**3.1. Methods to increase survival and viability of probiotics**

Researchers have long been encouraged to find new, efficient methods of improving the viability of probiotics in food products (especially fermented types), since viability can be affected by the acidic-bile conditions of the gastrointestinal tract (Mortazavian et al., 2007). The latest developments focus on fermentation technologies for producing probiotic bacteria; new approaches for enhancing the performance of these fastidious organisms during fermentation, downstream processing, and utilization in commercial products; and improving functionality in the gut. Processes to optimize survival and functionality in the gut include sublethal stress applications during cell production and new fermentation technologies, such as immobilized cell biolm-type fermentations, are promising in this respect (Lacroix & Yildirim, 2007).

### *3.1.1. Immobilized cell biofilm*

Cell immobilization in fermentations is an attractive and rapidly expanding research area because of its technical and economic advantages, compared to a free cell system (Stewart & Russell, 1986). The immobilization method is cheap, simple and easy (Kourkoutas et al., 2006). The technology of cell immobilization allows an increase in cell stability and a decrease of the lethal effect on the microbial cells, providing protection from the conditions of the environment (Champagne et al., 1994; Grosso & Fávaro-Trindade, 2004). Thus immobilization techniques could provide protection to acid-sensitive LAB and increase their survival rate during the shelf life of the yoghurt and during their passage through the gastrointestinal tract (Cui et al., 2000; Fávaro-Trindade & Grosso, 2002). Kushal et al. (2006) determined that the process of co-immobilization of probiotic strains of *L. acidophilus* NCDC 13 and *B. bidum* NCDC 255 resulted in better protection of the viability of the cultures during transit through the gastrointestinal tract. In another study conducted by Kourkoutas et al. (2006), *L. casei* cells were immobilized on apple pieces and the immobilized biocatalysts were used separately as adjuncts in producing probiotic fermented milk. The results showed that the immobilized biocatalyst was able to ferment after storage for 15, 98 and 129 days at 4 °C, while no infection was reported during storage periods. Denkova et al. (2007) determined that the immobilization of the cells of *L. acidophilus* A., *L. helveticus* H., *L. casei* subsp. *casei* C. and *L. plantarum* 226-15 in chitosan resulted in preparations with high concentration of viable cells. The immobilized LAB in the chitosan gel beads was resistant to the model conditions of digestion: low and neutral values of pH, enzyme presence, and high concentrations of bile salts.

### *3.1.2. Encapsulation*

Encapsulation is the process of forming a continuous coating around an inner matrix that is wholly contained within the capsule wall as a core of encapsulated material (Kailasapathy, 2002). Encapsulation occurs naturally when bacterial cells grow and produce exopolysaccharides. The microbial cells are entrapped within their own secretions that act as a protective structure or a capsule, reducing the permeability of material through the capsule, and making it less exposed to adverse environmental factors. Many LAB synthesise exopolysaccharides, but they produce insufficient amounts to encapsulate themselves fully (Shah, 2002). Encapsulating probiotics in hydrocolloid beads has been investigated as a means of improving their viability and survival in food products and in the intestinal tract (Picot & Lacroix, 2004). Other benefits of encapsulation include reduction of cell injury, protection of probiotics from bacteriophages (Steenson et al., 1987), increased survival during freeze-drying and freezing (Kim & Yoon, 1995), and greater stability during storage (Kebary et al., 1998). Several methods of encapsulation have been used on probiotics in fermented milk products and biomass production: emulsion or two phase systems, the extrusion or droplet method, and spray drying and spray coating (Mortazavian et al., 2007). The common materials used for microencapsulation of probiotics are alginate and its derivatives, starch, mixtures of xanthan-gelan, carrageenan and its mixtures, gelatin, cellulose acetate phethalate, chitosan, and miscellaneous compounds such as whey proteins, soybean oil, gums, wax, and calcium chloride (Rao et al., 1989, Picot & Lacroix, 2004, Chandramouli et al., 2004).

250 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

*3.1.1. Immobilized cell biofilm* 

concentrations of bile salts.

*3.1.2. Encapsulation* 

gut. Such potentially probiotic bacteria colonizing the intestinal mucosa provide a barrier effect against pathogens by using a variety of mechanisms, such as occupation of niches,

Researchers have long been encouraged to find new, efficient methods of improving the viability of probiotics in food products (especially fermented types), since viability can be affected by the acidic-bile conditions of the gastrointestinal tract (Mortazavian et al., 2007). The latest developments focus on fermentation technologies for producing probiotic bacteria; new approaches for enhancing the performance of these fastidious organisms during fermentation, downstream processing, and utilization in commercial products; and improving functionality in the gut. Processes to optimize survival and functionality in the gut include sublethal stress applications during cell production and new fermentation technologies, such as immobilized

cell biolm-type fermentations, are promising in this respect (Lacroix & Yildirim, 2007).

Cell immobilization in fermentations is an attractive and rapidly expanding research area because of its technical and economic advantages, compared to a free cell system (Stewart & Russell, 1986). The immobilization method is cheap, simple and easy (Kourkoutas et al., 2006). The technology of cell immobilization allows an increase in cell stability and a decrease of the lethal effect on the microbial cells, providing protection from the conditions of the environment (Champagne et al., 1994; Grosso & Fávaro-Trindade, 2004). Thus immobilization techniques could provide protection to acid-sensitive LAB and increase their survival rate during the shelf life of the yoghurt and during their passage through the gastrointestinal tract (Cui et al., 2000; Fávaro-Trindade & Grosso, 2002). Kushal et al. (2006) determined that the process of co-immobilization of probiotic strains of *L. acidophilus* NCDC 13 and *B. bidum* NCDC 255 resulted in better protection of the viability of the cultures during transit through the gastrointestinal tract. In another study conducted by Kourkoutas et al. (2006), *L. casei* cells were immobilized on apple pieces and the immobilized biocatalysts were used separately as adjuncts in producing probiotic fermented milk. The results showed that the immobilized biocatalyst was able to ferment after storage for 15, 98 and 129 days at 4 °C, while no infection was reported during storage periods. Denkova et al. (2007) determined that the immobilization of the cells of *L. acidophilus* A., *L. helveticus* H., *L. casei* subsp. *casei* C. and *L. plantarum* 226-15 in chitosan resulted in preparations with high concentration of viable cells. The immobilized LAB in the chitosan gel beads was resistant to the model conditions of digestion: low and neutral values of pH, enzyme presence, and high

Encapsulation is the process of forming a continuous coating around an inner matrix that is wholly contained within the capsule wall as a core of encapsulated material (Kailasapathy,

competition for nutrients, and production of antimicrobials (Ouwehand et al., 2001).

**3.1. Methods to increase survival and viability of probiotics** 

Hou et al. (2003) demonstrated that encapsulation of *L. delbrueckii* spp. *bulgaricus* increased their bile tolerance, and viability was elevated by approximately four log units after encapsulation within artificial sesame oil emulsions. Encapsulation in spray dried whey protein microcapsules improved survival of *B. breve* R070 but not that of *B. longum* R023 during refrigerated storage in yoghurt (Picot & Lacroix, 2004). Ding & Shah (2007) stated that encapsulation improved the survival of probiotic bacteria including *L. rhamnosus*, *B. longum*, *L. salivarius*, *L. plantarum*, *L. acidophilus*, *L. paracasei*, *B. lactis* type Bl-O4, and *B. lactis* type Bi-07 when exposed to acidic conditions, bile salts, and mild heat treatment. Capela et al. (2006) found improved viability of probiotic organisms encapsulated in 3% v/w sodium alginate in freeze-dried yogurt after 6 months of storage at 4 and 21°C. Ozer et al. (2009) studied the viability of encapsulated bacteria in whitebrined cheese; the researchers used *B. bifidum* BB-12 and *L. acidophilus* LA-5 that had been encapsulated in Na-alginate by either an extrusion or an emulsion technique. Both encapsulation techniques were found to be effective in keeping the numbers of probiotic bacteria higher than the level of the therapeutic minimum. While the counts of nonencapsulated probiotic bacteria decreased approximately by 3 logs, the decrease was more limited in the cheeses containing microencapsulated cells (approximately 1 log). Khater et al. (2010) tested the ability of twelve non-encapsulated and encapsulated lactic acid and bifidobacteria strains to assimilate cholesterol and to survive at a low pH and fairly high bile concentrations. The results obtained declared that encapsulation effectively protected the microorganisms from the hostile environment in the GIT, thus potentially preventing cell loss. The assimilative reductions of cholesterol by non-encapsulated and encapsulated strains were clearly different, varying from 32.6% to 89.3% and 27.9% to 85.1% respectively. Kim et al. (2008) stated that encapsulation reduces the ability of LAB to assimilate cholesterol.
