probiotics—from metchnikoff to bioactives
TRANSCRIPT
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Review
ProbioticsFrom Metchnikoff to bioactives
T. Vasiljevic , N.P. Shah
School of Molecular Sciences, Victoria University, PO Box 14428, Melbourne, Vic. 8001, Australia
a b s t r a c t
The benefits of probiotics have been recognized and explored for over a century. The pioneering work of
Tissier and Moro was elaborated in the Metchnikoffs theory of longevity and converted into commercial
reality by Shirota and Kellogg in 1930s and German nutritionists with their probiotic therapy in 1950s.Our knowledge about probiotics and their interactions with the host has grown ever since and many
potential and even proven mechanisms of action for probiotics have recently been published. Definitely,
there is enough clinical evidence to support certain health claims attributed to selected strains of
Lactobacillusand Bifidobacteriumspp. However, substantial work needs to be done to substantiate other
potentially beneficial properties including immunomodulation, hypocholesterolemic and anticarcino-
genic effects. The aim of this review is to pay the tribute to pioneers in the field and provide an overview
of the current state of knowledge about probiotics and their impact on our well-being.
&2008 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714
2. Evolution of the probiotic concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7153. Definition of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
4. Properties of lactic acid bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
5. Commercially important probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
6. Selection of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717
7. Technological challenges in the development of probiotic dairy products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
8. Health potential of probiotic foods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720
9. Health effects of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
9.1. Alleviation of lactose intolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
9.2. Prevention and reduction of diarrhoea symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
9.3. Treatment and prevention of allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
9.4. Reduction of the risk associated with mutagenicity and carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
9.5. Hypocholesterolemic effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
9.6. Inhibition ofHelicobacter pylori and intestinal pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
9.7. Prevention of inflammatory bowel disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
9.8. Modulation of the immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72510. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
1. Introduction
The increasing cost of health care, the steady increase in life
expectancy and the desire of the elderly for improved quality of
their lives are driving factors for research and development in the
area of functional foods. Although the concept of functional foods
was introduced long ago with Hippocrates and his motto Let food
be your medicine, fairly recently the body of evidence started to
support the hypothesis that diet may play an important role in
modulation of important physiological functions in the body.
Among a number of functional compounds recognized so far,
bioactive components from fermented foods and probiotics
certainly take the center stage due to their long tradition of safe
use, and established and postulated beneficial effects.
ARTICLE IN PRESS
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International Dairy Journal
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Corresponding author. Tel.: +613 9919 8062; fax: +613 9919 8284.
E-mail address: [email protected] (T. Vasiljevic).
International Dairy Journal 18 (2008) 714 728
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The fermentation of dairy foods presents one of the oldest
methods of long-term food preservation. The origin of fermented
milk can be traced back long before the Phoenician era and placed
in the Middle East. Traditional Egyptian fermented milk products,
Laban Rayeb and Laban Khad, were consumed as early as 7000 BC.
Their tradition claims that even Abraham owed his longevity to
the consumption of cultured milk (Kosikowski & Mistry, 1997).
Initially established in the middle and far east of Asia, thetradition of fermenting milk was spread throughout the east
Europe and Russia by the Tartars, Huns and Mongols during their
conquests. As a consequence, a wide range of fermented dairy
products still exists in these regions and some popular products
such as yoghurt and kefir are claimed to originate from the
Balkans and Eastern Europe.
2. Evolution of the probiotic concept
Although the preservation role of fermented dairy products was
widely recognized and appreciated early, scientists first realized in
the late 19th century that a wide range of traditional sour milk
products had additional benefits in addition to prolonged shelf-lifeand pleasant sensory properties. The work of numerous scientists,
mainly microbiologists, resulted in important developments and
expansion of knowledge pertaining to the microbiology of the human
body.Escherich (1885)was the first to recognize the importance of
examining bacteria appearing in normal faeces and the intestinal
tract, and consequently understanding the physiology of digestion
and the pathology and therapy of intestinal diseases of microbial
origin. In 1900, two microbiologists, Tissier and Moro, reported their
findings of isolates from the faeces of breast-fed infants. Tissier noted
that the anaerobically cultured organism had, in general, staining
reactions and morphological appearance similar to those of lactoba-
cilli; however, many of them appeared in bifurcated forms. Thus, he
named them Bacillus bifidus. Similarly, Moro (1900) postulated that
the isolate, which he termed Bacillus acidophilus due to its unusual
acid tolerance, was derived from the mothers breast and normally
resided in the neonates oral cavity and intestinal content. Later,
Tissier (1908) also showed that Bac. bifidus was the predominant
organism in the faeces of breast-fed infants approximately three days
postpartum as opposed to bottle-fed neonates, which predominantly
containedB. acidophilus (Moro, 1905).
At the same time, Nobel Laureate Ilya Metchnikoff noticed that
Bulgarian peasants had an average life-span of 87 years, exceptional
for the early 1900s, and that four out of every thousand lived past
100 years of age. One of the major differences in their lifestyle in
comparison with the contemporary diet was a large consumption of
fermented milk. In his well known auto-intoxication theory
(Metchnikoff, 2004), Metchnikoff suggested that a human body
was slowly poisoned by toxins present in the body produced by
pathogens in the intestine and bodys resistance steadily weakenedby proliferation of enteric pathogens, all of which were successfully
prevented by the consumption of sour milk and lactic acid
producing bacteria. His work was based on an organism previously
isolated by Grigoroff (1905), who cultivated it from podkvassa
used as a starter for production of the Bulgarian kiselo mleko
(sour milk or yahourth) and called it Lactobacillus bulgaricus. In
the process, Grigoroff also identified another organism, Streptococ-
cus thermophilus, which received no attention since it was
considered a pathogen at that time. Metchnikoffs experiments led
him to believe that L. bulgaricuscould successfully establish itself in
the intestinal tract and prevent multiplication and even decrease
the number of putrefactive bacteria. However, the work of Herter
and Kendall (1908) showed that this organism failed to establish
itself in the gut, although other substantial changes in the gutmicroflora were observed.
Despite the fact that these findings disputed Metchnikoffs theory,
scientists continued to investigate possible benefits of bacteria to the
human health. Consequently, certain strains of Lactobacillus acid-
ophiluswere isolated and found to be capable of colonizing human
digestive tract where they exerted appreciable physiological
activity. Rettger and Horton (1914) and Rettger and Cheplin
(1920a, 1920b)reported that feeding of milk or lactose to rats or
humans led to a transformation of the intestinal microfloraresulting in predominance of acidophilus and bifidus type culture.
These findings stimulated commercial interest in products
fermented by L. acidophilus (Burke, 1938). Other researches
followed suit with Minoru Shirota in Japan, who recognized the
importance of the preventive medicine and modulation of the
gastrointestinal microflora. In 1930, he succeeded isolating and
culturing a Lactobacillus strain capable of surviving the passage
through the gastrointestinal tract. The culture identified as
Lactobacillus casei strain Shirota was successfully used for the
production of the fermented dairy product called Yakult, which
initiated the foundation of the same company in 1935 ( Yakult,
1998). In the period between late 1930s and late 1950s, the
research in this area lost its pace likely due to extraordinary
conditions (depression, war) the world was facing at that time.The rejuvenated interest in the intestinal human microflora was
seen in the late 1950s and early 60s that led to the introduction of
the probiotic concept.
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Table 1
Some of the descriptions and definitions of probiotics commonly cited over the
years
Year Description Source
1953 Probiotics are common in vegetable food as
vitamins, aromatic substances, enzymes and
possibly other substances connected with vital
processes
Kollath
19 54 Probiot ics are opp osite of ant ib iotics V ergin
1955 Deleterious effects of antibiotics can be
prevented by probiotic therapy
Kolb
1965 A substance secreted by one microorganism
which stimulates the growth of another
Lilly and Stillwell
1971 Tissue extracts which stimulate microbial
growth
Sperti
1973 Compounds that build resistance to infection
in the host but do not inhibit the growth of
microorganisms in vitro
Fujii and Cook
1974 Organisms and substances that contribute to
intestinal microbial balance
Parker
1992 Live microbial feed supplement which
beneficially affects the host animal by
improving microbial balance
Fuller
1992 Viable mono- or mixed culture of live
microorganisms which, applied to animals or
man, have a beneficial effect on the host by
improving the properties of the indigenous
microflora
Havenaar and Huis
intVeld
1996 Live microbial culture or cultured dairy
product which beneficially influences the
health and nutrition of the host
Salminen
1996 Living microorganisms which, upon ingestion
in certain numbers, exert health benefits
beyond inherent basic nutrition
Schaafsma
1999 Microbial cell preparations or components of
microbial cells that have a beneficial effect on
the health and well-being of the host
Salminen,
Ouwehand, Benno
and Lee
2001 A preparation of or a product containing viable,
defined microorganisms in sufficient numbers,
which alter the microflora (by implantation or
colonization) in a compartment of the host and
by that exert beneficial health effect in this
host
Schrezenmeir and de
Vrese
2002 Live microorganisms that when administered
in adequate amount confer a health benefit on
the host
FAO/WHO
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3. Definition of probiotics
The word probiotics was initially used as an antonym of the
word antibiotic. It is derived from Greek words proand biotos
and translated as for life (Hamilton-Miller, Gibson, & Bruck,
2003). The origin of the first use can be traced back to Kollath
(1953), who used it to describe the restoration of the health of
malnourished patients by different organic and inorganic supple-ments. A year later, Vergin (1954) proposed that the microbial
imbalance in the body caused by antibiotic treatment could have
been restored by a probiotic rich diet; a suggestion cited by many
as the first reference to probiotics as they are defined nowadays.
Similarly, Kolb (1955)recognized detrimental effects of antibiotic
therapy and proposed the prevention by probiotics. Later on, Lilly
and Stillwell (1965)defined probiotics as substances produced by
one microorganism that promoted the growth of another micro-
organism. Similar to this approach, Sperti (1971) and Fujii and
Cook (1973) described probiotics as compounds that either
stimulated microbial growth or improved the immune response
of the host without inhibiting the growth of the culture in vitro.
Another definition offered byParker (1974)resembles more recent
description of probiotics. He defined them as organisms andsubstances, which contribute to intestinal microbial balance. This
definition was disputed by many authors since various substance
even antibiotics might have been included.
Late 1980s and 1990s saw a surge of different definitions of
probiotics. Most frequently cited definition is that of Fullers
(1992), who defined them as a live microbial feed supplement,
which beneficially affects the host animal by improving its
intestinal microbial balance. However his definition was more
applicable to animals than to humans. Other authors followed this
line offering their versions. Some of these definitions are listed in
Table 1.Although all cited authors agreed that probiotics include
live microorganisms,Salminen, Ouwehand, Benno, and Lee (1999)
offered their view incorporating non-viable bacteria in the
definition. Following recommendations of a FAO/WHO working
group on the evaluation of probiotics in food (2002), the
suggested definition describes probiotics as live microorganisms
that when administered in adequate amounts confer a health
benefit on the host. Consequently, a wide variety of species and
genera could be considered potential probiotics (Holzapfel,
Haberer, Snel, Schillinger, & Huisint Veld, 1998); commercially,
however, the most important strains are lactic acid bacteria (LAB).
4. Properties of lactic acid bacteria
LAB are usually described as Gram-positive microorganisms,
devoid of cytochromes and preferring anaerobic conditions but
are aerotolerant, fastidious, acid-tolerant, and strictly fermenta-
tive, producing lactic acid as a main product (Stiles& Holzapfel,1997). The most important genera are: Lactobacillus, Lactococcus,
Enterocococcus,Streptococcus,Pediococcus,Leuconostoc, andBifido-
bacterium. Based on their GC (guaninecytosine) pair content,
Gram-positive bacteria are divided into two major phylogenetic
branches. In contrast to other above-mentioned genera, bifido-
bacteria exhibit a relatively high G+C content of 5567 mol% in the
DNA and belong to the Actinomycetesbranch. Other genera have a
lower G+C content (o55mol% DNA) and form a part of the
Clostridium branch. However, Bifidobacterium shares certain
physiological and biochemical properties with typical LAB and
some common ecological niches such as the gastrointestinal tract.
Therefore, for practical and traditional reasons, bifidobacteria are
still considered a part of the LAB group (Stiles&Holzapfel, 1997).
Members of the LAB are usually subdivided into two distinctgroups based on their carbohydrate metabolism. The homofer-
mentative group consisting ofLactococcus,Pediococcus,Enterococ-
cus, Streptococcus and some lactobacilli utilize the Embden
MeyerhofParnas (glycolytic) pathway to transform a carbon
source chiefly into lactic acid. As opposed to homofermentors,
heterofermentative bacteria produce equimolar amounts of
lactate, CO2, ethanol or acetate from glucose exploiting phospho-
ketolase pathway. Members of this group include Leuconostoc,
Weissella and some lactobacilli. The species belonging to Enter-ococcus genus are frequently found in traditional fermentations
and may be included as a component of some mixed starters.
However, their deliberate utilization in dairy fermentations still
remains controversial, especially since some of the species have
been now recognized as opportunistic human pathogens asso-
ciated with hospital-acquired- and urinary tract infections (Franz,
Holzapfel,& Styles, 1999).
5. Commercially important probiotics
Probiotic cultures have been exploited extensively by the dairy
industry as a tool for the development of novel functional
products. While it has been estimated that there were approxi-mately 70 probiotic-containing products marketed in the world
(Shah, 2004), the list has been continuously expanding. Tradi-
tionally, probiotics have been incorporated in to yoghurt; how-
ever, a number of carriers for probiotics have been examined
recently including mayonnaise (Khalil & Mansour, 1998), edible
spreads (Charteris, Kelly, Morelli, & Collins, 2002) and meat
(Arihara et al., 1998) in addition to other products of dairy origin,
i.e., cheese (Ong, Henriksson,& Shah, 2006) or cheese-based dips
(Tharmaraj& Shah, 2004). Probiotic organisms are also available
commercially in milk, sour milk, fruit juices, ice cream, single shots
and oat-based products. Lunebest, Olifus, Bogarde, Progurt are only
some examples of commercial fermented dairy products with
probiotics available on the international market with a steady
increase in the market shares. The consumption of functional dairy
products across West Europe, United States and Japan rose by 12%
since 2005 (Zenith International, 2007). Probiotic products are very
popular in Japan as reflected in more than 53 different types of
probiotic-containing products on the market.
Commercial cultures used in these applications include mainly
strains ofLactobacillus spp. and Bifidobacterium spp. and some of
them are listed in Table 2. The probiotic strains are mainly used as
adjunct cultures due to their poor growth in milk which extends
the fermentation time (Shah, 2004). Lactobacilli are ubiquitous in
nature, found in carbohydrate rich environments. They are Gram-
positive, non-spore-forming microorganisms, catalase negative
with noted exceptions, appearing as rods or coccobacilli. They are
fermentative, microaerophylic and chemo-organotrophic. Consid-
ering the DNA base composition of the genome, they usually have
a GC content less than 54mol%. The genusLactobacillusbelongs tothe phylum Firmicutes, class Bacilli, order Lactobacillales, family
Lactobacillaceaeand its closest relatives are the genera Paralacto-
bacillusandPediococcus(Garrity, Bell,&Lilburn, 2004). This is the
most numerous genus, comprising 106 described species. Lacto-
bacillus acidophilus,L. salivarius,L. casei,L. plantarum,L. fermentum,
L. reuteri and L. brevis have been the most common Lactobacillus
species isolated from the human intestine (Mitsuoka, 1992). The
functional properties and safety of particular strains of L. casei,
L. rhamnosus,L. acidophilus, andL. johnsoniihave been extensively
studied and well documented.
Bifidobacteria were first isolated and visualized byTissier (1900)
from faeces of breast-fed neonates. These rod-shaped, non-gas
producing and anaerobic organisms were named B. bifidus due to
their bifurcated morphology. They are generally characterized asGram-positive, non-spore forming, non-motile and catalase-negative
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anaerobes with a special metabolic pathway, which allows them to
produce acetic acid in addition to lactic acid in the molar ratio of 3:2.
Due to their fastidious nature, these bacteria are often difficult to
isolate and grow in the laboratory. The taxonomy of bifidobacteria
has changed continuously since they were first isolated. They had
been assigned initially to the genera Bacillus, Bacteroides, Nocardia,
Lactobacillus and Corynebacterium, before being recognized as
separate genera in 1974. Due to their high (450 mol%) G+C content,
bifidobacteria are phylogenetically assigned in the actinomycete
division of Gram-positive bacteria. This family consists of five
genera: Bifidobacterium, Propionibacterium, Microbacterium, Coryne-
bacterium, andBrevibacterium. Presently, there are 32 species in the
genusBifidobacterium, 12 of which are isolated from human sources
(i.e., dental caries, faeces and vagina), 15 from animal intestinal tracts
or rumen, 3 from honeybees and remaining 2 found in fermented
milk and sewage. Bifidobacterium species found in humans are:B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum,
B. dentium,B. infantis, B. longum, andB. pseudocatenulatum. B. breve,B. infantis, andB. longumare found in human infants.B. adolescentis
andB. longumare found in human adults (Garrity et al., 2004).
6. Selection of probiotics
The importance of certain technological and physiological
characteristics of probiotic strains was recognized long time ago.
Gordon, Macrae, and Wheater (1957) noted that for achieving
successful outcome of the lactobacilli therapy was necessary for
the preparation to fulfil following requirements: the culture must
be a normal inhabitant of the intestine, non-pathogenic, and must
be capable of efficient gut colonization and delivered in
substantially high concentrations (107
109
cfumL1
of a product).Although numerous criteria have been recognized and suggested
(Mattila-Sandholm, Myllarinen, Crittenden, Fonden, & Saarela,
2002; Ouwehand, Kirjavainen, Shortt, & Salminen, 1999; Reid,
1999), a general agreement exists with regard to key selection
criteria listed inTable 3(FAO/WHO, 2002).
The first step in the selection of a probiotic is the determina-
tion of its taxonomic classification, which may give an indication
of the origin, habitat and physiology of the strain. All these
characteristics have important consequences on the selection ofthe novel strains (Morelli, 2007). The classification and related-
ness of probiotics (and other microorganisms) is based on the
comparison of highly conserved molecules, namely genes encod-
ing ribosomal RNA (rRNA). Major advances in molecular biology
methods have enabled sequencing of 16S/23S rRNA sequences and
consequently generation of large sequence databases, which may
facilitate a rapid and accurate classification of a desired probiotic
strain. Closely related strains nowadays are successfully distin-
guished using DNA-based methods such as plasmid profiling,
restriction enzyme analysis (REA), ribotyping, randomly amplified
polymorphic DNA (RAPD) and pulse-field electrophoresis (PFGE)
(Holzapfel, Haberer, Geisen, Bjorkroth, & Schillinger, 2001;
Vuaghan, Heilig, Ben-Amor,& de Vos, 2005).
Many authors (i.e., Ouwehand et al., 1999) advocated theimportance of origin in specific commercial applications. More
recently, an FAO/WHO (2001) expert panel suggested that the
specificity of probiotic action is more important than the source of
microorganism. This conclusion was brought forward due to
uncertainty of the origin of the human intestinal microflora since
the infants are borne with virtually sterile intestine. However, the
panel also underlined a need for improvement of in vitro tests to
predict the performance of probiotics in humans. Dairy and
probiotic cultures have been associated with a long tradition of
the safe use in commercial applications. Reports on the occur-
rence of harmful effects associated with consumption of probio-
tics are quite rare, although certainLactobacillusstrains have been
isolated from bloodstream and local infections (Ishibashi &
Yamazaki, 2001;Salminen et al., 2006). Another important safety
aspect is the antibiotic resistance of probiotics, since antibiotic
resistant genes, especially those encoded by plasmids, could be
transferred between microorganisms. The information in this
regard is rather contradictory; early reports indicated that certain
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Table 3
Key and desirable criteria for the selection of probiotics in commercial applications
(adapted fromShah, 2006; Morelli, 2007)
General Property
Safety criteria Origin
Pathogenicity and infectivity
Virulence factorstoxicity, metabolic activity and
intrinsic properties, i.e., antibiotic resistance
Technological criteria Genetically stable strains
Desired viability during processing and storage
Good sensory properties
Phage resistance
Large-scale production
Functional criteria Tolerance to gastric acid and juices
Bile tolerance
Adhesion to mucosal surface
Validated and documented health effects
Desirable
physiological
criteria
Immunomodulation
Antagonistic activity towards gastrointestinal
pathogens, i.e., Helicobacter pylori, Candida albicans
Cholesterol metabolism
Lactose metabolismAntimutagenic and anticarcinogenic properties
Table 2
Some of probiotic strains used in commercial applications (adapted from Holm,
2003; Shah, 2004)
Strain Source
L. acidophilusLA1/LA5 Chr. Hansen
L. delbrueckii ssp. bulgaricus Lb12
L. paracaseiCRL431
B. animalis ssp. lactis Bb12L. acidophilusNCFMs Danisco
L. acidophilusLa
L. paracaseiLpc
B. lactis HOWARUTM/Bl
L. acidophilusLAFTIs L10 DSM Food Specialties
B. lactis LAFTIs B94
L. paracaseiLAFTIs L26
L. johnsoniiLa1 Nestle
L. acidophilusSBT-20621 Snow Brand Milk Products Co. Ltd.
B. longumSBT-29281
L. rhamnosus R0011 Institute Rosell
L. acidophilus R0052
L. casei Shirota Yakult
B. breve strain Yakult
B. lactis HN019 (DR10) Foneterra
L. rhamnosus HN001 (DR20)
L. plantarum299V Probi ABL. rhamnosus 271
L. casei Immunitas Danone
B. animalisDN173010 (Bioactiva)
L. rhamnosus LB21 Essum AB
Lactococcus lactis L1A
L. reuteri SD2112 Biogaia
L. rhamnosus GG1 Valio Dairy
L. salivariusUCC118 University College Cork
B. longumBB536 Morinaga Milk Industry Co. Ltd.
L. acidophilusLB Lacteol Laboratory
L. paracaseiF19 Medipharm
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strains ofBifidobacterium(Matteuzzi, Crociani,&Brigidi,1983) andLactobacillus (Gupta, Mital, & Gupta, 1995) showed a strain
dependent resistance to tested antibiotics. On the other hand, a
recent study (Moubareck, Gavini, Vaugien, Butel, & Doucet-
Populaire, 2005) tested 50 strains belonging to eight Bifidobacter-
iumspp. and concluded that these strains were risk-free. The risk
of gene transfer depends on the nature of the genetic material
(plasmid, transposons), the nature and concentrations of thedonor and recipient strains and their interactions and the
environmental conditions, i.e., the presence of an antibiotic may
facilitate the growth of antibiotic resistant mutants (Marteau,
2001). Therefore, the probiotic strains need to be tested for their
natural antibiotic resistance to prevent the undesirable transfer of
resistance to other endogenous bacteria.
7. Technological challenges in the development of probiotic
dairy products
In order to exert their functional properties, probiotics need to be
delivered to the desired sites in an active and viable form. The
viability and activity of probiotics in the products have beenfrequently cited as a prerequisite for achieving numerous beneficial
health benefits. However, even non-viable cultures may exert certain
functional properties such as immunomodulation (Ouwehand et al.,
1999). Moreover, no general agreement has been reached on the
recommended levels and the suggested levels ranged from 106 cfu
mL1 (Kurman&Rasic, 1991) to over 107 and 108cfumL1 (Lourens-
Hattingh & Viljeon, 2001). These suggestions have been made to
compensate for the possible decline in the concentration of the
probiotic organisms during processing and storage of a probiotic
product as well as passage through the upper and lower parts of the
gastrointestinal tract. In Japan, a standard has been developed by
The Fermented Milks and Lactic Acid Bacteria Beverages Association
and this has advocated an approach in which at least 107 viable
bifidobacteria per gram of a product is required to constitute aprobiotic food for humans (Ishibashi&Shimamura, 1993). However,
numerous studies have demonstrated that probiotic strains grow
poorly in milk, resulting in low final concentrations in yoghurt and
even the loss of the viability during prolonged cold storage.
A number of commercial products of yoghurts have been
analyzed in Australia and Europe for the presence ofL. acidophilus
andBifidobacteriumover the years (Huys et al., 2006;Masco, Huys,
De Brandt, Temmerman, & Swings, 2005; Micanel, Haynes, &
Playne, 1997; Temmerman, Scheirlinck, Huys, & Swings, 2003;
Tharmaraj & Shah, 2003; Vinderola, Bailo, & Reinheimer, 2000).
Most of the products contained variable if not very low
concentrations of probiotics, especially bifidobacteria. Viability
and activity of the bacteria are important considerations, because
these bacteria must survive in food during shelf life, during transit
through the acidic conditions of the stomach, and resist degrada-
tion by hydrolytic enzymes and bile salts in the small intestine.
Furthermore, adequate enumeration techniques are required in
order to properly assess the viability and survival of probiotic
bacteria, especially in the light of the labeling requirements.
Several media for selective enumeration of L. acidophilus,
Bifidobacteriumspp. and L. casei were proposed in the 1990s, but
most of these methods were based on pure cultures of theseorganisms. Consequently, these methods were considered rather
inaccurate (Talwalkar & Kailasapathy, 2004). More recently,
Tharmaraj and Shah (2003) recommended media for selective
enumeration of S. thermophilus, L. delbrueckii ssp. bulgaricus,
L. acidophilus, Bifidobacterium spp., L. casei, L. rhamnosus and
propionibacteria in a mixture of probiotic bacteria. Their findings
are summarized inTable 4.
The viability and activity of probiotic cultures may be affected
during all steps involved in a delivery process through the
exposure to different stress factors (Table 5). In general, probiotics
are extremely susceptible to environmental conditions such as
water activity, redox potential (presence of oxygen), temperature,
and acidity (Siuta-Cruce & Goulet, 2001). In the initial phase,
probiotic cultures are selected based not only on the functionalcriteria but also on additional technological aspects including
enhanced yields during cultivation at the industrial scale and
improved survival during culture concentration and freeze drying.
The selection of adequate strains and improvement of various
technologies used in the preparation of probiotics are certainly
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Table 4
Recommended media for selective enumeration of S. thermophilus, L. delbrueckii ssp. bulgaricus, L. acidophilus, Bifidobacterium spp., L. casei, L. rhamnosus, and
propionibacteria in a mixture of bacteria (adapted from Tharmaraj& Shah, 2003)
Agar Bacteria Incubation conditions Colony morphology
S. thermophilus agar S. thermophilus Aerobic, 371C, 24 h 0.10.5 mm, round yellowish
MRSa agar (pH 4.58) L. delbrueckiissp. bulgaricus Anaerobic, 45 1C, 72 h 1.0 mm, white, cottony, rough, irregular
MRS-sorbitol agar L. acidophilus Anaerobic, 371C, 72 h Rough, dull, small (0.10.5), brownish
MRS-NNLPb agar Bifidobacteria Anaerobic, 371C, 72 h 1 mm, white, smooth, shiny
MRS-vancomycine agarc L. casei Anaerobic, 371C, 72 h 1.0 mm, white shiny, smooth
MRS-vancomycine agar L. rhamnosus Anaerobic, 43 1C, 72 h 1.02.0 mm, white shiny, smooth
Sodium lactate agar Propionibacteria4 Anaerobic, 301C, 7 9 d ays 1.0 2.5 mm, d ull b rown, l ighter m argin
a de man, Rogosa and Sharpe agar.b Nalidixic acid, neomycin sulfate, lithium chloride and paromomycin sulfate.c In case L. rhamnosus absent, if not then subtraction method required.
Table 5
Different stress vectors affecting viability of probiotic during processing
Processing step Stress vector
Production of probiotic
preparations
Presence of organic acids during cultivation
Concentrationhigh osmotic pressure, low water
activity, higher concentration of particular ions
Temperature
freezing, vacuum and spray dryingDrying
Prolonged storageoxygen exposure,
temperature fluctuation
Production of a probiotic
containing product
Nutrient depletion
Strain antagonism
Increased acidity
Positive redox potential (presence of oxygen)
Presence of antimicrobial compounds,
i.e., hydrogen peroxide and bacteriocins
Storage temperature
Gastrointestinal transit Gastric acid and juices
Bile salts
Microbial antagonism
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crucial elements. Probiotic cultures like other starter cultures are
delivered in frozen or dry form (freeze or spray dried) as ready-to
use cultures for the direct vat inoculation. The cultivation step
during production of probiotic cultures plays an important role in
the culture stability and activity during storage and food
applications (Carvalho et al., 2003; Reilly & Gilliland, 1999).
Unfortunately, exposure to high acidity, substrate limitations and
subsequently to low water activities and temperature (i.e., lowduring freezing or high during spray drying) leads to detrimental
changes that may affect the culture survival and activity not only
during cultivation but further application. In general, culture
survival throughout drying and storage depends on many factors
including initial cell concentration, growth conditions, growth and
drying medium, and rehydration conditions (Knorr, 1998). While
frozen and freeze dried probiotic cultures have been extensively
used commercially, industry has been seeking alternative ap-
proaches such as spray drying mainly due to several disadvan-
tages regarding handling of the frozen and freeze dried bacterial
materials including high transport and storage cost and detri-
mental effects of freeze thaw cycle on the viability (Gardiner et al.,
2000). Irrespective of the preservation method applied, probiotic
cultures are exposed to unfavorable environmental conditions dueto increased solute concentration, intracellular ice formation in
case of freezing and freeze drying, exposure to elevated tempera-
tures during spray drying and, in general, dehydration. To improve
the survival and preserve the activity of probiotics, protective
compounds (compatible solutes and cryoprotectants) are fre-
quently added either to the growth medium or before freezing or
dehydration step. Fairy recently, the stress response of LAB and
probiotics has become a focus of very intensive research efforts.
The reader is advised to consult Girgis, Smith, Luchansky, and
Klaenhammer (2002) for in-depth review of this subject. Briefly,
depending on stress factors encountered, LAB cultures are capable
of mobilizing a very sophisticated stress response system.
Induction of heat stress or cold stress genes provides enhanced
culture survival under abrupt temperature changes (Girgis et al.,
2002). Many microorganisms also accumulate compatible solutes
as metabolically inert stress compounds, which would in turn
protect the metabolic apparatus (Pichereau, Pocard, Hamelin,
Blanco, & Bernard, 1998). Bacterial compatible solutes are
accumulated either by de novo biosynthesis (endogenous osmo-
lytes, such as glutamate, proline, ectoine, trehalose and sucrose)
or by uptake from the environment (exogenous osmolytes such as
glycine betaine) (Csonka& Hanson, 1995). The compatible solutes
produced internally are highly soluble, pH neutral, and are usually
an end-product metabolite (Beales, 2004). More importantly,
compatible solutes do not alter metabolic processes and even
protect metabolic enzymes from denaturation brought about by
increased ionic strength as in case of freezing and drying (Baati
Fabre-Gea, Auriol,&Blanc, 2000). Like many other organisms, lactic
acid bacteria confronted with a decreased water activity (aw) over along period respond by accumulation of compatible solutes such as
betaine and carnitine (Kets, Galinski, de Wit, De Bont,&Heipieper,
1996). Therefore, the activation of required genes and intracellular
accumulation of compatible solutes may improve culture perfor-
mance under variety of conditions including freezing, heating,
drying and exposure to gastrointestinal environment.
Furthermore, the viability of probiotics in a delivery system
(i.e. food matrix) depends on a strain selected, interactions
between microbial species present, production of hydrogen
peroxide due to bacterial metabolism, and final acidity of the
product. Additionally, viability would also be affected by the
availability of nutrients, growth promoters and inhibitors, con-
centration of sugars, dissolved oxygen and oxygen permeation
through package (especially for Bifidobacteriumspp.), inoculationlevel, and fermentation time (Shah, 2000).L. acidophilushas a high
cytoplasmic buffering capacity (pH 3.727.74), which allows it to
resist changes in cytoplasmic pH and gain stability under acidic
conditions (Rius, Sole, Francis, & Loren, 1994). Thus, it is more
tolerant to acidic conditions than Bifidobacterium spp., whose
growth is significantly retarded below pH 5.0. The acid tolerance
of Bifidobacterium is very low and depends on the cultivation
conditions, strain and species (Charteris, Kelly, Morelli,& Collins,
1998; Collado, Moreno, Cobo, Hernandez, & Hernandez, 2005;Matsumoto, Ohishi, & Benno, 2004). Bifidobacterium animalis
subsp.lactis has the highest acid tolerance and is thus preferably
used in bifidus products. The use ofL. delbrueckiissp.bulgaricus
in yoghurt may affect survival ofL. acidophilusandBifidobacterium
due to acid and hydrogen peroxide produced during fermentation.
However, due to its proteolytic nature, L. delbrueckiissp.bulgaricus
may liberate essential amino acids, valine, glycine, and histidine
required to support the growth of bifidobacteria (Shihata&Shah,
2002). Additionally,S. thermophilus may stimulate the growth of
probiotic organisms due to consumption of oxygen.
The presence of oxygen (positive redox potential) in probiotic-
containing products can have a detrimental effect on the viability
of probiotics. Strains ofL. acidophilus and Bifidobacteriumspp. are
microaerophilic and anaerobic, respectively. They lack an elec-tron-transport chain, which results in the incomplete reduction of
oxygen to hydrogen peroxide. Furthermore, they are devoid of
catalase, thus incapable of converting hydrogen peroxide into
water. Bifidobacterium spp. is generally more susceptible to
deleterious presence of oxygen than L. acidophilus. To exclude
oxygen during the production of bifidus milk products, special
equipment is required to provide an anaerobic environment.
Oxygen can also enter the product through packaging materials
during storage. Oxygen may affect probiotic cultures in two ways.
Firstly, its toxicity to cells may be expressed directly due to culture
sensitivity to oxygen. This likely results in the intracellular
accumulation of hydrogen peroxide and consequently death of
the cell (Dave& Shah, 1997a, 1997b, 1997c). Secondly,L. delbrueckii
ssp. bulgaricus is known to produce hydrogen peroxide in the
presence of oxygen, which may affect probiotics indirectly (Dave &
Shah, 1997a;Villegas&Gilliland, 1998). A synergistic inhibition of
probiotic cultures due to acid and hydrogen peroxide was also
observed (Lankaputhra & Shah, 1996). Because of this reason,
removal ofL. delbrueckiissp.bulgaricusfrom some starter cultures
(i.e., ABT starter cultures) has had some success in improving
survival of probiotic organisms.
Due to their poor growth in milk, the inoculum size for
probiotics is usually greater (510%) than it is required, for
example for yoghurt starters, L. delbrueckii ssp. bulgaricus and
S. thermophilus, usually added at 1% (v/v). Starter antagonism also
can negatively affect the growth of probiotic strains due to the
production of inhibitory compounds (Vinderola, Mocchiutti, &
Reinheimer, 2002). On the other hand, starter cultures with a
proteolytic or oxygen scavenging ability may be beneficial for thegrowth of bifidobacteria (Ishibashi & Shimamura, 1993). Final
product pH appears to be the most crucial factor for the survival of
probiotic organisms. Below pH 4.4, probiotics do not thrive well
and a substantial decrease in number of probiotic bacteria is
usually observed. This process, frequently referred to as post-
acidification, usually occurs during production of yoghurt due to
acidophilic nature of Lactobacillus delbrueckii ssp. bulgaricus and
extended growth at low pH and low temperature (Donkor,
Henriksson, Vasiljevic, & Shah, 2006a). Most frequent approach
is the modification of an inoculation level (Dave&Shah, 1997a) or
the omission of a portion of the starter strains (Donkor et al.,
2006a). Another approach is the addition of probiotic organisms
after the fermentation of milk. This allows use of strains of
probiotic bacteria that cannot grow in the presence of otherorganisms. However, survival of probiotic organisms even in this
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case may not be warranted. Alternatively, initial fermentation may
be carried out with probiotic cultures followed by completion of
fermentation with starter cultures (Shah & Lankaputhra, 1997).
This two-step approach includes initial fermentation with
probiotic cultures for 2 h, followed by fermentation by yoghurt
starter bacteria for 4 h. This allows the probiotic organisms to be
in their final stage of lag phase or early stage of log phase resulting
in higher counts of probiotic organisms at the end of 6 h offermentation. The counts of probiotic bacteria have been found to
increase substantially in the product made using a two-step
fermentation process.
The numbers of probiotic bacteria in frozen fermented dairy
desserts or frozen yoghurt are reduced significantly by acid,
freeze-injury, sugar concentration of the product and oxygen
toxicity. For this reason, technologies such as enteric coating and
microencapsulation have been suggested and investigated as a
promising method for the efficient protection and delivery of the
physiologically active of probiotic strains. Microencapsulation is a
process where the cells are retained within the encapsulating
membrane in order to reduce the cell injury or cell loss. The use of
gelatine or vegetable gum as encapsulating materials has been
reported to provide protection to acid sensitive probiotic organ-isms. Another excipient, alginate, showed great potential due to
process requirements and overall costs. Furthermore, alginate is
non-toxic so that it may be safely used in foods. Alginate gels can
be solubilized by sequestering calcium ions thus releasing
entrapped cells. Encapsulated probiotic organisms when incorpo-
rated in fermented frozen dairy desserts, yoghurt or freeze dried
yoghurt showed improved viability in comparison with non-
encapsulated control organisms (Capela, Hay, & Shah, 2006;
Ravula& Shah, 2000).
Alternatively, the viability of probiotics in the product and
subsequently in the gastrointestinal tract can be improved by
addition of an appropriate prebiotic. Prebiotics are defined as
non-digestible food ingredients that beneficially affect the host
by selectively stimulating the growth and/or activity of one or a
limited number of bacteria in the colon that have the potential to
improve health (Gibson& Roberfroid, 1995). While their role by
definition is the selective stimulation of a limited number of
colonic and preferable beneficial bacteria, a range of prebiotics has
been used as a tool for improvement of probiotic activity and
survival in fermented foods during growth and storage (Bruno,
Lankaputhra, & Shah, 2002; Liong& Shah, 2005a). The approach
improved the survival of probiotic and had an effect on the
metabolic activity of the assessed cultures; however, the bacterial
response to these prebiotics was highly strain specific.
8. Health potential of probiotic foods
While various health claims have been associated with theconsumption of probiotics, they may in some instances be
influenced by composition of a delivery matrix. In dairy applica-
tions, probiotics are delivered with different fermented dairy
products, most notable yoghurt. Considering the nutritional
profile of these probiotic products, they resemble a dairy base
from which they are mademainly composed of skim milk non-
fat solids in a different ratio to milk fat. The natural function of
milk is to provide complete nutritional requirements to the
neonatal mammal. The composition of milk depends on many
factors such as genetic and individual mammalian differences,
feed, stage of lactation, age, and environmental factors such as the
season of the year. The nutritional value of the final product is also
affected by processing factors, including temperature, duration of
heat exposure, exposure to light, and storage conditions (Fox,2003). Furthermore, some of these milk constituents may be
modified by microbial action during fermentation which may
affect the nutritional and physiologic value of the final product.
In addition to exceptional nutritional attributes, milk and milk-
derived products such as fermented milk contain components that
possess a range of different bioactivities, some of them summarized
inTable 6. In their native form, milk proteins exert an appreciable
range of different physiological activities. Specific immunoglobu-
lins provide the first line of defense to suckling neonates throughpassively acquired immunity. Other non-specific antimicrobial milk
factors including iron-binding protein, lactoferrin, and several
enzymes such as lactoperoxidase and lysozyme prevent the
microbial proliferation (Florisa, Recio, Berkhout, & Visser, 2003).
The functionality of dairy proteins may also be enhanced via
liberation of bioactive peptides through proteolysis (Gobbetti,
Ferranti, Smacchi, Goffredi,& Addeo, 2000;Gobbetti, Minervini,&
Rizzello, 2004). Dairy starter cultures and some probiotics have
appreciable proteolytic activity, which is required for their rapid
growth in milk. During fermentation, milk proteins, namely
caseins, undergo a slight proteolytic degradation resulting in a
number of potentially bioactive peptides (Table 7). Casein- and
potentially whey protein-derived bioactive peptides released
through the proteolytic action of dairy starters may function asregulatory compounds or exorphins. These peptides with a
morphine-like activity may act as opioid agonists such as a- and
b-casomorphins and lactorphins or opioid antagonists presented by
casoxins. They have the ability to bind to opioid receptors on
intestinal epithelial cells exhibiting a range of physiological
functions such as modulation of social behavior, antidiarrheal
action and stimulation of endocrine responses (Clare&Swaisgood,
2000). Casomorphins appear to be resistant to digestion by
gastrointestinal enzymes expressing an appreciable activity in the
gut (Trompette et al., 2003), thus slowing down the rate of the
gastric emptying and enhancing the uptake rate of amino acids and
electrolytes by epithelial cells. Another group of bioactive peptides,
termed angiotensin I-converting enzyme (ACE, EC 3.4.15.1) inhibi-
tors, have been extensively studied due to their hypotensive role.
Most recently, a number of probiotic strains have been identified to
be capable of producing different peptides with a differing degree
of ACE-inhibitory activity in yoghurt and soy based yoghurt
(Donkor, Henriksson, Vasiljevic, & Shah, 2005; Donkor et al.,
2006a; Donkor, Henriksson, Vasiljevic, & Shah, 2006b). While the
observed activity was strictly strain dependent, it fluctuated with
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Table 6
Biogenic activity of native milk macro-components (fromVasiljevic&Shah, 2007)
Component Form Bioactivity
Protein Caseins Mineral carriers, antiosteoporotic, precursor
of bioactive peptides
a-Lactalbumin Modulation of lactose metabolism, Ca carrier,immunomodulation
b-Lactoglobulin Retinol carrier, fatty acid binder, presumed
antioxidative activity
Immunoglobulins Immune activity
Lactoferrin Antimicrobial, antioxidative,
immunomodulation, anticarcinogenic
Lactoperoxidase Antimicrobial
Ly sozym e Ant im icrobial
Fat Conjugated
linoleic acid
Anticarcinogenic, modulation of lipid and
protein metabolism, anti-inflamatory,
hypotensive, anti-atherosclerotic
Sphingolipids Anti-inflamatory, anticarcinogenic
Butyric acid Anticarcinogenic
Carbohydrates Oligosaccharides Prebiotic, antimicrobial (antiadhesive),
Ca absorption
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the storage time, which raised an important question regarding the
stability of these peptides during the prolonged storage.
9. Health effects of probiotics
Since Metchnikoffs era, a number of health benefits have been
contributed to products containing probiotic organisms. While
some of these benefits have been well documented and estab-
lished, others have shown a promising potential in animal models,
with human studies required to substantiate these claims. More
importantly, health benefits imparted by probiotic bacteria are
very strain specific; therefore, there is no universal strain that
would provide all proposed benefits, not even strains of the same
species. Moreover, not all the strains of the same species
are effective against defined health conditions. The strains
L. rhamnosus GG (Valio), Saccharomyces cerevisiae Boulardii
(Biocodex), L. casei Shirota (Yakult), and B. animalis Bb-12 (Chr.
Hansen) are certainly the most investigated probiotic cultures
with the established human health efficacy data against manage-
ment of lactose malabsorption, rotaviral diarrhoea, antibiotic-associated diarrhoea, and Clostridium difficile diarrhoea. Some of
these strain specific health effects are listed inTable 8.
9.1. Alleviation of lactose intolerance
The decline of the intestinal b-galactosidase (b-gal or com-
monly know as lactase) activity is a biological characteristic of the
maturing intestine in the majority of the worlds population. With
the exception of the inhabitants of northern and central Europe
and Caucasians in North America and Australia, over 70% of adults
worldwide are lactose malabsorbers (de Vrese et al., 2001).
Lactose upon ingestion is hydrolyzed by lactase in the brush
border membrane of the mucosa of the small intestine into
constitutive monosaccharides, glucose and galactose, which arereadily absorbed in the blood stream. However, the activity of
intestinal lactase in lactose intolerant individuals is usually less
than 10% of childhood levels (Buller & Grand, 1990). This decline,
termed hypolactasia, causes insufficient lactose digestion in the
small intestine, characterized by an increase in blood glucose
concentration or hydrogen concentration in breath upon ingestion
of 50 g lactose, conditions designated as lactose maldigestion
(Scrimshaw & Murray, 1988). Hypolactasia and lactose malab-
sorption accompanied with clinical symptoms, such as bloating,
flatulence, nausea, abdominal pain and diarrhoea, are termed
lactose intolerance. Symptoms are caused by undigested lactose in
the large intestine, where lactose is fermented by intestinal
microflora and osmotically increases the water flow into the
lumen. The severity of the symptoms depends primarily onthe size of the lactose load ingested. The development of the
intolerance symptoms also depends on the rate of lactose transit
to the large intestine, influenced by the osmotic and caloric load,
and the ability of the colonic microflora to ferment lactose
(Martini & Savaiano, 1988).
Numerous studies have shown that individuals with hypolac-
tasia could tolerate fermented dairy products better than an
equivalent quantity in milk (Hertzler & Clancy, 2003; Montalto
et al., 2005; Vesa et al., 1996). Various explanations have beensuggested in order to clarify this phenomenon. At least three
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Table 7
Some examples of the identified bioactive peptides in fermented milk and their corresponding physiological activity (adapted from Vasiljevic& Shah, 2007)
Sequence Microbial agent Precursor Bioactivity
Val-Pro-Pro L. helveticusCM4 and S. cerevisiae b- and k-casein Hypotensive
Ile-Pro-Pro
Val-Pro-Pro L. helveticus LBK16H b- and k-casein Hypotensive
Ile-Pro-Pro
Phe-Pro-Glu-Val-Phe-Glu-Lys Comme rcial products+digestion as1-Casein ACE inhibitionLys-Va l-Leu-Pro-Val-Pro -Gl u Comme rcial products+digestion b-Casein Antioxidative
Lys-Thr-Thr-Met-Pro-Leu-Trp Comme rcial products+digestion as1-Casein Possible immunomodulation
Asn-Leu-His-Leu-Pro-Leu-Pro-Leu-Leu L. helveticusNCC 2765 b-Casein ACE inhibition
Tyr-Pro-Phe-Pro-Glu-Pro-Ile-Pro-Asn L. helveticusNCC 2765 b-Casein Opioid
Tyr-Pro L. helveticusCPN4 Caseins ACE inhibition
Leu-Asn-Val-Pro-Gly-glu-Ile-Val-Glu L. delbrueckiissp. bulgaricus SS1 b-Casein ACE inhibition
Asn-Ile-Pro-Pro-Leu-Thr-Glu-Thr-Pro-Val L. lactisssp. cremoris FT4 b-Casein ACE inhibition
Table 8
Some of the established and potential health benefits of probiotic organisms
(adapted fromShah, 2006)
Health effect Mechanism
Scientifically established
Alleviation of lactose intolerance Delivery of intracellularb-galactosidase
into human gastrointestinal tract
Prevention and reduction of
symptoms of rotavirus and
antibiotic associated diarrhoea;
Competitive exclusion
Translocation/barrier effect
Improved immune response
Potential
Treatment and prevention of
allergy (atopic eczema, food
allergy)
Translocation/barrier effect
Immune exclusion, elimination and
regulation
Reduction of risk associated with
mutagenicity and carcinogenicity
Metabolism of mutagens
Alteration of intestinal microecology
Alteration of intestinal metabolic activity
Normalization of intestinal permeability
Enhanced intestinal immunity
Hypocholesterolemic effect Deconjugation of bile salts
Inhibition ofHelicobacter pylori
and intestinal pathogens
Competitive exclusion
Barrier effect
Production of antimicrobial compounds
Prevention of inflammatory bowel
diseases
Competitive exclusion
Improvement of epithelial tight junctions
Modification of intestinal permeability
Modulation of immune response
Production of antimicrobial products
Decomposition of pathogenic antigens
Stimulation of immune system Recognition by toll-like
receptorsinduction of innate and
adaptive immunity: downregulation of pro-inflammatory
cytokines and chemokines
upregulation of phagocytic activity
regulation of Th1/Th2 balance
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factors appear to be responsible for a better tolerance of lactose in
fermented milk including (a) starter culture, (b) intracellularb-galactosidase expressed in these cultures, and most importantly
(c) oro-caecal transit time. The traditional cultures used in dairy
fermentations utilize lactose as an energy source during growth,
thus at least, partially reducing its content in fermented products.
Furthermore, the bacterial lactase may resist luminal effectors
avoiding denaturation and can be detected in the duodenum andterminal ileum after consumption of products containing live
bacteria. The presence of this enzyme may lead to lactose
hydrolysis and improved lactose tolerance. On the other hand,
other studies not supporting this theory found no difference in
digestion and tolerance to lactose in several fermented dairy
products with substantially different lactase activities (Vesa et al.,
1996). It was suggested that increased viscosity of fermented milk,
in this case yoghurt, slowed gastric emptying and consequently
prolonged transit time through the gastrointestinal tract improv-
ing absorption and lactose tolerance.
9.2. Prevention and reduction of diarrhoea symptoms
One of the main applications of probiotics has been thetreatment and prevention of antibiotic-associated diarrhoea, which
is often caused by occurrence of C. difficile after an antibiotic
treatment. C. difficile is an indigenous gastrointestinal organism
usually encountered in low numbers in the healthy intestine;
however, the antibiotic treatment may lead to a disruption of
indigenous microflora and subsequently to an increase in the
concentration of this organism and toxin production, which causes
symptoms of diarrhoea. The administration of an exogenous
probiotic preparation is required to restore the balance of the
intestinal microflora. The application of probiotics in the clinical
setting significantly reduced antibiotic-associated diarrhoea by
52%, reduced the risk of travellers diarrhoea by 8% and that of acute
diarrhoea of diverse causes by 34%. Moreover, the associated risk of
acute diarrhoea among children was reduced by 57% and 26%
among adults. Interestingly, all strains evaluated including S.
boulardii, L. rhamnosus GG, L. acidophilus, L. bulgaricus, alone or in
combinations showed similar effect (Sazawal et al., 2006). The
strongest evidence of a beneficial effect of defined strains of
probiotics has been established for L. rhamnosusGG andB. animalis
Bb-12. Administration of oral rehydration solution containing
Lactobacillus GG to children with acute diarrhoea resulted in a
reduction of the duration of diarrhoea, lower chance of a protracted
course, and faster discharge from the hospital (Guandalini et al.,
2000). Similar to antibiotic and rotavirus associated diarrhoea,
probiotics may prevent and alleviate symptoms of travellers
diarrhoea, which is caused by bacteria, particularly enterotoxigenic
Escherichia coli. Several studies have assessed the effects of
probiotic preparations as prophylaxis for travellers diarrhoea,
however, the results have been conflicting due to methodologicaldeficiencies, which certainly limited the validity of their conclu-
sions (Marteau, Seksik,& Jian, 2002).
The mechanisms by which fermented dairy foods containing
probiotics or culture containing milks reduce the duration of
diarrhoea are still largely unknown. Several possible mechanisms
are listed inTable 8. A competitive exclusion is the mechanism by
which probiotics inhibit the adhesion of rotavirus by modifying the
glycosylation state of the receptor in epithelial cells via excreted
soluble factors (Freitas et al., 2003). The presence of probiotics also
prevents the disruption of the cytoskeletal proteins in the epithelial
cells caused by the pathogen, which leads to the improved mucosal
barrier function and failure prevention in the secretion of
electrolytes (Resta-Lenert& Barrett, 2003). Additionally, probiotic
strains may modulate the innate immune response both to anti-inflammatory and pro-inflammatory directions (Braat et al., 2004).
9.3. Treatment and prevention of allergy
The prevention and management of allergies is another area in
which probiotics may potentially exert their beneficial role. The
incidence of allergy is on the rise worldwide with a clear
difference between developed and developing countries. The
hygiene hypothesis postulates that limited childhood exposure to
bacterial and viral pathogens would affect the balance betweenT-helper cells by favoring the Th2 phenotype of the immune
system. An insufficient stimulation of Th1 cells cannot offset the
expansion of Th2 cells and results in a predisposition to allergy
(Yazdanbakhsh, Kremsner, & van Ree, 2002). A delayed coloniza-
tion ofBifidobacterium and Lactobacillus spp. in the gastrointest-
inal tract of children may be one of the reasons for allergic
reactions (Kalliomaki & Isolauri, 2003). Also, the difference in
gastrointestinal microbiota may play a role in susceptibility to
allergy. Infants with atopic dermatitis had a more adult type
Bifidobacterium microbiota. Healthy infants, on the other hand,
were colonized mainly byB. bifidum, typical for breast-fed infants
(Ouwehand et al., 2001). A recent study also indicated that early
consumption of probiotic preparations containingLactobacillusGG
may reduce prevalence of atopic eczema later in life (Gueimonde,Kalliomaki, Isolauri, & Salminen, 2006). Similarly, another study
suggested that treatment with Lactobacillus GG may alleviate
atopic eczema/dermatitis syndrome symptoms in IgE-sensitized
infants but not in non-IgE-sensitized infants (Viljanen, Savilahti,
et al., 2005), while a 4-week treatment with Lactobacillus GG
alleviated intestinal inflammation in infants with atopic eczema/
dermatitis syndrome and milk allergy (Viljanen, Kuitunen, et al.,
2005). The mechanisms of the protective effects of probiotics on
allergic reactions are not entirely known; although the reinforce-
ment of the different lines of gut defence including immune
exclusion, immune elimination and immune regulation has been
suggested (Isolauri, Ouwehand,& Laitinen, 2005).
9.4. Reduction of the risk associated with mutagenicity and
carcinogenicity
Antigenotoxicity, antimutagenicity and anticarcinogenicity are
important potential functional properties of probiotics, which
received much attention recently. Mutagens are frequently
formed during stress or due to viral or bacterial infections and
phagocytosis but also commonly obtained via foods. Endogenous
DNA damage is one of the contributors to ageing and age-related
degenerative diseases. The defence mechanism via leukocytes
liberates a range of compounds including NO, O2 and H2O2 thus
defending an individual from bacterial and viral infections, but
these may contribute to DNA damage and mutations. DNA
irreversible damage is a critical factor of carcinogenesis and
ageing. Antimutagencity could be described as a suppression ofthe mutation process, which manifests itself as a decrease in the
level of spontaneous and induced mutations. Some epidemiolo-
gical researches have emphasized that probiotic intake may be
related to a reduced colon cancer incidence (Hirayama & Rafter,
2000) and experimental studies showed the ability of lactobacilli
and bifidobacteria to decrease the genotoxic activity of certain
chemical compounds (Tavan, Cayuela, Antoine,& Cassand, 2002)
and increase in antimutagenic activity during the growth in
selected media (Lo, Yu, Chou,& Huang, 2004).
Antimutagenic effect of fermented milks has also been
detected against a range of mutagens and promutagens including
4-nitroquinoline-N0-oxide, 2-nitrofluorene, and benzopyrene in
various test systems based on microbial and mammalian cells.
However, antimutagenic effect might depend on an interactionbetween milk components and lactic acid bacteria. Lankaputhra
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and Shah (1998) studied the antimutagenic activity of organic
acids produced by probiotic bacteria against eight mutagens and
promutagens including 2-nitroflourene (NF), aflatoxin-B (AFTB),
and 2-amino-3-methyl-3H-imidazoquinoline (AMIQ). Among the
organic acids, butyric acid showed a broad-spectrum antimuta-
genic activity against all mutagens or promutagens studied.
Moreover, live bacterial cells showed higher antimutagenicity
than killed cells against the mutagens studied, which suggestedthat live bacterial cells were likely to be involved in metabolism of
mutagens. The results emphasized the importance of consuming
live probiotic bacteria and of maintaining their viability in the
intestine in order to provide efficient inhibition of mutagens.
Several factors have been identified to be responsible for
induction of colorectal cancer including bacteria and metabolic
products such as genotoxic compounds (nitrosamine, heterocyclic
amines, phenolic compounds, and ammonia). Epidemiological
studies have shown that diet plays a role in the etiology of most
large bowel cancers, implying that it is a potentially preventable
disease. Many studies confirm the involvement of the endogenous
microflora in the onset of colon cancer. This effect is mediated by
microbial enzymes such as b-glucuronidase, azoreductase, and
nitroreductase, which convert procarcinogens into carcinogens(Goldin & Gorbach, 1984). Experiments carried out in animal
models showed certain strains of L. acidophilus and Bifidobacter-
iumspp. were capable of decreasing the levels of enzymes such asb-glucuronidase, azoreductase, and nitroreductase responsible for
activation of procarcinogens. This inactivation consequently led to
a substantial decline of the risk associated with tumor develop-
ment. Several studies have shown that preparations containing
LAB inhibit the growth of tumor cells in experimental animals or
indirectly lower carcinogenicity by decreasing bacterial enzymes
that activate carcinogenesis (Rafter, 20 02). Short-chain fatty acids
produced by L. acidophilus and bifidobacteria were also reported
to inhibit the generation of carcinogenic products by reducing
enzyme activities. When incubated in vitro with 4-nitroquinoline-
1-oxide (4NQO), some probiotic strains inhibited the genotoxic
activity of 4NQO. L. casei was most effective, followed by
L. plantarum and L. rhamnosus (Cenci, Rossi, Trotta, & Caldini,
2002). The most convincing clinical data exist for L. casei Shirota,
in which the consumption of this organism was associated with
the decreased urinary mutagen excretion. Furthermore, it was
suggested that the habitual consumption of the fermented milk
with this strain reduced the risk of bladder cancer in the Japanese
population (Ohashi, 2000).
The mechanism of antimutagenicity and anticarcinogenicity of
probiotic bacteria has not been clearly understood. It has been
suggested that microbial binding of mutagens to the cell surface
could be a possible mechanism of antimutagenicity (Orrhage,
Sillerstrom, Gustafsson, Nord, & Rafter, 1994). Other proposed
mechanisms include alteration of intestinal microecology and
intestinal metabolic activity, normalization of intestinal perme-ability and enhanced intestinal immunity (Shah, 2006).
9.5. Hypocholesterolemic effect
It is well established that diet rich in saturated fat or cholesterol
would increase the serum cholesterol level, which is one of the
major risk factors for coronary heart diseases. Mann and Spoerry
(1974) were the first to observe a decrease in serum cholesterol
levels in men fed large quantities (8.33 L man1day1) of milk
fermented with Lactobacillus. As they suggested, this was possibly
due to the production of hydroxymethyl-glutarate by probiotic
bacteria, which was reported to inhibit hydroxymethylglutaryl-CoA
reductases required for the synthesis of cholesterol. Therefore,feeding of fermented milks containing very large numbers of
probiotic bacteria would likely cause a hypercholesterolemic effect
in human subjects. In vitro studies have postulated that the
hypocholesterolemic effect of probiotics might be exerted via
several possible mechanisms including assimilation by growing
cells or binding to the cell surface (Liong& Shah, 2005a, 2005b).
Another mechanism involving the deconjugation of bile by bile salt
hydrolase (BSH, cholylglycine hydrolase; EC 3.5.1.24) and co-
precipitation of cholesterol with the deconjugated bile has beenproposed (Begley, Hill,& Gahan, 2006). The cholesterol is excreted
via the faecal route and prior to its secretion the deconjugation of
bile results in free bile salts. They are less efficiently absorbed and
thus excreted in larger amounts in faeces. This effect is additionally
augmented by poor solubilization of lipids by free bile salts, which
limits their absorption in the gut leading to further decrease of
serum lipid concentration (Begley et al., 2006). The largest study
that assessed the ability of numerous species and strains of lactic
acid bacteria to hydrolyze bile salts showed that BSH activity was
common in Bifidobacterium and Lactobacillus but absent in
Lactococcus lactis, Leuconostoc mesenteroides, and S. thermophilus.
Almost all bifidobacteria species and strains possessed BSH activity,
while it was detected only in selected species of lactobacilli
(Tanaka, Doesburg, Iwasaki,&Mierau, 1999). Also the production ofshort-chain fatty acids has been implicated as another potential
mechanism for the cholesterol lowering effect of probiotics. In a
recent study (Liong & Shah, 2006), serum cholesterol level was
reduced via the alteration of lipid metabolism contributed by
short-chain fatty acids. This was supported by negative correlation
between serum cholesterol levels and caecal propionic acid, and
positive correlation with faecal acetic acid concentrations.
However, the findings of some in vivo studies have been rather
contradictory, i.e., either a lowering effect (Agerholm-Larsen et al.,
2000) or no effect was observed (De Roos, Schouten, & Katan,
1999; Lewis & Burmeister, 2005) even though in the latter the
strains were able to reduce cholesterol in vitro. Despite several
human studies, the reduction in serum cholesterol effect is still
not considered an established effect and double-blinded placebo-
controlled human clinical trials are needed to substantiate this
claim. Similarly, mechanisms involved in reducing cholesterol
level need to be clarified.
9.6. Inhibition of Helicobacter pylori and intestinal pathogens
Probiotic cultures produce a wide range of antibacterial
compounds including organic acids (e.g., lactic acid and acetic
acid), hydrogen peroxide, bacteriocins, various low-molecular-
mass peptides, and antifungal peptides/proteins, fatty acids,
phenyllactic acid, and OH-phenyllactic acid. Lactic and acetic acids
are the main organic acids produced during the growth of
probiotics and their pH lowering effect in the gastrointestinal
tract has a bacteriocidal or bacteriostatic effect. Low-molecular-mass compounds such as lactic acid have been reported to be
inhibitory towards Gram-negative pathogenic bacteria (Alakomi
et al., 2000). Moreover, a heat-stable, low-molecular-weight
antibacterial substance different from lactic acid was present in
the cell-free culture supernatant resulting in the inactivation of a
wide range of Gram-negative bacteria and inhibition of the
adhesion to and invasion of Caco-2 cells by Salmonella enterica
ser. typhimurium(Coconnier, Lievin, Lorrot,&Servin, 2000;Lievin-
Le Moal, Amsellem, Servin,&Coconnier, 2002). Also, probiotics like
many other lactic acid bacteria can produce various bacteriocins.
Bacteriocins are ribosomally synthesized antimicrobial peptides
effective against other bacteria, either in the same species (narrow
spectrum), or across genera (broad spectrum) with immunity to
their own bacteriocins (Cotter, Hill, & Ross, 2005). Recently,Corret al. (2007) showed that L. salivarius was capable of protecting
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mice against Listeria monocytogenes by direct antagonism
mediated by the bacteriocin Abp118. In some instances, the
inhibition of gastrointestinal pathogens is multifactorial including
all mentioned factors (Fayol-Messaoudi, Berger, Coconnier-Polter,
Lievin-Le Moal, & Servin, 2005). The production of these
antimicrobial compounds appeared to be stimulated by the
presence of pathogens (Rossland, Langsrud, Granum, & Sorhaug,
2005). In general, many mechanisms have been suggested bywhich probiotics prevent the detrimental effect of intestinal
pathogens including competition for limited nutrients, inhibition
of epithelial and mucosal adherence of pathogens, inhibition of
epithelial invasion by pathogens, production of antimicrobial
substances and/or the stimulation of mucosal immunity.Helicobacter pylori is an intestinal pathogen, long-term infec-
tion by which leads to chronic gastritis, peptic ulcer and increases
the risk of gastric malignancies (Plummer, Franceschi, & Munoz,
2004). Currently H. pylori infection is treated by a combined
therapy consisting of two antibiotics and a proton pump inhibitor,
which, although in many cases appeared very effective, presents a
very expensive treatment with many side effects including
antibiotic-associated diarrhoea and likelihood of induction of
the antibiotic resistance in intestinal pathogens (Malfertheineret al., 2002). The clinical outcome of H. pylori infection depends
on several factors including the strain of H. pylori, extent of
inflammation and cell density (Ernst & Gold, 2000). The risk
associated with the development of peptic ulcer and gastric
cancer is directly proportional to the level of infection (Tokunaga
et al., 2000). One of the measures, which may help reduce the rate
ofH. pylori infection, is a diet modulation with the inclusion of
probiotics (Khulusi et al., 1995).
Probiotic organisms do not appear to eradicate H. pylori, but
they are able to reduce the bacterial load and inflammation in
animal and human studies. It has been suggested that the
suppression effect is strain dependent (Sgouras et al., 2005).
L. caseiShirota strain showed a significant reduction in the levels
ofH. pylori colonization in the antrum and body mucosa in vivo
mouse model (Sgouras et al., 2004). This reduction was accom-
panied by a significant decline in the associated chronic and active
gastric mucosal inflammation observed at each time point
throughout the observation period. L. johnsonii La1 and L. gasseri
OLL2716 were also found to reduce H. pylori colonization and
inflammation (Felley et al., 2001). Similarly,L. acidophiluswas able
to inhibit the growth of H. pylori. In an intervention study, 14
patients infected with H. pylori received L. casei Shirota (21010
cfu day1) fermented milk for 6 weeks. Ureolytic activity was
reduced in 64% of the patients that consumed fermented products
containing probiotics, compared with 33% of the control group
(Cats et al., 2003). Similarly, a recent study concluded
that regular intake of yogurt containing B. animalis Bb12 and
L. acidophilus La5 may effectively suppress H. pylori infection in
humans (Wang et al., 2004). In the other studies in humanstreated either with lyophilized culture ofL. brevis(Linsalata et al.,
2004) or yogurts containing L. acidophilus and B. lactis (Wang
et al., 2004) or L. johnsonii La1 (Gotteland & Cruchet, 2003), a
decrease in theH. pyloribacterial load was observed indirectly via
the urea breath test. As many cited studies suggest, probiotic
administration alone would not lead to the eradication ofH. pylori
infection, however the use of probiotics as coadjuvants with the
H. pylori antibiotic treatment may resolve problems associated
with side effects. A number of studies conducted with varying
success and rather contradictory results may have been affected
by experimental design and applied controls (Sykora et al., 2005;
Tursi, Brandimarte, Giorgetti, & Modeo, 2004). Several mechan-
isms regarding the effect of probiotics on H. pylori have been
suggested including production of antimicrobial substances,enhanced gut barrier function and competition for adhesion sites;
however, the relative importance of these mechanisms is still
unclear.
9.7. Prevention of inflammatory bowel disease
Inflammatory bowel disease (IBD) comprises a spectrum of
disorders characterized by inflammation, ulceration and abnormalnarrowing of the gastrointestinal tract resulting in abdominal
pain, diarrhoea and gastrointestinal bleeding (Hanauer, 2006). It is
represented by two major phenotypes: ulcerative colitis and
Crohns disease, both of which are chronic, relapsing and
remitting diseases, predisposing affected individuals to the
development of colorectal cancer later in life (Itzkowitz&Harpaz,
2004). These two phenotypes have different pathogenesis, under-
lying inflammatory profiles, symptoms and treatment strategies.
Crohns disease is predominantly a Th1-driven immune response,
characterized by initial increase in interleukin (IL)-12 expression,
followed by interferon (IFN)-g and tumor necrosis factor (TNF)-a
(DHaens&Daperno, 2006). On the other hand, ulcerative colitis is
a Th2 immune response with predominant production of pro-
inflammatory cytokines including IL-5. The etiology of IBD is notwell understood with environmental, genetic and immunological
factors playing a role in the development of both diseases.
Several in vitro studies on cell models of IBD have shown the
ability of certain probiotic strains such as L. rhamnosus GG to
modulate the immune system by downregulating TNF-a-induced
IL-8 production (Zhang, Li, Caicedo, & Neu, 2005). The effect
clearly depended on cell concentration but not viability since dead
cells showed similar effects (Zhang et al., 2005). In contract to
these observations, the effects of L. reuteri were related to its
viability and, in addition to downregulation of IL-8 production,
up-regulation of the levels of the anti-inflammatory nerve growth
factor (Ma, Forsythe,&Bienenstock, 2004). In vivo animal studies
have indicated the importance of commensal bacteria in the
development of a functional immune system. B. lactis Bb12
initially elevated levels of IL-6 expression, but rats maintained
normal gut histology (Ruiz, Hoffmann, Szcesny, Blaut, & Haller,
2005). Furthermore, B. lactis Bb12 prevented the development of
significant intestinal inflammation caused by B. vulgatus (Ruiz
et al., 2005), indicating an important role for commensal bacteria
in initiating epithelial cell homeostasis. However, this effect
appears to be a strain specific (Schultz, Scholmerich, & Rath,
2003). Several studies have shown that probiotics might have had
beneficial effect on IBD patients (Gionchetti et al., 2000;
Guandalini, 2002). In one study, a possible effect ofLactobacillus
GG supplementation was investigated in four children with active
Crohns disease. Three of them treated with oral Lactobacillus GG
showed a significant improvement in terms of clinical outcome.
Although the results reported were very encouraging since
LactobacillusGG appeared to be effective in improving the clinicalstatus of children with Crohns disease, additional tests with a
larger sample size are required to substantiate this claim. In spite
of all the studies conducted, there is a lack of large, randomized,
double-blinded, placebo-controlled clinical trials assessing the
efficiency of probiotic strains and/or their combinations. The most
compelling evidence for the use of probiotics in IBD came from
randomized doubl