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
products competed with microorganisms that are injurious to health1.

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
2
the 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
6-9
colitis, 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
11
some 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,
race, socio economic circumstances, lifestyle can upset this balance12.

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
12
bacteria 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
intestine12 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.

Proximal small intestine Lactobacilli + Enterococcus faecalis (10 5 - 107 / ml of

fluid)

Distal small intestine Lactobacilli + Enterococcus faecalis + Coliforms +

8
Bacteroides (10 bacteria / ml of fluid)
11
Colon Bacteroides + Bifidobacteria (10 bacteria / ml of fluid)

Table I Composition of the human gastrointestinal microflora13

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
including breast milk or formula14. The infant tends to acquire the flora swallowed
from the vaginal fluid at the time of delivery. Because vaginal flora and intestinal flora
15
are 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
16
frequently 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
predominant17. 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
properties of the indigenous microflora12.

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.

The desirable properties of a probiotic dietary supplement are19

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 nontoxic
4. Contain a large number of viable cells
5. Be capable of surviving (should not be killed by gastric juice and bile
acids) and metabolizing in the gut
6. Remain viable during storage and use
7. 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
substrates such as the human intestinal mucosal membrane13. 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
13
most 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 million

Clostridium butyricum TO-A 2 million
Bacillus mesentericus TO-A 1 million
Lactobacillus 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 stomatitis
5. 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

Lactobacillus Saccharomyces
Properties Bifilac Species boulardii

1. Prebiotic + Yes Probiotic only Probiotic only

Probiotic

2. Natural gut Helps in Helps in It is a non-

flora supplementing supplementing colonizing yeast.
& in proliferation lactobacilli only. Does not alter
of predominant normal bowel
natural gut flora flora. Does not
organisms like colonize in the
lactobacilli and intestinal tract.
bifidobacteria.

3. Extent of Helps in Supplements Has

colonization proliferation of lactobacilli only predominant
of beneficial both lactobacilli which action in large
bacteria in (small intestine) predominantly intestine.
GI tract. and bifidobacteria colonize
(large intestine)& the small
thereby provides intestine.
beneficial effects
to the whole gut.
4. Possible side None None Danger of
effects Septicemia
when used in
immuno-
compromised
patients

5. Maintenance Intestinal mucosal No action. No action.

of intestinal integrity is
mucosal strengthened and
integrity maintained by
production of
butyrates and
acetates by the
three live bacteria
and through
glutamine
produced by
bifidobacteria.
6. Correction of The acetates and No action on No action on
abnormal butyrates abnormal bowel abnormal bowel
bowel produced by the movements. movements.
movements three live live
regulation bacteria help to
of bowel normalize
movements. abnormal bowel
movements.

7. Action on The three live live Lactobacillus Has some action

potentially bacteria have been species has some on inhibiting
pathogenic shown to inhibit action on growth of
bacteria in the growth of inhibiting growth potentially
intestine. many potentially of potentially pathogenic
pathogenic pathogenic intestinal micro-
intestinal intestinal organisms.
organisms microorganisms.
independently,
apart from a
similar action by
the probiotic
bacteria namely
lactobacillus
& bifidobacteria.
8. Nutritional Lactobacilli and Lactobacilli No such
benefit to bifidobacteria species nutritional
host benefit the host by to a certain extent benefit to host.
producing produce
B complex B complex
vitamins and vitamins and
Vitamin K. Vitamin K.

9. Dietary Both lactobacilli Lactobacilli to a No such role

intolerance / and bifidobacteria certain extent identified.
Malabsorption have been shown help in better
states to beneficially digestion of food a
affect dietary nd assimilation
intolerance or of nutrients.
malabsorption
states by helping
better digestion of
food and
assimilation
of nutrients.
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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