A Report On,

PRoBIOTIC AS A POTENTIAL ALTERNATIVE TO ANTIBIOTIC FOR MAINTAINING

HUMAN HEALTH

Submitted to
Dr. Bimal Karna
Department of Animal sCIENCE
IAAS, PG campus
Kirtipur

Submitted by
Shiva Khanal
Roll No: R-2018-lPM-01M
1STsemester
M.Sc.An.Sc.(Animal Nutrition)
IAAS, PG cAMPUS
Kirtipur

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1.0 Introduction
Probiotics are defend as non-pathogenic microorganisms which, when administered in adequate
amounts, confer a health benefit on the host and are able to prevent or improve some diseases.
(WHO, 2003). Probiotics are live bacteria’s and yeasts that are good health. We usually think of
bacteria as something that causes diseases. But our body is full of bacteria. Probiotics are often
called "good" or "helpful" bacteria because they help keep our gut healthy. The root of the word
probiotic comes from the Greek word “pro” meaning “promoting” and “biotic” meaning "life".
The discovery of probiotics came about in the early 20th century, when Elie Metchnikoff, known
as the "Father of Probiotics" had observed that rural dwellers in Bulgaria lived to very old ages
despite extreme poverty and harsh climate. He theorized that health could be enhanced and
senility delayed by manipulating the intestinal micro-biome with host-friendly bacteria found in
sour milk.

Probiotic has been redefined throughout the years as more scientific knowledge and better
understanding on its relationship between intestinal health and general well-being has been
gained. The following are definitions of “probiotics” derived through times.

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Lilly and Stillwell in 1965 (5) defined probiotics as “Growth promoting factors produced by
microorganisms.”

Parker in 1974 (7) suggested an interaction between microorganisms with the host: “Organisms
and substances with beneficial effects for animals by influencing the intestinal micro flora.”

Fuller in 1989 (3) defined it as “A live microbial feed supplement which beneficially affects the
host animal by improving its intestinal microbial balance.”

Havenaar and Huis Int Veld in 1992 (4) said probiotics are “A mono- or mixed culture of live
microorganisms which, applied to animal or man, affect beneficially the host by improving the
properties of the indigenous micro flora.” ILSI (International Life Sciences Institute) Europe
Working Group (1998) (9): “A viable microbial food supplement which beneficially influences
the health of the host.”

An effective probiotic is required to operate under a variety of different environmental conditions

and to survive in many different forms. It should, therefore have the following characteristics
(Fuller 1992)

 It should be capable of being prepared as a viable product on an industrial scale.

 It should remain stable and viable for long periods under storage and field
conditions.
 It should have the ability to survive (not necessarily grow) in the intestine.
 It must produce a beneficial effect in the host animal.
This definition has the following characteristics:

 Probiotics must deliver a measured physiological benefit, substantiated by studies

conducted in the target host.
 Probiotics need not be restricted to food applications or oral delivery.
 A definition of probiotics should not limit the mechanism of action. Probiotics must be
alive.
Antibiotics are chemical substances produced by microorganisms, which in small concentration
have the capacity of inhibiting the growth of other microorganisms. Antibiotics can enhance
human lives by treating or preventing diseases. However, a major public-health threat is the
resistance to antibiotics or the increased capability of bacteria to stay alive in the presence of
antibiotics. Moreover, antibiotics only treat bacterial infections and cannot be effectively used to
treat virus-related infections, such as colds (Cars et al., 2008; Costelloe et al., 2010). Antibiotics
have the greatest effect on human health among all medical developments achieved since the
beginning of the 20th century. Antibiotics are selective and specific in their target; thus, these
drugs can eradicate invading bacteria without inducing toxicity to the infected host (Guarner et
al., 2006). Antibiotics are frequently prescribed in most countries (Quigley, 2011). Antibiotics are
extensively used in farming, veterinary medicine and medicine in Australia. Almost 400 000 kg
of the total amount went into stock feed to stop diseases in intensively raised animals or for use

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as a growth promoter. In other words, over 20 g of antibiotics are used for agricultural purposes
per year for every man, woman and child in Australia. However, other countries consume a
significantly higher amount. For example, the mean for the USA is over 27 g per person each
year (Ellison, 2006; Friedman, 2012).

Meanwhile, probiotics are considered as living drugs that can reduce antibiotic consumption and
increase human health development. The application of probiotics in preventing and managing
gastrointestinal (GI) disorders has gained considerable interest over the past two decades. The
consumption of these products is increasing worldwide because probiotics are generally regarded
as safe. Anti- 3These authors contributed equally to this work. carcinogenic, anti-diabetic, anti-
allergic and anti-inflammatory Journal of Medical Microbiology (2015), 64, 137–146 DOI
10.1099/jmm.0.078923-0 078923 G 2015 The Authors Printed in Great Britain 137Downloaded
from www.microbiologyresearch.org by IP: 110.34.30.13 On: Fri, 29 Jun 2018 08:14:41
genetically modified probiotics, as well as oral vaccine development, are counted as new trends
of probiotic usage (Bouton et al., 2002). This review discusses the major future challenges for
antibiotics and probiotics, and the advantages and disadvantages of their use during medical
consumption.

To generate suitable alternatives, we first need to understand better why antibiotics are being
overused and to define the unintended consequences. Antibiotics have multiple purposes that

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suggest alternatives (Table 1). Many antibiotics are used to treat specific diseases, suggesting that
alternative therapies such as phages, bacteriocins, or predatory bacteria may be used similarly.
Additionally, antibiotics are used for growth promotion in food-producing animals, which is not
a therapeutic use. Finally, disease prevention accounts for much antibiotic use in both humans
and animals, where individuals are treated, arguably, at the potential cost of selecting for
antibiotic-resistant bacteria in the population. Here, we expand on our previous discussion of
alternatives for antibiotics. We will also discuss antibiotic adjuvants, with particular attention to
therapeutic combinations of antibiotics with certain alternatives.

General purpose Advantages Limitations

 Can be costly
to administer
 Prevent infections
to a human or
of viruses and
Disease prevention animal
bacteria
population
Vaccines  Promote
 Limited cross-
specificimmunolo
protection with
gical protection
some
pathogens
 Mixed efficacy
 Maintain or
of single
Probiotics, prebiotics, improve
probiotics
commensal gut
synbiotics, and  FDA
bacterial health
regulatory
competitive exclusion  Prevent pathogen
process is
colonization
complex
 Target specific
 Potential for
 Mixtures of
resistance
multiple phages
development
can be used to
 Specificity
reduce resistance
Disease treatment begets
development
technical
Phage therapy  Data show
limitation of
particular efficacy
administration
with topical
against
applications
multiple
 Can synergize
subspecies
with antibiotics

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  Potential for
expanded species
specificity over
Few options against
phages against
Endo- or exolysins Gram-negative
desired Gram-
bacteria
positive targets
 Can synergize
with antibiotics
 Diverse repertoire
 Potential for
 Target specific
resistance
 Many are already
development
food-grade safe
 Large
Bacteriocins compounds
bacteriocins
 Can synergize
could be
with and reduce
sensitive to
cytotoxicity of
proteolysis
antibiotics
 Interactions
with the host
 Effective against
and the
biofilms
commensal
Predatory bacteria  Can access
microbiota are
recalcitrant
unknown
infections
 Data are as yet
limited

Adapted from (Allen et al, 2014)

2.0 Rationale of the study

Antibiotics has been used for more than 80 years to treat bacterial infections. However, a major
public-health threat is the resistance to antibiotics or the increased capability of bacteria to stay
alive in the presence of antibiotics. Moreover various side effects and reduction in microbiome
population also limit its use. This study aims to investigate the positive benefits of probiotics on
gut health and beyond so that probiotics can be established as a potential alternative to antibiotics
for maintaining human health.

3.0 Objectives

3.1 General objective:

To review the beneficial effects of probiotic on human health as a potential alternative to
antibiotic without any residual effects.

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3.2 Specific objectives:
To study and understand the mechanism of action of probiotics
To study types and role of probiotics for maintaining human health
To study the effects of probiotic compounds on gut microflora

4.0 Methodology
Information and data for the topic were collected from different research articles, review papers
and scientific papers published on international journals which were available on internet. This
research work will be conducted by surfing internets, referring class notes, consulting with
subject teacher, scientists working in this sector in NARC and reviewing different research
papers, journals articles etc. published in the internet.

5.0 Literature Review:

Probiotics are commensal organisms that can be harnessed for therapeutic benefit (Fuller, 1989),
usually exerting their effects by positively influencing normal microbe-microbe and host-
microbe interactions. In acute infections, probiotics may augment the protection afforded by
commensal flora through competitive interactions, direct antagonism of pathogens, and/or
production of antimicrobial factors (Corr et al., 2007). While in other clinical conditions, such as
chronic infections and immunosuppression (associated, for example, with HIV/AIDS), microbe-
host signaling is probably more relevant to effective probiotic action. Gut homeostasis (the
maintenance of a “balanced” and beneficial flora) requires continual signaling from bacteria
within the gut lumen, maintaining the mucosal barrier while at the same time priming the gut for
responses to injury (Madara, 2004). Given the potential health promoting benefits of probiotics,
coupled with the fact that they are relatively simple and inexpensive to produce, transport, and
store, these microbes may herald a new era in health care, particularly for the developing world.
Herein, I review some of the major advances in the probiotic arena, dealing specifically with
some of the problems encountered in the developing world such as poverty, malnutrition, acute
and chronic enteric infections leading to complex diarrheal disorders, as well as the unshakable
scourge of HIVwhich continues to decimate sub-Saharan Africa. The World Health Organization
estimates that by the year 2015, 113.4 million children under five years of age will suffer from
malnutrition, the overwhelming majority of which will live in developing countries (De Onis et
al., 2004). With weakened immunity and little or no access to proper medical support the
malnourished in developing countries are increasingly susceptible to infections by enteric
pathogens, leading to bouts of debilitating and dehydrating diarrheawhich in turn worsens
nutritional status. This is compounded by HIV/AIDS, which has created a critical situation that
desperately needs to be addressed. We suggest that probiotic therapy, in combination with
conventional therapies, may help to alleviate some of the suffering, fight existing diseases, and
protect against future infections.

5.1 Mechanism of Action

Te exact mechanisms of action by which probiotics elicit their benefcial effects are not fully
understood. Researchers are working to better understand the specifcs of these proposed

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mechanisms by which probiotics appear to affect the health of humans. Here are some of the
known mechanisms by which probiotics can elicit their benefcial eff ects in the human
gastrointestinal tract.

Mode Process Mechanism Examples

Decreased apoptosis Decreased TNF-α Lactobacillus

Barrier Function
of epithelial cells production rhamnosus GG

Increasing mucin Increased expression

Lactobacillus sp
production of MUC 2

Host cell Increased up

Defensins (hBD E coli strain DSM
Antimicrobial regulation of
protein) 17252S2
Peptides Defensin

By butyrate
Cathelcidins
production

Probiotic
Lowering the luminal By secretion of Most of the
Antimicrobial
pH SCFA’s probiotics bacteria
Factors

Bacteriocin By Gram positive

production probiotics

By Gram negative
Microcin production
probiotics

Directly or indirectly
Epithelial By competing with
by producing protein
Adherence pathogens
that block adherence

By attenuating IL-8
Blocking pro secretion or blocking Salmonella
Immune Modulation Inflammatory the degradation of tyhimurium VSL#3
molecules the counter- probiotics
regulatory factor IκB

Increasing mucosal Increasing IgA

L. casei
immunity Production

Interference with Blocks the By secreting L. acidophilus

Quorum Sensing communication molecules which
Signaling between blocks quorum

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 pathogenic bacteria sensing signaling

Various mechanisms of probiotics action on human intestine cells. Abbreviations: TNF-α: Tumor
necrosis factor alpha; MUC 2: Mucin 2, hBD: Hemoglobin subunit delta; SCFA: Short chain
fatty acids; IL-8: Interleukin 8; and IκB: Inhibitor of kappa B

(Gomes et al, 2014)

Adapted from (Khaligh 2016)

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Adapted from Britio et al 2012

Schematic diagram illustrating potential or known mechanisms whereby probiotic bacteria might
impact on the microbiota. These mechanisms include (1) competition for dietary ingredients as
growth substrates, (2) bioconver‐ sion of, for example, sugars into fermentation products with
inhibitory properties, (3) production of growth substrates, for example, EPS or vitamins, for
other bacteria, (4)direct antagonism by bacteriocins, (5) competitive exclusion for binding sites,
(6) improved barrier function, (7) reduction of inflammation, thus altering intestinal properties
for colo‐ nization and persistence within, and (8) stimulation of innate immune response (by
unknown mechanisms). IEC : intra epithelial cells, DC: dendritic cells, T:T-cells.

5.1.1 Enhancement of the Epithelial Barrier

The intestinal epithelium is in permanent contact with luminal contents and the variable,
dynamic enteric flora. The intestinal barrier is a major defense mechanism used to maintain
epithelial integrity and to protect the organ-ism from the environment. Defenses of the intestinal
bar-rier consist of the mucous layer, antimicrobial peptides, secretory IgA and the epithelial
junction adhesion com-plex. Once this barrier function is disrupted, bacte-rial and food antigens
can reach the submucosa and can induce inflammatory responses, which may result in in-testinal
disorders, such as inflammatory bowel disease . Consumption of non-pathogenic bacteria can
contribute to intestinal barrier function, and probiotic bacteria have been extensively studied for
their involve-ment in the maintenance of this barrier.

Probiotics are capable of influencing many of the components of epithelial barrier function either
by decreasing apoptosis of intestinal cells or increased mucin production. Lactobacillus

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rhamnosus GG was able to prevent cytokine-induced apoptosis in intestinal epithelial cell models
by inhibiting tumor necrosis factor (TNF) . Lactobacillus species have been shown to increase
mucin expression in vitro in human intestinal epithelial cells, thus blocking pathogenic E. coli
invasion and adherence. Lactobacillus rhamnosus GG has shown to prevent inflammation and
programmed cell death of the lining intestinal epithelial cells shown to exert mitogenic effects
and enhancing mucosal regeneration.

5.1.2 Competition for adherence

Adhesion to intestinal mucosa is regarded as a prerequisite for colonization and is important for
the interac-tion between probiotic strains and the host. Adhesion of probiotics to the intestinal
mucosa is also im-portant for modulation of the immune system [43, 44] and antagonism against
pathogens. Probiotic bacteria compete with invading pathogens for binding sites to epithelial
cells and the overlying mucus layer in a strain-specific manner. Surface layer proteins purified
from L. Helveticas R0052 inhibited enterohemorrhagic Escherichia coli O157:H7 adherence and
the subsequent rise in permeability, without altering the growth of the pathogen . S. boulardii
secretes a heat-labile factor which has shown to be responsible for the decreased bacterial
adherence.

5.1.3 Competitive Exclusion of Pathogenic Microorganisms

In a report addressing the total exclusion of Salmo-nella typhimurium from maggots of blowflies
published in 1969, Greenberg [88] first used the ‘competitive exclu-sion’ term for the scenario in
which one species of bacte-ria more vigorously competes for receptor sites in the in-testinal tract
than another species. The mechanisms used by one species of bacteria to exclude or reduce the
growth of another species are varied, including the following mechanisms: creation of a hostile
microecology, elimina-tion of available bacterial receptor sites, production and secretion of
antimicrobial substances and selective me-tabolites, and competitive depletion of essential
nutrients [89].

Specific adhesiveness properties due to the interaction between surface proteins and mucins may
inhibit the col-onization of pathogenic bacteria and are a result of an-tagonistic activity by some
strains of probiotics against adhesion of gastrointestinal pathogens [90]. Lactobacilli and
bifidobacteria have been shown to inhibit a broad range of pathogens, including E. coli,
Salmonella, Helico-bacter pylori, Listeria monocytogenes and Rotavirus [91– 97]. Exclusion is
the result of different mechanisms and properties of probiotics to inhibit pathogen adhesion, in-
cluding the production of substances and the stimulation of IECs. Competitive exclusion by
intestinal bacteria is based on a bacterium-to-bacterium interaction mediated by competition for
available nutrients and for mucosal adhesion sites. To gain a competitive advantage, bacteria can
also modify their environment to make it less suitable for their competitors. The production of
antimicrobial substances, such as lactic and acetic acid, is one example of this type of
environmental modification [98]. Some lactobacilli and bifid bacteria share carbohydrate-bind-
ing specificities with some enteropathogens [99, 100], which makes it possible for the strains to
compete with specific pathogens for the receptor sites on host cells [101]. In general, probiotic
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strains are able to inhibit the attachment of pathogenic bacteria by means of steric hindrance at
enterocyte pathogen receptors.

Adapted from (Figueroa-González et al, 2016)

The effect of probiotic bacteria on the competitive exclusion of pathogens has been demonstrated
using hu-man mucosal material in vitro [45, 103] as well as chicken and pig mucosal material in
vivo. Hirano et al. showed that L. rhamnosus, a strongly adhering strain, is capable of inhibiting
the internalization of EHEC (enterohemorrhagic E. coli) in a human intestinal cell line showed
that L. rhamnosus, a strongly adhering strain, is capable of inhibiting the internalization of EHEC
(en-terohemorrhagic E. coli) in a human intestinal cell line.

5.1.4 Production of Antimicrobial Substances

One of the proposed mechanisms involved in the health benefits afforded by probiotics includes
the formation of LMW compounds (!1,000 Da), such as organic acids, and the production of
antibacterial substances termed bacteriocins (11,000 Da).

Production of Antimicrobial Substances Probiotics either by inducing host cells to produce

peptides or by directly releasing peptides interfere with pathogens, and prevent epithelial

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invasion. Defensins (hBD protein) and cathelicidins are the antimicrobial peptides expressed
constitutively by the intestinal epithelial cells and display antimicrobial activity against a wide
variety of bacteria, fungi and some viruses. Certain probiotic strains like E. coli strain DSM
17252 G2 (one of the three Symbioflor 2 genotype strains) and several Lactobacilli species have
shown to express certain defensins . Healthy volunteers who received probiotics had increased
fecal hBD protein and remained elevated for 9 weeks afer completion of 3 weeks of probiotic
treatment. Probiotics have been shown to suppress pathogen growth through the release of a
variety of antimicrobial factors like defensins, bacteriocins, hydrogen peroxide, nitric oxide, and
short chain fatty acids (SCFA), such as lactic and acetic acids, which reduce the pH of the
lumen . SCFA can disrupt the outer membranes of gram-negative pathogens causing inhibition of
pathogen growth. Bacteriocins can either permeabilize the inner membrane of gram-negative
bacteria, leading to disruption and formation of pores. Microcins (produced by gram negative
bacteria), on the other hand, can target the inner membrane, enzymes that are involved in DNA
or RNA structure and synthesis, or protein synthesis enzymes.

5.1.5 Interference with quorum sensing signaling

Bacteria communicate with each other as well as with their surrounding environment through
chemical signaling molecules called auto-inducers. Tis phenomenon is called quorum sensing.
The use of this cell-to-cell signaling mechanism facilitates the regulation of important traits of
enteric microbes that allow them to successfully colonize and/or start infection in their host.
Medellin-Pena et al. demonstrated that Lactobacillus acidophilus secretes a molecule that inhibits
the quorum sensing signalling or directly interact with bacterial transcription of E. coli O157
gene, involved in colonization and thus, bacterial toxicity is opposed.

5.1.6 Probiotics and the Immune System

It is well known that probiotic bacteria can exert an immunomodulatory effect. These bacteria
have the ability to interact with epithelial and dendritic cells (DCs) and with
monocytes/macrophages and lymphocytes. The immune system can be divided between the
innate and adaptive systems. The adaptive immune response depends on B and T lymphocytes,
which are specific for particular antigens. In contrast, the innate immune system responds to
common structures called pathogen-associated molecular patterns (PAMPs) shared by the vast
majority of pathogens. The primary response to pathogens is triggered by pattern recognition
receptors (PPRs), which bind PAMPs. The best-studied PPRs are toll-like receptors (TLRs). In
addition, extracellular Ctype lectin receptors (CLRs) and intracellular nucleotidebinding
oligomerization domain-containing protein (NOD)-like receptors (NLRs) are known to transmit
signals upon interaction with bacteria.

It is well established that the host cells that interact most extensively with probiotics are IECs. In
addition, probiotics can encounter DCs, which have an important role in innate and adaptive
immunity. Both IECs and DCs can interact with and respond to gut microorganisms through their
PPRs. Figure 2 shows a summary of how probiotics may interact and modulate the immune
system ells and endothelial cells. In mammals, the TLR family includes eleven proteins (TLR1–

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TLR11). However, there is a stop codon in the human TLR11 gene that results in a lack of
production of human TLR11. Activation of TLRs occurs after binding of the ligand to
extracellular leucinerich repeats. In humans, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are
outer membrane associated and primarily respond to bacterial surface-associated PAMPs. TLR3
TLR7, TLR8 and TLR9 are found on the surface of endosomes where they respond primarily to
nucleic acidbased PAMPs from viruses and bacteria [132]. Dimerization of TLRs and the highly
conserved toll-interleukin-1 (IL-1) receptor (TIR) domains leads to the recruitment of adaptor
molecules, such as myeloid differentiation primary response protein (MyD88), TIR domain-
containing adaptor protein and TIR domain-containing adapter inducing interferon (IFN)-
(TRIF), to initiate signaling activation. The TLR signaling pathway, except for TLR3, involves
the recruitment of MyD88, which activates the MAPK and nuclear factor (NF)-B signaling
pathways [133–135]. TLR3 utilizes the adaptor protein TRIF, leading to the expression of type 1
IFNs . Furthermore, TLR-mediated signaling has been shown to control DC maturation inducing
the upregulation of various maturation markers, such as CD80, CD83 and CD86, as well as the
CCR7 chemokine receptor. Moreover, commensal and probiotic microorganisms can create an
overall tolerant state mediated by the action of TLRs on DCs. It is clear that TLR9 signaling is
essential to mediate the antiinflammatory effect of probiotics. However, different studies have
implicated other TLRs, such as TLR3 and TLR7, in the tolerance induced by commensal and
probiotic bacteria. After activation by commensal and probiotic microorganisms, DCs initiate an
appropriate response, such as the differentiation of Th0 to Treg, which has an inhibitory effect on
Th1, Th2 and Th17 inflammatory responses.

It is well established that probiotics can suppress intestinal inflammation via the downregulation
of TLR expression, secretion of metabolites that may inhibit TNF- from entering blood
mononuclear cells and inhibition of NF-B signaling in enterocytes.

In this regard, cell wall components of lactobacilli can potentially signal through binding TLR2
in combination with TLR6. The diacylated membrane anchors of lipoproteins and lipoteichoic
acids bind to TLR2 and TLR6, thereby promoting dimerization and MyD88-mediated activation
of the canonical pathway of NF-B . Stimulation of TLR2 increases the production of cytokines,
and TLR2 activation has an important role in enhancing transepithelial resistance to invading
bacteria.

TLR2 recognizes peptidoglycan, which is the main component of Gram-positive bacteria,

including the Lactobacillus genus. Several studies have demonstrated that TLR2 is required for
some Lactobacillus strains to exert their immunomodulatory effects. Vinderola et al.
demonstrated that L. casei CRL 431 interacts with epithelial cells through TLR2 and that the
interaction between L. casei and gut-associated immune cells induces an increase in the number
of CD-206 and TLR2 receptors, mainly in the cells involved in the innate immune response.

In addition, Shida et al. [138] showed that L. casei induces a high level of IL-12 production in
both wild-type and TLR2-deficient macrophages, and that peptidoglycan induces low levels of

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IL-12 production in wild-type. Macrophages and even lower levels in TLR2-deficient
macrophages. They also suggested that the intact peptidoglycan of lactobacilli actually signals
via TLR2 to inhibit IL-12 production. Although the recognition by TLR2 is essential, 12–48% of
IL-12 production in TLR2- deficient macrophages is inhibited by peptidoglycan, thus suggesting
that other TLR2-independent mechanisms may also be involved. Furthermore, it has been
demonstrated that Lactobacillus strains, such as L. rhamnosus GG (LGG) and L. plantarum BFE
1685, enhance TLR2 in vitro in experiments using human intestinal cells, and more recently, L.
casei CRL 431 has been shown to exert a similar effect on healthy mice and mice infected with
S. enterica serovar typhimurium [139, 140]. For instance, probiotic administration to healthy
mice increases expression of TLR2, TLR4 and TLR9, and it improves the secretion of TNF-,
IFN- and IL-10 in Peyer’s patches.

Similarly, when porcine IECs encounter Lactobacillus jensenii TL2937, TLR2 may act
synergistically and cooperatively with one or more PRRs, which may result in a coordinated sum
of signals that induce the upregulation of several negative regulators of TLRs, including A20,
Bcl-3 and MKP-1

TLR2 also has an important role in the recognition of bifidobacteria. Hoarau et al.reported that a
fermentation product from Bifidobacterium breve C50 can induce maturation, high IL-10
production and prolonged survival of DCs via the TLR2 pathway.

Similarly, Zeuthen et al. [143] showed that TLR2–/– DCs produce more IL-2 and less IL-10 in
response to bifidobacteria, and they concluded that the immuno-inhibitory effect of
bifidobacteria is dependent on TLR2.

Recently, Kailova et al. [144] reported that oral administration of B. bifidum OLB 6378 to rats
with necrotizing enterocolitis (NEC) stimulates TLR2 expression in the ileal epithelium,
enhances epithelial expression of COX-2 and increases intestinal production of prostaglandin E2.
Indeed, pretreatment of IEC-6 cells with the probiotic strain stimulates TLR2 and COX-2
expression and blocks cytokine-induced apoptosis. However, there is no evidence of a clear link
between TLR2 activation and the upregulation of COX-2.
In contrast, it has been shown that the L. reuteri strains DSM 17938 and ATCC PTA 4659 have a
beneficial effect on preventing NEC in rats. In response to the probiotic, mRNA expression of
IL-6, and expression levels of TNF-, TLR4 and NF-B are significantly downregulated, and
mRNA levels of IL-10 are significantly upregulated. Moreover, L. reuteri treatment leads to de
creases in intestinal protein levels of TLR4, IL-1 and

TNF- in newborn rats with NEC. Furthermore, L. reuteri significantly increases survival rate,
reduces both the incidence and severity of NEC and decreases pro-inflammatory cytokine levels
in parallel with inhibition of TLR4 signaling via the NF-B pathway.

Moreover, TLR4 has a significant role in the host defense against Salmonella infection in vivo.
In healthy mice, L. casei CRL 431 activates this receptor and can be used as a surveillance

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mechanism against pathogenic bacteria [140]. Activation of TLR4 leads to the induction of pro-
inflammatory mediators, an increase in TLR2 expression, and a reduction in its own expression,
which leads to the recruitment of inflammatory cells and the initiation of the appropriate
responses in the spleen. Collectively, these events allow for the control of bacterial replication.

Similarly, heat-inactivated LGG and Lactobacillus delbrueckii subsp. bulgaricus can decrease
TLR4 expression similar to lipopolysaccharide (LPS) after 12 h in human monocyte-derived
DCs. Moreover, LGG downregulates p38 expression, and L. delbrueckii subsp. bulgaricus
reduces inhibitor protein B (IB) expression. In addition, these probiotic strains can modify the
immune response at the post-transcriptional level by modifying miRNA expression.

Another relevant TLR is TLR9, which recognizes bacterial CpG DNA and synthetic
unmethylated CpG oligonucleotide mimics (CpG-ODN). Unmethylated DNA fragments
containing CpG motifs that are released from probiotics in vivo have the potential to mediate
anti-inflammatory effects through TLR9 signaling at the epithelial surface. It is known that
Lactobacillus species differ in their C+G composition. Thus, the ability of different species to
stimulate TLR9 is likely to be different. TLR9 activation through apical and basolateral surfaces
activates different intracellular signaling pathways in polarized epithelial cells. Whereas
basolateral TLR9 triggers IB degradation and NF-B pathway activation, apical TLR9 induces
cytoplasmic accumulation of ubiquitinated IB and inhibition of NF-B activation.

Using polarized HT29 and T84 cell monolayers, Ghadimi et al. showed that binding of natural
commensal-origin DNA to the apical TLR9 initiates an intracellular signaling cascade in a
specific manner that is associated with the attenuation of TNF--induced NF-B activation and NF-
B-mediated IL-8 expression. When LGG DNA was apically applied, they showed a detracted
TNF--induced NF-B activation by reduced IB degradation and p38 MAPK phosphorylation,
thereby indicating that intracellular chemical signals may coordinately regulate multiple
properties of TLR9 expression that are relevant in multicellular functional responses of TLR9 to
bacterial DNA. They also showed that TLR9 silencing abolishes the inhibitory effect of natural
commensal-origin DNA on TNF--induced IL-8 secretion.

Similarly, B. breve (NumRes 204), L. rhamnosus (NumRes 1) and L. casei (DN-114 001) strains
induce different cytokine production levels by human and mouse primary immune cells. It has
been demonstrated that the B. breve strain induces lower levels of the pro-inflammatory cytokine
IFN- than L. rhamnosus and L. casei.Moreover, B. breve and lactobacilli induce cytokines in
aTLR9-dependent manner, and the lower inflammatory profile of B. breve is due to inhibitory
effects of TLR2 .

In addition, it has been shown that purified genomic DNA from L. plantarum (p-gDNA) does
not substantially stimulate pro-inflammatory cytokines. However, pgDNA inhibits LPS-induced
TNF- production by THP1 cells. Furthermore, p-gDNA reduces the expression of TLR2, TLR4
and TLR9, which induces the activation of NF-B through the LPS signaling pathway, leading to

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the upregulation of inflammatory cytokines . Pretreatment of p-gDNA inhibited the
phosphorylation of MAPKs and NF-B, and also inhibited LPS-induced TNF- production in
subsequent LPS stimulation. In this regard, L. plantarum genomic DNA-mediated inhibition of
signaling and TNF- was accompanied by the suppression of TLR2, TLR4 and TLR9, as well as
the induction of IL-1 receptor-associated kinase M (a negative regulator of TLR) .

5.1.7 NLRs and Probiotics

As mentioned before, there is another family of membrane-bound receptors: NLRs. They are
located in the cytoplasm and are important in tissues where TLRs are expressed at low levels.
The most thoroughly characterized members are NOD1 and NOD2, but currently more than 20
different NLRs have been identified .Unlike NOD1, which is ubiquitously expressed, the
expression of NOD2 is restricted to DCs, macrophages, Paneth cells, intestinal cells, lung cells
and oral epithelial cells, and it is expressed at low levels in T cells. NOD1 can sense
peptidoglycan moieties containing meso-diaminopimelic acid, which are associated with Gram-
negative bacteria, but NOD2 senses muramyl dipeptide motifs, which can be found in a wide
range of bacteria. Upon recogni tion of their agonist, both NOD1 and NOD2 self-oligomerize to
recruit and activate the adaptor protein RICK, a protein kinase that regulates CD95-mediated
apoptosis, which is essential for the activation of NF-B and MAPKs, resulting in the
upregulation of transcription and production of inflammatory mediators (e.g. cytokines,
chemoattractants, COX-2 and inducible nitric oxide synthase) .

There are a few studies showing the effect of probiotics on NLR. However, Fernandez et al.
recently demonstrated that the protective capacity of L. salivarius Ls33 correlates with local IL-
10 production, which is abolished in NOD2-deficient mice. Indeed, these authors showed that the
anti-inflammatory effect of Ls33 is mediated via NOD2.

Another important pathway activated by NLRs involves apoptosis-associated speck-like protein

with caspase recruitment to activated caspase 1, an adaptor protein which is necessary for the
cleavage of pro-IL-1 and pro-IL-18 into their mature and biologically active forms. NLRs
participate in the formation of inflammasomes, which leads to the activation of caspase-1. There
are three principal inflammasomes named after the NLR involved as follows: NOD-like receptor
family, pyrin domain containing protein (NLRP) 1, NLRP3 and NLRC4. NLRP3 detects LPS,
muramyl dipeptide, bacterial RNA and viral RNA.

The following two steps are required for the complete activation of the NLRP3 inflammasome: a
priming step to induce transcription of NLRP3 mRNA and a sequential step to recognize various
PAMPs and danger-associated molecular patterns by fully expressed NLRP3 itself. With regard
to probiotic mechanisms associated with NLRP3, Tohno et al. found that L. delbrueckii subsp.
bulgaricus NIAI B6 and L. gasseri JCM1131T are able to enhance NLRP3 expression in the
GALT of adult and newborn swine. Their results suggested that immunobiotic Lactobacillus
strains directly promote NLRP3 expression via TLR and NOD-mediated signaling, resulting in
the induction of appropriate NLRP3 activation in porcine GALT. Furthermore, their results

18
indicated that NLRP3 expression is upregulated by TLR2, TLR9, NOD1 and NOD2 agonists in
adult and newborn porcine GALT. It has been suggested that NLRP3 has an important role in the
regulation of human intestinal inflammation, such as in Crohn’s disease , and that dysregulated
NLRP3 expression results in the disruption of immune homeostasis associated with auto
inflammatory disease in humans . Because the potential expression level of NLRP3 is low in
immune cells, induction of cellular NLRP3 expression itself is a first step to evoke the
appropriate activation of the NLRP3- mediating signaling pathway in order to respond to danger-
associated molecular patterns and PAMP stimuli..

5.1.8 TLRs and Probiotics

TLRs are transmembrane proteins expressed on various immune and non-immune cells, such as
B cells, natural killer cells, DCs, macrophages, fibroblasts, epithelia

5.1.9 Probiotics Consume Available Nutrients

The intestines are the ideal environment for lactic acid bacteria to grow. Tere are lots of nutrients
available in the digestive tract that support the needs of lactic acid bacteria to grow. By
consuming a large portion of the available nutrients suitable for microbes, lactic acid bacteria
restrains the growth of bad microbes. With little to eat, pathogens fnd it hard to grow and
colonize in the intestines.

5.1.10 Probiotics Create an Acidic Environment

Lactobacilli and Bifdobacterium are bacteria families that excrete lactic acid and acetic acid into
their environment. Tis excretion of acid is part of their regular daily functions. In the
gastrointestinal tract, this causes the pH to lower. Many bad microbes do not like a low pH. As
such, lactic acid bacteria are capable of inhibiting the growth and colonization of bad microbes in
the gastrointestinal tract.

5.1.11 Probiotics Produce Benefi cial Enzymes

Probiotics make a variety of enzymes that offer health benefts to you. These enzymes include
lactase, which breaks down lactose, a sugar in milk that many people cannot digest. Some
probiotics produce enzymes that are harmful to bad microbes. Te enzyme activity of probiotics
has been found to help fght infectious disease, lactose intolerance, immune system defciencies,
and urogenital and vaginal diseases.

5.1.12 Probiotics Encourage Healthy Microflora

Probiotics may also work synergistically to create an environment that promotes probiotic
growth, not growth of bad microbes. Some probiotics excrete substances that enhance the growth
of fellow probiotics. Lactobacillus reuteri excretes AGGH, a protein that appears to encourage
growth of good bacteria in the small intestine. Tis is a fascinating area of research. In clinical
trials, the use of combinations of probiotic species has been found to offer greater health benefts
than any one of the probiotic species alone. As such, it is likely that probiotics work

19
synergistically in the intestines to promote the growth and colonization of fellow friendly
microbes.

7.0 Result and Discussion

The human gastrointestinal tract is home to diverse and vast communities of micro- organisms
representing over 400 cultivable species. The colonisation of the gastrointestinal tract begins
immediately after birth. The mode of delivery, use of antibiotics, and the level of hygiene are
known to exert a significant influence on the number and species of microorganisms that
colonise the gut. At first Escherichia coli and streptococcus dominates, but in breast fed infants
the number of bifidobacteria increases while those of E coli, streptococcus, and clostridia
decreases.1 A change to the adult flora occurs after weaning and by the second year of life the
intestinal flora becomes similar to that of an adult2 and remains relatively stable throughout life.
The density and diversity of microbes increases progressively from stomach (102–3 colony
forming units (cfu)/g lumenal contents) to colon (1011–12 cfu/g lumenal contents). In a healthy
adult, the gastrointestinal tract contains 10 times as many bacteria (1014 bacteria) as eukaryotic
cells in the entire body1; the combined genome of the intestinal flora is estimated to be 50–100
times the size of the human genome.3 As these organisms are metabolically active and interact
continuously with their environment (including other bacteria, the gut epithelium, mucosal
immune system, the central nervous system, and the endocrine system), they are able to exert a
significant influence on the postnatal development and the host physiology4; the metabolic
activity of microflora is considered to be equal to that of liver.

While a majority of the indigenous flora are benign or exhibit health promoting properties, some
possess the potential to cause disease. For example, bifidobacteria and lactobacilli are associated
with health, while clostridia are considered detrimental to health. Normally, a balance exists
between pro-health and anti-health organisms. However, when this delicate ecological balance is
perturbed by environmental or physiological factors, predisposition to infectious and immune
inflammatory diseases is enhanced. Research over the past two decades has provided evidence
that administration of probiotics could be used to optimise gut flora and to prevent and treat a
range of diseases. Probiotics are defined as live micro-organisms which when administered in
adequate amounts confer a health benefit on the host. Bifidobacteria and lactobacilli are
commonly used as probiotics. Consumption of specific strains of probiotics is associated with a
range of health benefits,6 although strong scientific evidence exists only for a small number of
conditions. The health benefits supported by adequate clinical data or promising animal data
include prevention and treatment of diarrhoeal disease (acute infantile diarrhoea, antibiotic
associated diarrhoea, and nosocomial infections), prevention of systemic infections, management
of inflammatory bowel disease, immunomodulation, prevention and treatment of allergies,
anticancer effects, treatment of cholesterolaemia, and alleviation of lactose intolerance.

7.1 PREVENTION AND TREATMENT OF DIARRHOEAL DISEASE

A number of clinical trials have tested the efficacy of probiotics in the prevention of acute
diarrhoeal conditions. Diarrhea is the most frequent side effect of both the short and long term

20
use of antibiotics, particularly during multiple antibiotic regimens. Coadministration of
probiotics to patients on antibiotic therapy has been shown to reduce the incidence of antibiotic
associated diarrhoea in children and in adults. In placebo controlled studies, diarrhoea occurred
at a rate of 15% to 26% in the placebo arms but only in 3% to 7% of patients receiving a
probiotic. Different strains have been tested including Lactobacillus rhamnosus strain GG,
Lactobacillus acidophilus, Lactobacillus bulgaricus, and the yeast Saccharomyces boulardii. Two
meta-analyses concluded that probiotics could be used to prevent antibiotic associated
diarrhoea.8 9

Nosocomial diarrhoea is a major problem in paediatric hospitals worldwide. Prophylactic use of

probiotics has proven useful for the prevention of acute diarrhoea in infants admitted into the
hospital ward for a chronic disease condition. In a double blind, placebo controlled trial,
Saavedra and coworkers showed that supplementation of an infant formula with Bifidobacterium
bifidum and Streptococcus thermophilus reduced the incidence of diarrhoea (7% v 31%) and
rotavirus shedding (10% v 39%) in hospitalised infants aged 5–24 months.10 In another placebo
controlled double blind study, oral administration of L rhamnosus strain GG to infants (1–36
months old), hospitalised for reasons other than diarrhoea, reduced the risk of nosocomial
diarrhoea (6.7% v 33.3%) and rotavirus gastroenteritis (2.2% v 16.7%).11 Prevalence of
rotavirus infection was not influenced by probiotic treatment but the risk of symptomatic
rotavirus enteritis was significantly reduced. A third published clinical trial on nosocomial
diarrhoea in infants (1–18 months old) showed no statistically significant benefit of Lactobacillus
GG intake,12 but the rate of symptomatic rotavirus enteritis in the probiotic arm (13.2%) was
found to be lower than in the placebo arm (20.8%).

Probiotics may also be useful in the prevention of community acquired diarrhoea. The study by
Oberhelman and coworkers13 included 204 infants (6–24 months old) from an indigent
periurban town who were followed up over a 15 month period. Significantly fewer episodes of
diarrhoea per child per year were observed in children given

Lactobacillus GG supplemented gelatin than in the placebo (control) group. In a multicentre,

randomised, double blind trial,14 conducted over four months with 928 healthy children aged 6–
24 months, the incidence of acute diarrhoea was significantly reduced by supplementation with
Lactobacillus casei fermented milk (15.9%) as compared with yogurt (22%).

Several studies have investigated the efficacy of probiotics in the prevention of travellers’
diarrhoea in adults, but methodological deficiencies, such as low compliance with the treatment
and problems with the follow up, limit the validity of their conclusions.15

The benefit of probiotics as a treatment for acute diarrhoea in children has also been
demonstrated. Probiotics such as Lactobacillus reuteri, Lactobacillus GG, L casei, and S
boulardii have proven useful in reducing the duration of acute diarrhoea in controlled clinical
trials. Three meta-analyses of controlled clinical trials have been published.16–18 The results of

21
the systematic reviews are consistent and suggest that probiotics are safe and effective. Probiotic
therapy shortens the duration of acute diarrhoeal illness in children by approximately one day.

7.1.1 Traveler’s diarrhea

Traveller’s diarrhea is estimated to affect more than 60% of travellers to developing countries
and, in terms of frequency and economic impact, is the number one health problem for
international travel. Travel is a risk factor for infectious gastroenteritis. A recent meta-analysis
revealed evidence of a protective effect by S. boulardi and by mixture of Lactobacillus
acidophilus and Bifidobacterium bifidum [19].

7.1.2 Rotavirus diarrhea

Rotavirus is a common cause of diarrhea amongst children in developing countries. Several
Lactobacillus species have been tested and an effect shown against Rotavirus associated diarrhea.
Rotavirus often causes gastro enteritis in hospitalised children but can also cause milder diarrhea
in children who are cared for in day care centres or similar institutions. Researchers in Denmark
studied the effect of Lactobacillus rhamnosus and Lactobacillus reuteri on acute diarrhea in
children in day care centres. They found that on average those receiving the probiotic had
diarrhea which lasted 40 hours less than those who received the placebo.

7.1.3 Antibiotic associated diarrhea

Multiple studies have evaluated a variety of probiotics in the treatment and prevention of
antibiotic associated diarrhea. At least two systematic reviews suggest that probiotics (including
various bacterial species and the yeast S. boulardii) are effective in reducing the incidence of
diarrhea in patients who are taking antibiotics. In a careful meta-analysis, DeSouza and
colleagues pooled data showed that probiotics were more effective than placebo. However,
discordant data have been published .

Because there is little harm from using a probiotic, it would seem that this therapy is important,
significantly effective and cost-effective although routine clinical use of probiotics for all cases
of antibiotic related diarrhoea cannot be justified. In addition, it remains unclear which probiotic
and what doses are most effective.

7.1.4 Clostridium difficile associated diarrhoea

Clostridium difficile has been associated with symptomatic diarrhea since it was identified as the
pathogen responsible for pseudomembranous colitis. As evidence that a normal bacterial flora
can suppress the growth of C. difficile, patients have successfully been treated with fecal enemas.
It is reasonable to conclude that probiotic therapy could treat C. difficile infection in a similar
fashion. Biller et al reported a series of four children with at least three recurrences of C. difficile
successfully treated with lactobacillus. The current largest, randomized, controlled trial with C.
difficile-associated colitis demonstrated that S. boulardii was able to prevent disease recurrence,
but only in those individuals who had more than one C. difficile sequential infection.

22
7.1.4 Radiation induced diarrhea
Diarrhea is a nearly constant adverse effect of irradiation of the pelvis. Three studies in the
literature report benefit of using probiotics for radiation-induced diarrhea. All improved the
patient status and the authors felt the probiotics were warranted and successful in decreasing
radiation diarrhea. Such potentially interesting therapeutic effects should be studied more
thoroughly.

7.2 CONSTIPATION
Constipation is common especially in elderly people. An increase in the number of bowel
movements or a decrease in transit time has been reported in controlled studies that employed
probiotics for treating constipation .The widely used laxative lactulose is a prebiotic, as it is not
attacked by human disaccharidases and is substrate for the bifidobacteria in the colonic flora, that
catabolise it to smaller molecules, creating an osmotic effect. However, one cannot draw any
conclusion, as there is a need for larger controlled studies using probiotics and prebiotics other
than lactulose.

7.3 TREATMENT OF HELICOBACTER PYLORI INFECTION

Probiotics have been tested as a new strategy for eradication of Helicobacter pylori infection of
the gastric mucosa in humans. Some strains of lactic acid bacteria are known to inhibit the
growth of H pylori in vitro. However, administration of a probiotic-containing yogurt was found
to be ineffective in the eradication of H pylori infection in 27 subjects.19 Two studies that
examined the use of probiotics as a supplement to the classical triple therapy with antibiotics also
failed to demonstrate any beneficial effect of probiotic therapy (see Sheu et al20). In contrast, in
a non-blinded trial, the triple therapy plus yogurt resulted in a higher H pylori eradication rate
than the triple therapy only (91% v 78%) by intention-to-treat analysis.20 It is important to note,
however, that H pylori eradication rates were similar for both groups of patients (93.5% v 89%,
not significant) by per protocol analysis—that is, when considering only the patients that
completed the seven day antibiotic therapy. Interestingly, a lower number of dropout events were
observed in the yogurt group. Since the trial was not blind, the consistency of this observation
needs to be confirmed.

7.3.1 Mechanisms of action

Several mechanisms (producing antimicrobial substances, stimulating mucus secretion,
strengthening gut barrier function, competing for adhesion sites, stimulating specific and non-
specific immune responses, etc) by which probiotics mediate their anti-infection effects have
been suggested.21 However, the relative importance of these mechanisms remains unknown.

7.4 Management of inflammatory bowel diseases

Crohn’s disease, ulcerative colitis, and pouchitis are chronic conditions of unknown aetiology.
Evidence suggests that abnormal activation of the mucosal immune system against the enteric
flora is the key event triggering inflammatory mechanisms that induce mucosal injury and
perpetuate intestinal lesions to chronicity. Patients show an increased mucosal secretion of IgG

23
antibodies against commensal bacteria,26 and mucosal T-lymphocytes are hyper-reactive against
antigens of the common flora, suggesting that local tolerance mechanisms are abrogated.27 In
Crohn’s disease, faecal stream diversion has been shown to prevent recurrence of mucosal
lesions, whereas infusion of intestinal contents activated the lesions.28 In ulcerative colitis,
treatment with an enteric coated preparation of broad spectrum antibiotics reduced metabolic
activity of the flora and mucosal inflammation.29 Experimental studies based on co-culture of
non-pathogenic bacteria with human intestinal mucosa have shown that different bacteria elicit
different types of cytokine response. In Crohn’s disease, a commensal E coli strain stimulates the
release of tumour necrosis factor-a (TNF-a) and interleukin (IL)-8 by the inflamed mucosa.30
However, some lactobacillus strains including L casei downregulate the spontaneous release of
TNF-a by inflamed tissue, and also the inflammatory response induced by E coli.30 31 The
antiinflammatory effect of L casei is transduced to the underlying tissue and results in reduced
expression of activation markers by lamina propria T-lymphocytes,30 suggesting that signals
generated at the mucosal surface can promote changes in the phenotype of lamina propria
lymphocytes. A balanced local microecology could restore immune homoeostasis in Crohn’s
disease.

Probiotics have been tested in animal models of bowel inflammation. Mice deficient of the IL-10
gene spontaneously develop colitis. Oral administration of either VSL#3, a mixture of eight
bacteria strains, or L plantarum significantly decreased histological colitis score in this animal
model.32 33 However, the same L plantarum strain failed to reduce the severity of colitis in the
TNBS model in the rat.34 A bacterium genetically engineered to secrete the anti-inflammatory
cytokine IL-10 prevented the onset of colitis in the IL10 knockout mouse model.35 The prebiotic
inulin increases counts of lactobacilli and bifidobacteria in the colonic lumen.

The effect of inulin was tested in the rat model of distal colitis induced by dextran sodium
sulphate, a model that resembles human ulcerative colitis.36 Oral inulin prevented mucosal
inflammation, as evidenced by lower colonic lesion scores, lower release of inflammatory
mediators, and lower tissue myeloperoxidase activity in test rats as compared with controls.

In ulcerative colitis, two randomised controlled trials investigated the effectiveness of an orally
administered enteric coated preparation of viable E coli strain Nissle 1917 as compared with
mesalazine, the standard treatment for maintenance of remission. These two studies concluded
that the non-pathogenic E coli strain has an equivalent effect to mesalazine in maintaining
remission. The same probiotic product was tested for efficacy in maintaining remission in 28
patients with colonic Crohn’s disease. This placebo controlled study showed a lower rate of
relapse (33% v 63%) in the probiotic group than in controls.

The VSL#3 mixture mentioned above has proven highly effective for maintenance of remission
of chronic relapsing pouchitis, after induction of remission with antibiotics.40 In this study, a
relapse occurred in only three out of 20 patients of the VSL#3 group and in all the 20 patients of
the placebo group. Of interest, all patients on remission in the probiotic arm had relapses within

24
four months after stopping treatment at conclusion of the trial. Treatment with VSL#3 is also
effective in the prevention of the onset of pouchitis after ileal pouch-anal anastomosis.41 Results
of a controlled trial published in abstract form suggest that VSL#3 is better than mesalazine in
the prevention of postoperative recurrence of Crohn’s disease at one year.

The efficacy of Lactobacillus GG in postoperative recurrence of Crohn’s disease has been tested
in a randomised, double blind trial. The probiotic showed no effect in the prevention of clinical
and endoscopic recurrence as compared with placebo.43 Lactobacillus GG also had no effect, as
a primary therapy, on clinical or endoscopic responses in patients with chronic pouchitis.44
Another study included 32 patients with quiescent Crohn’s disease and tested S boulardii for
maintenance of remission as a coadjuvant therapy added to standard mesalazine. Clinical relapse
rate was higher in the group treated with mesalazine alone.45 finally, the prebiotic inulin has
been tested in patients with an ileal pouch-anal anastomosis. Compared with placebo, three
weeks of dietary supplementation with inulin reduced endoscopic and histological parameters of
inflammation of the mucosa of the ileal reservoir. The effect was associated with an increase in
faecal butyrate and a decrease in bacteroides counts.

Although initial open label studies showed promising prospects, with the exception of the studies
on pouchitis, published results of controlled clinical trials in patients with inflammatory bowel
diseases are poor (see Tamboli for an extensive review of published trials47). We need further
research on mechanisms of action in order to optimise the use of probiotics or prebiotics for
these indications.

7.5 LACTOSE MALABSORPTION

Prevalence of lactose malabsorption in adult populations varies from 5% to 15% in northern
European and American countries and 50% to 100% in African, Asian, and South American
countries.112 It is well recognised that bacteria used as starter culture in yogurt (S thermophilus
and Lactobacillus delbrueckii subspecies bulgaricus) improve lactose digestion.113 The benefit
is due to the presence of microbial b-galactosidase (lactase) in the bacteria. A large number of
human studies in which consumption of fresh yogurt (with live yogurt cultures) was compared
with consumption of a pasteurised product (with heat killed bacteria), demonstrated better
lactose digestion and absorption in subjects that consumed yogurt with live cultures as well as
reduction of gastrointestinal symptoms.

7.6 IRRITABLE BOWEL SYNDROME

Symptoms of abdominal pain, bloating, flatulence, and diarrhoea are commonly seen in patients
with irritable bowel syndrome, and abdominal pain and bloating are difficult to treat and have a
significant impact on patients’ lives. Bacteria in the gut flora can produce intestinal gas but they
also consume gas. Theoretically, probiotics might improve the balance and reduce gas
accumulation within the bowel. In a double blind clinical trial, administration of a L plantarum
strain decreased pain and flatulence in patients with irritable bowel syndrome.122 Likewise, a
recent placebo controlled trial concluded that the probiotic mixture is useful for the relief of

25
abdominal bloating in patients with diarrhoea predominant irritable bowel syndrome, and the
effect is unrelated to an alteration in gastrointestinal or colonic transit.123 Probiotics need to be
evaluated further but they appear to be useful for the control of symptoms due to the altered
handling or perception of intestinal gas in this group of patients.

7.7 PREVENTION OF SYSTEMIC INFECTIONS

Translocation of viable or dead bacteria in minute amounts constitutes a physiologically
important boost to the immune system. However, dysfunction of the gut mucosal barrier may
result in the passage of large quantities of viable microorganisms, usually belonging to Gram
negative aerobic genera, which can disseminate throughout the body producing sepsis. Bacterial
translocation and its complications have been shown to occur in several pathological conditions
such as postoperative sepsis, severe acute pancreatitis, advanced liver cirrhosis, multisystem
organ failure, etc.

Probiotics have been used to prevent sepsis in patients with severe acute pancreatitis. In a
randomised double blind trial, patients were treated with either Lactobacillus plantarum or
placebo. Incidence of infected pancreatic necrosis and abscesses were observed at a significantly
lower rate in L plantarum treated patients than in the control group.

A randomised study involving 95 liver transplant patients compared the incidence of infections
among three groups of patients submitted to different prophylaxis procedures: selective bowel
decontamination with antibiotics, administration of live L plantarum with fermentable fibre, and
administration of heat killed L plantarum plus the fibre supplement. Postoperative infections
were recorded in 15 out of 32 patients (48%) in the antibiotics group, four out of 31 (13%) in the
live L plantarum group, and 11 out of 32 (34%) in the heat killed L plantarum group, being
significant the difference between antibiotics and live L plantarum groups.24 In a second study
with liver transplant patients recently concluded by the same group in Berlin, patients were
randomised to receive a synbiotic preparation (including four probiotic strains: Pediococcus
pentoseceus, Leuconostoc mesenteroides, Lactobacillus paracasei, and L plantarum and four
fermentable fibres: b-glucan, inulin, pectin, and resistant starch) or a placebo consisting of the
four fibres only.

Postoperative infection occurred in only one patient in the treatment group (n = 33), in contrast
to 17 out of 33 in the placebo group.25 Early administration of live probiotics may become a
useful and effective therapeutic alternative to prevent postoperative infections, as opposed to
prophylaxis with antibiotics.

7.8 PREVENTION AND TREATMENT OF ATOPIC DISEASES

Atopic diseases (for example, atopic dermatitis, allergic rhinitis, and asthma) represent an
exaggerated and imbalanced immune response to environmental or food allergens.

26
Recent studies have shown that prevalence of allergic diseases in western societies is increasing
at an alarming rate whereas the frequency of allergic diseases is much lower in developing
countries. It has been suggested that this may be the result of reduced microbial pressure48 or
exposure to inappropriate microbial stimulus (including overt infections) during infancy and
early childhood in industrialised countries due to improved hygienic conditions. Appropriate
microbial stimulus during early age is believed to be essential to counterbalance the skewed Th2
immune phenotype of the newborn and the maturation of the immune system to a nonatopic state
(education of the developing immune system).

Differences in the composition of microflora of infants with a high and a low prevalence of
allergy, and in infants in whom allergy was and was not developing support this hypothesis. The
balance between bifidobacteria and clostridia appears to be the key factor in determining
predisposition to allergies. Lower incidence of allergies in breast fed infants, compared with
formula fed infants, is associated with higher counts of bifidobacteria in their flora; formula fed
infants are known to have more clostridia. Several epidemiological and experimental studies
have indicated that stimulation of the immune system by certain microbes or microbial products
may be effective in the prevention and management of allergic diseases.51

7.9 Probiotics and the prevention of allergic diseases

The effectiveness of probiotic therapy in the prevention of allergic diseases has been
demonstrated in two randomised controlled trials. Lodinova-Zadnikova et al investigated the
preventive efficacy of at birth colonisation with a probiotic (non-enteropathogenic) E coli.52 The
incidence of allergies was assessed using a questionnaire both after 20 years (150 full term) and
10 years (77 preterm infants) after colonisation. Subjects colonised with E coli were found to
have significantly lower incidence of allergies, compared with the control subjects, both after 10
and 20 years. In the second randomised, double blind, placebo controlled study, Kalliomaki et al
demonstrated the effectiveness of Lactobacillus GG in the prevention of early atopic disease in
children at high risk.53 Lactobacillus GG supplementation prenatally to mothers (for two weeks)
with a family history of atopy and postnatally to their infants for six months significantly
reduced the incidence of atopic eczema (p = 0.008) during the first two years life, compared with
the placebo group. The frequency of atopic eczema in the probiotic supplemented group (15 of
64, 23%) was half that of the placebo group (31 of 68, 46%). Re-examination of the cohort at the
age of 4 years has further shown that the preventive effect of Lactobacillus GG on atopic eczema
extends beyond infancy54; 14 of 53 children receiving Lactobacillus GG had developed atopic
eczema, compared with 25 of 54 receiving placebo (relative risk 0.57, 95% confidence interval
0.33 to 0.97). Interestingly, however, supplementation had no effect on skin prick test and the
induction of IgE (as measured by in vitro IgE test). This may suggest that probiotic therapy does
not protect against IgE mediated sensitisation.

7.10 Probiotics and the management of allergic diseases

The ability of bacterial therapy to reduce the symptoms of food allergy was first highlighted by
Loskutova55 and Ciprandi et al.56 Since then, several well designed studies have provided

27
evidence that supplementation with specific strains of probiotics could be effective in the
management of atopic disorders.

Majamaa and Isolauri examined the efficacy of extensively hydrolysed whey formula
supplemented with Lactobacillus GG in infants with atopic eczema and cows’ milk allergy. In a
randomised, double blind, placebo controlled trial, subjects receiving formula with Lactobacillus
GG (n = 13) showed significant improvement in clinical symptoms (SCORing Atopic Dermatitis
(SCORAD) score) and markers of intestinal inflammation (indicated by significant decreases in
the concentrations of faecal a-antitrypsin and TNF-a) compared with the placebo group given
formula without probiotics (n = 14). Similar effects of probiotic therapy were reported in another
randomised, double blind, placebo controlled study by the same authors58 in which breast fed
infants (n = 27) with atopic eczema were weaned onto an extensively hydrolysed whey formula
containing Lactobacillus GG or Bb12 or without probiotics. After two months, infants receiving
whey formula with probiotics showed significant improvements in the extent and severity of
atopic eczema (SCORAD score: 0 for Lactobacillus GG, 1 for Bb 12 and 13 for placebo group)
and reduction in inflammatory responses (as indicated by reductions in the concentration of
soluble CD4 in serum and eosinophilic protein X in urine) compared with the placebo groups.58
Expression of cell surface receptors and the production of chemotactic factors remained
unchanged.

These findings have also been recently confirmed by Rosenfeldt et al.59 In a randomised, double
blind, placebo controlled crossover study, 56% of atopic children (to 13 years old) receiving L
rhamnosus 19070–2 and L reutri DSM 122460 for six weeks showed improvement of the eczema
compared with only 15% in the placebo group; during the active treatment the extent of eczema
decreased from a mean of 18.2% to 13.7% (p = 0.02). However, the total SCORAD index
showed no significant change. Interestingly, the treatment response was more pronounced in
subjects with raised IgE levels and a positive skin prick test response. From these studies, it
could be concluded that specific probiotic strains might be effective in the management of
allergies.

Effectiveness of viable but not heat inactivated probiotics in the management of atopic eczema
and cows’ milk allergy60 and an association between the consumption of fermented milk
products and a decrease in allergic symptoms has also been reported.61

However, little is known about the efficacy of probiotics in preventing allergic disorders in other
age groups. Helin et al examined the effect of probiotic supplementation in young adults or
teenagers with birch pollen or apple allergy.62 Lactobacillus GG therapy for 5.5 months (before,
during, and after the pollen birch season) failed to mitigate the symptoms of allergy or reduce the
use of medicine. No benefits were noted in subjects exposed to apple challenge test. In another
double blind crossover study involving 15 adults with moderate asthma, supplementation with
yoghurt with live L acidophilus or yoghurt with L acidophilus for one month was found to have
no beneficial effect on spirometric function.63 In addition to the induction of regulatory T (Tr)

28
cells and counter-regulation by Th1 cells,64 reduced immunogenicity of potential allergens
through modification of their structure,65 strengthening of the mucosal defences (production of
IgA), stabilisation of gut mucosal barrier and downregulation of inflammatory responses66 by
lactic acid bacteria has been suggested to contribute to antiallergy effects of probiotics. It was
also reported that the beneficial effects of Lactobacillus GG were associated with increases in
transforming growth factor-b,67 induction of IL-10 production68 and regulation of phagocytic
cell function.

7.11 ENHANCEMENT OF SPECIFIC PARAMETERS OF IMMUNE FUNCTION

The immune system employs non-specific and acquired immune mechanisms to mediate
protection against pathogenic organisms and the development and metastasis of cancers;
deficiencies or dysregulation of the immune system are associated with enhanced disease
susceptibility. The nonspecific immune responses constitute the first line of host defence and
operate non-selectively against foreign antigens/ substances. The major cellular effectors of
innate immune system include phagocytic cells (monocytes, macrophages, and
polymorphonuclear cells,), and natural killer (NK) cells.

Phagocytic cells play a major role in limiting the spread of infectious challenge, whereas NK
cells are critical for controlling viral infections and cancers. The humoral components of the
innate immune system include complement, acute phase proteins and cytokines (for example,
interferons). In addition, innate immune responses have an important role in initiating and
regulating the acquired immune responses.

The acquired immunity involves T-lymphocytes and Blymphocytes. B-cells secrete

immunoglobulins/specific antibodies, while T-cells provide help for B-cells to produce specific
antibodies, and mediate host defence by eliminating intracellular pathogens (that is, by activating
macrophages) and by killing virus infected cells. Which of these mechanisms are preferentially
activated depends on the nature of the disease causing agent. A successful immune response,
however, requires the coordination and participation of both non-specific and specific arms of
the immune system as most of the immune competent cells and their products have overlapping
and complementary functions.

7.12 Probiotics and immune function

The effect of probiotic intake on immune function has been the subject of several recent human
studies. There is strong evidence to suggest that specific strains of lactic acid bacteria, when
consumed in certain numbers, are able to modulate aspects of both natural and acquired immune
responses.

7.12.1 Probiotics and innate immune function

The ability of specific strains of probiotics to enhance aspects of natural immunity in human
subjects is well documented. Schiffrin and colleagues reported enhanced phagocytic capacity of
peripheral blood leucocytes (polymorphonuclear and monocytes)70 in healthy human adults

29
administered fermented milk supplemented with specific strains of probiotics (Lactobacillus
johnsonii La1 or Bifidobacterium lactis Bb12) for three weeks. The improvements in phagocytic

activity were sustained for several weeks after cessation of probiotic consumption, 70–72 and
granulocytes showed higher increases in phagocytic cell function compared with monocytes.70
73 Significant increases in the expression of receptors involved in phagocytosis (CR1, CR3,
FccRI and FcaR) in neutrophils, 69 phagocytic index,74 and oxidative burst73 75 or
microbicidal capacity71 in subjects receiving probiotics have also been reported. It has also been
observed that different lactic acid bacteria strains differ in their capacity to influence complement
receptor expression in phagocytic cells. For example, He et al found that Lactobacillus lactis was
more efficient at upregulating complement receptors on blood leucocytes than L rhamnosus.76
Furthermore, the immunostimulatory effect of probiotic intake was dose dependent and a
minimum dose of 109 cfu/day was found to be necessary to realise improvements in immune
function.73

Ageing is associated with a decline in immunocompetence. It has been suggested that

supplementation with probiotic could be used to correct age related decline in phagocytic cell
function in the elderly. Subjects receiving milk containing L rhamnosus (HN001) or B lactis
(HN019) for three to six weeks were found to exhibit significantly more phagocytically active
blood leucocytes (neutrophils and monocytes; fig 1) than subjects receiving milk without
probiotics.72 77 78 Notably, subjects with relatively poor preintervention phagocytic cell
function consistently showed higher relative increases in phagocytic activity than subjects with
adequate preintervention immune function.77 Furthermore, augmentation in phagocytic activity
was found to be correlated with age, with subjects older than 70 years exhibiting significantly
greater improvements in phagocytic activity than those under 70 years.77

Significant improvements in NK cell activity, and increases in the percentage of NK cells in the
peripheral blood of human volunteers fed yogurt, milk, or sausages containing probiotics have
also been reported.74 78–80 As with phagocytic cell function, augmentation of NK cell activity
in the elderly following administration of lactic acid bacteria (fig 2) was also correlated with age.

30
 Adapted from (Gill et al, 2004)

Polymorphonuclear cell phagocytic activity before and after consumption of L rhamnosus

HN001 in healthy elderly subjects (n = 13; five males, eight females; age 65–85 years). Subjects
consumed low fat, low lactose milk (200 ml twice/day) as a base diet during the run-in stage for
three weeks, followed by milk supplemented with L rhamnosus (5610 cfu daily) for three weeks
(intervention stage), before returning to non-supplemented low fat, low lactose milk for the final
three weeks. Data are mean (SEM); *significantly higher response than preceding time points
(p,0.001; repeat measure analysis of variance, Dunnett’s post hoc test for significance).

Contrary to the studies discussed above, however, a lack of any immunomodulatory effect of
probiotic intake on phagocytic cell function80 81 has also been observed in several studies.
Whether this was simply due to strain-dependent variation in the immunostimulatory capacity of
the probiotic bacteria used or some other reason is not clear. Differences in the ability of lactic
acid bacteria to influence immune function are well documented.

7.12.2 Humoral immune responses

A large number of human studies have also shown that the intake of specific strains of lactic acid
bacteria is able to potentiate humoral immune responses to natural infections and systemic or
oral immunisation. In a randomised, placebo controlled study involving infants with acute
rotavirus diarrhoea, Kaila et al observed that a reduced duration of diarrhoea after administration
of Lactobacillus GG fermented milk was associated with augmentation of both rotavirus specific
and non-specific antibody responses.83 At convalescence, 90% of the Lactobacillus GG group
compared with 46% of the placebo group exhibited rotavirus specific IgA antibody secreting cell
response. It is important to note, however, that a very low cut off level (0.05 antibody secreting
cells/106 cells) was used as an indicator of seroconversion.

Significantly higher IgG, IgA, and IgM immunoglobulin secreting cells in Lactobacillus GG fed
infants compared with those given a placebo were also observed. Comparison of different strains
further revealed that Lactobacillus GG was more efficient at stimulating IgA specific antibody
secreting cells to rotavirus and serum IgA responses than L casei subspecies rhamnosus or a
combination of S thermophilus and L delbruckii subspecies bulgaricus.84 Furthermore, viable
cells were found to be more efficacious at stimulating rotavirus specific immune response than
heat inactivated cells.

31
 Adapted from (Gill et al, 2004)

Relative increases in NK cell tumoricidal activity after consumption of milk supplemented with
L rhamnosus HN001. Data are mean (SEM) percentage increases in peripheral blood
mononuclear cell tumoricidal activity between time point T1 (before probiotic supplementation
—that is, after three week run-in period) and time point T2 (immediately after three weeks of L
rhamnosus supplementation). Adapted from Gill et al.

Effectiveness of probiotics in enhancing the immunogenicity of mucosal and systemic vaccines

has also been reported. Link-Amster and colleagues reported fourfold increase (p = 0.04) in
serum IgA antibody response to Salmonella typhi immunisation, in volunteers consuming
yoghurt containing B bifidum and L acidophilus La1 compared with a 2.5-fold increases in the
control group.86 Enhanced responsiveness (rotavirus specific IgM secreting cells and IgA
seroconversion rate) to live oral rotavirus vaccine in infants fed Lactobacillus GG, compared
with the control group, has also been reported.87 Supplementation with specific strains of
probiotics has also been shown to improve immune responses to immunisation with polioviruses.
In a randomised, double blind, placebo controlled study,88 subjects fed yoghurt containing L
rhamnosus and L paracasei showed significantly higher virus neutralising antibody responses
(mainly IgA) to a live attenuated polioviruses vaccine, compared with subjects given placebo
(chemically acidified milk); subjects receiving probiotic yoghurt also exhibited significantly
higher polio specific serum IgG and IgA responses. In another study, administration of a formula
supplemented with bifidobacteria significantly increased the levels of total faecal IgA and
antipoliovirus faecal IgA89 in infants immunised against poliovirus several months before
enrolment in the study.

Together these observations suggest that specific strains of lactic acid bacteria are endowed with
potent adjuvant properties that could be used for improving efficacy of oral vaccines and as
immunostimulants to promote recovery from infectious illnesses. Development of immunisation
strategies that avoid the use of needles, and non-toxic adjuvants is highly desirable as it will
simplify immunisation programmes and reduce barriers to large scale immunisation.

It is to be noted that probiotic administration is also known to induce antibody responses to

completely unrelated antigens and to themselves. Therefore, it is possible that some of specific
antibody activity detected in studies described above may in fact be due to non-specific cross
reacting antibodies. The mechanisms by which probiotics potentiate humoral immune responses
are not known but could be due to increased transport of antigenic materials across the gut and
improved antigen-presenting cell function (upregulation of antigen presenting molecules and
costimulatory molecules induced by proinflammatory cytokines91 and/or increased number of
B-cells.92 Although the studies reported above provide evidence of the immunoenhancing

32
effects of certain probiotic strains, further well designed studies are needed to unequivocally
demonstrate the efficacy of specific strains; only a small number of studies conducted to date
have been randomised, double blind, and/or placebo controlled. Whether consumption over long
periods will result in sustained improvements in immune capacity also remains to be determined.
Also, little is known about the effective probiotic dose for different population groups, and the
relevance of immunoenhancement to increased disease resistance. The precise mechanisms by
which probiotics influence the functioning of the immune system are not fully understood.

It appears that recognition of probiotic associated molecular patterns (for example,

peptidoglycans, lipotechoic acid, bacterial DNA) by pattern recognition receptors (such as toll-
like receptors) present on the surface of immunocompetent cells (monocytes, macrophages,
dendritic cells, etc), present in the Peyer’s patches, lamina propria or other sites triggers the
release of a range of cytokines that shape the developing immune response (unresponsiveness or
active immune response). Several studies have reported increased production of a vast array of
cytokines (for example, IL-1, IL2, IL-6, IL-10, IL-12, IL-18, TNF-a, interferon-c) following in
vitro stimulation of blood leucocytes with lactic acid bacteria and/or oral consumption of
probiotics.82 93 These cytokines are known to exert a range of modulatory effects on immune
cell function.21 For example, IL-12 and IL-18 induce interferon-c production by T-cells, B-cells
and NK cells, and interferon-c enhances phagocyte mediated clearance of microbes, augments
cytotoxic capacity of T-cells and NK cells, and stimulates helper T-cell function and augments
immunogenicity of vaccines. TNF-a further increases the microbicidal activity of macrophages
and exerts cytotoxic effect against tumours. Interferon-a mediates protection against viral and
microbial infections and cancers, while IL-1 stimulates proliferation of T-cells and B-cells and
IL-6 induces differentiation to antibody-secreting plasma cells. Transforming growth factor-b
and IL-10 possess potent anti-inflammatory properties and play an important part in immune
system homoeostasis. IL-2 influences induction and regulation of Tcell mediated immune
responses.

7.13 Anticancer Effects

Several animal studies have shown that supplementation with specific strains of lactic acid
bacteria (probiotics) could prevent the establishment, growth, and metastasis of transplantable
and chemically induced tumours.94 Studies in human subjects have also revealed that probiotic
therapy may reduce the risk of colon cancer by inhibiting transformation of procarcinogen to
active carcinogens, binding/inactivating mutagenic compounds, producing antimutagenic
compounds, suppressing the growth of pro-carcinogenic bacteria, reducing the absorption of
mutagens from the intestine, and enhancing immune function.95 96 An inverse relationship
between the consumption of fermented dairy products, containing lactobacilli or bifidobacteria,
and the incidence of colon and breast cancer has also been reported in epidemiological and
population based case-control studies.97 98 However, there is little ‘‘direct experimental
evidence’’ regarding the anticancer effectiveness (tumour suppression) of probiotic therapy in
humans. Aso and colleagues demonstrated the protective effect of L casei strain Shirota on the

33
recurrence of superficial bladder cancer in a randomised, controlled, multicentre study.99
Subjects were enrolled in the study within two weeks after removal of bladder tumours.

After one year, tumour recurrence rate was significantly lower in subjects receiving L casei
(57%) compared with the control group (83%). Treatment with L casei also delayed the onset of
tumour recurrence. Similar observations were made in a second, much larger, placebo controlled
study involving 125 patients by Aso and colleagues.100 Increases in the percentage of T-helper
cells and NK cells in adult colorectal cancer patients101 suggest that stimulation of the immune
system by L casei Shirota may have an important role in the suppression of tumour development.

Carefully designed, long term human studies are needed to verify findings of animal studies and
to establish a basis for probiotic therapy in cancer prevention.

7.14 Lowering of blood Cholesterol

Mann and Spoerry were the first to report that consumption of fermented milk was associated
with reduced serum cholesterol levels in the Maasai people. This stimulated much interest in the
cholesterol lowering effects of fermented milks and lactic acid bacteria. Several animal studies
have shown that administration of fermented milks or specific strains of lactic acid bacteria is
effective in lowering blood cholesterol levels. Studies in human subjects, however, have yielded
conflicting results. For example, Lin et al reported a reduction in serum cholesterol from 5.7 to
5.3 mmol/l after seven weeks and to 5.4 mmol/l after 16 weeks in subjects given L acidophilus
and L bulgaricus (3610 cfu/daily) for 16 weeks; serum cholesterol levels remained unchanged in
control subjects (without a placebo).103 However, in the second double blind, placebo controlled
(crossover design) trial involving 460 volunteers (334 subjects completed the study),
supplementation with these strains (two six week supplementation periods separated by a
washout period of three weeks) was found to have no effect on serum cholesterol levels.
Contradictory observations on the cholesterol lowering effects of yoghurt enriched with a
specific strain of L acidophilus have also been reported by others. While Shaafsma et al reported
a reduction in serum cholesterol in subjects receiving yoghurt supplemented with L acidophilus
and fructo-oligosaccharides for weeks,104 de Roos et al found no effect of the consumption of
yogurt containing L acidophilus L1 on serum cholesterol in a placebo controlled study (n =
78).105 Several factors, including differences in experimental design, differences in study
subjects, intakes of fermented milk, and strains of bacteria used have been suggested to account
for these conflicting observations. A meta-analysis of controlled short term (4–8 weeks) studies
(n = 6) involving 425 subjects (male and female with different initial cholesterol levels) has
shown that consumption of yoghurt containing Enterococcus faecium (Gaio) is effective in
reducing both total and low density lipoprotein cholesterol by 4% and 5%, respectively,
compared with the control group.106 Whether the effects are sustained over a long time remains
to be proven. Subjects given milk fermented with E faecium for 24 weeks showed reduction in
serum cholesterol levels at four and 12 weeks but not at 24 (end of the study) and 30 weeks (after
the follow up).107

34
Consumption of Pro Viva (Probi AB, Sweden) food product containing L plantarum 299v
(randomised, placebo controlled study) has also been reported to lower total and low density
lipoprotein cholesterol in subjects with moderately raised cholesterol levels.108

Furthermore, it has been reported that long term consumption of fermented products enriched
with specific strains of lactic acid bacteria may be effective in increasing high density lipoprotein
cholesterol. In a single blind parallel study, subjects consuming milk fermented with L casei
TMC 0409 and S thermophilus TMC 1543 (200 ml/day) showed significant increases, compared
with the pre-intervention levels, in high density lipoprotein levels after four and eight weeks
supplementation.109 The levels of triglycerides were also reduced significantly in subjects
receiving the fermented milk. No significant changes were noted in the control group.

Consumption of 300 g yoghurt supplemented with L acidophilus 145 and Bifidobacterium

longum 913 for seven weeks was also found to increase high density lipoprotein concentration
by 0.3 mmol/l (p = 0.002) and decrease ratio of low to high density lipoprotein from 3.24 to 2.48
(p = 0.001).110 However, the concentrations of total and low density lipoprotein cholesterol in
serum remained unaffected by the consumption of probiotic yoghurt; relatively high fat content
of the yoghurt (3.5%) was suggested to be the main reason for the lack of effect. The precise
mechanisms by which probiotics affect cholesterol levels are not fully understood. However, a
range of mechanisms include assimilation of cholesterol by bacterial cells, deconjugation of bile
acids by bacterial acid hydrolases (reduces cholesterol reabsorption, increases cholesterol
excretion of deconjugated bile salts, and increases cholesterol uptake by low density lipoprotein
receptor pathway in the liver as a compensatory response), cholesterol binding to bacterial cell
walls, and inhibition of hepatic cholesterol synthesis and/or redistribution of cholesterol from
plasma to the liver (through the action of short chain fatty acids, the end products of
carbohydrate fermentation in the gut).111

7.15 Prevention of recurrent vaginitis

A solution for the prevention and treatment of acute vaginal infections and recurrences caused by
Candida based on two different mechanisms of action

7.15.1 Physical primary activity

The production of CO2 creates an anaerobic environment able to significantly slow down
the respirative metabolism of Candida

7.15.2 Ancillary Specific activity

The simultaneous production of bacteriolysins and lactic acid by the two lactobacilli exerts
a specific inhibitory activity against Candida

Properties of lactobacilli Role in the vaginal colonization

Acidogenic (all) Lowering of vaginal pH, inhibition of competitive

35
 microorganisms

Production of lactic and

Bacteriostatic / bactericidal effect
acetic acid

Production of hydrogen
peroxide (L.
crispatus,
L. jensenii, L. gasseri, L. Bactericidal effect
delbrueckii, L. casei, L.
plantarum, L. vaginalis, L.
pentosus)

Biosurfactants production
- natural
surfactants - (L. Prevents the adhesion of contaminating
rhamnosus, L. fermentum, microorganisms to the epithelium
L.
acidophilus)

Competition for available nutrients and physical

Competitive exclusion (all)
occupation of receptors on epithelial cells

Production of bacteriocins
and bacteriolysis
(L. gasseri, L. plantarum, L. Inhibition of the growth of contaminant
fermentum, L. microorganisms
casei, L. delbrueckii, L.
reuteri, L. salivarius)

(Vicariotto et. al 2012)

Function/ characteristic Probiotic Antibiotic/Antimicoti c

Natural YES NO
Without side effects YES NO
Specific activity against YES YES (but it alter also
pathogens commensal/positive
bacteria)
Relapses prevention YES NO
Absence of possibile YES NO
phatogen resistance

36
development
Help to balance vaginal YES NO
microflora

3 7 months
Adapted from Vicariotto et. al, 2012

7.16 Liver Diseases

7.16.1 Hepatic encephalopathy

Alteration of gut flora (either with probiotics or with prebiotics such as fermentable fiber) has
been associated with improvement in hepatic encephalopathy in pilot studies [77,78]. Such
therapy appears to lower blood ammonia concentrations, possibly by favoring colonization with
acid-resistant, non-urease producing bacteria [79]. The role for this approach is still being
studied.

7.16.2 Nonalcoholic fatty liver disease (NASH)

Nonalcoholic steatohepatitis (NASH) is the term used to describe the distinct clinical entity in
which patients lack a history of significant alcohol consumption but have liver biopsy findings
indistinguishable from alcoholic hepatitis. Studies in rodent models of alcoholic fatty liver

37
disease have demonstrated that intestinal bacteria, bacterial endotoxin and TNF-α modulate
alcohol-induced liver damage. The concept that intestinal bacteria induce endogenous signals,
which play a pathologic role in nonalcoholic fatty liver disease, suggests a role for novel
probiotic therapy in this not so uncommon condition.

7.17 Pancreatitis
Pancreatic necrosis and associated pancreatic infection are determinants of poor outcome in
patients with severe acute pancreatitis. Colonization of the lower gastrointestinal tract and
oropharynx with gram-negative organisms often precedes contamination of the inflamed
pancreas. Human studies in which patients with acute pancreatitis received L. plantarum 299v
showed a decrease in occurrence of pancreatic infection/abscess and a shorter hospital stay
[82,83]. These human findings were supported by trials of probiotics (L. plantarum 299v and S.
boulardii) in animal models of acute pancreatitis in which intestinal microbial translocation was
reduced [84].

7.18 Probiotics in infants

Breast-feeding protects infants from infectious disease by multiple mechanisms. Components of
human milk may have an effect in modulating the composition of the intestinal flora and
bifidobacteria generally constitute a significant component of normal intestinal flora in breast-
fed infants [85, 86]. Breast-feeding can also affect the occurrence and virulence of colonizing
pathogens [87, 88]. Thus, it appears that a combination of increased bifidobacterial counts and
decreased concentrations of other enterobacteria and luminal host factors may play a role in
protecting premature babies and newborns from diarrheal disease.

Modification of the intestinal flora by increasing the predominance of specific nonpathogenic

bacteria would seem a reasonable means of attaining a prophylactic or therapeutic effect against
enteropathogens. Necrotizing enterocolitis is one devastating intestinal disorder known to occur
in 10-25% of premature infants and very low birth weight babies and has a high mortality of
2030%. Bacterial colonization or infection of the intestine by pathogens increases the risk of
necrotizing enterocolitis. Trials showing reduction of necrotizing enterocolitis in population of
premature newborns given supplements of Lactobacillus GG daily compared with historical
control subjects have been reported [89,90]. These findings suggested a correlation between the
reduction of lactobacilli and the increased risk of necrotizing enterocolitis.

7.19 Probiotics in pregnancy

Good nutrition during pregnancy improves the chances of having a healthy baby who will be at
lower risk of diseases later in life. Bacterial vaginosis, has been suggested as a factor that
increases risk of preterm labour and infant mortality and probiotics been shown to decrease risk
of bacterial vaginosis and maintain normal lactobacilli vaginal flora [107,108]. In animal studies,
these strains were found to be safe during pregnancy and to enhance the health of mothers and
newborns [109].

38
Another area of interest in the use of probiotics in pregnancy is to prevent allergic reactions.
Studies using L rhamnosus GG and B lactis BB12 have shown that atopic dermatitis, a condition
that causes severe skin rashes in up to 15% of babies, can be prevented in 50% of cases if
mothers ingest probiotics during pregnancy and newborns ingest them during the first 6 months
of life [97,110,111]. Probiotics during pregnancy also have an excellent safety record [112].

7.20 Probiotics and diabetes

The intestinal microbiota presents a vast set of antigens which may participate in the modulation
of immunological diseases. An intestinal barrier presenting full integrity ensures specific
interactions between the luminal antigens and the host. Functional disarrangements may
contribute to the autoimmune destruction of pancreatic β cells, which leads to T1D, and
increased expression of inflammatory cytokines may lead to insulin resistance and T2D.

The evidence available from experimental studies and clinical trials supports our suggestion that
the modulation of the intestinal microbiota by probiotics may be effective towards prevention
and management of T1D and T2D. The findings discussed here provide an insight into the
investigation of further hypotheses aiming to elucidate molecular mechanisms involved in the
modulation of intestinal microbiota by probiotic administration, their roles on the development
of T1D and T2D and potential effectiveness for clinical practice.

39
Gomes et al, 2014

7.21 Urogenital infections and HIV

Bacterial vaginosis, yeast vaginitis and recurrent urinary tract infections (UTIs) are common
urogenital problems. The normal vaginal flora in premenopausal women consists primarily of
lactobacilli, which are protective against infection, but many pathophysiologic factors can cause
unstable vaginal flora that may result in infection. There are only few studies with the use of
probiotics in vaginal infections [99,100]. The overwhelming experiences have revealed a positive
effect of L. acidophilus.

Evidence from the available studies also suggests that probiotics can be beneficial for preventing
recurrent UTIs in women [101]. The prevention or resolution of bacterial vaginosis is
particularly important in women at risk of human immunodeficiency virus (HIV) infection.
Studies have shown that women with bacterial vaginosis (no lactobacilli) are at significantly
increased risk of HIV [102,103]. Thus treatment of bacterial vaginosis and promotion of vaginal
lactobacilli may reduce a woman's risk of acquiring HIV-1, gonorrhea and trichomoniasis. A

40
recent publication has shown that a human vaginal probiotic strain (Lactobacillus reuteri RC-14)
can express potent functional viral inhibitors which may potentially lower the sexual
transmission of HIV [104].However, further research is needed to confirm these results before
the widespread use of probiotics for these indication can be recommended.

74.22 Regulation of bone health

The two well-known species of health-promoting probiotic bacteria residing in our intestinal
tract are lactobacilli and bifidobacteria. These creatures are known as “probiotics” and serve
many functions for us, including assisting in digestion, producing vitamins, and inhibiting
growth of harmful bacteria.

Focusing on osteoporosis and osteopenia, numerous studies suggest the bone-effects of

probiotics — such as increased bone mass, decreased bone breakdown and increased calcium
and phosphorus blood levels. Specifically, a healthful probiotic balance improves bone strength
by:

 Increasing calcium and magnesium absorption

 Making milk more digestible (reducing lactose)
 Reducing leaky gut and allergies
 Enhancing immunity
 Reducing the impact of dietary phytates which limit mineral absorption
 Enhancing absorption of phytoestrogens

Model of gut microenvironment signals regulating bone density (McCabe et al, 2016)

41
8.0 Conclusion:
The intestinal microbiota of the host can be modulated through the consumption or
administration of probiotic micro-organisms that establish an excellent means of remediation and
deterrence against a variety of intestinal disorders and infections. Given the lack and hazards of
antibiotics, including reduction of microbiome diversity and antibiotic resistance, the use of
probiotics instead of antibiotics is becoming increasingly acceptable.

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