**2.5 Mechanism of action of probiotics**

Probiotic bacteria beneficially affect the individual by improving the properties of the indigenous microflora and its microintestinal balance (Betoret et al., 2003; Frost & Sullivan, 2000; Matilla-Sandholm et al., 2002; Saarela et al., 2000). They compete with disease causing bacteria for villi attachment sites and nutrients (Chen & Yao, 2002). Probiotic bacterial cultures encourage growth of beneficial microorganisms and crowd out potentially harmful bacteria thereby reinforcing the body's natural defence mechanisms (Saarela et al., 2000). They provide specific health benefits by modifying gut microflora, strengthening gut mucosal barrier, e.g. adherence of probiotics to the intestinal mucosa thereby preventing pathogen adherence, pathogen inactivation, modification of dietary proteins by intestinal microflora, modification of bacterial enzyme activity, and influence on gut mucosal permeability, and regulation of the immune system (Betoret et al., 2003; Krasaekoopt et al., 2003; Salminen et al., 1998).

The probiotic effect is accredited to their production of metabolic by-products such as acid, hydrogen peroxide, bacteriocins, e.g. lactocidin, and acidophilin that manifest antibiotic properties and inhibit the growth of a wide spectrum of pathogens and/or potential pathogens such as *Escherichia coli*, *Klebsiella*, *Enterobacter*, *Pseudomonas*, *Salmonella*, *Serratia* and *Bacteroides* (Chen & Yao, 2002; Krasaekoopt et al., 2003). Lactic acid bacteria inhibit growth of pathogenic microorganisms by producing short chain fatty acids such as acetic, propionic, butyric as well as lactic and formic acids which reduces intestinal pH. Lactic acid produced by bifidobacteria in substantial amounts has antimicrobial activity against yeasts, moulds and bacteria (Adams & Moss, 2000; Percival, 1997). These species are also active in reducing the faecal activity of enzymes implicated in the production of genotoxic metabolites such as βglucoronidase and glycolic acid hydroxylase (Collins & Hall, 1984; Mombelli & Gismondo,

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

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.

*Lactobacillus acidophilus* were shown using clinical trials in humans (Fooks, 1999).

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

Probiotic bacteria beneficially affect the individual by improving the properties of the indigenous microflora and its microintestinal balance (Betoret et al., 2003; Frost & Sullivan, 2000; Matilla-Sandholm et al., 2002; Saarela et al., 2000). They compete with disease causing bacteria for villi attachment sites and nutrients (Chen & Yao, 2002). Probiotic bacterial cultures encourage growth of beneficial microorganisms and crowd out potentially harmful bacteria thereby reinforcing the body's natural defence mechanisms (Saarela et al., 2000). They provide specific health benefits by modifying gut microflora, strengthening gut mucosal barrier, e.g. adherence of probiotics to the intestinal mucosa thereby preventing pathogen adherence, pathogen inactivation, modification of dietary proteins by intestinal microflora, modification of bacterial enzyme activity, and influence on gut mucosal permeability, and regulation of the immune system (Betoret et al., 2003; Krasaekoopt et al.,

The probiotic effect is accredited to their production of metabolic by-products such as acid, hydrogen peroxide, bacteriocins, e.g. lactocidin, and acidophilin that manifest antibiotic properties and inhibit the growth of a wide spectrum of pathogens and/or potential pathogens such as *Escherichia coli*, *Klebsiella*, *Enterobacter*, *Pseudomonas*, *Salmonella*, *Serratia* and *Bacteroides* (Chen & Yao, 2002; Krasaekoopt et al., 2003). Lactic acid bacteria inhibit growth of pathogenic microorganisms by producing short chain fatty acids such as acetic, propionic, butyric as well as lactic and formic acids which reduces intestinal pH. Lactic acid produced by bifidobacteria in substantial amounts has antimicrobial activity against yeasts, moulds and bacteria (Adams & Moss, 2000; Percival, 1997). These species are also active in reducing the faecal activity of enzymes implicated in the production of genotoxic metabolites such as βglucoronidase and glycolic acid hydroxylase (Collins & Hall, 1984; Mombelli & Gismondo,

substantiate these claims.

**2.5 Mechanism of action of probiotics** 

2003; Salminen et al., 1998).

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, 2000; Kasper, 1998).

#### **2.6 Methods for quantification of probiotic cultures**

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

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., 2007; Mättö et al., 2006).

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).

Probiotics – What They Are, Their Benefits and Challenges 29

organisms per gram) was approved by countries of MERCOSUR which includes Argentina, Paraguay, Brazil and Uruguay (Krasaekoopt et al., 2003). In products containing multiple probiotic organisms, at least a million of each of them per gram should be present to produce required beneficial effects (Wahlqvist, 2002). In Japan, a minimum of 107 viable cells per millilitre of fresh dairy product is required. The South African legislation states that functional foods containing probiotic bacteria must deliver 1 x 108 bacterial cells per day. A daily intake of 109 to 1010 cfu viable cells is considered the minimum dose shown to have positive effects on host health (Fasoli et al., 2003). This could be achieved by consuming 100 g of a product containing between 106 and 107 viable cells g-1 daily (Boylston et al., 2004).

Low viability of probiotic cultures in yoghurt has been reported (Kailasapathy & Rybka,

Retention of viability of the probiotic bacteria presents a major marketing and technological challenge for application of probiotic cultures in functional foods (Desmond et al., 2002; Mattila-Sandholm et al., 2002). Many active cultures die during manufacturing, storage or transport of the finished product (Siuta-Cruce & Goulet, 2001) and also during the passage to the intestine (Sakai et al., 1987; Siuta-Cruce & Goulet, 2001; Park et al., 2002). Thus, the majority die even before the consumer receives any of the health benefits (Siuta-Cruce & Goulet, 2001). A serious problem of shelf instability had been encountered with dried cultures. Refrigerated products also have short lives due to negative effects of low temperature and formation of crystals on bacterial cells. The numbers of viable bacteria continually decrease with time during refrigerated storage (Porubcan et al., 1975). Market surveys have revealed much lower counts in the products even before the expiry date (Talwalkar et al., 2001). Shelf life for probiotics is thus unpredictable; hence, the industry has had difficulty backing up label claims (Siuta-Cruce & Goulet, 2001). Excesses of 50 to 200 % cells have been incorporated into products in an attempt to make-up for cells that die during storage. For example, in tablets containing dry cells, where the tablets are labelled as containing a certain minimum count of active cells per tablet, to be safe, the manufacturer must incorporate an excess of cells at the time the tablets are manufactured, thereby assuring that the labelling will remain accurate while the product is in stock by the retailers. This practice increases the cost and makes the use instructions inaccurate (Porubcan et al.,

Probiotics, after surviving food processing, are upon consumption then exposed to conditions prevailing in the stomach and small intestine before they reach their site which is the colon (Siuta-Cruce & Goulet, 2001; Hansen et al., 2002; Lian et al., 2002). The microbes may die during their transit through the upper intestinal tract to the colon and therefore they may not be able to colonize the colon (Talwakar et al., 2001). They must therefore survive gastric acidity and bile salts which they encounter during their passage through the GIT (Hansen et al., 2002; Lian et al., 2002; Sakai et al., 1987; Siuta-Cruce & Goulet, 2001;). Their survival in the GI T depends on the strain and species-specific resistance to low pH (pH values ranging from 1.3 to 3.0) in gastric juice and to bile salts found in the small

Probiotic bacteria can only perform when they find adequate environmental conditions and when they are protected against stresses (e.g extreme temperatures, high pressure, shear forces) they encounter during their production at the industry level or in the GIT (gastric

1997; Lourens-Hattingh & Viljoen, 2001; Shah, 2000).

intestine (Hansen et al., 2002; Lian et al., 2002).

1975).

All the above mentioned methods have their own disadvantages. For example, the viability kits and real time PCR are based on bacterial DNA which is not only present in live cells but can also be retained by dead cells in significant amounts. Both PCR and FISH are not independent as they require determination of a standard curve which is determined most of the times using standard plate counts. PCR requires expensive reagents which cannot be afforded by everyone in the industry. Detection limits for PCR and FISH are relatively high, being about 104 cells/ml and 106 cells/ ml, respectively. FISH is based on detection of 16s rRNA whose presence is not a direct proof of metabolic activity but rather an indication of potential viability (Biggerstaff, 2006). Real-time PCR and FISH have a limitation whereby counts of bacteria decreased but PCR and FISH results remained higher over the experimental period. This is due to detection of high levels of rRNA and DNA in dead cells. The intensity of rRNA in dead cells may still be strong enough for visually counting (detection) though it is expected to decrease upon cell death. Thus, the RNA content of the cell detected by fluorescent probes cannot be regarded as reliable indicator of cellular viability (Vives-Rego et al., 2000). Also, real time PCR detects both viable and non-viable bacteria, thus does not provide information on the condition of the cells and results in an overestimation of metabolically active cells (Kramer et al., 2009; Masco et al., 2007).

The appropriateness of PCR for quantification of viable cells can be improved by staining the samples with DNA binding dyes prior to DNA extraction and amplification. Treatment with DNA-binding dyes and subsequent PCR analysis uses membrane integrity as the criterion in determining viability of cells. Live cells are able to exclude DNA-binding dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), while dead cells or those whose membrane integrity has been compromised are able to pick-up these stains (Kramer et al., 2009). These dyes form covalent bonds with DNA upon exposure to visible bright light and thus inhibit subsequent PCR amplification. Only DNA from live cells with intact membranes is selectively amplified (Nocker et al., 2009).

Despite some of its drawbacks, the plate count method is traditionally used to assess cell viability in probiotic preparations (Alakomi et al., 2005). Though plate counting is arduous and time consuming, no method has yet been found that completely replaces it. Therefore it is still being routinely used in assessing viability of probiotic cultures in various foods, often in conjunction with culture-independent methods (Lopez-Rubio et al., 2009; Masco et al., 2005; Temmerman et al., 2003b).

#### **2.7 Probiotic challenges**

Commercially, viable probiotic strains are incorporated into fermented food products or are supplied as freeze-dried supplements or pharmaceutical preparations (Holzapfel & Schillinger, 2002). The basic requirement for probiotics is that products should contain sufficient numbers of microorganisms up to the expiry date (Fasoli et al., 2003). Thus, probiotics must contain specific strains and maintain certain numbers of live cells for them to produce health benefits in the host (Mattila-Sandholm et al., 2002). Different countries have decided on the minimum number of viable cells required in the probiotic product for it to be beneficial. In Australia, a minimum viable count of 106 organisms per gram should be available in fermented milk products at the end of the shelf life (Wahlqvist, 2002). However, according to Krasaekoopt et al. (2003), there are no specifications as to how many probiotics should be available in Australian fermented products. The same minimum count (106

All the above mentioned methods have their own disadvantages. For example, the viability kits and real time PCR are based on bacterial DNA which is not only present in live cells but can also be retained by dead cells in significant amounts. Both PCR and FISH are not independent as they require determination of a standard curve which is determined most of the times using standard plate counts. PCR requires expensive reagents which cannot be afforded by everyone in the industry. Detection limits for PCR and FISH are relatively high, being about 104 cells/ml and 106 cells/ ml, respectively. FISH is based on detection of 16s rRNA whose presence is not a direct proof of metabolic activity but rather an indication of potential viability (Biggerstaff, 2006). Real-time PCR and FISH have a limitation whereby counts of bacteria decreased but PCR and FISH results remained higher over the experimental period. This is due to detection of high levels of rRNA and DNA in dead cells. The intensity of rRNA in dead cells may still be strong enough for visually counting (detection) though it is expected to decrease upon cell death. Thus, the RNA content of the cell detected by fluorescent probes cannot be regarded as reliable indicator of cellular viability (Vives-Rego et al., 2000). Also, real time PCR detects both viable and non-viable bacteria, thus does not provide information on the condition of the cells and results in an

overestimation of metabolically active cells (Kramer et al., 2009; Masco et al., 2007).

intact membranes is selectively amplified (Nocker et al., 2009).

2005; Temmerman et al., 2003b).

**2.7 Probiotic challenges** 

The appropriateness of PCR for quantification of viable cells can be improved by staining the samples with DNA binding dyes prior to DNA extraction and amplification. Treatment with DNA-binding dyes and subsequent PCR analysis uses membrane integrity as the criterion in determining viability of cells. Live cells are able to exclude DNA-binding dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), while dead cells or those whose membrane integrity has been compromised are able to pick-up these stains (Kramer et al., 2009). These dyes form covalent bonds with DNA upon exposure to visible bright light and thus inhibit subsequent PCR amplification. Only DNA from live cells with

Despite some of its drawbacks, the plate count method is traditionally used to assess cell viability in probiotic preparations (Alakomi et al., 2005). Though plate counting is arduous and time consuming, no method has yet been found that completely replaces it. Therefore it is still being routinely used in assessing viability of probiotic cultures in various foods, often in conjunction with culture-independent methods (Lopez-Rubio et al., 2009; Masco et al.,

Commercially, viable probiotic strains are incorporated into fermented food products or are supplied as freeze-dried supplements or pharmaceutical preparations (Holzapfel & Schillinger, 2002). The basic requirement for probiotics is that products should contain sufficient numbers of microorganisms up to the expiry date (Fasoli et al., 2003). Thus, probiotics must contain specific strains and maintain certain numbers of live cells for them to produce health benefits in the host (Mattila-Sandholm et al., 2002). Different countries have decided on the minimum number of viable cells required in the probiotic product for it to be beneficial. In Australia, a minimum viable count of 106 organisms per gram should be available in fermented milk products at the end of the shelf life (Wahlqvist, 2002). However, according to Krasaekoopt et al. (2003), there are no specifications as to how many probiotics should be available in Australian fermented products. The same minimum count (106

organisms per gram) was approved by countries of MERCOSUR which includes Argentina, Paraguay, Brazil and Uruguay (Krasaekoopt et al., 2003). In products containing multiple probiotic organisms, at least a million of each of them per gram should be present to produce required beneficial effects (Wahlqvist, 2002). In Japan, a minimum of 107 viable cells per millilitre of fresh dairy product is required. The South African legislation states that functional foods containing probiotic bacteria must deliver 1 x 108 bacterial cells per day. A daily intake of 109 to 1010 cfu viable cells is considered the minimum dose shown to have positive effects on host health (Fasoli et al., 2003). This could be achieved by consuming 100 g of a product containing between 106 and 107 viable cells g-1 daily (Boylston et al., 2004).

Low viability of probiotic cultures in yoghurt has been reported (Kailasapathy & Rybka, 1997; Lourens-Hattingh & Viljoen, 2001; Shah, 2000).

Retention of viability of the probiotic bacteria presents a major marketing and technological challenge for application of probiotic cultures in functional foods (Desmond et al., 2002; Mattila-Sandholm et al., 2002). Many active cultures die during manufacturing, storage or transport of the finished product (Siuta-Cruce & Goulet, 2001) and also during the passage to the intestine (Sakai et al., 1987; Siuta-Cruce & Goulet, 2001; Park et al., 2002). Thus, the majority die even before the consumer receives any of the health benefits (Siuta-Cruce & Goulet, 2001). A serious problem of shelf instability had been encountered with dried cultures. Refrigerated products also have short lives due to negative effects of low temperature and formation of crystals on bacterial cells. The numbers of viable bacteria continually decrease with time during refrigerated storage (Porubcan et al., 1975). Market surveys have revealed much lower counts in the products even before the expiry date (Talwalkar et al., 2001). Shelf life for probiotics is thus unpredictable; hence, the industry has had difficulty backing up label claims (Siuta-Cruce & Goulet, 2001). Excesses of 50 to 200 % cells have been incorporated into products in an attempt to make-up for cells that die during storage. For example, in tablets containing dry cells, where the tablets are labelled as containing a certain minimum count of active cells per tablet, to be safe, the manufacturer must incorporate an excess of cells at the time the tablets are manufactured, thereby assuring that the labelling will remain accurate while the product is in stock by the retailers. This practice increases the cost and makes the use instructions inaccurate (Porubcan et al., 1975).

Probiotics, after surviving food processing, are upon consumption then exposed to conditions prevailing in the stomach and small intestine before they reach their site which is the colon (Siuta-Cruce & Goulet, 2001; Hansen et al., 2002; Lian et al., 2002). The microbes may die during their transit through the upper intestinal tract to the colon and therefore they may not be able to colonize the colon (Talwakar et al., 2001). They must therefore survive gastric acidity and bile salts which they encounter during their passage through the GIT (Hansen et al., 2002; Lian et al., 2002; Sakai et al., 1987; Siuta-Cruce & Goulet, 2001;). Their survival in the GI T depends on the strain and species-specific resistance to low pH (pH values ranging from 1.3 to 3.0) in gastric juice and to bile salts found in the small intestine (Hansen et al., 2002; Lian et al., 2002).

Probiotic bacteria can only perform when they find adequate environmental conditions and when they are protected against stresses (e.g extreme temperatures, high pressure, shear forces) they encounter during their production at the industry level or in the GIT (gastric

Probiotics – What They Are, Their Benefits and Challenges 31

use of oxygen-impermeable containers and microencapsulation (Özer et al., 2009). Of these techniques, microencapsulation is relatively new and has been investigated by various

Microencapsulation is a process by which solids, liquids or gases are packaged into miniature, sealed microcapsules that can release their contents at controlled rates under influences of specified conditions (Anal et al., 2006; Anal & Singh, 2007). It stabilizes the probiotics, increases their survival during processing and storage, controls the oxidative reaction, ensures sustained or controlled release at a specific target site (both temporal and time-controlled release) and improves shelf life (Anal & Singh, 2007; Dembczynski & Jankowski, 2002). The encapsulated probiotics are released from the microparticles as a result of many factors such as changes in pH and/or temperature (Gibbs et al., 1999; Vasishtha, 2003). These changes may cause microcapsule walls to swell and rupture or

Microparticles reduce loss of probiotic cell viability by blocking reactive components such as atmospheric moisture, oxygen (Kim et al., 1988; Krasaekoopt et al., 2003; Reid, 2002; Siuta-Cruise & Goulet, 2001; Vasishtha, 2003), high temperature, pressure, bacteriophage attack and cryoeffects (Krasaekoopt et al., 2003). Studies have indicated that probiotic cultures enclosed within solid fat microcapsules retain both their activity and vitality (Krasaekoopt et

Methods of microencapsulation used in pharmaceutical and food industries are classified as either physical or chemical. Physical methods include pan coating, air-suspension coating, centrifugal extrusion, vibrational nozzle and spray drying (Anal & Singh, 2007), spray coating, annular jet, spinning disk, spray cooling, spray drying and spray chilling (Versic, 1988), extrusion coating, fluidized bed coating, liposome entrapment, coarcervation, inclusion complexation, centrifugal extrusion and rotational suspension separation (Vasishtha, 2003). Chemical methods include interfacial polymerization, *in-situ* polymerization, matrix polymerization(Vidhyalakshmi et al., 2009) and extrusion. Extrusion and emulsion techniques are the mostly commonly used methods (Krasaekoopt

There is a diversity of materials used for encapsulation of probiotics. These include among others, alginate (Chandramouli et al., 2004; Dembczynski & Jankowski, 2002; Hansen et al., 2002; Krasaekoopt et al., 2003; Sultana et al., 2000), к-carrageenan, locust bean gum, cellulose acetate phthalate, chitosan, gelatin (Krasaekoopt et al., 2003), cellulose (Chan & Zhang, 2002), pectin, whey protein (Guerin et al., 2003) and rennet (Heidibach et al., 2009). These materials are used either as supporting materials or gelling agents by different investigators. Although generally there are positive effects of microencapsulation, this method is not without disadvantages. Some types of the resulting microparticles may shrink and lose mechanical strength due to their sensitivity to acids (Sun & Griffiths, 2000), may present problems for large scale production, others require use of potassium ions that should not be taken in large amounts in diet, (Sun & Griffiths, 2000), some of the polysaccharides used are prohibited in specific foods in other countries (Picot & Lacroix, 2004). Additionally and possibly the key disadvantage, is that the mentioned microencapsulation methods use water and other organic solvents whose use is less favoured due to their high costs and concerns

about their negative environmental impacts (Sihvonen et al., 1999).

dissolve (Franjione & Vasishtha, 1995; Brannon-Peppas, 1997).

researchers.

al., 2003).

et al., 2003).

acids and bile salts) (Siuta-Cruce & Goulet, 2001). Factors affecting viability during storage such as temperature, moisture, light and air should also be taken into consideration (Percival, 1997; Mattila-Sandholm et al., 2002). Oxygen toxicity is another major problem in the survival of probiotic bacteria in dairy foods. High levels of oxygen in the product are detrimental to the viability of these anaerobic bacteria (Talwakar et al., 2001).

Manufacturers of probiotics are facing the challenge that they should produce probiotic cultures that can survive for long periods, and are resistant to acidity in the upper intestinal tract so that they can reach the colon in high numbers to colonize the epithelium. Probiotic cultures should therefore be produced in a way that will protect these sensitive bacteria from unfavourable interactions with detrimental factors (Siuta-Cruce & Goulet, 2001).

In view of the health benefits associated with probiotics, it is not surprising that there is increasing interest in their viability. Probiotics do not have a long shelf life in their active form. Refrigeration is required in most cases to maintain shelf life as high temperatures can destroy probiotic cultures (Saxelin et al., 1999). However, most probiotics still have a short shelf-life even under low temperature storage (Lee & Salminen, 1995). There is low recovery of viable bacteria in products claiming to contain probiotic bacteria (Hamilton-Miller et al., 1999; Temmerman et al., 2003a).

The preservation of these probiotic microorganisms presents a challenge because they are affected by exposure to temperature, oxygen and light (Bell, 2001; Chen et al., 2006). Survival of most bifidobacteria in most dairy products is poor due to low pH and/or exposure to oxygen (Gomes & Malcata, 1999). Naturally many LAB may excrete exopolysaccharides to protect themselves from harsh conditions but this is usually not enough to give them full protection (Shah, 2002).
