. In the Indian subcontinent, making use of
fermented food using local food crops and other biological resources are very common. But the
nature of the products and base material vary from region to region (Sekar & Mariappan, 2007).
Fermented foods like idli and dahi were described as early as 700 BC. At present there are
hundreds of fermented foods with different base materials and preparation methodology. Each
fermented food is associated with unique group of microflora which increases the level of
proteins, vitamins, essential amino acids and fatty acids. However, fermented foods are still
produced traditionally by spontaneous fermentation and only limited knowledge has been
obtained regarding the microflora of these products (Jeyaram et al., 2009). Based upon the basic
ingredients used, fermented foods have been divided into 7 major types (Sekar & Mariappan,
2007): (i) cereal based (with/without pulses) fermented foods, (ii) cereal/pulse and butter milk

14

based fermented food, (iii) cereal based fermented sweets and snacks, (iv) milk based fermented
foods, (v) vegetable, bamboo shoot and unripe fruits based fermented foods, (vi) meat based
fermented foods, and (vii) pulse based fermented foods.

2.5.1. Cereal based (with/without pulses) fermented foods

Cereal based fermented foods are considered as staple foods in their respective regions. Cereals
such as rice (Oryza sativum), ragi flour (Eleusine coracana), wheat flour (Triticum spp.) and
barley flour (Hordeum vulgare) are predominantly used and pulses such as black gram dhal, red
gram, green gram dhal are used. These cereals and legumes are cultivated in India since Indus
valley civilization (9000-5500 BC) period (Samanta et al., 2011). They are considered as
effective substrates for the production of probiotic-incorporated functional food, as they can be
used as a source of non-digestible carbohydrates which stimulate the growth of Lactobacilli and
Bifidobacteria. They contain water soluble fibres like -glucan, arabinoxylan, galactooligosaccharides and fructooligosaccharides, which are prebiotics (Swennen et al., 2006).
Cereals and legumes are fermented by several groups of bacteria in the large intestine, yielding a
variety of fermentation products, particularly short-chain fatty acids (SCFA). The resulting
SCFA are known to provide an acidic environment in the large intestine, which stimulates the
proliferation of probiotic cultures (Roopashri & Vardaraj, 2009; Macfarlane et al., 2006). Mostly
batter is prepared from these basic ingredients and batter is left overnight at room temperature
for fermentation, occasionally sodium bicarbonate is added to provide anaerobic conditions for
the growth of yeast and lactic acid bacteria. During the preparation of Kallappam fermented
toddy is added as additional source of LAB. Fermented batter is either prepared as steamed cakes
(idli) or pan cakes (dosa, appam) before it gets too soured. Predominant microflora isolated from

15

batter of these foods include: Weissella paramesenteroides, Lactobacillus fermentum, L.

plantarum, Streptococcus faecalis, Pediococcus acidilactici, P. cerevisiae, Leuconostoc
mesenteroides (Table 1). L. plantarum AS1 isolated from south Indian fermented food
Kallappam

successfully

prevented

colonization

of

enterovirulent

bacterium

Vibrio

parahaemolyticus in HT-29 cell line (Satish et al., 2011).

2.5.2. Cereal/pulse and butter milk based fermented food

Buttermilk is an additional source of lactic acid bacteria in this type of fermented foods.
Although there are very few dishes reported in this category, these are very popularly consumed
in most part of India. Wiesella paramesenteroides isolated from Mor kuzhambhu showed
antibacterial activities towards food borne pathogens Salmonella typhi and Listeria
monocytogenes (Satish et al., 2010). Blandino et al., (2003) reported non-lactic acid bacteria
Bacillus sp., Micrococcus sp. in rabdi (Table 1).

2.5.3. Cereal based fermented sweets and snacks

These foods are consumed mostly during festival times and other special occasions. Wheat, rice
and barley flours are predominantly used cereals. Sugar or salt is added compulsorily in all food
items and this selects only those microbes which can survive low water activity. Pathogens are
discouraged and it allows growth of sugar/salt tolerant yeast and lactic acid bacteria. Fermented
sweets and snacks are popularly consumed throughout India, consequently many reports are
available for this category but only few reports are available on microflora isolation. L.
fermentum, L. buchneri, L. plantarum, L. acidophilus, L. mesenteroides, Lactococcus lactis,
Streptococcus lactis and S. faecalis were isolated from this class of fermented foods (Table 1).

16

2.5.4. Milk based fermented foods

Milk and milk based products are consumed most popularly due to their nutritive value. For the
same reason milk is easily spoiled by pathogenic microorganisms, hence fermentation of milk
using lactic acid bacteria is preferred for prevention. Lactic acid bacteria convert milk sugar
lactose into lactic acid and produces antibacterial substance bacteriocin to suppress spoilage
bacteria. Dahi or curd is most popular traditional Indian fermented product prepared by
fermentation of milk by lactic acid bacteria. Dahi differs from yogurt in its use of mixed starters
of mesophilic lactococci. A principle flavour-inducing metabolite is diacetyl, which is
appreciated more by people of South Asian origin compared to the acetaldehyde flavour in
yogurt (Yadav et al., 2007b). Yak (Bos grunniens; now Poephagus grunniens) is one of a few
domesticated animals capable of surviving in extreme environmental conditions. It is mainly
found in the highlands of the Nepalese Himalayas, India (Kashmir and Arunachal Pradesh),
China (Tibetan highlands), Mongolia and Bhutan. The composition of yak milk is 16.917.7 g/l
dry matter, 4953 g/l protein, 5572 g/l fat, 4550 g/l lactose and 89 g/l minerals (Prashant et
al., 2009). Yak milk is processed into a number of dairy products such as fermented milk
(Kurut), cheese (Chhurpi), Chhur churpen, Churkham, Chhu, Philuk, Shyow and Maa. The
chemical composition of yak cheese is around 68.2% of total solid (TS), 49.4% of butterfat on a
dry matter basis and 1.37% of salt. It is largely consumed in the Himalayan highland and its
industrial production is not yet standardized (Prashant et al., 2009).
LAB species isolated from fermented milk products include Streptococcus cremoris, S. lactis, S.
thermophilus, Lactobacillus bulgaricus, L. acidophilus, L. helveticus, L. cremoris, L. plantarum,
L. curvatus, L. fermentum, L. paracasei subsp. pseudoplantarum, L. alimentarius, L. kefir, L.
hilgardii, Enterococcus faecium, Leuconostoc mesenteroides, L. farciminis, L. brevis,

17

Lactococcus lactis subsp. cremoris, L. casei subsp. casei and L. bifermentans (Table 1). There
are reports that LAB isolated from dahi can be used to cure intestinal disease such as diarrhea
(Agarwal & Bhasin, 2002), intake of dahi has anti-cholesteremic (Sinha & Sinha, 2000), anticarcinogenic (Arvind et al., 2010), anti-diabetic (Yadav et al., 2007a), angiotensin-converting
enzyme inhibition effect (Harun-ur-Rashid et al., 2007) and anti-atopic dermatitis effect
(Watanabe et al., 2009).

Mitra et al., 2007, isolated Lactococcus lactis from dahi which

produced nisin like (Nisin Z) bacteriocin that inhibited important food pathogens Listeria
monocytogenes and Staphylococcus aureus.

2.5.5. Vegetable, bamboo shoot and unripe fruits based fermented foods
The lactic acid fermentation of vegetables, applied as a preservation method for the production
of finished and half-finished products, is considered as an important technology because of its
capability to improve the nutritive value, palatability, acceptability, microbial quality and shelf
life of the fermented product (Kingston et al., 2010). Moreover, this is a remarkable procedure to
store the perishable vegetable in absence of cold-storage or refrigeration, where majority of rural
people cannot afford canned or frozen foods. Certain fermented vegetable products (gundruk,
sinki, iniziangsang) are said to be good appetizers and the ethnic people use these foods for
remedies from indigestion (Tamang & Tamang, 2009). Fermented bamboo shoot (BSs) products
are consumed as a traditional food by ethnic people of North-Eastern states of India (Tamang et
al., 2009). In India, BSs are harvested annually in Sikkim (26.2 t), Meghalaya (435 t) and
Mizoram (426.8 t). Bamboo shoots are low in fat and cholesterol, but very high in potassium,
carbohydrates and dietary fibres. Many nutritious and active materials (vitamins and amino
acids) and antioxidants (flavones, phenols and steroids) can be extracted from bamboo shoots

18

(Choudhury et al., 2011). LABs are the dominant microorganisms in ethnic fermented vegetables
and bamboo shoot products (Tamang et al., 2009). Pediococcus pentasaceous, Lactobacillus
cellubiosus, L. plantarum, L. fermentum, L. brevis, L. mesenteroides, Lactococcus lactis,
Enterococcus faecium, and P. acidilactici are predominant LAB species found in fermented
vegetables (Table 1). Tamang et al., (2009) determined the functional properties of lactic acid
bacteria isolated from ethnic fermented vegetables (gundruk, sinki, khalpi and inziangsang) of
the Himalayas. LAB strains showed strong acidification and coagulation activities. They showed
antimicrobial activity, particularly a strain L. plantarum isolated from inziangsang, a fermented
leafy vegetable product, was inhibitory towards Staphylococcus aureus and Pseudomonas
aeruginosa. LAB strains showed various enzymatic activities such as alkaline phosphatase,
esterase, lipase, leucine arylamidase, valine arylamidase, cysteine-arylamidase, acid phosphatase,
napthol-AS-B1-phosphohydrolase,

-galactosidase,

-galactosidase,

-glucosidase,

glucosidase, N-acetyl--glucosaminidase and also degraded oligosaccharides. Some strains of L.

plantarum showed more than 70% hydrophobicity and adherence to the mucus secreting HT-29
MTX cells. L. plantarum isolated from ayurvedic medicinal food Kanji or Kanjika is a potential
source of Vitamin B12 (Madhu et al., 2010). During fermentation of radish tap root product sinki
L. plantarum utilizes mannitol to remove the bitter flavor from finished product (Tamang &
Sarkar, 1993). Bamboo shoot based fermented foods contain Lactobacillus plantarum, L. brevis,
L. corniformis, L. delbrueckii, L. fermentum, Leuconostoc fallax, Lactococcus lactis, L.
mesenteroides, Enterococcus durans, Streptococcus lactis, L. casei and Tetragenococcus
halophilus as predominant LAB species, they also showed functional probiotic properties
(Tamang et al., 2009)

19

2.5.6. Meat based fermented foods

Meat is highly susceptible to microbial spoilage. Drying, smoking or fermentation of meat is
critical steps in the traditional processing of meat (Oki et al., 2011). In India, North-Eastern
region people ferment meat of yak, goat, pig, fish and crab to preserve for longer period.
Kargyong is an ethnic sausage-like fermented product prepared from yak, beef and pork meat.
Three varieties of Kargyong are prepared and consumed: yak kargyong (prepared from yak
meat), lang kargyong (prepared from beef) and faak kargyong (prepared from pork). Yak
kargyong is a popular fermented sausage in Sikkim, Ladak, Tibet, Arunachal Pradesh and
Bhutan in the Himalayas (Rai et al., 2010). Fermented fish products are important dietary
components in the protein deficient far East especially in southeast Asia. Preservation of fish by
salt is an age old technology. This method of preservation still enjoys popularity in many
developing countries owning to its simplicity and low cost of processing. When fatty fishes are
salted there is usually a certain degree of fermentation involved. Fermentation of fish is brought
about by autocatalytic enzymes from fish and microorganisms in the presence of high salt
concentration (Majumdar & Basu, 2010). Lactococcus lactis subsp. cremoris, Lactococcus
plantarum, Enterococcus faecium, Lactobacillus fructosus, L. amylophilus, L. corneformis subsp.
torquens are predominant LAB species reported in fermented fish. Lactobacillus sake, L.
curvatus, L. divergens, L. carnis, L. sanfrancisco, Leuconostoc mesenteroides, E. faecium, L.
plantarum, L. brevis, Pediococcus pentosaceous are reported in fermented meat products of
Eastern Himalayas (Table 1). These LAB showed inhibitory activity towards Klebsiella
pnemoniae which is a contaminant of stored meat. Also, they demonstrated probiotic characters
such as enzymes production and hydrophobicity (Rai et al., 2010).

20

Table 1: Lactic acid bacteria isolated from Indian fermented foods

Fermented food
Usual
Place of
Lactic acid Bacteria Isolated
Composition/Ingredi Origin/Us
ents
age
Cereal Based (with/without pulses) Fermented Foods
Koozhu
Eleusine coracana
Tamil
Weissella paramesenteroides,
(ragi) flour, boiled
Nadu
Lactobacillus fermentum
rice, non-fat yoghurt
Pazhaiya soru
Rice, curd and salt
Tamil
Streptococcus faecalis,
Nadu
Pediococcus acidilactici
Idli
Rice, black gram
South
Leuconostoc mesenteroides, S.
dhal, table salt,
India
faecalis, P. cerevisiae
fenugreek seeds
Dosa
Rice, black gram dhal
South
L. mesenteroides, S. faecalis
(either raw or
India
parboiled rice), table
salt
Adai Dosa

Kallappam

Dhokla

Boiled rice, Bengal

gram, red gram, black
gram, green gram
Boiled or raw rice,
coconut toddy

South
India

Bengal gram dhal,

rice and leafy
vegetables

North
India

South
India

Ambali

Pediococcus sp.,
Streptococcus sp.,
Leuconostoc sp.
L. fermentum, L. plantarum

L. fermentum, L.
mesenteroides, S. faecalis

Ragi (Millet) flour

India
and rice
Cereal/pulse and butter milk based fermented food
Rabdi (Rabadi)
Flour of Barley, Pearl Rajasthan
millet, corn or
soybean and country
buttermilk
Mor Kuzhambhu
Butter milk, gram
Tamil
flour, vegetables,
Nadu
spices
Cereal based fermented sweets and snacks
Jilebi
Wheat, sugar and
South
curd
India

L. fermentum, L.
mesenteroides, S. faecalis

Bhaturu or
Indigenous bread

L. plantarum, L. acidophilus,
L. mesenteroides,
Lactococcus lactis

Wheat and starter

material Khameer/
Malera

Himachal
Pradesh

21

Reference

Satish et al.,
2010
Ramakrishnan,
1977; 1979
Mukherjee et al.,
1965, Steinkraus
et al., 1967
Labana, &
Kawatra, 1986,
Chavan, &
Kadam, 1989;
Steinkraus, 1996
Chavan, &
Kadam, 1989
Satish et al.,
2010
Ramakrishnan et
al., 1976;
Blandino et al.,
2003, Roy et al.,
2007
Ramakrishnan,
1977; 1979

Bacillus sp., Micrococcus sp.

Blandino et al.,
2003

Weissella paramesenteroides

Satish et al.,
2010

L. fermentum, S. lactis, L.
buchneri, S. faecalis

Ramakrishnan,
1977, Prakash et
al., 2004
Tamang, 1998,
Thakur et al.,
2004; Kanwar et

al., 2007
Milk based fermented foods
Curd (Dahi,
Milk
Thayir)

India

Chhurpi or Durkha
or churapi

Yak milk is preferred

for making this
cheese although any
other fresh milk may
be used

Arunachal
Pradesh

Chhu

Yak or cow milk

Sikkim

Philu or Philuk

Cow or yak milk

Sikkim

Shyow

Cow/yak milk

Sikkim

Mohi

Cow milk

Sikkim

Somar

Cow milk

Sikkim

Khadi

Buttermilk/curd

Gujrat

S. cremoris, S. lactis,
S.thermophilus,
L. bulgaricus, L. acidophilus,
L. helveticus, L. cremoris,
Pediococcus pentosaceous, P.
acidilactici, W. cibara, W.
paramesenteroides, L.
fermentum, L. plantarum,
Lactobacillus delbrueckii
subsp. indicus
L. plantarum, L.curvatus, L.
fermentum, L. paracasei
subsp. pseudoplantarum, L.
alimentarius, L. kefir, L.
hilgardii, Enterococcus
faecium and Leuconostoc
mesenteroides, L. helveticus

L. farciminis, L. brevis, L.
alimentarius, Lactococcus
lactis subsp. cremoris
L. casei subsp. casei,
L.bifermentans
and Enterococcus faecium
L.bifermentans, L. paracasei
subsp. pseudoplantarum
L. alimentarius, Lactococcus
lactis subsp. lactis, L. lactis
subsp. cremoris
L. paracasei subsp.
pseudoplantarum
Pediococcus sp.

Vegetable, Bamboo shoot and unripe fruits based fermented foods

Gundruk
Leaves of
Arunachal Pediococcus pentasaceous, L.
mustard/radish/
Pradesh
fermentum, L. casei, L. casei
cauliflower
subsp pseudoplantarum, L.
plantarum
Sinki

Radish root

Northeast
India

22

L. casei, L. brevis, L.
plantarum, Leuconostoc
fallax, L. fermentum

Srinivasan &
Banerjee, 1946;
Steinkraus, 1996;
Patil et al., 2010;
Davies, 1940;
Dellaglio et al.,
2005

Tamang &
Sarkar, 1988;
Tamang, 1998,
Tamang et al.,
2000; Singh et
al., 2007a;
Tiwari, &
Mahanta, 2007;
Prashant et al.,
2009
Dewan &
Tamang 2007b
Dewan &
Tamang 2007a
Dewan &
Tamang 2007a
Dewan &
Tamang 2007a
Dewan &
Tamang 2007a
Sukumar &
Ghosh 2010
Dahal et al.,
2005; Singh et
al., 2007b;
Tamang &
Tamang, 2009
Tamang &
Sarkar, 1993;
Singh et al.,

Sauerkraut or
Sauerkohi
Soibum or Soijim

Cabbage

India

Bamboo shoots

Manipur,
Nagaland

Soidon

Bamboo shoots

Manipur

Hiring

Bamboo shoots

Northeast
India

Ekung

Bamboo shoots

Manipur

Eup

Bamboo shoots

Mesu

Bamboo shoots

Arunachal
Pradesh
Darjeeling
hills and
Sikkim

Khalpi

Cucumber

Sikkim

Goyang

Wild plant maganesaag (Cardamine

macrophylla Willd.)
leaves
Mustard leaves

Darjeeling
hills and
Sikkim

Carrot or beet root,

rice, mustard

North
India

L. plantarum, L. brevis,
Lactococcus lactis,
Enterococcus faecium,
Pediococcus pentosaceus
Lactobacillus plantarum, L.
brevis, Pediococcus
acidilactici
L. pentosus, L.
paraplantarum, L. plantarum

Manipur

Enterococcus faecium, L.

Inziangsang

Kanji

Meat based fermented foods

Puntius sophore
Ngari

Nagaland,
Manipur

23

L. mesenteroides, L.
plantarum
Lactobacillus plantarum, L.
brevis, L. corniformis, L.
delbrueckii, Leuconostoc
fallax, L. lactis, L.
mesenteroides, Enterococcus
durans,
Streptococcus lactis, Bacillus
subtilis, B. licheniformis, B.
coagulans
Lactobacillus brevis,
Leuconostoc fallax, L. lactis,
L. plantarum, Carnobacterium
sp., E. faecium
L. plantarum, Lactococcus
lactis
Lactobacillus plantarum, L.
brevis, L.
casei, Tetragenococcus
halophilus
L. plantarum , L. fermentum
L. plantarum, L. brevis, L.
curvatus, Leuconostoc
citreum, Pediococcus
pentosaceus
L. brevis, L. plantarum

2007b
Steinkraus, 1996
Tamang &
Tamang,
2009; Jayaram et
al., 2009

Tamang &
Tamang, 2009;
Jeyaram et al.
2010
Singh et al.,
2007b; Tamang
& Tamang, 2009
Singh et al.,
2007b

Tamang &
Tamang, 2009
Tamang &
Tamang, 2009

Tamang, 1998;
Tamang &
Tamang, 2010
Tamang &
Tamang, 2009

Tamang &
Tamang, 2009
Reddy et al.,
2007; Madhu et
al.,2010;
Kingston et al.
2010
Thapa et al.,

(Phoubu) Fish

and Assam

Hentak

Esomus danricus
(Fish), petioles of
Alocasia macrorhiza

Manipur

Tungtap

Danio sp. (Fish)

Meghalaya

Lang kargyong

Meat of cattle

Eastern
Himalayas

Yak kargyong

Meat of yak

Eastern
Himalayas

Faak kargyong

Meat of pig

Kheuri
Lang satchu

Yak/beef meat
Red meat of beef

Eastern
Himalayas
Sikkim
Sikkim

Yak satchu
Suka Ko Masu

Red meat of yak

Red meat of buffalo
or goat

Chilu
Chartayshya

Yak/beef/lamb meat
Red meat of cattle

Geema

Red meat of cattle

Western
Himalayas

Arjia

Red meat of cattle

Western
Himalayas

Pulse Based Fermented Foods

Kinema
Soybeans

Tungrymbai

Soybeans

fructosus, L. amylophilus, L.
plantarum
Lactococcus lactis sub sp.
cremoris, L. plantarum,
Enterococcus faecium, L.
fructosus, L. amylophilus,
Lactococcus lactis sub sp.
cremoris, L. plantarum,
Enterococcus faecium, L.
fructosus, L. corneformis sub
sp. torquens,
Lactobacillus sake, L. curvatus,
L. divergens, L. carnis, L.
sanfrancisco, Leuconostoc
mesenteroides, E. faecium
L. plantarum, L. sake, L.casei,
L. curvatus, L. carnis, L.
divergens, L. sanfrancisco, Leu.
mesenteroides, E. faecium
L. brevis, L. plantarum, L.
carnis, L. mesenteroides
Not reported
L. casei, L. carnis, Pediococcus
pentosaceous
E. faecium, P. pentosaceous
L. plantarum, L. carnis, E.
faecium

2004; Jeyaram et
al., 2009
Thapa et al.,
2004; Jeyaram et
al., 2009

Not reported
Enterococcus hirae,
Pediococcus pentosaceous,
Weissella cibaria
Enterococcus durans, E. hirae,
Leuconostoc mesenteroides, L.
citreum, Pediococcus
pentosaceous
Enterococcus hirae, E. faecalis,
Pediococcus pentosaceous

Rai et al., 2009

Rai et al., 2009;
Oki et al., 2011

Darjeeling
Sikkim

Enterococcus faecium

Meghalaya

Enterococcus faecium

Kiers et al.,
2000; Sarkar et
al., 2002,
Tamang, 2003;
Singh et al.,
2007b
Dike, & Odunfa,
2003, Murughar,

Sikkim
Darjeeling
hills and
Sikkim
Sikkim
Western
Himalayas

24

Thapa et al.,
2004, Murugkar
&
Subbulakshmi,
2006
Rai et al., 2010

Rai et al., 2010

Rai et al., 2010

Rai et al., 2009
Rai et al., 2010
Rai et al. 2010
Rai et al., 2010

Oki et al., 2011

Oki et al., 2011

Wadi

Black gram and oil

Punjab,
West
Bengal

L. mesenteroides, L.
fermentum

Wari

Black bean and

soybean

Uttar
Pradesh

Lactobacillus bulgaricus
Streptococcus thermophilus

Masyaura

Blackgram or
Darjeeling
greengram, Colocosia hills and
tuber, ashgourd or
Sikkim
radish

Pediococcus pentosaceous,
Pediococcus acidilactic, and
Lactobacillus sp.

& Subblakshmi,
2006; Tamang et
al.,
2009; Sohliya et
al., 2009
Batra & Millner,
1974; Sandhu et
al., 1986;
Sandhu & Soni
1989; Aidoo et
al., 2006
Tewary &
Muller, 1989,
Tewary &
Muller, 1992;
Kulkarni et al.,
1997
Dahal et al.,
2005; Dahal et
al., 2003.

2.5.7. Pulse based fermented foods

Black gram, soyabean, Bengal gram, red gram and green gram are most commonly used pulses
in this type of fermented foods. Soyabean (Glycine max) is a summer leguminous crop, grown
under rain fed conditions in upland terraces as a sole crop as well as mixed crop with rice and
maize up to an elevation of 1,500 m in North-East India. Due to increased Mongolian population,
they consume different fermented foods of soyabean as a tradition. Food researchers have
documented number of soyabean based Indian fermented foods (Tamang et al., 2009).
Fermented soyabean food is an economical source of plant protein as compared to animal and
milk products on the basis of protein cost per kg, which is easily accessible to rural poor of
North-East region. A remarkable increase in free amino acids, mineral contents, vitamin-B
complex and antioxidant activity were reported during kinema fermentation (Sarkar et al., 1997;
Tamang & Nikkuni, 1998; Tamang et al., 2009). Increase in carotene and folic acid has been
25

reported in tungrymbai (Murungkar & Subbulakshmi, 2006). LAB reported includes

Enterococcus faecium, L. mesenteroides, L. fermentum, Lactobacillus bulgaricus, Streptococcus
thermophilus, Pediococcus pentosaceous and P. acidilactici (Table 1).

2.6. Adherence of lactic acid bacteria to intestinal cell line and inhibition of pathogen
adherence
HT-29 and Caco-2 cells are human intestinal cell lines expressing morphologic and physiologic
characteristics of normal human enterocytes and these have been exploited to elucidate the
mechanisms mediating enteropathogen adhesion. More recently, these cell lines were used to
select and subsequently assess lactic acid bacteria on the basis of their adhesion properties
(Dunne et al., 2001). Nevertheless, they have also been used to study inhibition of pathogen
adhesion by lactic acid bacteria. Spurbeck & Arvidson, (2010) studied the inhibitory effect of
lactic acid bacteria strain Lactobacillus jensenii over pathogenic bacterium Neisseria gonorrhoea
during adherence to epithelial cells. Inhibitory protein produced by Lactobacillus jensenii blocks
gonococcal binding to extracellular matrix component. Similarly, probiotics attenuated
Campylobacter jejuni association with and internalization into E12 cells and translocation to the
basolateral medium of transwells (Alemka et al., 2010). Also, the probiotic agents L. plantarum
299v and L. rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing
pathogenic E. coli to HT-29 cells (Mack et al., 1999). In another experiment, Lactobaillus
rhamnosus GG reduced morphological changes and diminished the number of A/E lesions
induced in response to EHEC 0157:H7 infection. With probiotic pre-treatment there was
corresponding attenuation of EHEC-induced drop in electrical resistance and the increase in
barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayer against
EHEC-induced redistribution of the Claudin-I and Zo-I tight junction proteins (Johnson-Henry et
26

al., 2008). Coconnier et al., (1992) with the help of scanning electron microscope determined
that L. acidophilus BG2F04 interacted with the well-defined apical microvilli of Caco-2 cells
with cell damage and with mucus secreted by the sub-population of HT-29MTX-cells.
Adlerberth et al., (1996) defined a mannose specific adherence mechanism in L. plantarum
conferring binding to the human colonic cell line HT-29.

2.7. Protective Roles of Probiotics on Colon Cancer

CRC is the second most common cause of mortality from malignant disease in Europe with 1,90,
000 new cases per year. Prognosis for advanced CRC is poor (Sant et al., 1995) and hence
prevention is required to control the incidence of the disease. Many studies confirm the
involvement of the endogenous microflora in the onset of colon cancer. This makes it reasonable
to think that changing the intestinal microflora could influence tumour development. Many
studies confirm the involvement of the endogenous microflora in the onset of colon cancer. This
makes it reasonable to think that changing the intestinal microflora could influence tumour
development. In one such experiment, Le Leu et al., (2005) used symbiotic combination of
resistant starch and probiotic bacteria to treat experimentally induced colon cancer. They used
Sprague-Dawley rats as their animal model and fed them semipurified diet containing resistant
starch, L. acidophilus and Bifidobacterium lactis (1 x 1010 CFU/g). The symbiotic combination
significantly facilitated the apoptotic response to a genotoxic carcinogen in the distal colon of
rats. In another such experiment, Femia et al., 2002 demonstrated the anti-tumorigenic activity of
the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus
rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in male
F344 rats. Similarly, Gallaher & Khil, (1999) used synbiotic combination of Bifidobacterium and
oligofructose that reduced aberrant crypt number in five of six experimental rats. More work on
27

Bifidobacterium strains employed to treat colorectal cancer was undertaken. Challa et al., (1997)
conducted anticancer trial on male Fisher 344 rats using Bifidobacterium longum. Feeding of B.
longum reduced the number of aberrant crypt foci to 143 9 as against untreated carcinogen
control with 187 9 aberrant crypts. Other noteworthy work on anti-colorectal cancer property
of Bifidobacterium longum was performed by Singh et al., (1997) and Reddy & Revenson,
(1993). Randomized trial of dietary fiber and Lactobacillus casei administration for prevention
of colorectal cancer was performed by Ishikawa et al., (2005).They reported that the occurrence
rate of tumors with a grade of moderate atypia or higher was significantly lower in group
administered L. casei. But no significant difference in the development of new colorectal tumor
was observed with administration of L. casei. McIntosh et al., (1999) fed rats with Lactobacillus
acidophilus (Delvo Pro LA-1), Lactobacillus rhamnosus (GG), Bidobacterium animalis (CSCC
1941) and Streptococcus thermophilus (DD145) and strains were examined for their influence on
colon cancer. There was 25% reduced colon cancer in the L. acidophilus treated rats compared to
untreated DMH control.

28