BIFILAC

INTRODUCTION

At the beginning of the last century, the Russian immunologist Elie Metchnikoff argued that life-long intake of yoghurt containing lactic acid-producing microorganisms could explain the differences in length of life between ethnic groups. The idea was that the bacteria in the fermented

1products competed with microorganisms that are injurious to health .

Today it is known that the normal human microflora is important as a barrier against colonization by exogenous pathogenic microorganisms and potentially pathogenic bacteria already present in small numbers in

2the microflora . The normal microflora influence several biochemical, physiological and immunological features of the host, particularly the gastrointestinal flora, which consists of the most dense and diverse

3.collection of bacteria

Disturbances in the normal microflora can be caused by several things, one 4

being the administration of antimicrobial agents . The normal microflora 5

is also disturbed in infectious conditions of the gastrointestinal tract and also when there is inflammation of the gastrointestinal tract (Ulcerative

6-9colitis, Crohn's disease, Chronic pouchitis) .

Probiotic microorganisms are thought to counteract disturbances and 10

thereby reduce the risk of colonization by pathogenic bacteria . Studies on strains of microorganisms used in probiotic dietary supplements have demonstrated that several strains produce antimicrobial substances such as organic acid, bacteriocins and peptide. In vitro and animal studies have further shown inhibitory effects of probiotic bacteria to be mediated by their interference with the adhesion of gastrointestinal pathogens or with toxins produced by the pathogenic microorganisms. Adjuvant-like effects on intestinal and systemic immunity have also been demonstrated for

11some strains .

A number of clinical trials using various probiotic strains in various gastrointestinal conditions are underway. Some of the completed trials have shown promising results in conditions like infectious diarrhoea, antibiotic associated diarrhoea, irritable bowel syndrome, inflammatory bowel disease (ulcerative colitis, and Crohn's disease). Therapy using prebiotics and probiotics as bacteriotherapy seems to be more realistic now and the future in treatment of many gastrointestinal disorders.

THE NORMAL GASTROINTESTINAL FLORA AND INTESTINAL ECOSYSTEM

Normal gastrointestinal flora

The condition and function of the gastrointestinal tract is essential to our well being. This largely depends on the maintenance of proper balance of the intestinal ecosystem. The human intestinal microflora contributes largely in maintaining a normal balance of the intestinal ecosystem.

The human intestinal microflora is highly important to the host for several reasons. Firstly, microflora benefits the host by increasing resistance to colonization by potentially pathogenic microorganisms ingested through food and water, causing gastrointestinal disorders, as well as by protecting against the overgrowth of already present potentially pathogenic organisms. Another function important to the host is the high metabolic activity of the intestinal flora, helping in the digestion process in the intestine. They also help in the synthesis of beneficial substances for the host like B complex vitamins and vitamin K. Indeed, it is now known that the normal gut flora plays an important role in maintaining good health by stimulating the immune system, aiding the digestion and assimilation of food and protecting the host from invading bacteria and viruses.

The composition of the gastrointestinal flora differs among individuals, and also during life within the same individual. Many factors, such as diet or climate, aging, medication (especially antibiotics), illness, stress, pH, infection geographic location,

12race, socio economic circumstances, lifestyle can upset this balance .

Interactions of the typical intestinal bacteria may also contribute to stabilization or destabilization of the gut flora. A state of balance within the microbial population within the GI tract can be called “eubiosis” while an imbalance is termed “dysbiosis”. For optimum “gut flora balance”, the beneficial bacteria, such as the gram - positive Lactobacilli and Bifidobacteria, should predominate, presenting a barrier to invading organisms. Around 85 % of the intestinal microflora in a healthy person should be good

12bacteria and 15% bad bacteria .

The intestinal ecosystem

The gastrointestinal tract of an adult human is estimated to harbor about 100 trillion viable bacteria. These live bacteria are known as intestinal or gut flora. Viruses, fungi and protozoa can also be present, but these normally form only a minor component of the total resident population of microorganisms in healthy individuals.

The density of microorganisms in the gut flora increases dramatically from 10 - 1,000 CFU/ml in the stomach to 10 - 100 billion CFU/gm in the large

12intestine and these belong to as many as 400 different species, and anaerobic bacteria outnumber aerobic bacteria by a factor of 1000:1. Anaerobic flora is dominated by bacteroides spp., bifidobacteria, lactobacillus, propionibacteria and clostridia. Among aerobic and anaerobic bacteria enterobacteria, mainly E. coli, and enterococci predominate.

The predominant microflora in the GI tract is as follows.

5 7Proximal small intestine Lactobacilli + Enterococcus faecalis (10 - 10 / ml of fluid)

Distal small intestine Lactobacilli + Enterococcus faecalis + Coliforms + 8

Bacteroides (10 bacteria / ml of fluid)

11Colon Bacteroides + Bifidobacteria (10 bacteria / ml of fluid)

13Table I Composition of the human gastrointestinal microflora

ESTABLISHMENT OF INTESTINAL MICROFLORA IN THE NEW BORN INFANT

Fetuses are sterile in the womb, but beginning with the birth process, infants are exposed to microbes that originate from the mother and the surrounding environment

14including breast milk or formula . The infant tends to acquire the flora swallowed from the vaginal fluid at the time of delivery. Because vaginal flora and intestinal flora

15are similar, an infant's flora may closely mimic the intestinal flora of the mother .

Another factor affecting the intestinal flora of the newborn is delivery mode. A normal vaginal delivery commonly permits transfer of bacteria from the mother to the infant. During cesarean deliveries, this transfer is completely absent. These infants commonly acquire and are colonized with flora from the hospital's environment and, therefore, their flora may differ from maternal flora. Thus infants delivered by cesarean section are colonized with more anaerobic bacteria, especially Bacteroides, than vaginally delivered infants. Clostridium perfringens is the anaerobic bacterium most frequently isolated after cesarean deliveries. When colonized, cesarean delivered infants less

16frequently harbor E. coli, and more often klebsiellae and enterobacteria .

The initial colonizing bacteria also vary with the food source of the infant. In breast fed infants, Bifidobacteria account for more than 90 % of the total intestinal bacteria. The low concentration of protein in human milk, the presence of specific anti - infective proteins such as immunoglobulin A, lactoferrin, lysozyme, and oligosacharides (prebiotics), as well as production of lactic acid, cause an acid milieu and are the main reasons for its bifidogenic characteristics. In bottle - fed infants, Bifidobacteria are not

17predominant . Instead enterobacteria and gram - negative organism dominate because of a more alkaline milieu and the absence of the prebiotic modulatory factors present in breast milk.

The establishment of an intestinal microbial ecology is very variable at the beginning but will become a more stable system similar to the adult microflora by the end of the breastfeeding period.

Mechanisms by which the normal intestinal flora protects the host against 18

intestinal disease

1. Production of inhibitory substances

Bacteria of the normal gut flora produce a variety of substances that are inhibitory to both gram - positive and gram - negative bacteria. They produce antimicrobial compounds (bacteriocins), volatile fatty acids, organic acids, and lactic acid, which reduces the intestinal pH. These compounds reduce the number of viable pathogenic organisms in the gastrointestinal tract.

2. Blocking of adhesion sites

The gut microflora compete directly with gut pathogenic organisms for epithelial attachment sites in the gastrointestinal tract, thereby preventing attachment and colonization of the GI tract by the potentially pathogenic organisms.

3. Competition for nutrients

The gut microflora compete directly with gut pathogenic organisms for the essential nutrients necessary for survival and multiplication, thereby inhibiting the growth and multiplication of potentially pathogenic organisms.

4. Stimulation of immunity

The underlying mechanisms of immune stimulation by the gut microflora are not well understood. However, local gut immunity enhancement by the gut microflora may be one possible mechanism of inhibiting growth of potentially pathogenic microorganisms.

PROBIOTICS

What are Probiotics?

The word probiotic is derived from the Greek meaning “for life”. A probiotic by general definition is a “Live microbial feed supplement, which beneficially affects the host by improving the host's intestinal microbial balance”. Probiotics are generally mono or mixed cultures of live microorganisms which otherwise form the major component of the gut microflora (e.g. lactobacilli, bifidobacteria). Probiotics, when ingested, beneficially affect the host by improving the

12properties of the indigenous microflora .

Ingestion of probiotics beneficially affects the host by

a. Replenishing the depleted gut microflora, which may have occurred due to use of antibiotics, illness, stress, travel or lifestyle changes.

b. Improving the properties of the indigenous microflora.

Probiotic bacteria are generally, though not exclusively, lactic acid bacteria and include

1. Lactobacillus species like Lactobacillus acidophilus, L. casei, L. bulgaricus, L. plantarum, L. salivarius, L. rhamnosus, L. reuteri,

2. Bifidobacterium species like Bifidobacterium bifidum, B. longum, B. infantis.

3. Saccharomyces boulardii (yeast)4. Streptococcus thermophilus.

19 The desirable properties of a probiotic dietary supplement are

1. Must be of human origin and be able to inhabit the small and large intestine

2. Exert a beneficial effect on the host by helping in proper digestion and assimilation of nutrients and synthesis nutrients like B complex Vitamins, and Vitamin K for the host (man)

3. Be nonpathogenic and nontoxic4. Contain a large number of viable cells5. Be capable of surviving (should not be killed by gastric juice and bile

acids) and metabolizing in the gut6. Remain viable during storage and use7. Be antagonistic to pathogens

Health benefits offered by probiotics are

1. Offers increased resistance to establishment of infection by potentially Pathogenic organisms in the intestine.

1. Decreased duration of diarrhoea (antibiotic associated, travelers', infective).

2. Use in lactose intolerance (promotion of intestinal lactose digestion).3. Increased nutritional value (better digestibility, increased absorption

of vitamins and minerals).4. Regulation of gut motility (constipation, irritable bowel syndrome). 5. Maintenance of mucosal integrity of the intestine.6. Reduction in serum cholesterol concentration.7. Reduction in allergy.8. Prevention of colon cancer.9. Reduction in carcinogen /co-carcinogen production.

IMPORTANT PROBIOTIC SPECIES

Lactobacilli and bifidobacteria are Gram - positive lactic acid producing bacteria that constitute a major part of the normal intestinal microflora in humans and animals. They play an important role in resistance to colonization against exogenous, potentially pathogenic organisms. They are essentially lactic acid producing bacteria. The lactic acid helps in reducing the pH in the intestine and thereby creates an environment, which is not conducible for growth of pathogenic organisms (pathogenic intestinal organisms prefer an alkaline environment for growth and proliferation).

Lactobacilli are Gram - positive, non - spore forming rods or coccobacilli. They have complex nutritional requirements, aerotolerant or anaerobic, acidophilic and are found in habitats rich in carbohydrate containing

13substrates such as the human intestinal mucosal membrane . They predominantly colonize the upper and lower small intestine.

Lactobacillus sporogenes is present predominantly in the small intestine and helps in synthesis of B complex vitamins and Vitamin K. It also produces enzymes required in the digestion of various carbohydrates, fats, and proteins and also aids in their absorption. They have the capability of transforming into the spore form, which gives it stability and the ability to withstand high temperature, gastric acid and bile acid. Once consumed by the host, these spores geminate in the upper small intestine and produce lactic acid thereby creating an environment, which will inhibit the growth of potentially pathogenic organisms. Lactobacillus sporogenes produces biologically active L (+) lactic acid, which is completely metabolized leading to glycogen synthesis. The WHO has recommended use of lactobacillus species that produce L (+) lactic acid, especially in infant nutritional formula. Some other strains of lactobacillus like lactobacillus acidophillus produce L (-) lactic acid, which cannot be metabolized by the body and so is not preferable as a nutritional supplement.

Bifidobacteria are nonmotile, non - spore forming, Gram - positive rods with varying appearance. Most strains are strictly anaerobic. B. longum may be considered as the

13most common species of bifidobacteria, being found both in infant and adult feces . They constitute a major part of the normal intestinal microflora in humans throughout life. Their number tends to decrease with age. They predominantly colonize the large intestine.

Saccharomyces Boulardii belongs to the yeast species. It is non - pathogenic and non - colonizing. It is considered a transient yeast in the human intestines, which means that it does not set up residence in the mucosal membrane of the intestinal tract like the lactobacillus and bifidobacteria. As it travels to the intestines, this yeast has the ability to aggressively displace species of pathogenic yeast and bacteria and at the same time it does not harm the normal intestinal flora. S. boulardii is described as a probiotic which has the ability to beneficially affect the delicate balance of the intestinal bacteria, and also has the capacity to prevent or reduce the effects of harmful pathogenic organisms. The mechanism of probiotic action is probably by production of acetic and lactic acid, which lower the intestinal pH and thereby inhibiting the growth of pathogenic yeast and bacteria. This also encourages a good environment for the intestinal resident bacteria (lactobacillus and bifidobacteria). There are some reports in medical literature of infections (septicemia) in immnuocompromised patients after treatment with S. boulardii.

Streptococcus Thermophilus are lactic acid producing aerobic gram-positive cocci. They are mainly found in the upper and lower small intestine and produce lactase enzyme, which is helpful in digestion of lactose, a sugar found in milk.

PREBIOTICS

What are prebiotics?

Prebiotics are range of non-digestible dietary supplements, which modify the balance of the intestinal micro flora, stimulating the growth and / or activity of beneficial organisms and suppressing potentially deleterious bacteria.

These supplements include lactulose, lactitol, a variety of oligosaccharides (especially fructo-oligosaccharides or FOS), and inulin. In particular, prebiotics promote the proliferation of bifidobacteria in the colon. Some of them also help in promoting the proliferation of lactobacilli in the small intestine to a certain extent.

To be effective, prebiotics should escape digestion in the upper gut, and reach the large bowel, and be utilized selectively by a restricted group of microorganisms that have been clearly identified to a health promoting properties (e.g. lactobacillus, bifidobacteria).

Three live bacteria that act as prebiotic and probiotic agents

The three live bacteria that act as pre and probiotic are a. Streptococcus faecalis T-110 (lactic acid bacteria)b. Clostridium butyricum TO-A (butyric acid bacteria)c. Bacillius mesentericus TO-A (amylolytic bacteria)

Streptococcus faecalis T-110 are live gram-positive, aerobic, non-spore forming cocci. They proliferate actively through the symbiotic action with B. mesentericus TO-A and C. butyricum TO-A to yield lactic acid with inhibition of growth of harmful bacteria. This lactic acid bacteria is found in the region from the upper to lower part of small intestine.

Clostridium butyricum TO-A are live gram-positive, anaerobic, spore forming bacilli. They proliferate actively through the symbiotic action with streptococcus faecalis T-110 to yield short chain fatty acids such as butyric acid and acetic acid with a resultant decrease in intestinal pH and inhibition of growth of harmful bacteria. The short chain fatty acids, in addition, help to regularize abnormal bowel movements. The short chain fatty acids also help in adjustment of water and electrolyte concentration of the intestinal tract. They also serve as source of nutrient for intestinal mucosal cells. It is found predominantly in the region from the upper small intestine to the colon.

Bacillius mesentericus TO-A are live gram-positive, aerobic, spore forming bacilli. They proliferate actively through the symbiotic action with streptococcus faecalis T-110. It is a spore forming bacteria and produces an amylolytic enzyme (amylase) and protease to activate proliferation of streptococcus TO-A. It is also

responsible for production of a nutrient which helps in increasing the count of bifidobacteria. It is found predominantly in the small intestine.

Important properties of the three live bacteria

The three bacteria proliferate actively throughout the intestinal tract through symbiosis, which is defined as the biological association of two or more species to their mutual benefit.

The three activated bacteria strongly inhibit the growth of potentially pathogenic bacteria in the gastrointestinal tract (probiotic effect).In vitro studies using bacterial cultures have shown that when the three live bacteria are grown together with potentially pathogenic bacteria like entero toxigenic Escherichia coli, Clostridium perfringers, Salmonella Typhi, Vibrio parahaemolyticus, Campylobacter, Yersinia enterocolitica, the three live bacteria significantly inhibited the growth of the above mentioned potentially pathogenic organisms.

The three bacteria facilitate the proliferation of bifidobacterium, thereby increasing their count significantly in the intestine. To a lesser extent they also facilitate proliferation of lactobacilli in the intestine (prebiotic effect).The growth acceleration of bifidobacteria in the intestine by the three live bacteria is through the production of a growth factor by the bacteria bacillus mesentericus TO-A.

The three bacteria normalize the intestinal flora, prevent colonization of the gastrointestinal tract by potentially pathogenic organisms and help regulate abnormal bowel movements.Intestinal flora is normalized through its prebiotic action that helps in the proliferation of bifidobacteria and lactobacillus.Prevention of colonization of the gastrointestinal tract by potentially pathogenic organisms is by lowering of intestinal pH by production of lactic acid (by Streptococcus faecalis T-110), butyric acid and acetic acid (by Clostridium butyricum TO-A).Regulation of abnormal bowel movements is done by the action of short chain fatty acids such as butyric acid and acetic acid, produced by Clostridium butyricum TO-A, on the bowel wall.Additionally, acetic acid and butyric acid, produced by Clostridium butyricum, help in adjustment of water and electrolyte concentration of the intestinal fluid and also serve as a source of nutrient for the intestinal mucosal cells.

The three live bacteria have been shown to be resistant to the action of gastric juice and intestinal juice including bile. They can therefore pass unaffected Through the upper GI tract (stomach and duodenum) and

colonize in the lower GI tract (upper and small intestine and colon), when taken orally.

It has been shown that these live bacteria help in normalizing the intestinal flora by promoting the growth of beneficial bacteria and preventing the growth of harmful bacteria. In cases of intestinal infection with pathogenic bacteria, intake of these three live bacteria can lower the counts of the pathogenic bacteria, while simultaneously increasing the count of beneficial bacteria. This is shown by a reversal of ratio of predominant aerobic : anaerobic bacteria to a predominant anaerobic : aerobic bacterial ratio. Here the aerobic bacteria signify the potentially pathogenic organisms whereas, the anaerobic bacteria signify the beneficial resident bacteria in the intestine.

The increased count of bifidobacteria, generated in the intestine through the action of the three live bacteria, produces Glutamine from NH4 + and glutamic acid in the intestine. Glutamine is the fuel for the intestinal cells and helps in maintaining the integrity of the intestinal mucosal barrier. In this manner, the colonization by potentially pathogenic microorganisms in the intestine is inhibited.

What is synbiotic?

Synbiotic is a combination of prebiotic and probiotic. So, when a preparation contains both prebiotic and probiotic, it is known as synbiotic.

What is bacteriotherapy?

Bacteriotherapy by general definition is using harmless and beneficial bacteria to displace pathogenic organisms. It is an alternative and promising way of combating infections.

THE RELATIONSHIP BETWEEN PROBIOTIC AND PREBIOTIC BACTERIA AND POTENTIALLY PATHOGENIC MICROORGANISM (PPMS).

BIFILAC (PREBIOTIC + PROBIOTIC)

Preparations

Bifilac is available as granules (in sachets) and as capsules. Each sachet / capsule contains

Streptococcus faecalis T-110 30 millionClostridium butyricum TO-A 2 millionBacillus mesentericus TO-A 1 millionLactobacillus sporogenes 50 million

Streptococcus faecalis T-110, Clostridium butyricum TO-A, Bacillus mesentericus TO-A are the three live bacteria which proliferate together by a process of symbiosis in the gastrointestinal tract and act as a prebiotic and probiotic. Lactobacillus sporogenes is a probiotic.

Dosage

Children one sachet three times a day or as directed by the physician.Adults one to two capsules three times a day or as directed by the physician.

Duration of treatment As advised by the physician.

Indications

1. Infective diarrhoea (viral, bacterial, protozoal). 2. Antibiotic associated diarrhoea.3. Lactose intolerance.4. Recurrent aphthous ulcers and stomatitis5. Inflammatory bowel disease (ulcerative colitis, Crohn's disease)6. Irritable bowel syndrome.7. Travelers' diarrhoea.8. Diverticular disease of colon.9. Post operative state.

Safety

The three live bacteria along with Lactobacillus sporogenes in Bifilac colonize in the intestinal tract and do not enter into systemic circulation. Bifilac is therefore safe to use, as the ingredients of Bifilac confine themselves only to the lumen of the gastrointestinal tract. Bifilac must not be administered to infants less than 3 months of age.Safety of Bifilac in pregnancy and lactation has not been estabilished.

Superiority of Bifilac over other probiotic preparations available

Properties BifilacLactobacillus

SpeciesSaccharomyces

boulardii

1. Prebiotic + Probiotic

Yes Probiotic only Probiotic only

2. Natural gut flora

Helps in supplementing & in proliferation of predominantnatural gut flora organisms like lactobacilli and bifidobacteria.

Helps in supplementing lactobacilli only.

It is a non- colonizing yeast. Does not alter normal bowelflora. Does not colonize in the intestinal tract.

3. Extent of colonization of beneficial bacteria in GI tract.

Helps in proliferation of both lactobacilli (small intestine) and bifidobacteria(large intestine)& thereby provides beneficial effects to the whole gut.

Supplements lactobacilli only which predominantly colonize the small intestine.

Has predominant action in largeintestine.

4. Possible side effects

None None Danger of Septicemia when used in immuno-compromised patients

5. Maintenance of intestinal mucosal integrity

Intestinal mucosalintegrity is strengthened and maintained by production of butyrates and acetates by thethree live bacteria and through glutamine produced by bifidobacteria.

No action. No action.

6. Correction of abnormal bowel movements regulation of bowel movements.

The acetates and butyrates produced by the three live live bacteria help to normalize abnormal bowel movements.

No action on abnormal bowel movements.

No action on abnormal bowel movements.

7. Action on potentially pathogenic bacteria in intestine.

The three live live bacteria have been shown to inhibit the growth of many potentially pathogenic intestinal organisms independently, apart from a similar action by the probiotic bacteria namely lactobacillus& bifidobacteria.

Lactobacillus species has some action on inhibiting growth of potentially pathogenicintestinal microorganisms.

Has some actionon inhibiting growth of potentially pathogenic intestinal micro-organisms.

8. Nutritional benefit to host

Lactobacilli and bifidobacteria benefit the host by producing B complex vitamins and Vitamin K.

Lactobacilli species to a certain extent produceB complex vitamins and Vitamin K.

No suchnutritional benefit to host.

9. Dietary intolerance / Malabsorption states

Both lactobacilli and bifidobacteriahave been shown to beneficially affect dietary intolerance or malabsorption states by helping better digestion of food and assimilation of nutrients.

Lactobacilli to a certain extent help in better digestion of food and assimilation of nutrients.

No such role identified.

SPECIAL NOTE: The efficacy of oral administration of probiotic species of Bifidobacteria and Streptococcus thermophilus is questionable as these species are not able to survive passage through the GI tract. They are easily killed and made ineffective on contact with gastric acid and bile acid.

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