Probiotics—From Metchnikoff to bioactives

ARTICLE IN PRESS International Dairy Journal 18 (2008) 714– 728

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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj

Review

Probiotics—From 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 Metchnikoff’s 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 Lactobacillus and Bifidobacterium spp. However, substantial work needs to be done to substantiate other potentially beneficial properties including immunomodulation, hypocholesterolemic and anticarcinogenic 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. 2. 3. 4. 5. 6. 7. 8. 9.

10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714 Evolution of the probiotic concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715 Definition of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716 Properties of lactic acid bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716 Commercially important probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716 Selection of probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717 Technological challenges in the development of probiotic dairy products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718 Health potential of probiotic foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720 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 of Helicobacter pylori and intestinal pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723 9.7. Prevention of inflammatory bowel disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724 9.8. Modulation of the immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725 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

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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.

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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, the tradition 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-life and 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 lactobacilli; 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 mother’s breast and normally resided in the neonate’s 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 contained B. 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 body’s resistance steadily weakened by 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, Streptococcus thermophilus, which received no attention since it was considered a pathogen at that time. Metchnikoff’s experiments led him to believe that L. bulgaricus could 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 gut microflora were observed.

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Despite the fact that these findings disputed Metchnikoff’s theory, scientists continued to investigate possible benefits of bacteria to the human health. Consequently, certain strains of Lactobacillus acidophilus were 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 microflora resulting 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. 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 Probiotics are opposite of antibiotics Deleterious effects of antibiotics can be prevented by probiotic therapy A substance secreted by one microorganism which stimulates the growth of another Tissue extracts which stimulate microbial growth Compounds that build resistance to infection in the host but do not inhibit the growth of microorganisms in vitro Organisms and substances that contribute to intestinal microbial balance Live microbial feed supplement which beneficially affects the host animal by improving microbial balance 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 Live microbial culture or cultured dairy product which beneficially influences the health and nutrition of the host Living microorganisms which, upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition Microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being of the host 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 Live microorganisms that when administered in adequate amount confer a health benefit on the host

Kollath

1954 1955 1965 1971 1973

1974 1992

1992

1996

1996

1999

2001

2002

Vergin Kolb Lilly and Stillwell Sperti Fujii and Cook

Parker Fuller

Havenaar and Huis int’Veld

Salminen

Schaafsma

Salminen, Ouwehand, Benno and Lee Schrezenmeir and de Vrese

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 pro and 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 supplements. 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 microorganism. 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 by Parker (1974) resembles more recent description of probiotics. He defined them as organisms and substances, 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 Fuller’s (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, & Huisin’t 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 fermentative, producing lactic acid as a main product (Stiles & Holzapfel, 1997). The most important genera are: Lactobacillus, Lactococcus, Enterocococcus, Streptococcus, Pediococcus, Leuconostoc, and Bifidobacterium. Based on their GC (guanine–cytosine) pair content, Gram-positive bacteria are divided into two major phylogenetic branches. In contrast to other above-mentioned genera, bifidobacteria exhibit a relatively high G+C content of 55–67 mol% in the DNA and belong to the Actinomycetes branch. Other genera have a lower G+C content (o55 mol% 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 distinct groups based on their carbohydrate metabolism. The homofer-

mentative group consisting of Lactococcus, Pediococcus, Enterococcus, Streptococcus and some lactobacilli utilize the Embden– Meyerhof–Parnas (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 phosphoketolase pathway. Members of this group include Leuconostoc, Weissella and some lactobacilli. The species belonging to Enterococcus 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 associated 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 approximately 70 probiotic-containing products marketed in the world (Shah, 2004), the list has been continuously expanding. Traditionally, probiotics have been incorporated in to yoghurt; however, 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 of Lactobacillus 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 Grampositive, non-spore-forming microorganisms, catalase negative with noted exceptions, appearing as rods or coccobacilli. They are fermentative, microaerophylic and chemo-organotrophic. Considering the DNA base composition of the genome, they usually have a GC content less than 54 mol%. The genus Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae and its closest relatives are the genera Paralactobacillus and Pediococcus (Garrity, Bell, & Lilburn, 2004). This is the most numerous genus, comprising 106 described species. Lactobacillus 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, and L. johnsonii have been extensively studied and well documented. Bifidobacteria were first isolated and visualized by Tissier (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 as Gram-positive, non-spore forming, non-motile and catalase-negative

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Table 2 Some of probiotic strains used in commercial applications (adapted from Holm, 2003; Shah, 2004) Strain

Source

L. acidophilus LA1/LA5 L. delbrueckii ssp. bulgaricus Lb12 L. paracasei CRL431 B. animalis ssp. lactis Bb12 L. acidophilus NCFMs L. acidophilus La L. paracasei Lpc B. lactis HOWARUTM/Bl L. acidophilus LAFTIs L10 B. lactis LAFTIs B94 L. paracasei LAFTIs L26 L. johnsonii La1 L. acidophilus SBT-20621 B. longum SBT-29281 L. rhamnosus R0011 L. acidophilus R0052 L. casei Shirota B. breve strain Yakult B. lactis HN019 (DR10) L. rhamnosus HN001 (DR20) L. plantarum 299V L. rhamnosus 271 L. casei Immunitas B. animalis DN173010 (Bioactiva) L. rhamnosus LB21 Lactococcus lactis L1A L. reuteri SD2112 L. rhamnosus GG1 L. salivarius UCC118 B. longum BB536 L. acidophilus LB L. paracasei F19

Chr. Hansen

Danisco

DSM Food Specialties

Nestle Snow Brand Milk Products Co. Ltd. Institute Rosell Yakult Foneterra Probi AB Danone Essum AB Biogaia Valio Dairy University College Cork Morinaga Milk Industry Co. Ltd. Lacteol Laboratory Medipharm

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, Corynebacterium, and Brevibacterium. Presently, there are 32 species in the genus Bifidobacterium, 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, and B. pseudocatenulatum. B. breve, B. infantis, and B. longum are found in human infants. B. adolescentis and B. longum are found in human adults (Garrity et al., 2004).

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(Mattila-Sandholm, Mylla¨rinen, Crittenden, Fonde´n, & Saarela, 2002; Ouwehand, Kirjavainen, Shortt, & Salminen, 1999; Reid, 1999), a general agreement exists with regard to key selection criteria listed in Table 3 (FAO/WHO, 2002). The first step in the selection of a probiotic is the determination 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 of the novel strains (Morelli, 2007). The classification and relatedness of probiotics (and other microorganisms) is based on the comparison of highly conserved molecules, namely genes encoding 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 distinguished 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, Bjo¨rkroth, & Schillinger, 2001; Vuaghan, Heilig, Ben-Amor, & de Vos, 2005). Many authors (i.e., Ouwehand et al., 1999) advocated the importance 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 occurrence of harmful effects associated with consumption of probiotics are quite rare, although certain Lactobacillus strains 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 Table 3 Key and desirable criteria for the selection of probiotics in commercial applications (adapted from Shah, 2006; Morelli, 2007) General

Property

Safety criteria

Origin Pathogenicity and infectivity Virulence factors—toxicity, 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 metabolism Antimutagenic and anticarcinogenic properties

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 cfu mL1 of a product). Although numerous criteria have been recognized and suggested

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strains of Bifidobacterium (Matteuzzi, Crociani, & Brigidi, 1983) and Lactobacillus (Gupta, Mital, & Gupta, 1995) showed a strain dependent resistance to tested antibiotics. On the other hand, a recent study (Moubareck, Gavini, Vaugien, Butel, & DoucetPopulaire, 2005) tested 50 strains belonging to eight Bifidobacterium spp. 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 the donor 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 been frequently 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 108 cfu mL1 (LourensHattingh & 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 a probiotic 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 of L. acidophilus and Bifidobacterium over 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 degradation 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, Bifidobacterium spp. and L. casei were proposed in the 1990s, but most of these methods were based on pure cultures of these organisms. 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 in Table 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 functional criteria 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

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 Concentration—high osmotic pressure, low water activity, higher concentration of particular ions Temperature—freezing, vacuum and spray drying Drying Prolonged storage—oxygen 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

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 MRSa agar (pH 4.58) MRS-sorbitol agar MRS-NNLPb agar MRS-vancomycine agarc MRS-vancomycine agar Sodium lactate agar

S. thermophilus L. delbrueckii ssp. bulgaricus L. acidophilus Bifidobacteria L. casei L. rhamnosus Propionibacteria4

Aerobic, 37 1C, 24 h Anaerobic, 45 1C, 72 h Anaerobic, 37 1C, 72 h Anaerobic, 37 1C, 72 h Anaerobic, 37 1C, 72 h Anaerobic, 43 1C, 72 h Anaerobic, 30 1C, 7–9 days

0.1–0.5 mm, round yellowish 1.0 mm, white, cottony, rough, irregular Rough, dull, small (0.1–0.5), brownish 1 mm, white, smooth, shiny 1.0 mm, white shiny, smooth 1.0–2.0 mm, white shiny, smooth 1.0–2.5 mm, dull brown, lighter margin

a b c

de man, Rogosa and Sharpe agar. Nalidixic acid, neomycin sulfate, lithium chloride and paromomycin sulfate. In case L. rhamnosus absent, if not then subtraction method required.

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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., low during 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 approaches such as spray drying mainly due to several disadvantages regarding handling of the frozen and freeze dried bacterial materials including high transport and storage cost and detrimental 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 due to increased solute concentration, intracellular ice formation in case of freezing and freeze drying, exposure to elevated temperatures during spray drying and, in general, dehydration. To improve the survival and preserve the activity of probiotics, protective compounds (compatible solutes and cryoprotectants) are frequently 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 osmolytes, 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 a long 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 performance 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, concentration of sugars, dissolved oxygen and oxygen permeation through package (especially for Bifidobacterium spp.), inoculation level, and fermentation time (Shah, 2000). L. acidophilus has a high

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cytoplasmic buffering capacity (pH 3.72–7.74), 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, Herna´ndez, & Herna´ndez, 2005; Matsumoto, Ohishi, & Benno, 2004). Bifidobacterium animalis subsp. lactis has the highest acid tolerance and is thus preferably used in ‘‘bifidus’’ products. The use of L. delbrueckii ssp. bulgaricus in yoghurt may affect survival of L. acidophilus and Bifidobacterium due to acid and hydrogen peroxide produced during fermentation. However, due to its proteolytic nature, L. delbrueckii ssp. 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 probioticcontaining products can have a detrimental effect on the viability of probiotics. Strains of L. acidophilus and Bifidobacterium spp. are microaerophilic and anaerobic, respectively. They lack an electron-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 of L. delbrueckii ssp. bulgaricus from 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 (5–10%) 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 the growth 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 postacidification, 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 other organisms. 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 of fermentation. 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 organisms. 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 incorporated in fermented frozen dairy desserts, yoghurt or freeze dried yoghurt showed improved viability in comparison with nonencapsulated 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 the consumption of probiotics, they may in some instances be influenced by composition of a delivery matrix. In dairy applications, 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 made—mainly composed of skim milk nonfat 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 milkderived products such as fermented milk contain components that possess a range of different bioactivities, some of them summarized in Table 6. In their native form, milk proteins exert an appreciable range of different physiological activities. Specific immunoglobulins provide the first line of defense to suckling neonates through passively 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 as regulatory 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) inhibitors, 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

Table 6 Biogenic activity of native milk macro-components (from Vasiljevic & Shah, 2007) Component

Form

Protein

Caseins

Fat

Conjugated linoleic acid

Bioactivity

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 Lysozyme Antimicrobial

Sphingolipids Butyric acid Carbohydrates Oligosaccharides

Anticarcinogenic, modulation of lipid and protein metabolism, anti-inflamatory, hypotensive, anti-atherosclerotic Anti-inflamatory, anticarcinogenic Anticarcinogenic Prebiotic, antimicrobial (antiadhesive), Ca absorption

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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 Ile-Pro-Pro Val-Pro-Pro Ile-Pro-Pro Phe-Pro-Glu-Val-Phe-Glu-Lys Lys-Val-Leu-Pro-Val-Pro-Glu Lys-Thr-Thr-Met-Pro-Leu-Trp Asn-Leu-His-Leu-Pro-Leu-Pro-Leu-Leu Tyr-Pro-Phe-Pro-Glu-Pro-Ile-Pro-Asn Tyr-Pro Leu-Asn-Val-Pro-Gly-glu-Ile-Val-Glu Asn-Ile-Pro-Pro-Leu-Thr-Glu-Thr-Pro-Val

L. helveticus CM4 and S. cerevisiae

b- and k-casein

Hypotensive

L. helveticus LBK16H

b- and k-casein

Hypotensive

Commercial products+digestion Commercial products+digestion Commercial products+digestion L. helveticus NCC 2765 L. helveticus NCC 2765 L. helveticus CPN4 L. delbrueckii ssp. bulgaricus SS1 L. lactis ssp. cremoris FT4

as1-Casein b-Casein as1-Casein b-Casein b-Casein Caseins b-Casein b-Casein

ACE inhibition Antioxidative Possible immunomodulation ACE inhibition Opioid ACE inhibition ACE inhibition ACE inhibition

the storage time, which raised an important question regarding the stability of these peptides during the prolonged storage.

9. Health effects of probiotics Since Metchnikoff’s era, a number of health benefits have been contributed to products containing probiotic organisms. While some of these benefits have been well documented and established, 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 management of lactose malabsorption, rotaviral diarrhoea, antibioticassociated diarrhoea, and Clostridium difficile diarrhoea. Some of these strain specific health effects are listed in Table 8. 9.1. Alleviation of lactose intolerance The decline of the intestinal b-galactosidase (b-gal or commonly know as lactase) activity is a biological characteristic of the maturing intestine in the majority of the world’s 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 are readily 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 malabsorption 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 on the size of the lactose load ingested. The development of the

Table 8 Some of the established and potential health benefits of probiotic organisms (adapted from Shah, 2006) Health effect Scientifically established Alleviation of lactose intolerance

Mechanism

Delivery of intracellular b-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 of Helicobacter 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 receptors—induction of innate and adaptive immunity:  downregulation of pro-inflammatory cytokines and chemokines  upregulation of phagocytic activity



regulation of Th1/Th2 balance

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 hypolactasia 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 been suggested in order to clarify this phenomenon. At least three

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factors appear to be responsible for a better tolerance of lactose in fermented milk including (a) starter culture, (b) intracellular b-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 and terminal 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 improving absorption and lactose tolerance. 9.2. Prevention and reduction of diarrhoea symptoms One of the main applications of probiotics has been the treatment 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. rhamnosus GG and B. 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 traveller’s diarrhoea, which is caused by bacteria, particularly enterotoxigenic Escherichia coli. Several studies have assessed the effects of probiotic preparations as prophylaxis for traveller’s diarrhoea, however, the results have been conflicting due to methodological deficiencies, which certainly limited the validity of their conclusions (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 in Table 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 antiinflammatory 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 between T-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 colonization of Bifidobacterium and Lactobacillus spp. in the gastrointestinal tract of children may be one of the reasons for allergic reactions (Kallioma¨ki & 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 by B. bifidum, typical for breast-fed infants (Ouwehand et al., 2001). A recent study also indicated that early consumption of probiotic preparations containing Lactobacillus GG 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 reinforcement 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, O 2 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 of the mutation process, which manifests itself as a decrease in the level of spontaneous and induced mutations. Some epidemiological 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 interaction between 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 antimutagenic activity against all mutagens or promutagens studied. Moreover, live bacterial cells showed higher antimutagenicity than killed cells against the mutagens studied, which suggested that 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 Bifidobacterium spp. were capable of decreasing the levels of enzymes such as b-glucuronidase, azoreductase, and nitroreductase responsible for activation of procarcinogens. This inactivation consequently led to a substantial decline of the risk associated with tumor development. 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, 2002). 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-nitroquinoline1-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 permeability 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 man1 day1) 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

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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 coprecipitation of cholesterol with the deconjugated bile has been proposed (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 of short-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 placebocontrolled 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-molecularmass 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-molecularmass 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; Lie´vinLe 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, Corr et 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 by which 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 infection by which leads to chronic gastritis, peptic ulcer and increases ˜ oz, the risk of gastric malignancies (Plummer, Franceschi, & Mun 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 (Malfertheiner et 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 of H. 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. casei Shirota strain showed a significant reduction in the levels of H. pylori colonization in the antrum and body mucosa in vivo mouse model (Sgouras et al., 2004). This reduction was accompanied 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. acidophilus was able to inhibit the growth of H. pylori. In an intervention study, 14 patients infected with H. pylori received L. casei Shirota (2  1010 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 humans treated either with lyophilized culture of L. 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 the H. pylori bacterial load was observed indirectly via the urea breath test. As many cited studies suggest, probiotic administration alone would not lead to the eradication of H. 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 mechanisms 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 abnormal narrowing of the gastrointestinal tract resulting in abdominal pain, diarrhoea and gastrointestinal bleeding (Hanauer, 2006). It is represented by two major phenotypes: ulcerative colitis and Crohn’s 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, underlying inflammatory profiles, symptoms and treatment strategies. Crohn’s 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 (D’Haens & Daperno, 2006). On the other hand, ulcerative colitis is a Th2 immune response with predominant production of proinflammatory cytokines including IL-5. The etiology of IBD is not well 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 of Lactobacillus GG supplementation was investigated in four children with active Crohn’s 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 Lactobacillus GG appeared to be effective in improving the clinical status of children with Crohn’s 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 double-blind placebo-controlled trials of VSL#3 (a mixture of four species of lactobacilli, three species of bifidobacteria and S. thermophilus) in patients with pouchitis. In a study by Gionchetti et al. (2000), the efficacy of VSL#3 was assessed as a maintenance treatment in forty patients with chronic relapsing pouchitis. After 4 months fewer relapses were found to occur in the intervention group than in the control group. Moreover, all patients were subsequently found to relapse 3 months after cessation of VSL#3. Another study (Mimura et al.,

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2004) confirmed these findings by showing the effectiveness of VSL#3 as maintenance therapy in patients with recurrent or chronic pouchitis. In 2003, Gionchetti expanded his initial study to determine whether VSL#3 treatment was able to prevent the onset of acute pouchitis and improve the quality of life in patients in the year immediately following surgical resection (Gionchetti et al., 2003). Only 10% of the VSL#3 treatment group developed pouchitis compared to 40% of the placebo group. While a number of mechanisms have been suggested (Table 8), further elaboration and confirmation of previous studies is required.

9.8. Modulation of the immune system The complexity of the immune system is secondary only to that of the central nervous system and includes two principal components: innate and adaptive immunity, which work in concert to protect us from external and internal insults. The innate system is ancestral and is neither anticipatory nor clonal and does not respond to environmental changes. It represents the first line of defense with natural killer (NK) cells as the primary cells involved in the identification and spontaneous lysis of offensive targets (virus-infected cells, tumor cells, bone marrow stem cells and embryonic cells). An inverse relationship exists between the rise and fall of NK cells and the incidence of tumor growth (Dussault & Miller, 1996). In contrast, the adaptive system is acquired through interactions with the environment. It is subject to induction, anticipation (immune memory) and clonal expansion. Understanding these responses is the key to understanding the mechanisms of allergy, autoimmunity, vaccination and carcinogenicity. The innate and adaptive systems are highly integrated and interdependent (Hoebe, Janssen, & Beutler, 2004). Humans as mammals have developed an extremely sophisticated adaptive immune system of both systemic and mucosal (local) type. Intestinal epithelial cells are in direct contact with the intestinal microflora and also interface and segregate the immune system. It has been suggested that the immune system might be beneficially affected in the presence of probiotics through the action of recognition receptors expressed on the surface of epithelial cells (Isolauri, Su¨tas, Kankaanpa¨a¨, Arvilommi, & Salminen, 2001). The innate immune system via toll-like receptors (TLRs) recognizes a large group of chemical structures in pathogens such as lipopolysaccharides (LPS) and lipoteichoic acids which enables them to recognize foreign objects which trigger a cascade of immunological defence mechanisms, such as the production of pro- and anti-inflammatory cytokines (Anderson, 2000). TLRs are expressed mainly by macrophages and dendritic cells (DCs), but also include a variety of other cell types such as B cells and epithelial cells (Pasare & Medzhitov, 2005). The activation of TLRs results in the initiation of the response of the DCs which leads to the production of cytokines and upregulation or downregulation of cell-surface molecules (Granucci & Ricciardi-Castagnoli, 2003). These signals critically influence further induction of both innate and adaptive immunity. The suppression of the formation of pro-inflammatory cytokines and chemokines in the presence of probiotics has been reported in several in vitro studies. The response of the immune system to a probiotic was weaker than in the presence of a Grampositive pathogen. More importantly, the study showed that human monocyte-derived DCs responded differently to different Gram-positive bacteria (Veckman et al., 2004). The different immune response to different bacteria was confirmed in a different study, in which Gram-negative Klebsiella pneumoniae and L. rhamnosus were compared (Braat et al., 2004). Both cultures induced DC maturation, but resulted in a different cytokine profile. K. pneumoniae activated the expression of T-helper (Th) 1

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type cells, where L. rhamnosus reduced the production of proinflammatory cytokines (TNF-a) and interleukins (IL-6 and IL-12) by immature DCs as well as IL-12 and IL-18 by mature DCs. Moreover, the cytokine response may vary greatly in the presence of different probiotics. The mixture of eight different probiotic and LAB strains including L. acidophilus, L. delbrueckii ssp. bulgaricus, L. casei, L. plantarum, B. longum, B. infantis, B. breve and S. thermophilus upregulated production of IL-10 and downregulated production of IL-12 by DCs derived from human blood and lamina propria. The pro-inflammatory effect was reduced by suppression of IL-12 production in the presence of probiotics, while maintaining high production of IL-10, which was regulated by bifidobacteria that upregulated IL-10 production. Furthermore, most of the strains suppressed IL-12 production (Hart et al., 2004; Lammers et al., 2003). Despite many studies showing the immunomodulatory potential of probiotics, there are still concerns with regard to validity of results due to poor study designs, lack of appropriate controls, inadequate probiotic administration, reliance on in vitro indicators, and short duration of the in vivo studies. Furthermore, the consumption of probiotics over a prolonged period has not been assessed with regard to sustained improvements of the immune system and the immunomodulatory potential of probiotics in relation to different age groups including children and elderly (Gill & Guarner, 2004). The reader is invited to consult more recent references (i.e. Ezendam & van Loveren, 2006; Gill & Guarner, 2004) for more in-depth information on this subject.

10. Conclusions This review covered the knowledge of probiotics that spans over a hundred years. More work is needed to establish mechanisms by which probiotics affect our well-being at the molecular level. Some of these developments have been provided in this review. Similarly, more work is needed to validate host-probiotic interactions in human studies. While scientists continue to work on elucidation of the mechanisms of the well-established strains targeting the general population, it should be remembered that all effects are strain specific. These effects can be enhanced in the presence of a mixture of probiotics or with addition of prebiotics. References Agerholm-Larsen, L., Raben, A., Haulrik, N., Hansen, A. S., Manders, M., & Astrup, A. (2000). Effect of 8-week intake of probiotic milk products on risk factors for cardiovascular diseases. European Journal of Clinical Nutrition, 54, 288–297. Alakomi, H. L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., & Helander, I. M. (2000). Lactic acid permeabilizes Gram-negative bacteria by disrupting the outer membrane. Applied and Environmental Microbiology, 66, 2001–2005. Anderson, K. V. (2000). Toll-like receptors: Critical proteins linking innate and acquired immunity. Current Opinion in Immunology, 12, 13–19. Arihara, K., Ota, H., Itoh, M., Kondo, Y., Sameshima, T., Yamanaka, H., et al. (1998). Lactobacillus acidophilus group lactic acid bacteria applied to meat fermentation. Journal of Food Science, 63, 544–547. Baati, L., Fabre-Gea, C., Auriol, D., & Blanc, P. J. (2000). Study of the cryotolerance of Lactobacillus acidophilus: Effect of culture and freezing conditions on the viability and cellular protein levels. International Journal of Food Microbiology, 59, 241–247. Beales, N. (2004). Adaptation of microoganims to cold temperatures, weak acid preservatives, low pH, and osmotic stress: A review. Comprehensive Reviews in Food Science and Food Safety, 3, 1–20. Begley, M., Hill, C., & Gahan, C. G. M. (2006). Bile salt hydrolase activity in probiotics. Applied and Environmental Microbiology, 72, 1729–1738. Braat, H., de Jong, E. C., van den Brande, J. M., Kapsenberg, M. L., Peppelenbosch, M. P., van Tol, E. A., et al. (2004). Dichotomy between Lactobacillus rhamnosus and Klebsiella pneumoniae on dendritic cell phenotype and function. Journal of Molecular Medicine, 82, 197–205. Bruno, F. A., Lankaputhra, W. E. V., & Shah, N. P. (2002). Growth, viability and activity of Bifidobacterium spp. in skim milk containing prebiotics. Journal of Food Science, 67, 2740–2744. Buller, H. A., & Grand, R. J. (1990). Lactose intolerance. Annual Reviews in Medicine, 41, 141–148.

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