European Journal of Pharmaceutical Sciences 15 (2002) 19

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Review article

Probiotics: potential pharmaceutical applications

Indu Pal Kaur*, Kanwaljit Chopra, Amarpreet Saini
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160 014, India
Received 10 April 2001; received in revised form 5 October 2001; accepted 17 October 2001

Abstract
Realisation of the importance of human gut microbiota in health restoration and maintenance has kindled an interest in probiotics.
Probiotics are defined as the microbial food supplements, which beneficially affect the host by improving its intestinal microbial balance.
Probiotics are the health enhancing functional food ingredients used therapeutically to prevent diarrhea, improve lactose tolerance and
modulate immunity. They may also have potential to prevent cancer and lower serum cholesterol levels. Lactobacillus, Bifidobacterium
and several other microbial species are perceived to exert such effects by changing the composition of the gut microbiota. However, it is
important that exogenously administered bacteria reach and establish themselves in the large intestine in an intact form. The use of
non-digestible oligosaccharides (prebiotics) can fortify intestinal microflora and stimulate their growth. The present review encompasses
information regarding the probiotics and their proposed uses. It addresses the concepts of prebiotics and synbiotics, the application of
genetic engineering to produce newer probiotics. Finally, the list of commercially available products are reviewed with discussion of
questions regarding the reliability, utility and the safety of these products. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Probiotics; Prebiotics; Microbiota; Recombinant probiotics

1. Introduction
Probiotics are defined as the viable microorganisms that
exhibit a beneficial effect on the health of the host by
improving its intestinal microbial balance. The term probiotics was first coined by Lilly and Stillwell in 1965 in
reference to substances produced by protozoa, which
stimulated the growth of other organisms. Interest in
probiotics has been spurred by the growing abundance of
modern disorders such as neoplasms, atherosclerosis,
cardiac diseases, hypertension and HIV infection. Probiotic
consumption is reported to exert a myriad of beneficial
effects including: enhanced immune response, balancing of
colonic microbiota, vaccine adjuvant effects, reduction of
fecal enzymes implicated in cancer initiation, treatment of
diarrhea associated with travel and antibiotic therapy,
control of rotavirus and Clostridium difficile-induced colitis and prevention of ulcers related to Helicobacter pylori.
Probiotics are also implicated in the reduction of serum
cholesterol, the antagonism against food-borne pathogens
and tooth decay organisms, the amelioration of lactose
malabsorption symptoms as well as candidiasis and urinary
*Corresponding author. Tel.: 191-172-534-107; fax: 191-172-541142.
E-mail address: indupalkaur@yahoo.com (I.P. Kaur).

tract infections (Saavedra, 2001). Promising probiotic

strains include the members of genera Lactobacillus,
Bifidobacterium, and Enterococcus. The representative
species include Lactobacillus acidophilus, L. johnsonii, L.
casei, L. gasseri, L. plantarum, L. rhamnosus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium
bifidum, Bifidobacterium infantis, Enterococcus faecalis
and Enterococcus faecium (Fuller, 1991; Gordin and
Gorbach, 1992). They have considerable potential for
further inclusion in functional foods and health-related
products. Even though these health claims are generally
accepted by scientists and consumers, however the underlying molecular mechanisms remain controversial. An in
depth investigation of the molecular basis of probiotic
traits will give a vital reinforcement to the probiotic
concept and is a pre-requisite for their rational development.

2. Properties and proposed mechanism of action

A group of requirements have been identified for a
microorganism to be defined as an effective probiotic
(Salminen et al., 1996). These include the ability to: (a)
adhere to cells; (b) exclude or reduce pathogenic adherence; (c) persist and multiply; (d) produce acids, hydrogen

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I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

improving general gut health, (b) improving bodys natural

defenses, and (c) lowering blood cholesterol.

3.1. Gastrointestinal uses

Fig. 1. Purported mechanisms of action of probiotics.

peroxide, and bacteriocins antagonistic to pathogen

growth; (e) be safe, noninvasive, noncarcinogenic and
nonpathogenic; and (f) coaggregate to form a normal
balanced flora.
Emerging evidences have revealed that prevention of
gastrointestinal tract (GIT) colonization by a variety of
pathogens is a primary mechanism of beneficial effects
mediated by probiotics (Lu and Walker, 2001; Forestier et
al., 2001). Probiotic bacteria attach to enterocytes and thus
inhibit the binding of enteric pathogens to the intestinal
mucosa by production of inhibitory substances (competitive exclusion of pathogens) (Nemcova, 1997; KoppHoolihan, 2001). These inhibitory substances include
bacteriocins, lactic acid and toxic oxygen metabolites (Fig.
1). Of the toxic oxygen metabolites, the production of
hydrogen peroxide is of prime importance as, in combination with lactoperoxidasethiocyanate milk system, it
exerts a bactericidal effect on most pathogens. Therefore,
inclusion of probiotic bacteria in fermented dairy products
enhances their value as better therapeutic functional foods
(Kailasapathy and Chin, 2000). Attachment of probiotic
bacteria to cell surface receptors of enterocytes also
initiates signalling events that result in the synthesis of
cytokines. Furthermore, the production of butyric acid by
some probiotic bacteria affects the turnover of enterocytes
and neutralizes the activity of dietary carcinogens, such as
nitrosamines, the latter being generated by the metabolic
activity of commensal bacteria in subjects consuming a
high-protein diet (Wollowski et al., 2001).

3. Expected therapeutic uses

The commercial interest in functional foods that contain
probiotics, is paralleled by increasing study of their role in
the digestive tract. To date, interest in probiotic containing
foods has centered around three health propositions (a)

Beneficial aspects of the human gut flora need definitive

confirmation and mechanistic explanations. Historically,
there has always been an interest in modulating the
composition of gut flora such that a more favourable
balance of bacteria resides in the gut (Marteau et al.,
2001). Competent clinical studies appear to suggest that
specific probiotic microbes can (a) alleviate or prevent
diverse intestinal diarrhea-inducing disorders such as lactose indigestion and cause prophylaxis of intestinal and
urogenital infections; and (b) inhibit the mutagenicity of
intestinal contents and reduce the incidence of intestinal
tumors (Ferencik et al., 1999). Beneficial probiotic traits
also include the demonstration of bile tolerance, acid
resistance, and adherence to host epithelial tissue to act as
antagonists to potentially pathogenic microorganisms.
It is generally agreed that a probiotic must be capable of
colonizing the intestinal tract to influence human health.
This requirement may disqualify many of the strains
currently used in fermented dairy products. In contrast,
Lactobacillus GG (LGG), a variant of Lactobacillus casei
subsp. rhamnosus, is one of the most extensively studied
probiotic which has proved benefits in reducing the
severity and duration of diarrhea. LGG-fermented milk
reduces the intestinal permeability defects caused by
exposure to cows milk or rotavirus infection. When
consumed as a dairy product or as a lyophilized powder,
LGG colonizes the gastrointestinal tract for 13 days in
most individuals and up to 7 days in about 30% of
subjects. Consumption of foods containing LGG may
shorten the course of rotavirus infection causing diarrhea
(de Roos and Katan, 2000), travellers diarrhea and
antibiotic-associated diarrhea. Clostridium difficile is the
leading cause of nosocomially acquired intestinal infection
in the United States, affecting virtually all cases of pseudomembranous colitis and up to 20% of cases of antibioticassociated diarrhea. Even after receiving antibiotic treatment with either metronidazole or vancomycin, 20% of
patients show recurrent Clostridium difficile diarrhea. LGG
reduces the recurrence rate of Clostridium difficile, and
patients report disappearance of abdominal cramps and
diarrhea (Pochapin, 2000).
LGG modulates the intestinal immunity by increasing
the number of IgA- and other immunoglobulin-secreting
cells in the intestinal mucosa. Furthermore, it stimulates
the local release of interferons and facilitates antigen
transport to underlying lymphoid cells, which serves to
increase antigen uptake in Peyers patches. LGG also acts
as an immunoadjuvant for oral vaccines. Few of its major
immunological effects are depicted in Fig. 2.
An aggressive immunological response by the resident
luminal bacteria seems to be responsible for the develop-

I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

Fig. 2. Beneficial effects of Lactobacillus GG on intestinal immunity.

ment and chronicity of inflammatory bowel diseases. The

results of recent studies with probiotics in animal models,
suggest that these agents antagonise the pathogenic organisms implicated in chronic inflammatory bowel disease,
pouchitis, and ulcerative colitis (Schultz and Sartor, 2000).
There is circumstantial evidence linking Escherichia coli
with ulcerative colitis. Administration of a non-pathogenic
strain of Escherichia coli may have an effect equivalent to
mesalazine, a standard therapy for remission of ulcerative
colitis (Rembacken et al., 1999). Similarly, 5-aminosalicylic acid (5-ASA) is the standard maintenance treatment for ulcerative colitis but its use can be limited by
associated adverse effects. A new probiotic preparation,
VSL[3 (Table 1), has been found to be useful in the
patients intolerant or allergic to 5-ASA (Venturi et al.,
1999). Recently, Sakamoto et al. (2001) have reported that
Lactobacillus gasseri is effective both in suppressing H.
pylori and reducing gastric mucosal inflammation in 31
human subjects.

3.2. Pediatric gastroprotection

In humans, microbial colonization of a previously germfree gut begins just after birth. Development of the normal
flora is a gradual process, which is determined by factors
such as composition of the maternal gut microflora, diet,
environment and possibly also by genetic aspects. A
number of variables, such as the degree of hygiene, mode
of delivery, use of antibiotics or other medication and a
need for nursing in incubators, can all have a substantial
effect on the microbial colonization and development in
infants. The pattern of bacterial colonization in the premature neonatal gut is different from that in the healthy,
full-term infant gut. Those infants requiring intensive care
acquire intestinal organisms slowly, and this delayed
bacterial colonization of the gut with a limited number of
bacterial species tends to be virulent. The aberrant colonization of the premature infant may contribute to the
development of necrotizing enterocolitis. Hence, the control and manipulation of the bacterial colonization in the
neonatal gut may be a novel approach to the prevention

and treatment of intestinal infectious diseases of various

etiologies (Dai and Walker, 1999). It is postulated that in
the infant gut, an elevated Bifidobacterial count may be
associated with health advantages that breast-fed infants
have over formula-fed infants. Oligosaccharides and
glycoconjugates, the natural components in human milk,
may further prevent intestinal attachment of enteropathogens by acting as receptor homologues (Gopal
and Gill, 2000).
Numerous probiotic agents have been studied for the
management of diarrheal disease in pediatric patients
(Friedrich, 2000; Davidson and Butler, 2000). In particular, the prevention and management of acute viral
diarrhea, the treatment of recurrent Clostridium difficile
diarrhea, as well as the control of antibiotic-associated
diarrhea seem to be the areas of significant potential
benefit. LGG is the key probiotic being explored and
Lactobacillus reuteri and Saccharomyces boulardii seem
to be the other promising agents. In general, in pediatric
populations, the effect of probiotic agents appears to be
most significant against viral (rotaviral) diarrhea, suggesting an immunological mechanism for these effects.
The probiotic agents are thus becoming an important part
of the armamentarium against gastrointestinal problems in
infants and children (Saavedra, 2000).

3.3. Food allergy reduction

Human beings are exposed to numerous environmental
antigens through food. The intestinal mucosa is efficient in
assimilating antigens encountered by the enteric route
(Heyman and Desjeux, 1992), but high-level antigen
exposure during the first few months of life may predispose individuals to allergic sensitization. Intestinal
inflammation seems to be a predisposing factor in increased sensitization of a subject (Holt, 1994).
The intestinal microflora is an important constituent of
the gut mucosal barrier (Wells et al., 1988; Fuller, 1991).
In the absence of intestinal microflora, antigen transport is
increased. Intact cows milk proteins have been shown to
stimulate peripheral blood mononuclear cells to release
pro-inflammatory cytokines in patients with cows milk
allergy (Heyman et al., 1995). However, it has been shown
that cows milk proteins degraded by Lactobacilli, but not
by trypsin or pepsin, may generate tolerogenic peptides
from the native protein. These findings tend to substantiate
the hypothesis that specific strains of intestinal microflora
may aid in host protection against allergic sensitization
(Hatakka et al., 2001).
Current approach in the management of food allergy
aims at complete avoidance of foods proven to cause
symptoms. In infants with cows milk allergy, extensively
hydrolyzed formulas are used to eliminate cows milk
antigens from the diet. However, even extensive pepsin
trypsin hydrolysis does not render the formula non-antigenic; and even minute quantities of immunoreactive

I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

Table 1
Commercially marketed formulations of probiotics
Product name

Probiotic bacteria present

Description and properties of the product

Kyo-Dophilus capsules
(Wakunaga Probiotics)

Lactobacillus acidophilus,
Bifidobacterium bifidum,
Bifidobacterium longum

Contains human strains of bacteria. Each

capsule contains about 1.5 billion live cells.

Kyo-Dophilus tablets
(Wakunaga Probiotics)

Lactobacillus acidophilus

Stable preparation not requiring refrigeration,

completely vegetarian, dairy and sugar free,
chewable, tasty and convenient for travel.

Acidophilas
(Wakunaga Probiotics)

Lactobacillus acidophilus,
Lipase, protease, amylase
and lactase enzymes

Enzymes assist in breakdown of fats, proteins

and carbohydrates, lactase assists in digestion
of milk sugar lactose in lactose intolerance

Probiata tablets

An internationally accepted
strain of Lactobacillus
acidophilus

Bacteria pre-adapted for growth in human intestine.

Each tablet contains one billion live cells per tablet,
preparation requires no refrigeration and is heat
resistant. Survives when taken with food and
resistant to acid of stomach (pH 34)

Flora Grow
(Arise & Shine)

Bifidobacterium infantis,
Bifidobacterium longum,
Bifidobacterium bifidum

Generate a pH between 6.5 and 7.0 instead of

pH 4.5, as produced by Lactobacillus acidophilus.
At the latter pH the beneficial bacteria of the human
bowel begin to die off.

Bifa 15
(Eden Foods)

Bifidobacterium longum

Contains microencapsulated Bifidobacterium longum

designed to get past the stomach acids and reach the
colon. Produces a very hostile environment for HIV,
Candida and other infections of the large intestine.

TH1 Probiotics
(Jarrow Formulas)

Bifidobacterium longum,
Saccharomyces boulardi,
Lactobacillus casei,
Lactobacillus plantarum

Contains one billion Bifidobacterium longum per

serving along with 1 / 2 billion Saccharomyces boulardi,
both Lactobacillus casei and Lactobacillus plantarum
are heat treated so as to be safe for use in severely
immune compromised persons who have leaky gut.

Replenish
(Innercleanse 2000)

Lactobacillus acidophilus,
Lactobacillus plantarum,
Lactobacillus bifidus,
Lactobacillus bulgaricus,
Lactobacillus rhamnosus,
Lactobacillus casei,
Lactobacillus brevis, FOS
(Fructo-oligosaccharides)

Causes an increase in production of beneficial short-chain

fatty acids, reduction of serum cholesterol and blood
pressure, improved liver function and improved elimination
of toxic compounds. FOS helps promote the growth of
friendly bacteria, while simultaneously reducing the colonies
of detrimental bacteria.

Flora BacE
(PDI labs)

Stabilized Bifidobacteria
and Acidophilus,
Lactobacillus sporogenes,
Bacillus laterosporus
BOD, enzymatic bacteria
BaCCineE.

VSL[3
(CSL)

5310 11 cells / g of three strains

of Bifidobacteria, four strains
of lactobacilli and one strain
of Streptococcus salivarius
ssp. thermophilus

Subalin

Recombinant Probiotic
(Bacillus subtilis)

Colinfant, Mutaflor

Non-enteropathogenic
Escherichia coli

Colonize the intestine and help in the

remission of ulcerative colitis in the
patients intolerant or allergic to
5-aminosalicylic acid.

Live oral vaccines, which colonize the

intestines of full term and pre-term
infants and establish themselves as
resident strains. Stimulate antibody
production in gut, saliva and serum of
colonized infants. Decrease the presence
of pathogenic bacterial strains in the
intestines as well as on other mucosal
surfaces of the body.

I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

Table 1. Continued
Product name

Probiotic bacteria present

Description and properties of the product

Symbiotik capsules

(Le Sante)

Lactobacillus sporogenes,
antibiotics amoxycillin (as
trihydrate) and cloxacillin
(as sodium)

Each capsule contains 250 mg amoxycillin,

250 mg cloxacillin and 60 million spores
of Lactobacillus sporogenes, useful for
respiratory tract infections, ENT infections,
skin and soft tissue infections, urinary tract infections.

Symbiotik-P tablets

(Le Sante)

Lactobacillus sporogenes,
antibiotics amoxycillin (as
trihydrate) and cloxacillin
(as sodium)

Each tablet contains 125 mg amoxycillin,

125 mg cloxacillin and 30 million spores
of Lactobacillus sporogenes, it has the
same indications as above.

Primal DefenseE
capsules

Contains HSOE
(homeostatic soil organisms
including Lactobacillus acidophilus,
Bifidobacterium bifidum,
Bacillus licheniformis,
Bacillus subtilus,
Lactobacillus lactis,
Lactobacillus bulgaricus)
Each capsule contains 250 mg
HSO, 160 mg certified organic
green grass juices (kamut grass
juice, alfalfa grass juice, oat
grass juice, barley grass juice),
80 mg phytosterol / sterolin blend
sprouts (wild african potato,
oilseed plant, lupins, fenugreek,
barley, wheat, soybean and african
sunflower sprouts)

Food Supplement. HSOE are dormant in the

capsule and activated by fluids so the product
has at least a 3-year, non-refrigerated shelf life.
Bifidobacterium subtilus and Bacillus licheniformis
are extremely effective against viruses. Phytosterols
and sterolins are involved in repair and maintenance
of the immune system and also useful in allergies,
viruses, fibromyalgia, tumors, BPF (enlarged prostate)
and are proven to lower LDL (bad) cholesterol.
Grass juices contain minerals, vitamins A, B, C and K,
the antioxidant-enzyme superoxide dismutase (SOD),
P4D1, and the Grass Juice Factor. These slow cellular
deterioration and mutation and may even help reverse
aging. The Grass Juice Factor has a potent
anti-inflammatory effect also.

components of the native protein are capable of eliciting

the allergic reaction. Oral introduction of probiotics can
help in treatment of such food allergies by alleviating
intestinal inflammation. It has also been suggested that
intestinal microorganisms could down-regulate the allergic
inflammation by counter-balancing T-helper cell Type 2
responses and by enhancing antigen exclusion through
induction of an IgA response (Kirjavainen and Gibson,
1999).
Majamaa and Isolauri (1997) suggest that probiotic
bacteria may promote endogenous barrier mechanisms in
the patients with atopic dermatitis and food allergy. They
also report a decrease in a 1 -antitrypsin and the fecal tumor
necrosis factor-a in infants with atopic eczema and cows
milk allergy, upon treatment with LGG fortified extensively hydrolyzed whey formula. These results suggest that
probiotics, by alleviating intestinal inflammation, may act
as a useful tool in the treatment of food allergy.

3.4. Immunomodulatory activity

Immunomodulation by probiotics is a subject of growing
interest. Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS have shown a
specific dose- and duration-dependent immunomodulatory
effects on the proliferative activity of murine B and T
lymphocytes (Kirjavainen et al., 1999). In another study
the effects of heat-killed cells, cell walls, and cytoplasmic

extracts of Bifidobacterium, Lactobacillus acidophilus, L.

bulgaricus, L. casei, L. gasseri, L. helveticus, L. reuteri,
and Streptococcus thermophilus, on cell proliferation and
cytokine and nitric oxide (NO) production, were examined
using the RAW 264.7 macrophage cell line and in murine
cultures composed of peritoneal, spleen, and Peyers patch
cells. Both the cell wall and cytoplasmic fractions were
able to stimulate cloned macrophages to produce significant amounts of tumor necrosis factor-a, interleukin-6
(IL-6), and NO. Pronounced enhancement of IL-6 production by peritoneal cells was observed when cultured
with these extracts. Based on the results, it appears that
lactic acid bacteria appear capable of stimulating macrophages and possibly other immune cells to produce
cytokines and NO. Probiotic cell walls and cytoplasm
contribute to these capacities (Tejada-Simon and Pestka,
1999).
Lactobacillus acidophilus UFV-H2b20 has been reported to stimulate a non-specific immune response in
germ-free Swiss mice as indicated by the stimulation of the
host mononuclear phagocytic activity. The latter was
demonstrated by the clearance of a Gram-negative bacterium innoculated intravenously, and a two-fold increase
in the amount of Kupffer cells, responsible for the clearance of circulating bacteria (Neumann et al., 1998).
Oral administration of Bifidobacterium breve YIT4064
has been reported to activate the humoral immune system
by augmenting anti-rotavirus IgA production or anti-in-

I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

3.5. Antimutagenic /anticarcinogenic activity

Fig. 3. Reported immunomodulatory mechanisms of different probiotic

strains.

fluenza virus, IgG production, and thus protect against

rotavirus infection or influenza infection, respectively (Fig.
3). Furthermore, when these organisms were administered
to infants there was a significant reduction in the frequency
of rotavirus shedding in stool samples. The oral administration of Lactobacillus casei strain in mice stimulated
type 1 helper T (Th1) cells, activated the cellular immune
system and inhibited incidence of tumors and IgE production. (Yasui et al., 1999). It is also reported to modify
the humoral and cellular immune responses to type II
collagen (CII), and these modifications could result in the
reduction of the development of CII-induced arthritis in
DBA / 1 mice (Kato et al., 1998).
The cell wall of Lactobacillus casei CRL 431, presents
lectin like surface molecules which stimulate the immune
system. Given the role that lectins and lectin like substances may play in the adhesion phenomenon, it is
probable that this is an initial stage in the immunostimulation produced by this bacterium (Morata de Ambrosini et al., 1999). The administration of yoghurt supplemented with Lactobacillus acidophilus, Bifidobacterium
bifidum and Bifidobacterium infantis has been shown to
enhance mucosal and systemic IgA responses to the
cholera toxin immunogen, in mice. (Tejada-Simon et al.,
1999).
Unheated and heat-treated homogenates of probiotic
bacteria, including Lactobacillus rhamnosus GG,
Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus
thermophilus possess heat-stable anti-proliferative component(s) which suppress phytohemagglutinin-induced proliferation of mononuclear cells (Pessi et al., 1999). Thus,
these bacteria may be used to generate microbiologically
nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.
Fukushima et al. (1999) indicate that feeding of Bifidobacteria to lactating mice can enhance local production of IgA
in milk and the intestine, which may protect the young
ones from exposure to food antigens.

Cancer is one of the most important causes of morbidity

and mortality in Western countries. Although there is no
direct experimental evidence for cancer suppression in
humans as a result of consumption of lactic acid cultures in
fermented or unfermented dairy products yet, there is a
wealth of indirect evidence, based largely on laboratory
studies, in the literature (Hirayama and Rafter, 1999).
Anti-mutagenic activities of live and killed cells of different strains of Lactobacillus acidophilus, Bifidobacteria and
of organic acids usually produced by these probiotic
bacteria have been demonstrated using potent chemical
mutagens and promutagens. Live probiotic bacteria
showed a higher antimutagenic activity and their efficiency
in inhibiting the mutagens was better than that of killed
bacterial cells (Lankaputhra and Shah, 1998). Some of the
lactic acid bacteria have also been reported to inhibit colon
cancer. The precise mechanism of action is not yet known.
However, a variety of mechanisms may be taking place
including: enhancing the hosts immune response, binding
and degrading potential carcinogens, quantitative and / or
qualitative alterations in the intestinal microflora Other
effect may include the alteration of the metabolic activities
of intestinal microflora and alteration of physicochemical
conditions in the colon (Hirayama and Rafter, 1999).
Reddy (1999) reports that the dietary administration of
lyophilized cultures of Bifidobacterium longum strongly
suppressed colon and mammary tumor development. This
was associated with a decrease in the colonic mucosal cell
proliferation. Two lactic acid bacteria strains isolated from
human faeces have also been reported to possess an antiproliferative effect on a tumour cell line (Zabala et al.,
2001).
The consumption of either Bifidobacterium longum or
inulin was also associated with a decrease in azoxymethane (colon carcinogen)-induced small colonic aberrant crypt-foci (ACF). ACF are putative neoplastic lesions
from which adenomas and carcinomas may develop in the
colon. The combined administration of the Bifidobacterium
and inulin resulted in more potent inhibition of ACF than
administration of the two separately. Consumption of diets
containing Bifidobacterium longum, inulin or both was also
associated with a decrease in beta-glucuronidase activity
and ammonia concentration in the caecal contents. Both
these factors have been associated with carcinogenesis of
the colon in experimental animal models (Rowland et al.,
1998). Some chicory-derived fructans have also been
reported to stimulate apoptosis in rat colon and demonstrate chemopreventive effects (Hughes and Rowland,
2001).

3.6. Cholesterol lowering effect of probiotics

The administration of Lactobacillus reuteri CRL 1098
(10 4 cells / day) to hypercholesterolemic mice for 7 days

I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

decreased total cholesterol by 38%, producing serum

cholesterol concentrations similar to that of the control
group. This dose of Lactobacillus reuteri caused a 40%
reduction in triglycerides and a 20% increase in the ratio of
high density lipoprotein to low density lipoprotein without
bacterial translocation of the native microflora into the
spleen and liver (Taranto et al., 1998).

4. Recombinant probiotic subalin

A new probiotic class based on recombinant strains of
bacteria has been designed to produce predetermined
therapeutic proteins. The biological properties of Bacillus
subtilis 2335 strain were transformed by the plasmid
encoding the synthesis of human interferon alpha-2. The
recombinant strain was demonstrated to preserve the high
antagonistic activity of the parent culture (biosporin) and
to acquire marked antiviral properties due to interferon
synthesis. Using this strain, Sorokulova et al. (1997)
designed the new probiotic possessing a combination of
antibacterial and antiviral properties. Clinical and bacteriological investigations during administration and after
termination of the course of oral administration of subalin
have suggested innocuousness as well as to the absence of
any side-effects of subalin (Sorokulova, 1998). Both
biosporin and subalin have been proven to be safe when
injected intravenously and intraperitoneally into animals at
a dose of 5310 9 cells per 0.5 ml of physiological saline
(Osipova et al., 1998).
Subalin has been tested for its ability of increasing the
effectiveness of anti-tumor therapy with cyclophosphamide. The combined treatment resulted in a significantly
higher inhibition of primary tumor growth and metastatic
spreading, as compared with control receiving cyclophosphamide alone. The cytostatic activity of peritoneal macrophages was higher in mice treated with subalin. It is
suggested that the chemo-sensitizing and antitumor effects
of subalin are due to its ability to induce endogenous
interferon production (Cherdyntseva et al., 1997). Furthermore, the application of gene engineering methods may aid
in designing a new generation of probiotics with predictable biological properties.

5. Prebiotics
It is a well-established fact that resistance to pathogens
and an immune stimulation can be achieved by probiotic
therapy. However, it is important that these exogenous
bacteria (i.e. probiotics) reach the large intestine in an
intact and viable form. Upon ingestion, these probiotics are
confronted by a number of physical and chemical barriers
such as gastric and bile acids. As a result only a small
proportion of the probiotic microorganisms are able to
reach and establish themselves in the colon. Once the

probiotic has established itself by adhering itself to the

intestinal epithelium, it will have to compete for nutrients
and ecological sites of colonization with already established microflora comprising of several hundred other
bacterial species. Furthermore, it has been indicated that
when the product containing probiotics is discontinued, the
added bacteria are rapidly washed out of the colon.
A recent approach that partly overcomes these limitations of probiotics is the use of prebiotics, which are not
viable entities but rather are growth substrates that fortify
the beneficial intestinal flora. Prebiotics are non-digestible
food ingredients that beneficially affect the host by selectively stimulating the growth and / or activity of one or a
limited number of bacteria in the colon (Roberfroid, 2001).
In order for a food ingredient to be classified as a prebiotic,
it must (a) be neither hydrolyzed nor absorbed in the upper
part of the GIT, (b) be able to alter the colonic flora in
favour of a healthier composition, (c) induce luminal or
systemic effects that are beneficial to the host health.
Increasingly, probiotics and prebiotics are being used in
combination, this being termed a synbiotic. Synbiotics or
eubiotics are defined as a mixture of probiotics and
prebiotics that improve the survival and implantation of
live microbial dietary supplements in GIT, either by
stimulating growth or by metabolically activating the
health promoting bacteria.
The prebiotics developed so far are the non-digestible
oligosaccharides, especially the non-digestible fructans.
The feeding of oligofructose and inulin to human volunteers alters the gut flora composition in favor of
Bifidobacteria, a purportedly beneficial genus. Future
human studies that exploit the use of modern molecularbased detection methods for bacteria will determine the
efficacy of prebiotics and it may be possible to address
certain gastrointestinal complaints prophylactically through
the selective targeting of gut bacteria (Gibson, 1999).

6. Conclusions and future perspectives

The global market for functional foods is growing at a
very fast rate and the probiotic products represent a
potential growth area. Intense research efforts are under
way to develop products into which probiotic organisms
such as Lactobacillus and Bifidobacterium species are
incorporated. The long term exploitation of probiotics
would depend on scientifically proven clinical evidence of
health benefit, of consumer expectation and of effective
marketing strategies (Stanton et al., 2001).
One approach that should be encouraged for future
research is a combination of probiotics and prebiotics (as
synbiotics) which can result in an increased number of
ingested bacteria reaching the colon in a viable form.
Another approach would be to use these probiotics in
combination with food enzymes and antibiotics (Table 1).
However, no probiotic can be described as useful unless it

I.P. Kaur et al. / European Journal of Pharmaceutical Sciences 15 (2002) 1 9

can be prepared in a viable manner on a large scale,

remains viable and stable during use and storage and is
able to survive the intestinal ecosystem. It must also
remain metabolically active in the gut, must not produce
any biochemical changes such as proteolysis, sugar metabolism and organic acids in the food products in which it is
incorporated
While the development of foods containing probiotic
bacteria are being investigated for their positive effects
both on general health and for specific disease states, a
rational selection and design of probiotics remains an
important challenge for scientific community. There is also
an urgent need for the development of correct formulations
and novel ways of their administration in different clinical
situations (Mombelli and Gismondo, 2000). The purported
benefits for any probiotic must pass the highest standards
of scientific scrutiny before the claims for its usefulness
can be accepted.
Safety factors that must be addressed in the evaluation
of safety of probiotics include pathogenicity, infectivity
and virulence factors comprising toxicity, metabolic activity and intrinsic properties of microbes. Members of the
genera Streptococcus and Enterococcus have been recognized as opportunistic pathogens causing bacteremia, endocarditis, urinary tract infection and they can act as potential
recipients of vancomycin resistance genes (Lund and
Edlund, 2001). However, many intrinsically vancomycinresistant strains of Lactobacilli have a long history of safe
use as probiotics. There is no indication that vancomycinresistant Lactobacilli could transfer the resistance to other
bacteria (Saavedra, 2001). Ishibashi and Yamazaki (2001)
have highlighted the frequent isolation of probiotic bacteria
from infective sources which influences their intrinsic
properties as well as pathogenicity. Except for one or two
reports in literature including formation of liver abscess by
LGG in an elderly lady with a history of hypertension and
diabetes mellitus (Rautio et al., 1999) and induction of
endocarditis in an elderly male with use of L. rhamnosus
(Mackay et al., 1999), probiotics have been not produced
significant side-effects when administered in large quantities over the past two decades. One area of concern seems
to be the administration of probiotics in immunocompromised patients in whom the benefit-to-risk ratio has to be
clearly established. Molecular biological characterization
of the bacteria used as probiotics and the possibility of
strain-specific infection, require further studies. On the
other hand, documentation of several clinical trials evaluating different probiotic strains in humans reveals virtual
absence of adverse effects with both short term as well as
long term use.
Thus probiotics, prebiotics and synbiotics need to be
further established as functional foods, which affect the
functions of the body in a targeted manner. In addition,
genetic characterization of probiotic cultures to identify the
beneficial genotypes resulting in strain selection and
differentiation requires more research.

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