Review of Literature
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Review of Literature
Introduction
History and the Definition of Probiotic
The word ‘probiotic’ comes from Greek language ‘pro-bios’ which means ‘for
life’. The use of lactic acid bacteria as feed supplements goes back to pre-Christian times
when fermented milks were consumed by humans. It was not until the beginning of this
century that Metchnikoff, working at the Pasteur Institute in Paris, started to put the
subject on to a scientific basis. In 1908, he proposed the beneficial effects of probiotic
microorganisms on human health. Metchnikoff hypothesized that Bulgarians are healthy
and lived long because of the consumption of fermented milk products which consists of
Lactobacillus spp. Therefore, these bacteria affect the gut microflora positively and
decrease the microbial toxic activity (Gismondo et al., 1999; Chuayana et al., 2003). The
term ‘probiotic’ was used in 1965 for the first time by Lilley and Stillwell to describe
substances which stimulate the growth of other microorganisms. The word ‘probiotic’
was used based on its mechanism and the effect on human health. Parker (1974) defined
‘probiotic’ as ‘substances and organisms which contribute to intestinal microbial
balance’. According to Fuller (1989) probiotic is a live microbial supplement which
affects host’s health positively by improving its intestinal microbial balance. Guarino et
al. (1998) defined probiotics as “living microorganisms, which upon ingestion in certain
numbers exert health benefits beyond inherent basic nutrition”. Later a lot of researchers
defined probiotics as:
Naidu et al. (1999) - ‘A microbial dietary adjuvant that beneficially affects the host
physiology by modulating mucosal and systemic immunity, as well as improving
nutritional and microbial balance in the intestinal tract’.
Salminen et al. (1998)- ‘A live microbial food ingredient that is beneficial to health’.
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Schrezenmeir and de Vrese, (2001)- ‘A preparation of or a product containing viable,
defined microorganisms in sufficient numbers, which alter the microflora in the host and
exert beneficial health effects in the host’.
FAO/WHO (2002)- ‘Live microorganisms which when administered in adequate
amounts confer health benefit on the host’.
Probiotics are used in a large variety of fields relevant to human and animal
health. Probiotic products contain one or several species of probiotic bacteria. Most of
the products are dairy based like fermented milk or as lyophilized powders in the form of
tablets. The oral consumption of probiotic microorganisms produces a protective effect
on the gut flora. Lots of studies suggest that probiotics have beneficial effects on
microbial disorders of the gut. They are known to have a positive therapeutic effect
against traveller’s diarrhoea, antibiotic associated diarrhoea and acute diarrhoea
(Gismondo et al., 1999; Ouwehand et al., 1999). More than 400 bacterial species exist in
human intestinal tract. Lots of factors may change the balance from potentially beneficial
or health promoting bacteria like Lactobacilli and Bifidobacteria to potentially harmful
or pathogenic microorganisms like Clostridia species. It makes the host more susceptible
to illnesses. In this case the prevalence of the beneficial bacteria must be supported. Use
of probiotics helps to protect the host from various intestinal disorders by increasing the
number of beneficial bacteria (Fooks et al., 1999). The most commonly used
microorganisms in probiotic products are the lactic acid bacteria (LAB) and it is
important to know how these LAB affect the immune status of the consumer. The
probiotic approach is attractive because it is a reconstitution of the natural condition
which means repairing a deficiency rather than the addition of foreign chemicals to the
body which may have toxic consequences or, as in the case of antibiotics induce
resístance and compromise subsequent therapy. The discovery that probiotics can
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stimulate an immune response (Fuller and Perdigon, 2000) provides a scientific basis for
some of the observed probiotic effects. The probiotics which are used to feed both man
and animals are given in Table 1.
Table 1. Microorganisms used in probiotic products
Homofermenter
Facultative homofermenter
Obligate heterofermenter
Enterococcus faecium
Lactobacilus bavaricus
Lactobacillus brevis
E. faecalis
L. casei
L. buchneri
Lactobacillus acidophilus
L. coryniformis
L. cellobiosus
L. lactis
L. curvatus
L. confuses
L. delbrueckii
L. plantarum
L. coprophilus
L. leichmannii
L. sake
L. fermentatum
L. salivarius
L. sanfrancisco
Streptococcus bovis
Leuconostoc dextranicum
S. thermophilus
Leu. mesenteroides
Pediococcus acidilactici
Leu. paramesenteroides
P. damnosus
P. pentosaceous
Lactic acid bacteria as probiotics
Lactic acid bacteria are non-spore forming, gram positive and catalase negative
without cytochromes, non-aerobic or aerotolerant, fastidious, acid-tolerant and strictly
fermentative bacteria with lactic acid as the major end product during sugar
fermentation. LAB genera isolated from various fermented foods are Lactobacillus,
Pediococcus, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Streptococcus,
Tetrazenococcus, Carnobacterium, Vagococcus and Weissella (Stiles and Holzapfel,
1997; Carr et al., 2002; Salminen et al., 2004). LAB produce organic acids during
fermentation, mostly lactic acid which is the characteristic fermentative product and
which reduces the pH of the substrate to a level where the growth of pathogenic,
putrefactive, and toxinogenic bacteria are inhibited (Holzapfel et al., 1995). LAB are
conferred the GRAS (generally recognized as safe) status in foods (Donohue and
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Salminen, 1996). Many species of LAB can also act as “biopreservers” and some of
them are exploited commercially (Holzapfel et al., 2003).
Lactic acid bacteria: Sources
Lactic acid bacteria are widely distributed in the nature. In this group are
included representatives of the genus Lactobacillus, Lactococcus, Pediococcus and
Leuconostoc. They could be isolated from soil, water, plants, silages, waste products, and
also from the intestinal tract of animals and humans. The lactic acid fermentation plays
an essential role in the production of all dairy products and is involved in the production
of many foods and drinks – sausages, pickles, boza etc. They should possess stable
fermentation characteristics and should be resistant to bacteriophages (Lee, 1996).
Lactobacilli are known to be involved in many locally fermented foods. They have been
isolated from fermented cassava products e.g. gari, fufu, lafun, fermented cereal grains
and milk curd (Okafor and Okafor, 1977; Oyewole et al., 1988).
Identification of Probiotic Strains
Probiotics in human gastrointestinal tract are identified by colony morphology,
fermentation
patterns,
serotyping
or
some
combination
of
these.
Classical
microbiological techniques are really important for selection, enumeration and
biochemical characterization but it is not efficient to classify a culture by classical
methods. With the developing technology about the molecular typing it is getting more
reliable to identify and differentiate bacterial strains. Molecular characterization methods
are powerful even between closely related species. There are number of alternative
taxonomic classification methods which were well known including hybridization with
species-specific probes and generation of profile PCR applicants by species-specific
primers (Klaenhammer and Kullen, 1999). The most powerful and accurate methods is
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sequencing (Coeuret et al., 2003). The application of 16S rRNA is one of the best and
most reliable approaches to identify bacteria on a phylogenetic basis.
Mechanism of Probiotic Action
Probiotic microorganisms affect host health by different mechanisms. Many
studies have been trying to explain how probiotics could protect the host from the
intestinal disorders. These mechanisms are listed below briefly (Salminen et al., 1999;
Castagliuoluolo et al., 1999; Rolfe, 2000).
Production of inhibitory substances: Production of some organic acids,
hydrogen peroxide and bacteriocins
Blocking of adhesion sites: Probiotics inhibit the pathogens by adhering to the
intestinal epithelial surfaces by competitively blocking the adhesion sites.
Competition for nutrients: Despite lack of studies in vivo, probiotics inhibit the
pathogens by consuming the nutrients which pathogens need.
Stimulation of immunity: Stimulation of specific and nonspecific immunity
Degradation of toxin receptor: Because of the degradation of toxin receptor on
the intestinal mucosa, it was shown that S. boulardii protects the host against
C.difficile intestinal disease.
Selection Criteria for Probiotic lactic acid bacteria
In order to exert its beneficial effects, a successful potential probiotic strain is
expected to have a number of desirable properties. The selection criteria are enlisted in
Table 2 (Ouwehand et al., 1999).
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Table 2. Selection criteria for probiotics
Probiotic Strain
Remarks
Properties
Human origin
Although Saccharomyces
boulardii is not of human origin, this
criterion is important for species dependent health effects
Acid and bile
Important for oral consumption, survival through the intestine,
tolerance
adhesiveness and metabolic activity
Adhesion to mucosal Important to improve immune system, competition with pathogens,
surface
maintain metabolic activity, prevent pathogens colonization.
Safe for food
Identification
and
characterization
of
strains
accurately
documented safety. No invasion of intestinal mucus
Clinically validated
and documented
health effects
Good technological
properties
Minimum effective dosage has to be known for each particular
strain and in different products
Survival in products if viable organisms are required, strain
stability, culturable in large scales, oxygen resistance, have no
negative effects on product flavor
The selection criteria can be categorized in four basic groups. Appropriateness,
suitability for technical application, competitiveness, performance and functionality
(Klaenhammer and Kullen, 1999). Strains which have these criteria should be used in
order to get effective results on health and functional probiotic lactic acid bacteria.
Saarela et al. (2000) proposed the properties of probiotics in three basic groups as; safety
aspects, aspects of functionality and technological aspects. Some major selection criteria
will be discussed in detail as follows.
Acid and Bile Tolerance
The first criteria for probiotic strains are their tolerance to acid and bile. Bacteria
used as probiotic strains are joined in the food system with a journey to the lower
intestinal tract via the mouth. In this food system, probiotic bacteria should be resistant
to the enzymes like lysozyme in the oral cavity. It goes to the stomach and enters the
upper intestinal tract which contains bile. The strains should have the ability to resist the
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digestion process. The viability and activity of probiotics are needed at the lower
digestive tract, these organisms should withstand the adverse conditions encountered in
the host’s upper gastrointestinal tract (Ding and Shah, 2007). Strains need to be resistant
to the stressful conditions of the stomach (pH 1.5-3.0) and upper intestine which contain
bile (Chou and Weimer, 1999). To show probiotic property, they should reach to the
lower intestinal tract and maintain themselves there. Bile acids are synthesized in the
liver from cholesterol and sent to the gallbladder and secreted into the duodenum in the
conjugated form (500-700 ml/day). In the large intestine these acids suffer some
chemical
modifications
(deconjugation,
dehydroxylation,
dehydrogenation
and
deglucuronidation) due to the microbial activity. Conjugated and deconjugated bile acids
show antimicrobial activity especially on Escherichia coli subspecies, Klebsiella spp.,
and Enterococcus spp. The deconjugated acid forms are more effective on gram positive
bacteria (Dunne et al., 2001). Chou and Weimer (1999) demonstrate some of the L.
acidophilus strains resistant to acid at pH 3.5 for 90 min and were able of grow in
medium at pH 3.5 containing mixed bile salts (0.2%). In another study a large culture
collection of lactic acid bacteria was screened to select strains to be used as probiotics.
For this, over 200 strains of Lactobacillus and Bifidobacterium were examined according
to their ability of resistance to bile and acid and four of them were selected. Three of
them were from dairy origins and the last one was from human origin. They were
compared with the two commercial probiotic strains namely Lactobacillus rhamnosus
GG and Lactobacillus acidophilus LA-1. The isolated strains were analyzed for a series
of pH between 1 and 3 and also for tolerance against bile at final concentrations of 0, 0.5
and 1% w/v (Prasad et al., 1998). In another research, twenty nine Lactobacillus strains
of dairy origin were tested in vitro for their probiotic potential. The resistance of bacteria
was examined in between pH 1.0 and pH 3.0. Tolerance to bile salt was tested against
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(0.3%) oxgall. All the examined strains were resistant to pH 3.0 for 3h, but most of them
lost their viability within 1h at pH 1.0. Also all of them tolerated 0.3% bile salt
concentration for 4 h. An experiment was performed on three Lactobacillus species
isolated from human milk if they can be used as potential probiotic strains. They were
identified as Lactobacillus gasseri and one of them as Lactobacillus fermentum. Survival
in low pH and gastrointestinal environment were examined for comparison with
commercial probiotic strains of L. rhamnosus GG, L. casei and L. johnsonii La1. The
strains especially L.gasseri showed that it can be used as a potential probiotic strain
(Martin et al., 2004).
Antimicrobial Activity
Antimicrobial activity is one of the most important selection criteria for
probiotics. Antimicrobial activity targets the enteric undesirables and pathogens
(Klaenhammer and Kullen, 1999). Antimicrobial effects of lactic acid bacteria are
formed by producing some substances such as organic acids (lactic, acetic, propionic
acids), carbon dioxide, hydrogen peroxide, diacetyl, low molecular weight antimicrobial
substances and bacteriocins (Ouwehand and Vesterlund, 2004). Here are some examples
of these substances: Lactobacillus reuterii (a member of normal microflora of human
and many other animals) produces a low molecular weight antimicrobial substance
reuterin; subspecies of Lactococcus lactis produces a class I bacteriocin, nisin A;
Enterococcus feacalis DS16 produces a class I bacteriocin cytolysin; Lactobacillus
plantarum produces a class II bacteriocin plantaricin S; Lactobacillus acidophilus
produces a class III bacteriocin acidophilucin A (Ouwehand and Vesterlund, 2004).
Production of bacteriocins is highly affected by the factors of the species of
microorganisms, ingredients and pH of the medium, incubation temperature and time.
Nisin, produced by L. lactis subsp. lactis is a well known bacteriocin and is permitted in
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food preparations. Lactobacilli and Bifidobacteria isolated from human ileum were
assayed for antimicrobial activity against a range of indicator microorganisms, Listeria,
Bacillus, Enterococcus, Staphylococcus, Clostridium, Pseudomonas, Escherichia coli,
Lactobacillus, Streptococcus, Bifidobacterium and Lactococcus. Antimicrobial activity
of Lactobacillus salivarius UCC118 was measured against the bacteria listed above. The
study showed that Lactobacillus salivarius UCC118 is significantly capable of inhibiting
in vitro growth of both gram positive and gram negative bacteria such as; L. fermentum
KLD, B. longum, B. bifidum, Bacillus subtilus, B. cereus, B.thuringensis, E. faecalis, E.
faecium and is not effective against some of Lactobacillus, Lactococcus, Leuconostoc,
Streptococcus species (Dunne et al., 2001). Some milk products were used to isolate
potential probiotic bacteria and determine their possible antimicrobial activities.
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi,
Serratia marcescens and Candida albicans were used as indicator microorganisms. After
the study, the results showed that, Yakult and Ski D’ Lite probiotics inhibited all the test
indicator microorganisms. Nestle yogurt probiotics were bactericidal for S.aureus and P.
aeruginosa but inhibitory for S. typhi , Neslac probiotics killed E. coli and S. typhi while
they were only inhibitory for S.aureus and C. albicans (Chuayana et al., 2003). In
another study eight lactic acid bacteria strains producing bacteriocins were isolated from
Burkina Faso fermented milk and they were examined for the antimicrobial activity
against Enterococcus faecalis 103907 CIP, Bacillus cereus 13569 LMG, Staphylococcus
aureus ATCC 25293, Escherichia coli 105182 CIP. The lactic acid bacteria strains were
identified as Lactobacillus fermentum, Pediococcus spp., Lactococcus spp., Leuconostoc
mesenteroides subsp. mesenteroides. The diameters of inhibition zones were obtained
between 8 mm and 12 mm. Lactobacillus fermentum (S1) gave the biggest zone of 12
mm against Enterococcus faecalis while the smallest one was obtained with Leuconostoc
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mesenteroides subsp. mesenteroides (S5) on the same strain Enterococcus faecalis
(Savadogo et al., 2004). In a research which was aimed to test the production of
bacteriocin in vaginal lactobacilli flora and characterization of this flora was also made.
First antimicrobial activity was assayed for 100 vaginal Lactobacilli isolates. Six of them
were determined for the production of bacteriocin. In this study, common human
pathogens Gardneralla
vaginalis, Pseudomonos aeruginosa, Proteus vulgaris,
Escherichia coli, Enterobacter cloacae, Streptococcus milleri, Staphylococcus aureus
and Candida albicans were used as indicator microorganisms. Six of the strains had
bacteriocin activity against eight of ten different Lactobacillus species an also S.milleri,
P. vulgaris, P. aeruginosa, E. coli, E. cloacae and G. vaginalis. But none of the isolated
strains showed efficiency on test organisms S. aureus and C. albicans. Some
characteristics of bacteriocins were obtained from the research (Karaoglu et al., 2003). In
another research, potential probiotic lactobacilli strains (L.reuteri, L. plantarum,
L.mucosae, L. rossiae strains), were used as additives in pelleted feeding and examined
according to their antibacterial activity against Salmonella typhimurium ATCC 27164, E.
coli, C. perfringens 22G, S. aureus ATCC 25923, B.megaterium F6, L. innocua DSM
20649 and B. hyodysenteriae ATCC 27164. Generally the cell free extracts of
lactobacilli were able to inhibit all potential pathogens except B. hyodysenteriae ATCC
27164. The study showed that, neutralization and treatment with catalase affect the
antibacterial activity (De Angelis et al., 2006). In another similar study four
Lactobacillus strains (L. salivarius CECT5713, L. gasseri CECT5714, L. gasseri CECT
5715 and L. fermentum CECT5716) isolated from human milk were investigated for the
antimicrobial activity. All the strains showed antibacterial properties against pathogenic
bacteria (Salmonella choleraesuis CECT4155, CECT409 and CECT443, Esherichia coli
CECT439 and E. coli O157:H7 serover CECT4076, Staphylococcus aureus CECT4013
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and CECT9776, Listeria monocytogenes Scott A and the spoilage strain Clostridium
tyrobutyricum CECT4011). However, the antimicrobial properties of Lactobacilli strains
varied and L. salivarius CECT5713 revealed not only the best in vitro antibacterial
activity, but also the highest protective effect against a Salmonella strain in the murine
infection model (Olivares et al., 2006).
LAB have been used as natural preservatives because of their antimicrobial capacity
•
Fermentative processs: Antimicrobial activity can be exerted through the reduction
of pH or production of organic acids (lactic acid, acetic acid), CO2, reuterin, diacetyl,
2-pyrrolidone, 5-carboxylic acid (PCA) (Mayra-Makinen and Bigret, 1998).
Effective starter culture activity can prevent the pathogen and contaminant growth
that may occur during cheese making process.
•
Bacteriocins production: Bacteriocins can be defined as protein antibiotics of
relatively high molecular weight and mainly affecting the same or closely related
species. These bacteriocins can potentially be used to control the growth of spoilage
and pathogenic organisms in food (Cardinal et al., 1997). Bacteriocin producing
lactococcal strains have been used successfully as starter cultures for cheese making
in order to improve the safety and quality of the cheese. In recent work, 79 wild
lactococci have been studied and 32 of these have been found to be antimicrobially
active (Wouters et al., 2002). In 17 of these strains, the well-known antimicrobial
peptide nisin has been found, whereas the others produced diplococcin, lactococcin
or an unidentified bacteriocin-like compound. Moreover, the use of nisin as an
effective preservative in processed cheese has been widely accepted.
Proteolytic Activity
The ability to produce extracellular proteinases is a very important feature of
LAB. These proteinases catalyse the initial steps in the hydrolysis of milk proteins,
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providing the cell with the amino acids that are essential for growth of LAB. The
proteolytic activities of LAB including yoghurt starter bacteria and probiotic organisms
were studied extensively and proteolytic enzymes were isolated and characterized.
Bacterial enzymatic hydrolysis was shown to enhance the bio-availability of protein and
fat (Friend and Shashani, 1984). Bacterial protease can increase the production of free
amino acids which can benefit the nutritional status of the host particularly if the host has
a deficiency in endogenous protease production. Proteolytic system of LAB in cheese
ripening and rapid growth in milk during fermentation as well as improved survival
during storage. This ability of dairy LAB and probiotics has become even more
important upon realising that a range of bioactive peptides may be liberated due to
microbial action. Biologically active peptides are generated during milk fermentation by
proteolytic enzymes produced by L. helveticus, L. lactis subsp. cremoris FT4 and L.
delbrueckii ssp. bulgaricus SS1.
Flavor and Aroma Formation
Proteolytic and lipolytic activities of probiotic cultures degrade proteins and
lipids which is responsible for the characteristic taste and flavor of dairy products. For
example the popular probiotic L. acidophilus is responsible for the tangy flavor of
cultured dairy products, including yogurt. The quality of cheese and other fermented
food products is dependent on the ability of flavor and aroma production of
microorganisms which include starter culture. L. lactis ssp lactis and L. lactis ssp
cremoris are known to produce organic acids such as lactic acid and acetic acid in
fermented milk and L. lactis ssp. lactis biovar diacetylactis and Leuconostoc sp produce
acetaldehyde, diacetyl, acetoin, and 2-3 butylene-glycol which are responsible for
flavour formation in cheese. It has been reported that these aroma compounds might be
produced to avoid pyruvate accumulation in the cell. Moreover, improved knowledge of
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proteolysis and peptidolysis in cheese, analysis on enzymatic systems of LAB and
evaluation of different strains, will provide better understanding between flavor
development and starter activity. A number of different LAB have been evaluated for
their ability to degrade amino acids to aroma compounds. L. lactis subsp. Lactis, L.
lactis supsp. cremoris, Lactobacillius lactis, L. helveticus, L. bulgaricus, L. casei are
capable of degrading methionine to methonethiol, dimethyledisulphide (DMDS) and
dimethyltrisulphide (DMTS) (Yvon and Rijnen, 2001).
Exopolysaccharide production
Many strains of LAB produce exopolysaccharides (EPS). These compounds can
be produced as capsules which are tightly attached to the bacterial cell wall or as a loose
slime (ropy) which is liberated into the medium (Mayra-Makinen and Bigret, 1998).
Although differences are observed in EPS production by this strain (L. helveticus)
regarding the energy sources (lactose or glucose), the monomeric composition of the
polymers produced is independent of the carbohydrate use. Due to the increase in
viscocity and stabilizing properties, EPS producing starter cultures are beneficial for
industrial usage. They contribute to the texture development.
Probiotics in Health
Probiotic bacteria provide specific health benefits when consumed as a food
component (Gaurner and Malagelada, 2003). There are lots of studies on the health
benefits of probiotic fermented foods. These health-related effects can be listed as below
(Dugas et al., 1999; Scherezenmeir and de Vrese, 2001; Dunne et al., 2001).
Prevention of gastrointestinal problems
Improving immune system
Reduction of cholesterol
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Lowering blood pressure
Reduction of allergic symptoms
Suppression of pathogenic microorganisms (antimicrobial effect)
Prevention of osteoporosis
Prevention of urogenital infections
Prevention of gastrointestinal problems
Lactose Intolerance
A large number of population become lactose intolerant after weaning. These
lactose intolerant people cannot metabolize lactose due to the lack of essential enzyme βgalactosidase. When they consume milk or lactose-containing products, symptoms
including abdominal pain, bloating, flatulence, cramping and diarrhoea ensue. The
studies provide that the addition of certain starter cultures to milk products allows the
lactose intolerant people to consume those products without the usual rise of breath
hydrogen or associated symptoms (Lin et al., 1991; Scheinbach, 1998; Ouwehand and
Salminen, 1998; Fooks et al., 1999). The beneficial effects of probiotics on lactose
intolerance are due to the high lactase activity of bacterial preparations used in the
production and increased lactase enzyme (Salminen et al., 2004). Furthermore, the LAB
which is used to produce yogurt, Lactobacillus bulgaricus and Streptococcus
thermophilus, are not resistant to gastric acidity. Hence, the products with probiotic
bacteria are more efficient for lactose intolerant humans because of their ability to
withstand low pH and capability to improve digestibility of lactose by β-galactosidase
production.
Anti-diarrheal effect
Diarrhea is caused by many different pathogens and it is difficult to evaluate the
effects of probiotics on diarrhea. But there are lots of researches and evidence that
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probiotics have beneficial effects on some types of diarrhea. In the treatment of rotavirus
diarrhea, Lactobacillus GG is reported to be effective (Pant et al., 1996; Guandalini et
al., 2000). Also Lactobacillus acidophilus LB1, Bifidobacterium lactis and Lactobacillus
reuterii are reported to have beneficial effects on shortening the duration of diarrhea
(Salminen et al., 2004). Another type of diarrhea is traveller’s diarrhea which affects the
healthy traveller’s not only in developing countries but also in European countries.
Oksanen et al. (1990) evaluated the efficacy of Lactobacillus GG in preventing diarrhea
in 820 people travelling from Finland to Turkey. Antibiotic therapies are also known to
cause mild and severe outbreaks of diarrhea. The normal microflora may be suppressed
during the microbial therapy resulting in more pathogenic strains. The changes in
microflora may also encourage the resistant strains like Clostridium difficile which is the
main reason for antibiotic associated diarrhea (AAD).
Probiotics and Immune System
Probiotic bacteria are known to have positive effects on the immune system of
their hosts (Mombelli and Gismondo, 2000). Some in vitro and in vivo studies have been
carried out in mice and in humans. Data indicate that probiotic feeding supported the
immune system against some pathogens (Scheinbach, 1998; Dugas et al., 1999).
Probiotics enhance immune system by producing cytokines, stimulating macrophages
and increasing secretory IgA concentrations (Scheinbach, 1998, Dugas et al., 1999).
Some of these effects are related to adhesion while some of them are not (Ouwehand et
al., 1999). Link-Amster et al. (1994) examined whether eating fermented milk containing
Lactobacillus acidophilus La1 and Bifidobacteria could modulate the immune response
in humans. They give volunteers the test fermented milk over a period of three weeks
during which attenuated Salmonella typhi Ty21a was administered to mimic an
enteropathogenic infection. After three weeks, the specific serum IgA titre rise to S. typhi
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Ty21a in the test group was >4-fold and significantly higher (p=0.04) than in the control
group which did not consume fermented foods but received S. typhi Ty21a. The total
serum IgA increased. These results showed that LAB which can survive in the
gastrointestinal tract and act as an adjuvant to the humoral immune response (LinkAmster et al., 1994; Ouwehand et al., 1999). Perdigon et al. (1986) fed the mice with
Lactobacilli or yogurt which stimulated macrophages and increased secretory IgA
concentrations (Scheinbach, 1998). Also in a human trial, Halpern et al. (1991) fed
humans with 450 g of yogurt per day for 4 months and observed a significant increase in
the production of γ-interferon. Mattila-Sandholm and Kauppila (1998) showed that
Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb-12 derived extracts suppress
lymphocyte proliferation in vitro. Children suffering with severe atopic eczema resulting
from food allergy were fed with Lactobacillus rhamnosus GG and Bifidobacterium lactis
Bb-12. An improvement in clinical symptoms was observed (Saarela et al., 2000).
Anti-cancer effect
Epidemiological studies point out that if the consumption of saturated fats
increases in the diet the occurrence of colon cancer increases. Bacterial enzymes (βglucoronidase, nitroreductase and azoreductase) convert precarcinogens to active
carcinogens in the colon. It is thought that probiotics could reduce the risk of cancer by
decreasing the bacterial enzyme activity. Although the exact mechanism for the anti
tumor action is not known, some suggestions were proposed by many authors as follows
(Scheinbach, 1998; Fooks et al., 1999):
Carcinogen/precarcinogens are suppressed by binding, blocking or removal.
Suppressing the growth of bacteria with enzyme activities that may convert the
procarcinogens to carcinogens.
Changing the intestinal pH thus altering microflora activity and bile solubility.
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Altering colonic transit time to remove fecal mutagens more efficiently.
Stimulating the immune system.
There are in vitro and in vivo evidences not only from animal studies but also
from human studies that probiotics have beneficial effects on suppression of cancer. Oral
administration of lactic acid bacteria has been shown to reduce DNA damage caused by
chemical carcinogens, in gastric and colonic mucosa in rats. The consumption of
Lactobacilli by healthy volunteers has been demonstrated to reduce the mutagenicity of
urine and feaces associated with the ingestion of carcinogens in cooked meat. When it
comes to epidemiological studies, they show an association between fermented dairy
products and colorectal cancer. The consumption of a large quantity of dairy products
especially fermented foods like yogurt and fermented milk containing Lactobacillus or
Bifidobacterium may be related to a lower occurrence of colon cancer (Hirayama and
Rafter, 2000; Rafter, 2003). A number of studies have shown that predisposing factors
like “increase in enzyme activity that activate carcinogens, increase procarcinogenic
chemicals within the colon or alter population of certain bacterial genera and species” are
altered positively by consumption of certain probiotics (Brady et al., 2000).
Anti-cholesteremic activity
Lots of researchers proposed that probiotics have cholesterol reduction effects.
They are known to bind or incorporate cholesterol directly into the cell membrane or
produce bile salt hydrolase enzyme which deconjugate the bile salts and increases
cholesterol breakdown (Scheinbach, 1998). A study on the reduction of cholesterol has
shown that Lactobacillus reuteri CRL 1098 decreased total cholesterol by 38% when it is
given to mice for 7 days at the rate of 104 cells/day. This dose of Lactobacillus reuteri
caused a 40 % reduction in triglycerides and a 20 % increase in the ratio of high density
| 21
lipoprotein to low density lipoprotein without bacterial translocation of the native
microflora into the spleen and liver (Kaur et al., 2002).
Probiotic Lactic Acid Bacteria: Strain Improvement
Due to the considerable importance of LAB, culture improvement studies have
been accelerated in recent years. Progress in gene technology has allowed this
development by introducing new genes to improve bacteria that better fitted to
technological processes or enhanced organoleptic properties. It is expected that better
understanding of the genetics and physiology of LAB will give rise to better strain use,
selection and improvement (Ross et al., 2000). Construction of bacteriophage resistant
strains is very important. The resistant mechanisms are often carried out by plasmids and
transposons. Due to large number of multiple genes insertional mutagenesis is not
practical for protection against low pH. John and Nampoorthiri (2008) used nitrous acid
to improve a strain of L. delbrueckii NCIM 2025 for lactic acid productivity, acid
tolerance and sugar tolerance.
Safety Aspects of Probiotics
Today, there are evidences that probiotic strains used as commercial bacteria are
safe. The safety of the probiotic products is appraised with the phenotypic and genotypic
characteristics and the use of microorganisms. Safety aspects of probiotic bacteria should
fulfil the following requirements (Saarela et al., 2000).
1. Strains for human use are preferred to be of human origin
2. They are isolated from healthy human gastrointestinal tract
3. They have to be non-pathogenic
4. They have no history of relationship with diseases like, infective endocarditis or
Gastro-intestinal tract disorders
5. They do not deconjugate bile salts
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6. They should not carry transmissible antibiotic resistance genes.
Probiotic in functional fermented foods
Steinkraus (1996) defined indigenous fermented foods as “foods where
microorganisms bring about some biochemical changes in the substances during
fermentation and enrichment of the human diet through the development of a flavour,
aroma and texture in the foods”. The remarkable aspects of fermented foods is that they
have biological functions enhancing several health promoting benefits for the consumers
due to the functional microorganisms associated with them. Bioenrichment of nutritional
value, protective properties, bioavailability of minerals, therapeutic value, production of
antioxidants and immunological effects are some of the biological functions of fermented
foods (Tamang, 2007).
Cereal based fermented foods
Cereals are high in starch, dietary fiber, vitamins and minerals, but typical
amounts and quality of protein present do not fulfil the nutritional requirements of
animals (McDonald et al., 2002). Thus, cereal based feeds are amended with additional
protein sources to attain the required protein levels. The addition and proliferation of
microorganisms in feed may improve the nutritional quality for the animals (Ravindra,
2000).
Wheat (Triticum aestivum) is an excellent fodder cereal, with high energy and
low bran content, but the protein quality is generally poor ( Shewry, 2007). The high
starch content also limits the use of wheat in ruminant feeds, as it might perturb the
rumen fermentation (McDonald et al., 2002). More specifically, a validation of the
protein quality is necessary, as the proportion of essential amino acids often decreases
when crude protein content increases (Simonsson, 1995; Shewry, 2007).
| 23
Tamang (2010) defined ethnic, fermented foods as foods from locally available
raw materials of plant or animal sources either naturally or by adding starter cultures
containing functional microorganisms that modify the substrates biochemically and
organoleptically into edible products that are culturally and socially acceptable to the
consumers.
Types of fermented foods
Fermented foods are of two types
I.
II.
Natural fermented food
Controlled fermented food
In natural fermented food, raw materials get fermented by natural microflora present on
the raw ingredients or in the environment, without addition of starter cultures. e.g.,
Gundruk
Controlled fermented foods are prepared by using starter cultures. In this fermentation
there are two types:
I.
Mono culture fermented food: A single, pure culture strain of microorganisms
used for the preparation of food. e.g., Natto
II.
Multicultural fermented foods: Using a biculture or multicultural strains of
microorganisms or mixed inocula. e.g., Cheese, Sausages
On the basis of fermentation (substrates, nonalcoholic) foods are categorized into
Fermented milk products
Fermented cereal products
Fermented vegetable products
Fermented fish products
Fermented meat products
Fermented legume products
Miscellaneous fermented products
| 24
Varieties of fermented products are prepared and eaten in different parts of the world
(Table 3).
Table 3. Fermented cereal based food products around the world
Fermented
Cereal
cereals
substrate used
Ambali
Millet, rice
Bahtura
Wheat flour
Ben-saalga Pearl millet
Sensory
property
Acidic,
Bread
Gruel
Jalebi
Wheat,barley,
buckwheat
Rice and black
gram
Weat flour
Mildly acidic,
soft, spongy
Crispy, sweet
Kinky
Maize
Acidic, solid
Maheu
Mawe
Maize,
sorghum, millet
Maize
Naan
Natto
Wheat flour
Soybean
Sour,
nonalcoholic
Sour,
nonalcoholic
Baked bread
Alkaline
Ogi
Maize,
sorghum, millet
Wheat
Chilra
Idli
Pizza
dough
Puda/pudla
Rabadi
Maize, Bengal
gram
Cereals, pulses
with cow milk
Sourdough
Wheat, Rye
Trahana
Rice, wheat,
soybean and
milk
Maize,
sorghum, millet
Uji
Source: Tamang (2010).
Dough
Acidic
Leavened
dough
Pancake
Thick product
with slight
acidic
Leavened
bread
Sweet and
sour
Sour
Type of
product
pan cake
pancake
Weaning
food
Staple
Microorganism
Country
used
LAB
India
LAB, yeasts
India
LAB, yeasts
Ghana
LAB, yeasts
India
Breakfast
food
Deep
fried
snacks
Steamed,
staple
Beverage
LAB, yeasts
India,Srilanka,
Malaysia
India, Pakistan,
Nepal
LAB, yeasts
LAB, yeasts
Ghana
LAB
South Africa
Porridge
Yeasts, LAB
Togo, Benin
Staple
breakfast
LAB, yeasts
Bacillus natto
Staple
porridge
Used as
base
Snack
food
Drink
LAB, yeasts
India,Afghanistan,
Pakistan
Nigeria
Baker’s yeast
worldwide
LAB, yeasts
India
Yeasts, LAB
India, Pakistan
breakfast
LAB, yeasts
Australia, Europe
soup
Yeasts, LAB
Turkey
Staple
LAB
Kenya, Tanzania
| 25
Quorum sensing
Signalling mechanisms that govern physiological and morphological responses to
changes in cell density are common in bacteria. These signal transduction processes are
called quorum sensing, and involve the production, release and response to hormone-like
molecules that accumulates in the external environment when the cell population grows
(Fuqua et al., 1994; Pappas et al., 2004). The cooperation of microorganisms that would
form a multicellular structure on a surface appears to be controlled by mechanisms that
are, in some cases, not functioning in individual microbial cells grown in shaken liquid
cultures (Palkova and Vachova, 2006). Quorum sensing allows microorganisms to
monitor their density and consequently leads to a specific response by the whole
population. Farnesol has been identified as a quorum sensing molecule responsible for
hyphal formation in stationary phase cells of Candida albicans (Hornby et al., 2001).
The system of quorum sensing allows the amount of a signalling compound to increase
more efficiently than could be achieved by a simple increase in the number of noninduced cells. Therefore, the population can change its behaviour before reaching a
critical density (Chen et al., 2004; Palkova, 2004). Chen and Fink (2006) showed that the
quorum sensing molecules regulate transcription of a small set of genes (~150). About
70% of these genes have previously been shown to be up-regulated upon entry into
stationary phase. The induction of these genes suggests that quorum sensing molecules
may also play a role in signalling the transition to stationary phase.
Autoinducer II
The biological properties of piperazine have attracted attention in recent times
(McCleland et al., 2004). Their heterocyclic system constitutes a rich source of new
biologically active compounds (Li and Yang, 2002). They are known to have activities as
antitumor (Kanzaki et al., 2000), antiviral (Sinha et al., 2004), antifungal (Byun et al.,
| 26
2003), antibacterial (De Kievit and Iglewski, 2000; Fdhila et al., 2003; Uhegbu and
Trischman, 2005) and antihyperglycaemic (Kwon et al., 2000). For medical chemistry
properties of diketopiperazine, a derivative of piperazine as donor and acceptor groups
for hydrogen bonding is very interesting. They are considered ideal for the development
of new therapeutic agents (Niidome et al., 2003). Piperazine is the smallest cyclic
peptides commonly biosynthesized from aminoacids by different organisms and are
considered to be secondary functional metabolites. Cyclo diketo piperazine has shown
antitumor activity by significantly reducing the viability over 50% oh Hela, WHCO3 and
MCF-7 cells (McCleland et al., 2004). Several antimicrobially active diketopiperazines
have been isolated from microorganisms like marine fungus belonging to Ascomycota
which have been shown to have powerful activity against filamentous fungus Pyricularia
oryzae (Byun et al., 2003). Fungus Chaetomium globosum is found active against
Mycobacterium tuberculosis (Kanokmedhakul et al., 2002). Several bacterial strains
produce pathogenic factors when spread as biofilms where they are protected against
toxic agents. To achieve an organized status in these aggregates, bacteria affect a quorum
sensing process through small water soluble molecules, which act as auto-inducers.
Piperazines comprise a novel family of signalling compounds, identified cell-free
supernatants of gram negative and gram positive bacteria (Abraham, 2005).
Diketopiperazines are the smallest cyclic peptide derived from the folding head to tail of
a linear dipeptide. Due to their chiral, rigid and functionalized structure, they bind to a
large variety of receptors with high affinity, giving a broad range of biological activities.
The combinational use of natural and unnatural aminoacids a large library of compounds
may contribute to an understanding of the structural requirements for receptor
interactions and will open new perspectives for drug discovery processes (Martins and
Carvalho, 2007). Antifungal activity has also been demonstrated by piperazine propanol
| 27
derivative of 1,3 –β-D-glucosynthase inhibitor (Kondoh et al., 2005). Malaria is one of
the major diseases of developing countries caused by protozoan parasite Plasmodium.
The parasite is transmitted by the female anopheles mosquito and transits through the
liver and the blood of the mammalian host (Maitland and Marsh, 2004). Bisquinolines
have been used to target the erythrocytic stage of the parasite (Poulain and Melnyk,
2005). In this regard, 1,4 bis piperazine is used as a new antimalarial drug.
Lactococcus lactis, a lactic acid bacterium
Lactococcus are gram-positive cocci, occurring in pairs and form D (-) lactic acid
and carbon dioxide from the fermentation of glucose. These microorganisms form small,
grey, flat colonies on agar media. Lactococcus lactis and Lactococcus cremoris are of
vital importance as starter cultures in the diary industry. They produce diacetyl, a flavor
compound in the manufacture of dairy products like butter, buttermilk, cheeses (Jay,
1982). Strains belonging to the Lactococcus lactis, are the most important organisms in
the manufacture of fermented dairy products such as sour milk, cream, butter, fresh
cheeses and many varieties of semi-hard cheeses. Research on the genetic and
physiological properties of these bacteria has expanded rapidly in the last decade.
Lactococcus lactis can be isolated from various environmental sources and are
mainly targeted as industrial starter cultures for the manufacture of a wide range of
fermented dairy products including fermented milks, sour cream, soft and hard cheeses,
and lactic casein (Ward et al., 2002). The taxonomy of L. lactis has changed many times
and currently it is based phenotypically (Schleifer et al., 1985; van Hylckama et al.,
2006; Rademaker et al., 2007). This includes two subspecies (subsp. lactis and subsp.
cremoris) and one biovar (subsp. lactis biovar diacetylactis). The lactis and cremoris
phenotypes are differentiated on the basis of arginine utilization, growth temperature,
| 28
and salt tolerance, whereas the biovar diacetylactis strains have the additional ability to
metabolize citrate. Numerous studies including DNA–DNA hybridization, 16S rRNA,
and gene sequence analysis have demonstrated the existence of two main genotypes.
These two genotypic groups have also been called L. lactis subsp. lactis and L. lactis
subsp. cremoris, but unfortunately the genotype and phenotype designations do not
necessarily correspond, thus introducing a degree of confusion into the taxonomy of this
species (Tailliez et al., 1998). L. lactis isolates of dairy and plant origin using various
genomic fingerprinting methods and multilocus sequence analysis has clearly
demonstrated that two major lineages exist (Rademaker et al., 2007). One of these
comprises those strains with a L. lactis subsp. cremoris genotype and includes strains
with both lactis and cremoris phenotypes. The other comprises those strains with a L.
lactis subsp. lactis genotype that includes strains with the lactis phenotype as well as
biovar diacetylactis. Comparative genome hybridization (CGH) using 39 L. lactis strains
of dairy or plant origin (Bayjanov et al., 2009) provides further evidence confirming the
unusual taxonomic structure in this species. As a result, it is necessary to specify a
genotype (cremoris or lactis) and a phenotype (cremoris, diacetylactis, or lactis) to
adequately describe individual strains. Strains that show both the subsp. cremoris
genotype and phenotype cluster closely together and form a definite subgroup that shows
limited diversity relative to the other L. lactis strains examined (Rademaker et al., 2007;
Taibi et al., 2010). These L. lactis subsp. cremoris strains are favored for use as defined
strain starter cultures for Cheddar cheese production because they are less likely to cause
bitterness and other flavor defects (Heap, 1998). The citrate metabolizing biovar
diacetylactis strains contribute to the flavor and aroma profile of a range of fermented
dairy products and are also a component of the starter blends used for lactic casein
manufacture (Heap and Lawrence, 1984). These strains have long been distinguished
| 29
taxonomically (Kempler and McKay, 1981), but with the description of the genus
Lactococcus (Schleifer et al., 1985), they were incorporated into L. lactis subsp. lactis.
Biovar diacetylactis dairy starter strains have been genotypically (Kohler et al., 1991;
Beimfohr et al., 1997) and phenotypically (Bachmann et al., 2009) distinguished from
other L. lactis cultures, suggesting that these cultures may also form a separate subgroup.
Both L. lactis subspecies have been isolated from a variety of environmental sources but
are most commonly associated with fresh or fermented plant material or with milk and
milk products. Strains that show the genotype of lactis subspecies can be readily isolated
from these environments, whereas isolation of cultures with the cremoris subspecies
genotype are comparatively rare (Klijn et al., 1995; Salama et al., 1995). Attempts to
isolate new cremoris or diacetylactis phenotype strains from environmental sources have
met with little success as wild-type strains of both subspecies show the lactis phenotype
(Klijn et al., 1995; Salama et al., 1995; Ward et al., 1998). Because of its industrial
relevance, L. lactis has become the best studied of the lactic acid bacteria and is regarded
as a model organism for this bacterial group, although most work has been focused on a
small number of laboratory strains of dairy origin. Complete genome sequences have
been published for four strains. These include the two plasmid-cured strains (IL1403 and
MG1363) on which much of the detailed biochemical and genetic knowledge of L. lactis
is based (Bolotin et al., 2001; Wegmann et al., 2007). Both IL1403 and MG1363 belong
to L. lactis subsp. lactis phenotypically, but the parent strain of IL1403 (CNRZ157) has a
citrate permease plasmid and is able to metabolize citrate placing it with L. lactis subsp.
lactis biovardiacetylactis, whereas MG1363 has a lactis phenotype and a cremoris
genotype. The third genome-sequenced strain (SK11) has been used as a cheese starter
culture and belongs to the subgroup of strains with both the subsp. cremoris genotype
and phenotype (Makarova et al., 2006). The fourth genome is from a L. lactis subsp.
| 30
lactis strain of plant origin (KF147), and a partial sequence is also available for a second
plant strain (KF282) (Siezen et al., 2008; 2010). Comparison of the genomes from plant
and dairy isolates has highlighted the differences in gene content that can occur between
individual strains in the same species (Siezen et al., 2008) and shows that sequencing of
one representative genome does not give a complete picture of a species. Attempts to
describe this intraspecies diversity have led to the term species genome and pangenome
(Medini et al., 2005) being defined to cover all the genes present in the characterized
strains of a species. Under both definitions, the genome has a core of genes responsible
for the basic aspects of the biology of the species and a set of auxiliary or dispensable
genes that contribute to species diversity and may provide a selective advantage in
certain environments.
Antioxidant activity of LAB
Oxidative stress can be defined as an excess of reactive oxygen species (ROS)
that have strong oxidizing potential for cells (Farr and Kogoma, 1991; Fridovich, 1998).
ROS cause damage to macromolecular constituents such as DNA, RNA, proteins and
lipids (Berlett and Stadtman, 1997). Toxicity occurs when the degree of oxidative stress
exceeds the capacity of cell defence systems (Farr and Kogoma, 1991). ROS originate
from partial reduction of molecular oxygen (O2) to superoxide, hydrogen peroxide
(H2O2) and hydroxyl radical (OH) (Storz and Imlay, 1999). The biological sources of
ROS are numerous, e.g. they can be generated in aerobiosis by flavoproteins (Condon,
1987), and by macrophages during inflammatory reactions (Ross, 1991). Thus, oxidative
stress plays an important role in pathologies of the gastrointestinal tract of humans such
as inflammatory bowel diseases (Kruidenier and Verspaget, 2002; Kruidenier et al.,
2003) and in the radio-induced tissue injury that may occur during radiotherapy (Sun et
al., 1998). Bacterial species are more or less sensitive to oxygen according to the
| 31
enzymic equipment they possess to prevent or repair ROS damage. Some lactic acid
bacteria, such as Lactococcus lactis, widely used in the production of fermented food
products, produce a superoxide dismutase, which degrades O2 to generate H2O2 (Sanders
et al., 1995). However, they lack catalases, antioxidant metalloenzymes that catalyse the
reaction in which toxic H2O2 is reduced to two H2O molecules and O2. But catalase
activity has been reported in several Lactobacillus species during the last decade (Knauf
et al., 1992; Igarashi et al., 1996). In the absence of catalase, H2O2 produced by the cell
or present in the environment accumulates in presence of iron for the production of more
toxic hydroxyl group (Fridovich, 1998; Imlay, 2003). Catalases have been distinguished
in two classes according to their active-site composition: one is haem-dependent, and the
other, also named pseudocatalase, is manganese-dependent for the reduction of H2O2
(Abriouel et al., 2004; Noonpakdee et al., 2004). Strains of lactic acid bacteria
expressing high levels of catalases could be useful in both traditional food applications
and new therapeutic uses. A probiotic antioxidant strain able to eliminate ROS in the
digestive tract of animals and humans could have applications for treatment of
inflammatory diseases or post-cancer drug treatments.
Preservation of lactic acid bacteria culture viability by different methods
According to the proposed concept on the efficacy of probiotic application, the
probiotic bacteria must be viable at the time of consumption to achieve beneficial
function. Official standards for the minimum suggested level for probiotics in the food to
attain this viability require a minimum of 106-107 cfu/g, which have been introduced by
several food organizations worldwide (Talwalkar and Kailasapathy, 2004). Drying is
widely used as a means of preservation of bacterial cells. Because high water activity
dramatically decreases the viability of probiotics, removal of water can effectively
extend the shelf life of probiotic products. Drying is widely used as a means of
| 32
preservation of bacterial cells. Because high water activity dramatically decreases the
viability of probiotics, removal of water can effectively extend the shelf life of probiotic
products. Freeze-drying is another practicable method to improve the viability of
probiotics is to immobilize the bacteria in an external protective matrix, which can
improve their resistance to adverse conditions and facilitate better survival in specific
food products (Adhikari, et al., 2003; Godward and Kailasapathy, 2003). Freeze-drying
is a common method used to incorporate probiotics in foods. However, the viability of
freeze-dried probiotic bacteria is affected during processing and storage. Freeze-dried
probiotic organisms are protected by adding cryoprotectants, and the identification of
protective agents that enhance cellular survival during storage and application in food is
the key challenge (Hubalek, 2003). The technique of microencapsulation based on
complex (w/o/w) dispersion offers several advantages for the immobilization of
probiotics.
| 33
Aim and Scope
The aim of this study was to isolate novel LAB with antimicrobial and high
antioxidant properties. Further, the aim was to prevent the deterioration of probiotic
cereal blend product.
The present study also focused on the probiotic properties of L. lactis MTCC
5441 isolated from green gram dhal. The probiotic properties investigated included
tolerance to acid and bile conditions, adhesion to epithelial cells, hydrophobicity,
antimicrobial property and antioxidant activity. This strain has been used as a starter
culture for the preparation of probiotic cereal blend. During the storage a quorum sensing
signalling molecule was produced which helped in keeping the food quality during the
storage period. Encapsulation and freeze drying methods were used to improve its
survival.
Future Prospective
In this study the first step was taken to study the probiotic properties of the
isolate. The production of signalling molecule was also identified during storage of the
probiotic product. The work can be taken in detail to study the mechanism and mode of
action.
Proposed objectives of the thesis:
1. Isolation and characterization of lactic acid bacteria from cereals and legumes.
2. Elucidation of functional properties
3. Preparation of cereal based probiotic foods with potential lactic acid bacteria and
evaluation of their probiotic functional properties during storage
4. Identification of quorum sensing signalling molecules produced in the probiotic
foods during storage.