**1. Introduction**

20 New Advances in the Basic and Clinical Gastroenterology

Zoetendal, E.G.; Collier, C.T.; Koike, S.; Mackie, R.I. & Gaskins, H.R. (2004) Molecular

This chapter reviews literature on probiotics. Probiotics are defined and different microbial cultures used as probiotics will be considered. It further discusses delivery vehicles for probiotic cultures, with their advantages and disadvantages. Since the presence of viable probiotic cultures in products is vital to their functionality, different methods used for their detection in products will be examined. The beneficial health effects of probiotics, the methods that are currently used in an attempt to overcome some of the challenges faced will also be discussed. Different strategies for protection of probiotic cultures and challenges for the probiotic industry are highlighted. In addition to these, alternative strategies increasing numbers of beneficial microorganisms through administration of prebiotics and synbiotics are briefly mentioned.

#### **2. What are probiotics?**

The definition of probiotics has been modified with increasing knowledge in the field of how they function. The term is derived from the Greek language meaning 'for life'. In the past there have been many attempts to define the term 'probiotic', one of the first being described by Lilly & Stillwell in 1965. They defined probiotics as "substances secreted by one microorganism, which stimulates the growth of another". The focus of this definition was to distinguish them from and make it clear that they are the opposite of antibiotics. Subsequently, in 1974, Parker defined them as "organisms and substances which contribute to intestinal microbial balance" (Schrezenmier & de Vrese, 2001). In 1989, Fuller tried to improve on Parker's definition by proposing the following definition: "live microbial feed supplement, which beneficially affect the host (animal or human) by improving its intestinal microbial balance" (Salminen et al., 1999; Vilsojevic & Shah, 2008). Then, Havenaar & Huis In't Veld (1992) defined probiotics acceptably as 'a viable mono- or mixed culture of microorganisms which applied to animal or man, beneficially affects the host by improving the properties of the indigenous microflora'. Schrezenmeir & de Vrese (2001) defined the term probiotic as "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 effects on host health". Among these descriptions and definitions, there were many others, until the Food and Agriculture Organization of the United Nations-World Health Organization (FAO-WHO) officially

Probiotics – What They Are, Their Benefits and Challenges 23

probiotic should survive the environmental conditions in their target site of action and proliferate in this location (Havenaar & Huis int'Veld, 1992). That is, they should be able to adhere to and colonise the epithelial cell lining to establish themselves in the colon (Guarner & Schaafsma, 1998; Parracho et al., 2007). The ability to adhere to the epithelium secures the strain from being easily flushed out by peristaltic movements (Gupta & Garg, 2009). Technologically, a good probiotic strain should be easily, inexpensively reproducible (Charteris et al., 1998; Havenaar & Huis int'Veld, 1992). It must be able to withstand stress during processing and storage, with process and product application robustness (Charteris et al., 1998). The organism should be able to survive, in particular, the harsh environmental conditions of the stomach and small intestine (e.g. gastric and bile acids, digestive enzymes)

In addition, technological aspects must be taken into account before selecting a probiotic strain. Strains should be capable of being prepared on a large scale and should be able to multiply rapidly, with good viability and stability in the product during storage. The strains must not produce off flavours or textures once incorporated into foods. They should be metabolically active within the GIT and biologically active against their identified target. Probiotic strains must be resistant to phages and have good sensory properties (Collins et al., 1998; Kolida et al., 2006; Lacroix & Yildirim, 2007; Mattila-Sandholm et al., 2002; Saarela et al., 2000;). Therefore probiotic containing foods and products need to be of good quality and must have high enough numbers of viable probiotic cells to ensure that consumers get the optimal benefits from the product (Alakomi et al., 2005). Probiotic strains have to be good vehicles for specific target delivery of peptides and recombinant proteins within the

A number of microorganisms are currently used as probiotics. However, the most commonly used are bacteria belonging to the genera *Lactobacillus,* the first and largest group of microorganisms to be regarded as probiotics (Mombelli & Gismondo, 2000; Wolfson, 1999) and *Bifidobacterium*. These bacteria are indigenous to the human GIT (Bielecka et al., 2002; Tannock, 2001). They are known to have no harmful effects, which is in contrast to other gut bacteria (Kimoto-Nira et al., 2007). Species of Lactobacilli include *L*. *acidophilus*, *L*. *rhamnosus , L*. *casei*, *L*. *delbrueckii* ssp. *bulgaricus*, *L*. *johnsonii , L*. *reuteri*, *L*. *brevis*, *L*. *cellobiosus*, *L*. *curvatus*, *L*. *fermentum*, *L*. *gasseri* and *L*. *plantarum* (Krasaekoopt et al., 2003; Meurman & Stamatova, 2007). The most recognized bifidobacteria species used are *Bifidobacterium breve*, *B*. *animalis* subsp *lactis* formerly *B*. *lactis* (Masco et al., 2004) and *B*. *longum* biotypes *infantis*

Probiotics now include other lactic acid bacteria (LAB) from genera such as *Streptococcus*, *Lactococcus*, *Enterococcus*, *Leuconostoc*, *Propionibacterium*, and *Pediococcus* (Krasaekoopt et al., 2003; O'Sullivan et al., 1992; Power et al., 2008; Vandenplas et al., 2007; Vinderola & Reinheimer, 2003). Some countries are, however, concerned about the possible transfer of antibiotic resistance genes by some members of the *Enterococcus* (Lund & Edlund, 2001). Other non-related microbes used include bacteria such as non-pathogenic *E*. *coli* Nissle 1917 and *Clostridium butyricum* (Harish & Varghese, 2006), yeasts (*Saccharomyces cereviciae*, *Saccharomyces boulardii*), filamentous fungi (*Aspergillus oryzae*), and some spore forming

bacilli (Fuller, 2003; Mombelli & Gismondo, 2000; Wolfson, 1999).

(Dunne et al., 2001; Parracho et al., 2007).

human GIT (Dunne et al., 2001; Parracho et al., 2007).

**2.2 Common probiotic microorganisms** 

and *longum* (Masco et al., 2005).

defined probiotics as: "live microorganisms that when administered in adequate amounts confer a significant health benefit on the host" (FAO, 2001). This definition was later endorsed by the International Scientific Association for Probiotics and Prebiotics (ISAPP) and is currently the most accepted definition of probiotics by scientists worldwide (Reid, 2006).

Probiotic food cultures have become popular due to appreciation of their contribution to good health (Desmond et al., 2002). In probiotic therapy, these beneficial microorganisms are ingested and thereby introduced to the intestinal microflora intentionally. This results in high numbers of beneficial bacteria to participate in competition for nutrients with and starving off harmful bacteria (Mombelli & Gismondo, 2000). The probiotics take part in a number of positive health promoting activities in human physiology (Chen & Yao, 2002).

The beneficial effects of the ingested probiotic bacteria are provided by those organisms that adhere to the intestinal epithelium (Salminen et al., 1998). The presence and adherence of probiotics to the mucous membrane of the intestines build up a strong natural biological barrier for many pathogenic bacteria (Chen & Yao, 2002). Adhesion is therefore regarded as the first step to colonization. Adhesion to the epithelium can be specific, involving adhesion of bacteria and receptor molecules on the epithelial cells, or non-specific, based on physicochemical factors.

#### **2.1 Desirable properties for a probiotic strain**

A microbial strain has to fulfil a number of specific properties or criteria for it to be regarded as a probiotic. These criteria are classified into safety, performance and technological aspects (Gibson & Fuller, 2000). The criteria are further dependent on specific purpose of the strain and on the location for the expression of the specific property. With regards to safety, the probiotic strain must be of human origin, isolated from the gastrointestinal tract (GIT) of healthy individuals. They should possess GRAS (generally regarded as safe) status, be nonpathogenic, and without previous association with diseases such as infective endocarditis or gastrointestinal disorders. Probiotic strains must not deconjugate bile salts and they should carry no antibiotic resistance genes that can be transferred to pathogens (Collins et al., 1998; Saarela et al., 2000). The strain must not induce an immune reaction in the host, i.e. the host must be immuno-tolerant to the probiotic (Havenaar & Huis int'Veld, 1992). The strain itself, its fermentation products or its cell components after its death, should be non-pathogenic, non-toxic, non-allergic, non-mutagenic or non-carcinogenic even in immunocompromised individuals (Collins et al., 1998; Havenaar & Huis int'Veld, 1992). It must have antimutagenic and anticarcinogenic properties and not promote inflammation in individuals (Collins et al., 1998). A probiotic strain should possess a desirable antibiogram profile. It must also be genetically stable with no plasmid transfer mechanism (Havenaar & Huis int'Veld, 1992; Ziemer & Gibson, 1998).

With respect to their performance, potential probiotic strains should be acid-tolerant and therefore survive human gastric juice and bile. They must be able to survive in sufficient numbers and adhere to the intestinal mucosal surface in order to endure the GIT. They should have antagonistic activity against pathogens such as *Salmonella* species, *Clostridium difficile* and *Listeria monocytogenes* that adhere to mucosal surfaces. Lastly, probiotic strains should also stimulate an immune response, thereby positively influencing the host (Biavati et al., 2000; Kolida et al., 2006; Mattila-Sandholm et al., 2002; Saarela et al., 2000;). The

defined probiotics as: "live microorganisms that when administered in adequate amounts confer a significant health benefit on the host" (FAO, 2001). This definition was later endorsed by the International Scientific Association for Probiotics and Prebiotics (ISAPP) and is currently the most accepted definition of probiotics by scientists worldwide (Reid, 2006).

Probiotic food cultures have become popular due to appreciation of their contribution to good health (Desmond et al., 2002). In probiotic therapy, these beneficial microorganisms are ingested and thereby introduced to the intestinal microflora intentionally. This results in high numbers of beneficial bacteria to participate in competition for nutrients with and starving off harmful bacteria (Mombelli & Gismondo, 2000). The probiotics take part in a number of positive health promoting activities in human physiology (Chen & Yao, 2002).

The beneficial effects of the ingested probiotic bacteria are provided by those organisms that adhere to the intestinal epithelium (Salminen et al., 1998). The presence and adherence of probiotics to the mucous membrane of the intestines build up a strong natural biological barrier for many pathogenic bacteria (Chen & Yao, 2002). Adhesion is therefore regarded as the first step to colonization. Adhesion to the epithelium can be specific, involving adhesion of bacteria and receptor molecules on the epithelial cells, or non-specific, based on

A microbial strain has to fulfil a number of specific properties or criteria for it to be regarded as a probiotic. These criteria are classified into safety, performance and technological aspects (Gibson & Fuller, 2000). The criteria are further dependent on specific purpose of the strain and on the location for the expression of the specific property. With regards to safety, the probiotic strain must be of human origin, isolated from the gastrointestinal tract (GIT) of healthy individuals. They should possess GRAS (generally regarded as safe) status, be nonpathogenic, and without previous association with diseases such as infective endocarditis or gastrointestinal disorders. Probiotic strains must not deconjugate bile salts and they should carry no antibiotic resistance genes that can be transferred to pathogens (Collins et al., 1998; Saarela et al., 2000). The strain must not induce an immune reaction in the host, i.e. the host must be immuno-tolerant to the probiotic (Havenaar & Huis int'Veld, 1992). The strain itself, its fermentation products or its cell components after its death, should be non-pathogenic, non-toxic, non-allergic, non-mutagenic or non-carcinogenic even in immunocompromised individuals (Collins et al., 1998; Havenaar & Huis int'Veld, 1992). It must have antimutagenic and anticarcinogenic properties and not promote inflammation in individuals (Collins et al., 1998). A probiotic strain should possess a desirable antibiogram profile. It must also be genetically stable with no plasmid transfer mechanism (Havenaar &

With respect to their performance, potential probiotic strains should be acid-tolerant and therefore survive human gastric juice and bile. They must be able to survive in sufficient numbers and adhere to the intestinal mucosal surface in order to endure the GIT. They should have antagonistic activity against pathogens such as *Salmonella* species, *Clostridium difficile* and *Listeria monocytogenes* that adhere to mucosal surfaces. Lastly, probiotic strains should also stimulate an immune response, thereby positively influencing the host (Biavati et al., 2000; Kolida et al., 2006; Mattila-Sandholm et al., 2002; Saarela et al., 2000;). The

physicochemical factors.

**2.1 Desirable properties for a probiotic strain** 

Huis int'Veld, 1992; Ziemer & Gibson, 1998).

probiotic should survive the environmental conditions in their target site of action and proliferate in this location (Havenaar & Huis int'Veld, 1992). That is, they should be able to adhere to and colonise the epithelial cell lining to establish themselves in the colon (Guarner & Schaafsma, 1998; Parracho et al., 2007). The ability to adhere to the epithelium secures the strain from being easily flushed out by peristaltic movements (Gupta & Garg, 2009). Technologically, a good probiotic strain should be easily, inexpensively reproducible (Charteris et al., 1998; Havenaar & Huis int'Veld, 1992). It must be able to withstand stress during processing and storage, with process and product application robustness (Charteris et al., 1998). The organism should be able to survive, in particular, the harsh environmental conditions of the stomach and small intestine (e.g. gastric and bile acids, digestive enzymes) (Dunne et al., 2001; Parracho et al., 2007).

In addition, technological aspects must be taken into account before selecting a probiotic strain. Strains should be capable of being prepared on a large scale and should be able to multiply rapidly, with good viability and stability in the product during storage. The strains must not produce off flavours or textures once incorporated into foods. They should be metabolically active within the GIT and biologically active against their identified target. Probiotic strains must be resistant to phages and have good sensory properties (Collins et al., 1998; Kolida et al., 2006; Lacroix & Yildirim, 2007; Mattila-Sandholm et al., 2002; Saarela et al., 2000;). Therefore probiotic containing foods and products need to be of good quality and must have high enough numbers of viable probiotic cells to ensure that consumers get the optimal benefits from the product (Alakomi et al., 2005). Probiotic strains have to be good vehicles for specific target delivery of peptides and recombinant proteins within the human GIT (Dunne et al., 2001; Parracho et al., 2007).

#### **2.2 Common probiotic microorganisms**

A number of microorganisms are currently used as probiotics. However, the most commonly used are bacteria belonging to the genera *Lactobacillus,* the first and largest group of microorganisms to be regarded as probiotics (Mombelli & Gismondo, 2000; Wolfson, 1999) and *Bifidobacterium*. These bacteria are indigenous to the human GIT (Bielecka et al., 2002; Tannock, 2001). They are known to have no harmful effects, which is in contrast to other gut bacteria (Kimoto-Nira et al., 2007). Species of Lactobacilli include *L*. *acidophilus*, *L*. *rhamnosus , L*. *casei*, *L*. *delbrueckii* ssp. *bulgaricus*, *L*. *johnsonii , L*. *reuteri*, *L*. *brevis*, *L*. *cellobiosus*, *L*. *curvatus*, *L*. *fermentum*, *L*. *gasseri* and *L*. *plantarum* (Krasaekoopt et al., 2003; Meurman & Stamatova, 2007). The most recognized bifidobacteria species used are *Bifidobacterium breve*, *B*. *animalis* subsp *lactis* formerly *B*. *lactis* (Masco et al., 2004) and *B*. *longum* biotypes *infantis* and *longum* (Masco et al., 2005).

Probiotics now include other lactic acid bacteria (LAB) from genera such as *Streptococcus*, *Lactococcus*, *Enterococcus*, *Leuconostoc*, *Propionibacterium*, and *Pediococcus* (Krasaekoopt et al., 2003; O'Sullivan et al., 1992; Power et al., 2008; Vandenplas et al., 2007; Vinderola & Reinheimer, 2003). Some countries are, however, concerned about the possible transfer of antibiotic resistance genes by some members of the *Enterococcus* (Lund & Edlund, 2001). Other non-related microbes used include bacteria such as non-pathogenic *E*. *coli* Nissle 1917 and *Clostridium butyricum* (Harish & Varghese, 2006), yeasts (*Saccharomyces cereviciae*, *Saccharomyces boulardii*), filamentous fungi (*Aspergillus oryzae*), and some spore forming bacilli (Fuller, 2003; Mombelli & Gismondo, 2000; Wolfson, 1999).

Probiotics – What They Are, Their Benefits and Challenges 25

1999; Shah, 2007). Positive effects of probiotics are not confined to the gut only, but can extend to other parts of the body. For instance, probiotics are known to have anti-

Lactose malabsorption (also referred to as lactose intolerance or lactose indigestion) is the inability to hydrolyze lactose (Adams & Moss, 2000; Salminen et al., 1998a). It is caused by a deficiency of the enzyme β-D-galactosidase (lactase) (Buller & Grand, 1990). The undigested lactose passes to the colon where it is attacked by resident lactose fermenters (Adams & Moss, 2000). Colonic lactose fermentation results in high levels of glucose in blood and hydrogen gas in breath (Buller & Grand, 1990; Mombelli & Gismondo, 2000; Scrimshaw & Murray, 1988; Shah, 1993; Vesa et al., 2000). Probiotics strains and the traditional yoghurt cultures, *Lactobacillus delbrueckii* spp. *bulgaricus* and *Streptococcus thermophilus* produce β-Dgalactosidase thereby improving tolerance to lactose (Adams & Moss, 2000; Fooks et al.,

Constipation, a disorder of motor activity of the large bowel characterized by bowel movements that are less frequent than normal (Salminen et al., 1998b), pain during defecation, abnormal swelling and incomplete emptying of colon contents (Salminen et al., 1998a), can also be relieved by probiotic use. *Lactobacillus reuteri, Lactobacillus rhamnosus* and *Propionibacterium freudenreichii* are probiotic strains shown to improve the condition

Incidences of antibiotic associated diarrhoea caused by *Clostridium difficile* (Fuller, 2003; Tuohy et al., 2003; Vasiljevic & Shah, 2008) and rotavirus diarrhoea (Salminen et al., 1998a) can also reduced by administration of probiotics. Strains associated with reduction of diarrhoea include *Bifidobacterium* spp, *B*. *animalis* Bb12 (Fuller, 2003, Guandalini et al., 2000), *L*. *rhamnosus* GG, *L*. *acidophilus, L. bulgaricus* (Fuller, 2003; Goldin, 1998; Gorbach, 2000; Sazawal et al., 2006) and *Saccharomyces boulardii* (Kotowska et al., 2005, Sazawal et al., 2006). The effect of probiotics against diarrhoea is the most researched and substantiated claim, with documented clinical applications (BergogneBérézin, 2000; Cremonini et al., 2002; Marteau et al., 2001, McNaught & MacFie, 2001; Reid et al., 2003;

Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are other intestinal disorders that can be treated with varying degrees of success using probiotics. IBD is a collection of disorders including ulcerative colitis, Crohn's disease and pouchitis, characterized by chronic or recurrent inflammation, ulceration and abnormal narrowing of the GIT resulting in abdominal pain, diarrhoea and gastrointestinal bleeding (Hanauer, 2006; Marteau et al., 2001). IBS is typically characterized by abdominal pain, excessive flatus, variable bowel habit and bloating (Madden & Hunter, 2002). Several studies have been conducted to investigate the efficacy of probiotics in treatment of IBD (Guandalini, 2002; Ma et al., 2004; Zhang et al., 2005). The tested strains against IBD include among others VSL#3 probiotic (Gionchetti et al., 2000), *Bifidobacterium longum (*Furrie et al., 2005) and *Lactobacillus rhamnosus* GG *(*Gupta et al., 2000). Combination of *Lactobacillus acidophilus and Bifidobacterium infantis* (Hoyos, 1999) and of *Bifidobacterium bifidus*, *Bifidobacterium infantis* and *Streptococcus thermophilus* were shown to reduce incidences of ulcerative colitis (Bin-Nun et al., 2005). Several studies reported the success of bifidobacteria for the alleviation of IBS (O'Mahony et al., 2005; Brenner et al., 2009; Jankovic et al., 2010). Alfredo (2004)

inflammatory benefit when administered parenterally (Shiel et al., 2004).

1999, Shah, 2000c)

(Ouwenhand et al., 2002).

Sullivan & Nord 2005).
