Probiotics: A New Concept In Prophylaxis

Research and Reviews: A Journal of Biotechnology

Volume 3, Issue 1, ISSN: 2231- 3826

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RRJoB (2013) 1-10 © STM Journals 2013. All Rights Reserved

Probiotics: A New Concept In Prophylaxis

Neha Agarwal, Rakhi Thakur, Rajarshi Banerjee* Haldia Institute of Technology, Haldia, WB, India

Abstract Foods that have health benefits beyond the traditional nutrients possessed by them are called functional foods or nutraceuticals. These are generally characterized as foods

similar in appearance to conventional foods, consumed as part of usual diets and elicit health benefits beyond meeting the basic nutritional requirements. Functional foods

reduce risk of diseases but they do not act as a vehicle to address deficiency. They are a

source of mental and physical wellbeing, enhancing the immunocompetence of the body.

They must be foods and not drugs and their over-intake could lead to nutritional

imbalance. Food is considered functional because it provides nutrients but nutraceuticals contain natural components that provide health benefits to the body. Traditional

nutraceuticals are natural whole food that deliver benefits beyond basic nutrition, such

as lycopene in tomatoes. Non-traditional nutraceuticals are food resulting from agricultural breeding or added nutrients, e.g., vitamin enhanced soybeans. Probiotics,

prebiotics, symbiotic are considered as functional foods. In recent years the overuse of

antibiotics, which although treated many diseases but also wiped-off the useful microflora of the intestine, has led to the development of resistant strains of bacteria and

thus hindering treatment of several diseases. Herein, comes the need to develop an alternative method for treatment of diseases. Probiotics, i.e., useful bacteria have been

used in foods since ages, though their properties in alleviating symptoms of disease were

unknown. This paper throws light on this field of research for a better understanding of

the topic.

Keywords: Nutraceuticals, immunocompetence, nutritional imbalance, probiotics,

prebiotics, symbiotic

*Author for Correspondence E-mail: [email protected]

INTRODUCTION Foods that have health benefits beyond the

traditional nutrients possessed by them are

called functional foods or nutraceuticals.

These are generally characterized as foods

similar in appearance to conventional foods,

consumed as part of usual diets and elicit

health benefits beyond meeting the basic

nutritional requirements. Functional foods

reduce risk of diseases but they do not act as a

vehicle to address deficiency. They are a

source of mental and physical wellbeing,

enhancing the immunocompetence of the

body. They must be foods and not drugs and

their over-intake could lead to nutritional

imbalance. Food is considered functional

because it provides nutrients but nutraceuticals

contain natural components that provide health

benefits to the body. Traditional nutraceuticals

are natural whole food that delivers benefits

beyond basic nutrition, such as lycopene in

tomatoes. Non-traditional nutraceuticals are

food resulting from agricultural breeding or

added nutrients, e.g., vitamin enhanced

soybeans. Probiotics, prebiotics and

symbiotics are considered as functional foods

[17, 50, 57, 88].

In recent years, the overuse of antibiotics,

which although treated many diseases but also

wiped-off the useful microflora of the

intestine, has led to the development of

resistant strains of bacteria and thus hindering

treatment of several diseases. Herein, comes

the need to develop an alternative method for

treatment of diseases. Probiotics, i.e., useful

bacteria have been used in foods since ages,

though their properties in alleviating

symptoms of disease were unknown. This

paper throws light on this field of research for

a better understanding of the topic.

[email protected]

Probiotics Concept in Prophylaxis Rajarshi Banerjee

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PROBIOTICS: A NEW CONCEPT IN

PROPHYLAXIS In recent years, bacterial antibiotic resistance

has been considered a problem due to the

extensive use of classical antibiotics in

treatment of human and animal diseases. As a

result, multiple resistant strains developed

which restricted the use of antibiotics. So the

development of a new class of antimicrobial

agent has gained importance. According to

WHO/FAO definition, probiotics are the live

microorganisms which when administered in

adequate amounts confer a health benefit to

the host organisms. Probiotics modulate

systemic and mucosal immunity of the host

and improve nutritional and microbial

imbalance in the intestinal tract. It competes

and suppresses the growth of pathogens by

multiple mechanism of action and also

produces a number of beneficial health effects

of their own. They can be used as

complementary and alternative medicine

(CAM). Probiotics are commonly consumed

as part of fermented foods with specially

added active live cultures, such as in yoghurt,

fermented and unfermented milk, juices or as

dietary supplements. Most probiotics are

bacteria similar to those found naturally in the

gut of human beings. The probiotics most

commonly used are Lactic Acid Bacteria

(LAB) and Bifidobacterium. Few yeast

probiotics different from bacteria have been

found, e.g., Saccharomyces boulardii [9, 77,

89].

Probiotics-containing products are used for

improved nutrition and growth of humans, as

animal feed supplement, for aquaculture.

Majority of probiotic products are available as

vegetative cells of bacteria that are non-spore

formers. But, products containing Bacillus

spores show more advantage than LAB

products, since they can be stored indefinitely

in a dessicated and dehydrated form and can

germinate in significant numbers in jejunum

and ileum [99].

To achieve a probiotic status, microorganisms

must fulfil a number of criteria related to

safety, functional effects and technological

properties (FAO/WHO, 2001). From the safety

point of view, probiotics must not be

pathogenic and must not have the ability to

transfer antibiotic resistance genes. The

functional aspects must include acid-bile

stability, antagonistic activity against human

pathogens, adhesion to intestine surfaces, anti-

carcinogenic and anti-mutagenic properties.

The technological properties are – good

survival during freeze drying or spray-drying,

proper growth and viability in foods, phage

resistance, and stable during long-term storage

[96]. Bacillus subtilis 3 was identical to

amicoumacin A and B which have anti-

inflammatory and anti-stress ulcer properties

and act in an additive manner. Oral

administration of LAB probiotics also

enhances innate immunity, NK cell functions,

macrophage phagocytosis and production of

lysosomal enzymes [3, 20, 25, 32, 39, 43, 44,

52, 61, 71, 97].

We now know that probiotics isolated from

foods, animal models or plant sources have

been helpful in treating diseases in humans

and strengthening the gut microflora that are

beneficial for them. Now the question is, are

probiotics also useful to animals and poultry

birds?

POTENTIAL OF PROBIOTIC USE IN

PISCICULTURE, LIVESTOCK AND

POULTRY INDUSTRIES Antibiotics have been used in animal

husbandry as therapeutic agents and growth

promoters. The antibiotic resistance and

multiple drug resistant (MDR) strains of

bacteria have thus increased. Probiotics have

been proposed as a cost-effective alternative

for improved breeding performance and

controlling diseases in animals. Some spores

isolated from heat and ethanol treated faecal

material of organically reared broilers showed

antimicrobial activity against a broad range of

bacteria and were also tolerant to

gastrointestinal (GI) tract conditions. A strain

of B. subtilis isolated in laboratory was shown

to suppress poultry colonization by different

avian pathogens [61]. In livestock and poultry

industries, Paciflor and Biogrow which are

combination of B. subtilis and Bacillus

licheniformis spores are used extensively as

probiotics. It was observed in murine model

that spores do not disseminate in significant

numbers beyond GI tract but may persist there

and germinate in its anaerobic environment

although it is an aerobic saprophyte [25, 71,

93]. Bacillus spp. were supplemented in diets



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of broilers as direct-fed microbial (DFM), a

commercial product. DFM was shown to

provide protection to the host against enteric

pathogens and had immunomodulating effects

[35, 58].

Probiotic treatment of fish during fish-farming

to reduce their mortality rate due to bacterial

diseases is an alternative to the use of

antibiotics. Pseudomonas fluorescens (P.

fluorescens) strain of aquatic origin (AH2) has

been used as antagonists of fish pathogen

Vibrio anguillarum (V. anguillarum).

Fluorescent pseudomonads are iron-chelating

siderophores, while fish with iron overload are

more prone to attack by V. anguillarum. Thus,

under iron-limited conditions P. fluorescens

proved to be inhibitory to V. anguillarum in

vitro [62, 78].

So long we have been saying that probiotics

treat diseases and are beneficial to man and

animals alike. But what are the diseases that

are treated or the symptoms reduced by using

probiotics?

PROBIOTICS ATTRIBUTES Antibiotic-associated Diarrhea (AAD) In 2008, a survey showed that diarrhea killed

more children that AIDS, malaria and measles

combined. A survey done on 3000 children by

NICED, Kolkata, showed that Lactobacillus

casei strain Shirota prevented acute diarrhea

by 14% [12]. Diarrhea is induced by direct

damage to the epithelium caused by parasites.

Antibiotic therapy causes colonic microbiota

imbalance thus changing carbohydrate

metabolism with fatty acid malabsorption and

osmotic diarrhea. Probiotics thus reduce the

incidence and severity of AAD by avoiding

the overgrowth of pathogenic organisms.

Inactivated Lactobacillus GG has been shown

to reduce symptoms of diarrhea while the live

cultures induced antigen-specific IgA response

[64]. Caution must however be taken while

administering probiotics to patients with

compromised intestinal barrier. AAD was also

treated with VSL#3 which showed a consistent

and beneficial effect on intestinal microbial

profiles [6].

Lactose Intolerance LAB converts lactose to lactic acid, thus

probiotic therapy may help patients to tolerate

lactose at high concentrations. Individuals

with lactose intolerance can tolerate lactose

present in yoghurt than in raw milk even if

they are in the same concentration, due to the

high levels of lactase released by yoghurt

bacteria when lysed by bile salts of the GI

tract. Bifidobacterium longum present in curd

thus reduces lactose intolerance in individuals

suffering from the disease [86].

Irritable Bowel Syndrome (IBS) and Colitis Commercial strains of both Bifidobacterium

infantis and Lactobacillus plantarum have

been shown to reduce the symptoms of IBS in

women which are associated with increased

epithelial permeability. Inflammation in this

disease is caused by bacterial products such as

superantigens, peptidoglycans, and

lipopolysaccharide [28, 90]. Probiotics help by

maintaining the appropriate bowel transit time,

inducing interleukin-10 (IL-10) production

and by removing the toxins [10, 86].

Cholesterol Reduction Some strains of LAB reduce serum cholesterol

level. Probiotics inhibit the reabsorption of

bile in the blood as cholesterol by breaking it

down in the gut itself. Several strains of

Lactobacilli and Bifidobacteria isolated from

the gut of humans, as for example

Lactobacillus fermentmKC5b, reduced serum

cholesterol level and could tolerate bile and

acid concentrations found in the upper

gastrointestinal tract of humans. Further the

strain being of human origin provided added

benefit of competing with the indigenous

microflora [27].

Immune Functions and Infections LAB prevents pathogen overgrowth by

competitive inhibition. It increases the IgA

producing plasma cells, T lymphocytes and

NK cells, thus improving the immune

functions. Probiotics have antibacterial,

antifungal and antiviral properties. It controls

overgrowth of Candida sp. and prevents food

poisoning [7].

Inflammatory Bowel Disease (IBD) IBD caused by two overlapping phenotypes,

Crohn’s disease and ulcerative colitis, thought

to occur due to genetic disposition and

intestinal microflora may be reduced by using

probiotics. The symptoms include disruption


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in bowel habits and mucosal inflammation

which may be alleviated by using a

combination of strains of Bifidobacterium

[42]. IBD caused by Helicobacter hepaticus

was treated with Lactobacillus paracasei and

Lactobacillus reuteri combination [41]. IBD

treatment by VSL#3 (consisting of four strains

of LAB, three strains of Bifidobacterium and

one strain of Streptococcus) has also been

done [21, 37, 67].

CDI Clostridium difficile (C. difficile) induced

infections (CDI) cause fatal symptomatic

diarrhea and are responsible for

pseudomembranous colitis occurring mainly in

hospitals by overgrowth of the bacteria (also

known as “hospital superbug”) in the GI tract.

It can be treated by VSL#3 since it normalizes

barrier integrity and inhibits proteosome

function (146, 148). Some probiotics by

colonization resistance suppress the growth of

C. difficile by increasing acidity, producing

inhibitory metabolites, or by competing for

nutrients. Probiotics such as Saccharomyces,

Lactobacilli, Enterococci, Bacteroides, and

Bifidobacteria have shown antagonistic

activity against C. difficile infections and

antibiotic-associated diarrhea [66].

Constipation

This disease is common in the elderly people

and causes an increase in the number of bowel

movements or a decrease in the transit time.

Lactulose, a prebiotic has been widely used to

treat this disease. But probiotics like

Bifidobacteria sp. are mainly used for curing

this disease.

Cancer High activity of some fecal enzymes like

urease, azoreductase, nitroreductase, beta-

glucuronidase convert procarcinogens to

carcinogens due to alteration in the intestinal

microflora composition and increase the risk

of colorectal cancer (CRC). Bifidobacterium

longum (B. longum) and Bifidobacterium

breve produce conjugated linoleic acid which

has anti-carcinogenic activity and thus

prevents DNA-induced damage by

carcinogens. The mechanisms of the mode of

action have not been determined yet and

further studies are being done on that.

UTI Probiotics treat urinary tract disorders like,

ulcers, yeast vaginitis, bacterial vaginosis and

other infections of the urinary tract [13].

COMMENSALS OF THE INTESTINE Human large intestine consists of a huge

variety of bacterial species which show the

propensity to grow in communities in matrix

enclosed surfaces in natural environments. The

ideal balance of bacteria in our GI tract should

be 85% good and 15% bad; the foundation of

which is given by mothers to their babies

through normal birth and breast feeding [11].

The human colon is sterile at birth while

breastfed infants have intestine largely

dominated by Bifidobacterium. On the other

hand, formula-fed infants have a complex

microbiota. Upon weaning, the differences in

the complexity of flora diminishes and an

adult type complex flora becomes established.

Colonization of the GI tract starts immediately

after birth, initially with maternal vaginal and

intestinal flora. In “normally” born children,

colonization occurs in the first week after birth

while colonization takes more time in

caesarean births and the species are also

different. A mutualism exists between the

bacteria residing in the mucus layer and the

host colonocytes. Microflora that produces

short chain fatty acids by fermentation is used

by host cells as nutrients while the bacteria use

the mucus secretions as energy source [46].

Normal motor propulsive activity of the GI

tract and lysozyme limits the growth of

organisms in the small intestine [14, 68].

Bifidobacteria is the most common genera in

the human colon and among the species

known today B. longum is the most studied

commensal promoting beneficial effects to the

host. Bacteria like LAB, Streptococci and some

yeast present in stomach are acid-tolerant. The

ileum and colon have same bicarbonate and

chloride concentrations and similar pH but

jejunum has low bicarbonate and high chloride

concentrations with a low pH. The rate of

secretions was greater in ileum and jejunum

than in colon. The concentration of Na, K, and

Mg was same in all intestinal fluids.

Duodenum is unfavorable to bacteria while

downstream of small intestine the amount of

bacteria rises due to favourable conditions.

Bifidobacteria, Fusobacterium and

Bacteroides are found in this region. In large

intestine which has higher favorable



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conditions than small intestine, species like

Clostridia, Proteus, Staphylococcus,

Veillonella, yeast, protozoa are found [14, 30,

54, 80].

Probiotics like LAB and Bifidobacteria are

considered as key commensals in human-

microbe interactions. They improve the

microbial ecology of an organism and play an

important role in production of ferments,

polysaccharides, glycoprotein and other

bioactive compounds, thus playing an essential

role in improving the protein, lipid and

mineral metabolism [65]. It is generally

accepted that except Streptococci and

Enterococci, LAB are rarely pathogenic to

humans and animals [69].

PURPORTED MECHANISMS OF

ACTION OF PROBIOTICS The physiological effects related to probiotics

include production vitamins, digestive

enzymes, and antibacterial substances like

organic acids, hydrogen peroxide, lactones,

bacteriocins and some other unidentified

substances. They increase the nutrient

bioavailability of calcium, magnesium,

phosphorus, zinc and copper. Probiotics

reduce gut pH, stimulate immune functions,

remove carcinogens, and reduce faecal

enzyme activity [8, 85, 96].

Short Chain Fatty Acids (SCFA): Probiotics increase the SCFA production

which modulates gut functions.

Oligosaccharides: Oligosaccharides are

used in conjunction with probiotics to

enhance their beneficial actions.

Β-galactosidase Activity: Probiotics

produce B-galactosidase which breaks

lactose into B-galactose and glucose to

produce energy.

Cholesterol Assimilation: Probiotics

reduce bile salt reabsorption in the blood

and thus reduce hypercholesterolemia.

Antioxidant: Probiotics reinforce defense

systems of normal mucosal cells by

exerting their antioxidant properties.

Immunostimulatory: Probiotics help in

improving immunity of the body by

producing cytokines and interferons,

active against viral infections.

Neutralization of Dietary Carcinogens: Probiotics neutralize the faecal enzymes

like urease and nitroreductase which

convert procarcinogens to carcinogens.

Survival and Adhesion Competitions

with Pathogenic Bacteria: Probiotics

survive in the intestine by the method of

competitive exclusion of the pathogens

Bacteriocins: They are toxins present in

bacteria which restrict the growth of other

similar bacterial strains due to high

demand for nutrients from same sources of

food available.

Thus, probiotics act by various mechanisms

and alleviate symptoms of diseases. Here, the

authors would concentrate on their property of

producing bacteriocins and its attributes.

BACTERIOCINS Bacteriocins, first discovered by A. Gratia in

1925, are proteinaceous toxins produced by

bacteria to inhibit the growth of closely related

bacterial strains due to nutritive demand for

the same scarce sources, thus they have a

narrow host range. However, Lactobacillus

plantarum F1 and Lactobacillus brevis OG1

isolated from Nigerian fermented foods

produced bacteriocins that had broad spectrum

of inhibition against pathogenic, food spoilage

organisms [90]. Bacteriocins, found in a large

number of bacteria are used by them in their

struggle for survival and bacterial

communication. The most studied bacteriocins

are colicins, produced from E. coli. The most

common colicin is Colicin V which is a small

peptide secreted differently from the classic

colicins. The bacteriocins produced by LAB

(GRAS – generally recognized as safe) have

potential applications to prevent the growth of

harmful bacteria in humans and are used in

food preservation and feed production. They

are a part of the natural microbial flora in

foods human have consumed for centuries, and

they constitute a significant part of the

indigenous microbiota of mammals including

humans. Evidence that many intestinal

bacteria such as Fusobacterium mortiferum

isolated from chicken ceca synthesize

bacteriocin in vitro supports the notion that

bacteriocin may be required for survival [79].

Lactobacillus salivarius K7 isolated from

chicken intestine produced bacteriocin which

inhibited both Gram-positive and negative

bacteria, like Lactobacillus mesenteroides,

Lactobacillus sakei, Bacillus coagulans,

Staphylococcus aureus, Enterobacter feacium,


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P. fluorescens [19, 26, 31, 38, 48, 51, 55, 73,

85, 92].

Bacillus thermoleovorans SII and NR-9, two

endospore-forming obligate thermophiles were

isolated from mud and water samples.

Bacteriocins thermoleovorin-S2 and NR-9

were effective against a wide range of Gram-

positive bacteria. There bacteriocins are

capable of utilizing hydrocarbons for their

growth and can also inhibit Thermus

aquaticus. Their stability under a wide range

of temperature and pH make them suitable for

use as both a food and feed grade additive

[45].

Bacillus sp. MTCC 43 isolated from the

rhizosphere of radish showed inhibition

against S. aureus and Aeromonas hydrophila.

This bacteriocin was used as a biopreservative

in milk because it increased its shelf life and

suppressed growth of various milk-borne

pathogens and spoilage organisms [4, 70].

CLASSIFICATION OF BACTERIO-

CINS AND THEIR PROPOSED

MODE OF ACTION Bacteriocins are a diversified class of peptides

with varying characteristics, mode of action,

structure and target cell specificities.

Bacteriocins can be classified into five groups

on the basis of their molecular mass,

thermostability, enzymatic sensitivity,

presence of post-translationally modified

amino acids, and mode of action [87, 95].

Class I Bacteriocins This group comprises lantibiotics, i.e.,

antibiotics containing unusual post-

translationally modified amino acids such as

lanthionine or β-methyl-lanthionine. They

undergo posttranslational modification such as

formation of dehydrated residues and

lanthionine bridges [85].

(i) Group Ia consists of amphipathic, flexible,

screw shaped, small cationic peptides that

produce pores in the cell membrane of the

target cell. The largest lantibiotic of this class

is Carnocin UI49. The prototypic lantibiotic

nisin is a member of this group.

Staphylococcus warneri ISK-1 produces

nukacin ISK-1, a type Ia lantibiotic which

consists of linear N-terminal region and C-

globular region containing lanthionine rings. It

was found that some unusual amino acids were

responsible for the binding of nukacin ISK-1

to nukH expressing cells [56].

(ii) Group Ib consists of neutral or anionic

rigid peptides having a globular shape. They

exert their actions by interfering with the

enzymatic reactions of the sensitive bacteria.

Class I bacteriocins have mainly been shown

to be produced by several oral Streptococcal

species like Streotococcus pyogenes,

Streptococcus salivarius (S. salivarius),

Streptococcus sanguis, Streptococcus mutans

(S. mutans). Salivaricin A (SalA), encoded by

the SalA gene in S. salivarius strain 20P3 was

the first S. salivarius lantibiotic to be

characterized [76].

S. mutans which causes dental caries in

humans was isolated clinically and its

bacteriocin C3603 which is a basic protein was

shown to be stable over a wide range of pH

(1–12). It was not affected by pronase, trypsin,

papain, -amylase. But pancreatin and

-chymotrypsin showed some partial activity.

It was active against Streotococcus species and

also some other Gram-positive bacteria.

However it was ineffective against some

strains of Escherichia coli, Candida albicans,

Klebsiella pneumoniae. Cornflour, skim milk,

cellulose, cornstarch were found to reduce the

activity of the bacteriocin due to loss of

activity by binding or adsorption of the

components to the protein [91].

Variacin 8 and 12 produced from Micrococcus

varians MCV8 and MCV12 respectively

isolated from meat fermentations in laboratory

were compared with lacticin 481 and found to

have a broad spectrum of activity, though

ineffective against Gram-negative bacteria.

The plasmid profiles of the lanthionines

showed that the primary sequences of variacin

and lacticin 481 were similar but there were

differences in their leader sequence,

processing site and transport enzymes.

Lacticin 481 produced from Lactococcus lactis

(L. lactis) SL2 was sensitive to variacin which

proves the difference in processing mechanism

of lanthionines. [2, 24, 49, 72, 74].

For Hispanico cheese manufacture, a starter

culture of L. lactis subsp. lactis INIA 639, L.



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lactis subsp. INIA 437, or a combination of the

strains were used. Lactobacillus helveticus (L.

helveticus) LH92 having high aminopeptidase

and dipeptidase activity was also added but it

was found to be sensitive to lacticin 481

produced by L. lactis subsp. lactis INIA 639

and the lysis of L. helveticus enhanced the

taste and flavor of the cheese. This method is

an inexpensive way to enhance flavor and

reduce bitterness, thereby accelerating cheese

ripening [18, 83].

Class II Bacteriocins

This group comprises small peptides of

molecular-masses smaller than 10kDa. These

are matured by a simple cleavage of a leader

peptide and have a conserved sequence

YGNGVXC at the N-terminus. These peptides

contain 37–48 residues and show potent

activity against related Gram-positive bacteria,

e.g., Listeria spp. The peptides of this class are

heat-stable and do not contain any modified

amino acids. It can be further sub-divided into

three sub-groups [47, 75, 85, 94].

(i) Group IIa: They consist of anti-listerial

peptides that disrupt the integrity of the

cell membrane thus producing ionic

imbalance and leakage of organic

phosphate to exert their killing action.

They thus act as bactericides. These

bacteriocins are co-expressed with

cognate-immunity proteins, i.e., pediocin-

like immunity proteins. Based on their

primary structure, the peptide chains are

roughly divided into two regions: a

hydrophilic, cationic and highly conserved

N-terminal region and a less conserved

hydrophobic/amphiphilic C-terminal

region, e.g., pediocin secreted by

Pediococcus spp. This class of

bacteriocins has a narrow spectrum of

activity and inhibit Listeria species even at

low nanomolar concentrations. They form

pores in the membranes and thus depletion

of ATP occurs. They are active against

food-borne pathogens, human pathogens

like vancomycin-resistant Enterococci,

opportunistic pathogens S. aureus [22, 23,

29, 40].

Carnobacterium piscicola (C. piscicola)

CS526 was active against Enterococcus,

Pediococcus, Listeria, Leuconostoc and was

inactivated by proteolytic enzymes. The

YGNGV consensus motif common in Class

IIa bacteriocins was not found in this

bacteriocin. C. piscicola CS526 inhibited

Listeria monocytogenes (L. monocytogenes)

growth in cold-smoked salmons [53].

Name of the bacteria Bacteriocin produced

Pediococcus acidilactici Pediocin PA-1

Leuconostoc mesenteroides Mesentericin Y105

Carnobacterium piscicola Carnobacteriocin B2

Lactobacillus sakei Sakacin P

Enterococcus faecium strains Enterocin A and Enterocin P

Leuconostoc gelidum Leucocin A

Lactobacillus curvatus Curvacin A

Listeria innocua Listeriocin 743A

(ii) Group IIb: They require two different

unmodified peptides for their activity. The two

peptides may be active individually but must

act synergistically. The peptides must be

present in equal amounts so that the

bacteriocins exert optimal antimicrobial

activity.

(iii) Group IIc: These are also known as

circular bacteriocins. It consists of all

bacteriocins that do not fall in groups IIa and

IIb. It is of two types: antibiotics with one or

two cysteine residues (e.g., thiolbiotics) and

antibiotics without cysteine residues (e.g.,

lactococcin A). They have a wide range of

effects on membrane permeability, cell wall

formation and pheromone actions of target

cells [60].

Organisms of the genus Enterococcus have

been shown to produce type II bacteriocins

mainly. Enterococcus faecalis (E. faecalis)

produces pAD1-encoded bacteriocin-

hemolysin (cytolysin) and peptide AS-48, both

of which are encoded by transferable plasmids.

Bacteriocin reduction confers an ecological

advantage on the producer strain.


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Enterococcal isolates of different origin highly

inhibit Listeria species, mainly L.

monocytogenes, moderately inhibit Lactic acid

bacteria and does not show any activity

towards Bacillus or Staphylococci. This shows

high specificity of bacteriocins produced by

different Enterococcal populations towards

different species of bacteria [82].

Enterococci are Gram-positive organisms that

reside in the human gut but have the capability

to cause disease in immunocompromised

people. Despite their pathogenic properties,

Enterococci are potential probiotics that can

treat gastroenteritis in both humans and

animals by maintaining the normal microbial

balance of the intestine [63]. Class IIa

bacteriocin produced by Enterococcus faecium

(E. faecium) T8, isolated from the vaginal

secretions of children affected with HIV virus

was studied and compared with the other class

IIa bacteriocin produced by Enterococcus

species. Bacteriocin T8 has bactericidal

properties and differs from Enterocin P and

bacteriocin 31 produced by E. faecium P13

and E. faecalis Y1717 respectively.

Group III Bacteriocins These are non-bacteriocin lytic proteins

termed as bacteriolysins. This group consists

of peptides that are heat-labile and have a

molecular-mass larger than 30kDa. Most of

them are produced by the bacteria of genus

lactobacillus and are thermosensitive proteins.

(i) Group IIIa comprises those peptides that

kill bacterial cells by cell-wall degradation,

thus causing cell lysis. The best studied

bacteriolysin is lysostaphin, a 27kDa peptide

that hydrolyses several Staphylococcus spp.

cell walls, principally S. aureus.

(ii) Group IIIb, in contrast, comprises those

peptides that do not cause cell lysis, killing the

target cells by disrupting the membrane

potential, which causes ATP efflux.

Group IV Bacteriocins This group requires non-protein moieties for

their activities and consists of glycoprotein or

lipoproteins.

Group V Bacteriocins This group consists of circular/cyclic

bacteriocins and their N- and C-termini are

covalently linked. Circular bacteriocin,

Gassericin A, was isolated from a food-

producer Lactobacillus gasseri LA39 that

showed activity towards a wide range of

Gram-positive food-borne pathogens. Another

cyclic bacteriocin, Circularin A, was produced

by Clostridium beijerinckii ATCC25752 and

showed activity against the cheese-spoilage

bacterium Clostridium tyrobutyricum. Both the

cyclic bacteriocins were resistant to several

proteases and peptidases [98].

THE FIRST COMMERCIALLY

MARKETED BACTERIOCIN: NISIN Nisin is a mixture of closely related polycyclic

antibacterial peptides with 34 amino acid

residues produced by L. lactis subsp lactis. It

is a lantibiotic that as a result of post

translational modification contains some

unusual amino acid residues like,

dehydrobutyrine, dehydroalanine, lanthionine

and β-methyl-lanthionne. The unusual amino

acids originate from serine and threonine and

the thioester bridges which form five ring-like

structures in nisin molecules are formed due to

the enzyme-catalyzed addition of cysteine

residues to the didehydro amino acids. The

primary and tertiary structures of nisin show

that it has amphiphilic character. The N-

terminal end has a high number of

hydrophobic residues while the C-terminal end

has hydrophilic residues containing the

positively charged side chains of lysine and

histidine residues. Nisin is positively charged

over a wide range of pH due to the absence of

residues with negatively charged side chains.

However at neutral pH its charge is reduced

due to deprotonation of histidine. NMR studies

show that nisin molecules have a well-defined

structure within the rings formed by

lanthionines. Nisin exhibits high solubility and

stability at low pH. Two naturally synthesized

nisin are Nisin A and Nisin Z which differ by

one amino acid residue. Nisin A has His while

Nisin Z has Asn at position 27. Both types are

equally distributed in nisin producing strains

of L. lactis. Nisin F and Nisin Q have also

been isolated from L. lactis and types U and

U2 have also been isolated from Streptococci

species. NisI, an amino acid protein with a

lipoprotein signal sequence provides immunity

to nisin by inhibiting its pore formation.

nisFEG gene cluster encodes ABC transporter

which transports nisin from the cytoplasmic

membrane of the immune cells, thus

conferring immunity [36]. Bacteriocin



__________________________________________________________________________________________


produced from certain strains of L. lactis by

fermentation using substrates like milk,

dextrose has been used extensively in the food

industry as a preservative for cheese, meats,

and beverages by suppressing Gram-positive

spoilage and pathogenic bacteria. In foods, it is

used at a range of 1–25 ppm. Nisin cannot be

synthesized chemically. Nisin is a rare

example of broad-spectrum bacteriocin that is

effective against many Gram-positive bacteria.

Nisin shows no effect on Gram-negative

bacteria, yeasts, molds. Bacilli and Clostridia

are more sensitive to nisin. It is effective

against Gram-negative bacteria when used

along with the chelating agent EDTA. Nisin is

considered safe for consumption because the

protease of the digestive system breaks it

down into amino acids and it can resist the

acidic environment of the stomach. One major

limitation of using nisin in foods is the allergic

response of some people due to lactose

intolerance, since the major substrate for nisin

production is milk [5, 15, 16, 18, 34, 36, 81].

The use of bacteriocin in food industry can

help to reduce the addition of chemical

preservatives, intensity of heat treatments, thus

resulting in foods that are more naturally

preserved and are rich in nutritional properties.

This can thus be considered as an alternative

to satisfy the consumer demands for safe,

ready-to-eat, minimally processed foods and

also to develop “novel” food products.

Bacteriocins may be added to foods in the

form of concentrated preparations as food

preservatives, shelf-life extenders, or as

additives. In addition to commercial

preparations of nisin, some other bacteriocins

that offer promising perspectives are pediocin

PA-1/AcH, lacticin, variacin, enterocin [1, 55].

Bacteriocins offer potential advantages in food

preservation by reducing the chemical

additives and intensive heat treatments which

decreases the nutritional properties.

Bacteriocins can be added in foods in

concentrated forms, in immobilized forms or

synergistically with other bacteriocins or

chemicals in low concentrations. Ex situ

produced bacteriocins are added as raw

concentrates by adding cultivated producer

strain in food-grade substrates. In immobilized

form the bacteriocins are bound to the carrier

which acts as a reservoir, allowing continuous

supply of bacteriocins. Efficacies of

bacteriocins in foods depend on environmental

factors thus, the conditions for application of

each bacteriocin must be known properly [1].

In recent years, consumer demand for non-

dairy-based probiotics has increased and thus

it has been incorporated into drinks as also

marketed in the form of tablets, capsules and

freeze-dried preparations. Fruits and

vegetables have components of functional

foods and also avoid dairy allergen which

limits the use of a wide range of dairy products

by a segment of the population. Cabbage

which consists of vitamins, minerals and

phytochemicals was used to produce probiotic

cabbage juice by fermentation with

Lactobacillus casei, Lactobacillus delbrueckii,

L. plantarum. This was advantageous since it

could be used by vegetarians and lactose-

allergic people [59].

INDIAN SCENARIO The interest in probiotics has gained

importance worldwide in the recent years. The

concept is gaining momentum in India too,

due to the gaining awareness and health

conscious Indians, the only hindrance arising

due to misconceptions and myths regarding

probiotics being safe for use. Use of probiotics

does not fit into the healthcare scenario of

India as effectively as it does in other

countries where people have a broader mind-

set towards accepting new methodologies of

disease treatment. ICMR (Indian Council for

Medical Research) in collaboration with the

DBT (Department Of Biotechnology) has

launched the “Indian Guidelines for Probiotic

Cultures and Foods” but it has not yet been

implemented at the governmental level. Thus

there is a high chance of spurious and

ineffective products entering the market and

further shatter the confidence of Indian

consumer on probiotic foods. Thus, with the

help of doctors, scientists, clinicians, medical

practitioners, and industry personnel the

confusion can be minimized. The Probiotic

Association of India (PAI) was registered as a

society in 2010 with the support of the

members of the National Core Group on

Probiotics. It is a scientific society committed

on its mission to promote probiotic concept

with transparency so that these “good bugs”

reach the target population without any

discrimination [9].


__________________________________________________________________________________________


GUIDELINES AND REQUIREMENTS FOR PROBIOTIC PRODUCTS ISSUED BY

ICMR-DBT

Traditional Indian Foods Comprising

Probiotics Dahi (Indian Yoghurt)

Milk

Buttermilk

Lassi

Idli

Dosa

Commercial Probiotic Products

Available in India Yakult Danone (probiotic drink)

Nestle (b-Activ probiotic dahi, lassi, curd,

Nutrifit: strawberry and mango)

AMUL (Ice-cream, lassi is being test-

marketed)

Mother Dairy

In recent years, there has been an increasing

globalization of food trade. In spite of the

formidable challenges, the prospects of the

Indian probiotic market expanding in a

steadfast way looks bright. The combined

efforts of the Indian pharmaceutical and food

industries to diversify their products for

catering to domestic and foreign needs with

the right kind of awareness can pave the way

for the probiotic industry to make a giant

stride in the global market. With the

availability of these products increasing

exponentially and multiple claims being put

forward regarding their beneficial health

effects, there is need to protect consumers

from adverse effects and issue regulations and

standardize commercial products as soon as

possible.

SCOPE

MANUFACTURING

& HANDLING

PROCEDURES

LABELLING

REQUIREMENTS

EFFECTIVE DOSAGE

OF PROBIOTIC

STRAINS

EVALUATION OF

EFFICACY STUDIES

IN HUMANS

EVALUATION OF

SAFETY FOR

HUMAN USE

IN VIVO EFFICACY

STUDIES IN ANIMAL

MODELS

IN VIVO SAFETY

STUDIES IN ANIMAL

MODELS

IN VITRO TESTS TO

SCREEN POTENTIAL

PROBIOTIC STRAINS

GENUS, SPECIES,

STRAIN

IDENTIFICATION

PROBIOTICS

DEFINITION

ICMR-DBT

GUIDELINES



__________________________________________________________________________________________


1. List of Characterized Probiotic Strains

Strain Source

L. plantarum 299V Probi AB (Sweden)

L. rhamnosus 1091 Danlac (Canada)

L. reuteri MM53 Biogaia (USA)

L. bulgaricus 1261 Danlac (Canada)

Streptococcus thermophilus 1131 Kenko-dontokoi (Japan)

Bifidobacterium breve strain Yakult Yakult (Japan)

B. lactis Bb-12 Chr. Hansen, Inc. Denmark

E. faecium SF68 Cerbios Pharma (Switzerland)

L. acidophilus CK120 Matsutani Chemical Product (Japan)

L. casei01 Chr. Hansen, Inc. Denmark

L. fermentum RC-14 Urex Biotech (Canada)

L. paracasei F19 Arla Dairy (Sweden)

L. salivarius UCC118 Uni. College Cork (Ireland)

Lactococcus lactis L1A Essum AB (Sweden)

Streptococcus thermophilus F2 Danlac (Canada)

L. rhamnosus R0052 Institute Rosell (Canada)

L. rhamnosus VTT E -97800 Research Strain VTT, Finland

L. casei Imunitass (Defensis, DN114, DN-

014001)

Danone (France)

L. casei Shirota (YIT 0918) Yakult (Japan)

L. crispatus CTV05 Gynelogix, Colorado (USA)

L. acidophilus SBT-2062 Snow Brand Milk Products Co. Ltd (Japan)

L. johnsonii La-1(Lj 1) Nestec Ltd. (Switzerland)

B. longum BB 536 Morinaga Milk Industry Co. Ltd.(Japan)

B. lactis LKM 512 Fukuchan Milk (Japan)

B. species 420 Danlac (Canada)

Ref. [84].


__________________________________________________________________________________________


2. Beneficial Effects Attributed by LAB Health benefit Proposed mechanism

Strain example References

Alleviates lactose tolerance Action of bacterial β-

galactosidase on lactose

Lactobacillus

rhamnosus GG

67

Improvement of the immune

system IgA production

Increased phagocytosis

Cytokine synthesis

induced

Lactobacillus reuteri

CT 7, Lactobacillus

gasseri CT 5

100

Reduction of allergic reaction Regulation of cytokine

synthesis

Prevention of antigen

translocation in the blood

stream

Lactobacillus

rhamnosus GG,

Bifidobacterium

lactis Bb -12

67

100

Prevention of GIT infection Competition for nutrients

Gut flora alteration

Competitive exclusion

(CE)

Lactobacillus

rhamnosus GG,

Enterococcus

faecium

144

Influence on intestinal flora Antibacterial

characteristics

Reduction of toxic

metabolite production

Lactobacillus

reuteri, Lb.

rhamnosus GG

67

100

Anti-colon cancer effect Carcinogen deactivation

Immune response

Colonic microbes

activity altered

Bifidobacterium

bifidum,

Bifidobacterium

infantis

100

Urogenital infections CE

Adhesion to urinary tract

Inhibitor production:

biosurfactants

Lactobacillus lactis,

Pediococcus

acidilactici

67

3. Immune Modulation of Probiotic Strains

Probiotic

Commercial

probiotic

Target Measure Effect Treatment

Bifidobacteria,

Lactobacillus lactis

ABx support PMBC In vitro TNF- (i), IL-6,

IL-12, IL-18

Acute diarrhea, supports

normal GI flora during

antibiotic therapy

Lactobacillus acidophilus Probiotic

complex caps

and powder

Murine

M

In vitro IFN- (i) Acute diarrhea in

infants, traveler’s

diarrhea

Bifidobacterium bifidum,

Lactobacillus johnsonii

La 1

Probiotic

complex caps

and powder

Human

(oral)

In vivo

Ex vivo

Phagocytosis (i)

of granulocytes,

monocytes

Prevents diarrhea

Lactobacillus rhamnosus Ther-biotic

factor 1

Mice

(oral)

Ex vivo Phagocytosis (i)

of blood and

peritoneal cells

Prevents non-bloody

acute diarrhea,

erythromycin-associated

diarrhea

[41, 100]

Abbreviations: i-induction, PMN-polymorphic nuclear cells, M -macrophage, PBMC-peripheral

blood mononuclear cells, NK-Natural killer



__________________________________________________________________________________________


CONCLUSIONS Let Food be Thy Medicine and Medicine be

Thy Food – Hippocrates

The growing interest in safe-care and

integrative medicine coupled with the health-

embracing population, the link between food

and medicines or rather between diet and

health has never been stronger. Thus the

market for functional foods, which promotes

health beyond basic nutrition, has been

flourishing. The use of fermented milk, curd,

and other home-made fermented food products

for curing stomach ailments was known since

ages. But the mode of their action has been

recently elucidated. Understanding probiotic

action may allow modulation of the immune

system. Knowledge of probiotics action on

host immune system has entered a fascination

phase of research and its further progression

would allow treatment for a wide variety of

human diseases. Its application in drug-

delivery is also being looked upon. The major

problem in usage of probiotics lies in the fact

that maintaining their viability while transport

or storage is a big issue. It should also undergo

proper controlled studies to estimate their

suitability for the severely ill or

immunocompromised patients. Another area to

be focused is that the commercial probiotic

should properly mention probiotics intended

for medical use and those meant for healthy

individuals, their composition must be

different. They must also maintain their

potency and effectiveness until they are

consumed. This requires a responsible

approach by both the consumer and the

producer. Thus, a systematic approach based

on the guidelines issued by the WHO/FAO

expert consultation should be adopted by the

researchers and industry personnel to produce

commercial products of probiotics which

could prove to be a boon to the mankind.

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Probiotics: A New Concept In Prophylaxis

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