probiotics—from metchnikoff to bioactives

7/25/2019 ProbioticsFrom Metchnikoff to Bioactives

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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

Contents lists available atScienceDirect

journal homepage: www.elsevier.com/locate/idairyj

International Dairy Journal

0958-6946/$- see front matter& 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.idairyj.2008.03.004

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.

ARTICLE IN PRESS

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

ARTICLE IN PRESS

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



5/15

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

ARTICLE IN PRESS

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

ARTICLE IN PRESS



7/15

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

ARTICLE IN PRESS

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



8/15

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

ARTICLE IN PRESS

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


Barrier effect

Production of antimicrobial compounds

Prevention of inflammatory bowel

diseases


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



9/15

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

probiotics—from metchnikoff to bioactives

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