**2.4.1 Nutritional benefits**

Microbial action in the gut, specifically by beneficial cultures, has been shown to enhance the bioavailability, quantity and digestibility of certain nutrients (Parvez et al., 2006). Ingestion of probiotics is associated with improved production of riboflavin, niacin, thiamine, vitamin B6, vitamin B12 and folic acid (Gorbach, 1997; Hargrove & Alford, 1978). Probiotics play a role in increasing bioavailability of calcium, iron, manganese, copper, phosphorous (Alm, 1982; McDonough et al., 1983) and increase the digestibility of protein and fat in yoghurt (Fernandes et al., 1987). Enzymatic hydrolysis of protein and fat leads to an increase in free amino acids and short chain fatty acids (SCFAs). Organic acids such as acetate and lactate produced during fermentation by LAB lower the pH of intestinal contents thereby creating undesirable conditions for harmful bacteria (Mack et al., 1999; Parvez et al., 2006).

#### **2.4.2 Therapeutic benefits**

Patients prefer medicine with little or no side effects for treatment of their ailments. Probiotics provide such an alternative, being living, non-pathogenic organisms, which are extremely safe as indicated by their GRAS status. Probiotic bacteria are claimed to alleviate and prevent conditions such as lactose intolerance, allergies, diarrheal diseases, lowering of serum cholesterol, reduction of the risk associated with mutagenicity and carcinogenicity and inhibition of pathogens, as well as stimulation of the immune system (Collins & Gibson,

Probiotics can be consumed either as food components or as non-food preparations (Stanton et al., 1998). Foods containing probiotics are referred to by others as functional foods. This refers to foods with nutrient or non-nutrient components that affect targeted function(s) in the body resulting in a positive health effect (Bellisle et al., 1998). Thus, functional foods have a physiological or psychological effect beyond basic nutritional value (Clydesdale, 1997). Several probiotic LAB strains are available to consumers in both traditional fermented foods and in supplemented form (Kourkoutas et al., 2005). The majority of probiotics are incorporated into dairy products such as milk powders, yoghurt, soft-, semi-hard and hard cheeses and ice cream (Desmond et al., 2005; Dinakar & Mistry, 1994; Stanton et al., 2001; Stanton et al., 2005). These products offer a suitable environment for probiotic viability and growth (Özer et al., 2009; Ross et al., 2002). There is an increase in use of other foods as vehicles for probiotics. This is partly due to allergenicity of some consumers to milk products. Non-dairy products such as malt-based beverages and fruit juices (Champagne & Raymond, 2008; Rozada-Sanchez et al., 2007; Sheehan et al., 2007), meat sausages (Ruiz-Moyano et al., 2008), capsules, and freeze-dried preparations (Berni-Carnani et al., 2007) are among these alternatives. Growing vegetarian alternatives have also led to soy-based probiotic foods (Farmworth et al., 2007). Recently, Aragon-Alegro et al. (2007) added

The benefits attributed to probiotics can either be nutritional or therapeutic (Prasad et al.,

Microbial action in the gut, specifically by beneficial cultures, has been shown to enhance the bioavailability, quantity and digestibility of certain nutrients (Parvez et al., 2006). Ingestion of probiotics is associated with improved production of riboflavin, niacin, thiamine, vitamin B6, vitamin B12 and folic acid (Gorbach, 1997; Hargrove & Alford, 1978). Probiotics play a role in increasing bioavailability of calcium, iron, manganese, copper, phosphorous (Alm, 1982; McDonough et al., 1983) and increase the digestibility of protein and fat in yoghurt (Fernandes et al., 1987). Enzymatic hydrolysis of protein and fat leads to an increase in free amino acids and short chain fatty acids (SCFAs). Organic acids such as acetate and lactate produced during fermentation by LAB lower the pH of intestinal contents thereby creating undesirable conditions for harmful bacteria (Mack et al., 1999;

Patients prefer medicine with little or no side effects for treatment of their ailments. Probiotics provide such an alternative, being living, non-pathogenic organisms, which are extremely safe as indicated by their GRAS status. Probiotic bacteria are claimed to alleviate and prevent conditions such as lactose intolerance, allergies, diarrheal diseases, lowering of serum cholesterol, reduction of the risk associated with mutagenicity and carcinogenicity and inhibition of pathogens, as well as stimulation of the immune system (Collins & Gibson,

1998). Benefits associated are, however, strain specific (Saarela et al., 2000).

**2.3 Probiotic products** 

probiotic chocolate mousse to the list of alternatives.

**2.4 Beneficial effects of probiotics** 

**2.4.1 Nutritional benefits** 

Parvez et al., 2006).

**2.4.2 Therapeutic benefits** 

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

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., 1999, Shah, 2000c)

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 (Ouwenhand et al., 2002).

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; Sullivan & Nord 2005).

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)

Probiotics – What They Are, Their Benefits and Challenges 27

2000). Probiotic organisms produce enzymes that help in digestion of proteins, fats and lactose (Frost & Sullivan, 2000). They also produce β-galactosidase, an enzyme that aid lactose intolerant individuals with breaking down or digestion of lactose (Krasaekoopt et al., 2003).

Production of short chain fatty acids in the colon during fermentation by colonic microflora is the main process that prevents colorectal cancer (Holzapfel & Schillinger, 2002). Probiotic strains also reduce levels of some colonic enzymes such as glucoronidase, β-glucoronidase nitroreductase, azoreductase (Adams & Moss, 2000; Chen & Yao, 2002; Fooks et al., 1999; Gorbach, 2000) and glycoholic acid hydrolase. These enzymes convert procarcinogens to carcinogens such as nitrosamine or secondary bile acids (Chen & Yao, 2002). Low levels of these enzymes therefore decrease chances of cancer development in the colon (Gorbach,

The methods used for detection of viable probiotic cells include conventional plate counts (culture dependent) and molecular techniques (culture-independent). The culture dependent method has been criticized for underestimation of counts due to bacteria forming chains and/or clumping and unsuitability (inappropriateness) of media for growing of viable but non-culturable cells (Auty et al., 2001; Lahtinen et al., 2006; Veal et al., 2000). Isolation media used may be insufficiently selective, affecting the reproducibility of results (Roy, 2001). These limitations of plate counting techniques prompted the use of molecular techniques and other alternative methods (Vitali et al., 2003). New methods include molecular based techniques such as quantitative real-time polymerase chain reaction (PCR), fluorescent *in situ* hybridization (FISH) (Boulos et al., 1999; Veal et al., 2000), confocal scanning laser microscopy (CSLM) (Auty et al., 2001; Gardiner et al., 2000; Palencia et al., 2008), flow cytometry (Alakomi et al., 2005) and microplate scale fluorochrome staining

Flow cytometry is a rapid and sensitive technique that measures physiological characteristics such as membrane integrity, enzyme activity, respiration, membrane potential and intracellular pH (Bunthof et al., 2001) of each cell individually (Bunthoff & Abee, 2002). Microplate scale fluorochrome staining assay is appropriate for assessing viability of fresh, freeze-dried and stressed cells. It can detect changes in the condition of probiotic cells earlier than can be done with conventional cultivation methods (Filoche et al.,

The fluorescence based molecular techniques are used in conjunction with viability staining techniques. A number of commercial techniques are available. LIVE/DEAD® *Bac*Light™ and BD Cell viability assay kit (BD Biosciences, Oxford, UK) are some examples. LIVE/DEAD® *Bac*Light consists of two nucleic acid stains SYTO® 9 and propidium iodide (PI). Green-fluorescent SYTO9 (excitation and emission maxima, 480 and 500 nm, respectively) penetrates both viable and nonviable cells. Red-fluorescent PI (excitation and emission maxima, 490 and 635 nm, respectively) penetrates cells with damaged cell membranes (Auty et al., 2001). The BD Cell viability assay kit (BD Biosciences, Oxford, UK) contains the stains, thiazole orange and propidium iodide (Doherty et al., 2009). Cells stained using these kits can also be assessed using microscopes, which will also distinguish between 'live' (e.g. green-stained) from 'dead' (e.g. red-stained) cells (Berney et al., 2007).

2000; Kasper, 1998).

**2.6 Methods for quantification of probiotic cultures** 

assay (Filoche et al., 2007; Mättö et al., 2006).

2007; Mättö et al., 2006).

demonstrated the efficacy of *Lactobacillus plantarum* LP01 and *Bifidobaccterium breve* BR0 as short-term therapy for IBS. Although some of the results obtained were very encouraging, there is need for larger, randomized, double-blinded, placebo-controlled clinical trials to substantiate these claims.

Hereditary allergic conditions of increasing importance in developing countries such as eczema, asthma, atopic dermatitis and rhinitis can be treated with probiotics (Holgate, 1999; Kalliomaki et al., 2003; Salminen et al., 1998a). Tested probiotics with antiallergenic properties include *Bifidobacterium lactis* Bb-12 (Isolauri et al., 2000) and *Lactobacillus* GG (Isolauri et al., 2000; Kalliomaki et al., 2001; Kalliomaki et al., 2003; Lee et al., 2008; Mirkin, 2002; Vanderhoof & Young, 2003). However, contradictory studies report on the poor efficiency of probiotics in allergy alleviation (Helin et al., 2002; Vliagoftis et al., 2008) and highlight the need for more convincing and conclusive research in allergy treatment.

Probiotics have the ability to lower levels of cholesterol in serum, contributing to the prevention of cardiovascular disease (Fooks et al., 1999; Proviva, 2002). This ability has been shown for *Lactobacillus johnsonii* and *L. reuterii* using animal models (Mombelli & Gismondo, 2000). They also reduce the risk of cancer (Sanders, 1999) due to their activity against certain tumors (Chen & Yao, 2002). Several studies indicated that probiotics in a diet reduces the risk of cancer (Sanders, 1999). Anticarcinogenic effects of *Bifidobacterium bifidum* and *Lactobacillus acidophilus* were shown using clinical trials in humans (Fooks, 1999).
