**2.8 Methods for improving probiotic viability**

In view of the health benefits associated with probiotics, it is not surprising that there is an increasing interest in their viability. The common practice is storage at refrigerated temperatures to prolong their shelf life (Saxelin et al., 1999). Nevertheless, most of them still have a short shelf-life (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).

Viability of probiotics is reduced as a result of their exposure to high temperature, oxygen, low pH and light (Bell, 2001; Chen et al., 2006; Gomes & Malcata, 1999). Naturally many LAB may excrete exo-polysaccharides (EPS) to protect themselves from harsh conditions. However, protection afforded by these EPS is not sufficient (Shah, 2002). As a result, researchers are continuously searching for ways to improve survival of probiotic cultures during processing, storage and GIT transit. Different approaches are used in an attempt to preserve viability of probiotic cultures. These include among others, pre-exposure of probiotic cultures to sub-lethal stresses (Desmond et al., 2002) and incorporation of micronutrients such as peptides and amino acids (Shah, 2000). The disadvantage of pre-exposure to sublethal stresses is that it may result in significant decreases in cellular activity, cell yield and process volumetric productivity (Doleyres & Lacroix, 2005). Other alternative methods for improving probiotic viability are genetic modification (Sheehan et al., 2006; Sheehan et al., 2007; Sleator & Hill, 2007), immobilization (Doleyres et al., 2004), two-step fermentations,

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

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.,

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

In view of the health benefits associated with probiotics, it is not surprising that there is an increasing interest in their viability. The common practice is storage at refrigerated temperatures to prolong their shelf life (Saxelin et al., 1999). Nevertheless, most of them still have a short shelf-life (Lee & Salminen, 1995). There is low recovery of viable bacteria in products claiming to contain probiotic bacteria (Hamilton-Miller et al., 1999; Temmerman et

Viability of probiotics is reduced as a result of their exposure to high temperature, oxygen, low pH and light (Bell, 2001; Chen et al., 2006; Gomes & Malcata, 1999). Naturally many LAB may excrete exo-polysaccharides (EPS) to protect themselves from harsh conditions. However, protection afforded by these EPS is not sufficient (Shah, 2002). As a result, researchers are continuously searching for ways to improve survival of probiotic cultures during processing, storage and GIT transit. Different approaches are used in an attempt to preserve viability of probiotic cultures. These include among others, pre-exposure of probiotic cultures to sub-lethal stresses (Desmond et al., 2002) and incorporation of micronutrients such as peptides and amino acids (Shah, 2000). The disadvantage of pre-exposure to sublethal stresses is that it may result in significant decreases in cellular activity, cell yield and process volumetric productivity (Doleyres & Lacroix, 2005). Other alternative methods for improving probiotic viability are genetic modification (Sheehan et al., 2006; Sheehan et al., 2007; Sleator & Hill, 2007), immobilization (Doleyres et al., 2004), two-step fermentations,

detrimental to the viability of these anaerobic bacteria (Talwakar et al., 2001).

1999; Temmerman et al., 2003a).

al., 2003a).

to give them full protection (Shah, 2002).

**2.8 Methods for improving probiotic viability** 

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

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

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 al., 2003).

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 et al., 2003).

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).

Probiotics – What They Are, Their Benefits and Challenges 33

Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of colonic bacteria and thereby improve host health (Femia et al., 2002; Gibson & Roberfroid, 1995; Roberfroid, 1998; Theuer et al., 1998; Tuohy et al., 2003; Young, 1998). Gibson et al. (2004) redefined them as 'a selectively fermented ingredient that allows specific changes; both in the composition and/or activity in the gastrointestinal microflora that confers benefits to host well-being and health'. The refined definition takes into account both the microbial changes and the nutritional and physiological benefits attributed to prebiotics. Just like probiotics, they modulate the composition of the natural ecosystem though probiotics does so through introduction of exogenous bacteria into the colon (Bouhnik et al., 2004). Prebiotics pass through the upper GIT unfermented and are only utilized in the colon and are therefore called 'colonic food' (Roberfroid, 2000). Non-digestibility can be demonstrated *in vitro* by subjecting the carbohydrates to pancreatic and small intestinal enzymes. It can be shown *in vivo* on human subjects with an ileostomy (i.e people who have had their large intestine removed and have a stoma at the end of the ileum) (van Loo et al., 1999). Compounds that are not digested and absorbed by the host but are preferentially fermented by *Bifidobacterium* species in the colon are called 'bifidogenic' factors (Shah, 2007). Prebiotics are not readily digested by pathogenic bacteria (Annika et al., 2002; Farmer, 2002; Femia et al., 2002). They favour or promote growth of potentially health-promoting bacteria such as lactobacilli and bifidobacteria, thereby allowing them to be predominant (Bouhnik et al., 2004; Flamm et al., 2001; Gibson et al., 1999; Roberfroid, 1998; Wang, 2009). This subsequently leads to predominant numbers of the stimulated endogenous bacteria in faeces as well (Femia et al., 2002; Losada & Olleros, 2002). Scientific studies in Japan indicated that consumption of prebiotics increases the populations of bifidobacteria and other beneficial microorganisms even in the absence of probiotics in diet. The selective stimulation of growth of bifidobacteria by prebiotics is characterized by a substantial decrease in numbers of

The following criteria are used for selection of a carbohydrate as a prebiotic: It must not be absorbed in the stomach or small intestine; It must be selectively fermented by the beneficial gut microflora; It should also stimulate the growth and/or activity of beneficial bacteria; Its fermentation should induce the beneficial luminal or systemic effects within the host; It must be resistant to gastric acidity and mammalian enzyme hydrolysis (Kolida, et al., 2002;

Classes of non-digestible oligosaccharides (NDOs) commercially available are cyclodextrins, fructooligosaccharides (FOS), gentiooligosaccharides, glycosylsucrose and isomaltulose (also known as palatinose). Other classes include lactulose, lactosucrose and maltooligosaccharides (Sako et al., 1999). NDOs such as inulin, fructooligosaccharide, lactulose and dietary fibre are common prebiotics (Davis & Milner, 2009). There are conflicting views on the prebiotic classification of resistant starch. Some researchers classify it as a prebiotic (Douglas & Sanders, 2008) while others (Shah, 2004) differ, arguing that resistant starch is not digested by some beneficial bacteria, and therefore cannot be classified as a prebiotic. Inulin and FOS are the only NDOs that have been sufficiently studied to give adequate data to analyze their functional properties (Roberfroid, 2000). Galacto-oligosaccharides (GOS), glucooligosaccharides, lactulose, isomalto-oligosaccharides, raffinose, transgalacto-oligosaccharides,

potentially pathogenic bacteria (Losada & Olleros, 2002).

Manning and Gibson, 2004).

**3. Prebiotics** 

Progress towards elimination of use of organic solvents has been made with development of microencapsulation technique using supercritical technology (Moolman et al., 2006). This microencapsulation technique is based on the formation of an interpolymer complex between poly (vinyl pyrrolidone) (PVP) and poly (vinyl acetate-co-crotonic acid) (pVA-CA) in supercritical carbon dioxide (scCO2). A supercritical substance is neither a gas nor a liquid but possesses properties of both, making it unique (Moshashaee et al., 2000). Since supercritical fluids have a wide spectrum of solvent characteristics, they can be used as solvents in different techniques (Frederiksen et al., 1997). Microparticles produced using this method have suitable morphological characteristics, encapsulation efficiency and affords encapsulated probiotic cultures protection in simulated gastrointestinal fluids (Mamvura et al., 2011; Thantsha et al., 2009).
