**3. Technological usage of probiotics**

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

activity, as demonstrated by *in vitro* competition test.

(202.4%).

& Wind, 2003).

logarithmic and stationary phases. The researchers found that the % of recovery in the logarithmic phase ranged from 1.0% to 43.8% in water for all tested strains, and from 80.5% to 197% in milk. They observed higher survival rates in the stationary phase for all strains. *L*. *acidophilus* subsp. *johnsonii* showed the highest survival rate in both water (93.9%) and milk

The safety of probiotic bacteria must be carefully assessed, with particular attention to transferable antibiotic resistance (Mathur & Singh, 2005). In the last decade, increasing concern has arisen about the safe use of LAB cultures for food and feed applications, in light of the latest knowledge about their possible role as an antibiotic-resistant gene reservoir. Particular concern is due to evidence of widespread occurrence in this bacterial group of conjugative plasmids and transposons (Clementi & Aquilanti, 2011). It is known that lactobacilli have a high natural resistance to bacitracin, cefoxitin, ciprofloxacin, fusidic acid, kanamycin, gentamicin, metronidazole, nitrofurantoin, norfloxacin, streptomycin, sulphadiazine, teicoplanin, trimethoprim/sulphamethoxazole, and vancomycin (Danielsen

One of the primary benets associated with probiotic bacterial cultures is that they can exclude pathogenic bacteria from the small and large intestine (Kos et al., 2008). Another benefit is that in food products, antimicrobial activity of probiotic bacteria may contribute to an improvement in the quality of fermented foods. This may result from control of spoilage and pathogenic bacteria, extension of shelf life, and improvement of sensory quality (Wei et al., 2006; Siripatrawan & Harte, 2007). Kos et al. (2008) used overnight cultures and cell-free supernatants of the three probiotic strains *L. acidophilus* M92, *L. plantarum* L4, and *E. faecium* L3 for determining the antagonistic effect against *Listeria monocytogenes*, *Salmonella typhimurium*, *Yersinia enterocolitica*, and *Acinetobacter calcoaceticus*. The researchers determined that probiotic strains *L. acidophilus* M92, *L. plantarum* L4, and *E. faecium* L3 demonstrated anti-*Salmonella* activity. *L. acidophilus* M92 was also shown to have antilisterial

Production of antimicrobial compounds, which may take part in the inhibition of intestinal pathogens, is another criterion for classifying a potentially probiotic bacteria **(**Hutt et al., 2006). The inhibition of pathogenic microorganisms by selected probiotic strains may occur via a) production of antibiotic-like substances, b) bacteriocins and bacteriocin-like inhibitory substances such as acidophilin and reuterin, c) lowering of pH by producing organic acids such as acetic, lactic and phenyllactic acid, d) production of hydrogen peroxide and short chain fatty acids, e) decreasing the redox potential, and f) consumption of available

The ability of LAB to adhere to epithelial cells and mucosal surfaces is thought to be an important property of many bacterial strains used as probiotics (FAO/WHO, 2001). Cell adhesion is a complex process involving contact between the bacterial cell membrane and interacting surfaces. Difficulties experienced in studying bacterial adhesion *in vivo*, especially in humans, have stimulated interest in the development of *in vitro* models for preliminary screening of potentially adherent strains (Duary et al., 2011). Attachment and

nutrients (Holzapfel et al., 1995; Ouwehand, 1998; Tharmaraj & Shah, 2009).

The use of starter cultures in the production of fermented food is necessary for guaranteeing safety and standardizing properties. LAB functions primarily to drop the pH of the batter; lower pH a) promotes product safety by inactivating pathogens, b) creates the biochemical conditions to attain the nal sensory properties through modication of the raw materials, and c) improves the product stability and shelf life by inhibiting undesirable changes brought about by spoilage microorganisms or abiotic reactions (Ammor & Mayo, 2007).

Functional starter cultures are dened as microbes that possess at least one inherently functional property aimed at improving the quality of the end product (De Vuyst, 2000). The use of probiotics in food has reinforced the acclaimed healthy properties and given rise to an increased consumption of these products in Europe and the USA (Kristo et al., 2003). Probiotics have been evaluated as functional starter cultures in various types of fermented food products such as yoghurt, cheese, dry sausage, salami, and sourdough. They have also been studied in therapeutic preparations to assess their positive effects on physico-chemical properties of foods and their impact on the nutritional quality and functional performance of the raw material (Knorr, 1998; Rodgers, 2008).

Fermented dairy products are widely-accepted, healthy food products and valued components of diets. The incorporation of probiotic bacteria as adjuncts in various fermented milk products is currently an important topic with industrial and commercial

consequences. A number of dairy products containing probiotic bacteria are currently on the market. Fermented milk and cheeses have been described as the most suitable carriers, because they enhance the transit tolerance of bacteria (Saarela et al., 2000; Lourens-Hattingh & Viljoen, 2001). Some strains of *Lactobacillus* and *Bidobacterium* have been shown to tolerate acidic stress when ingested with milk products (Mater et al., 2005). Lactobacilli (e.g. *L. acidophilus*, *L. casei* subsp. *casei*, *L. gasseri*, *L*. *paracasei*, *L*. *reuteri* and *L. rhamnosus*) and bifidobacteria (e.g. *Bifidobacterium adolescentis*, *B*. *bifidum*, *B. breve*, *B. infantis* and *B. longum*) constitute a significant proportion of probiotic lactic acid bacterium cultures used in the dairy industry (Wood & Holzapfel, 1995; Klein et al., 1998). It is also important to determine the technological features of the strains because they could greatly affect food quality. Further, probiotic starter cultures need to be tested for large-scale production feasibility in regard to acidification, proteolysis, and aroma formation. They must accomplish this without losing viability and functionality or creating unpleasant flavor or texture (De Vuyst, 2000; Lacroix & Yildirim, 2007).

Highlights in Probiotic Research 249

Critical characteristics included acidication and enzymatic activities responsible for nal sensory properties; also important were safety characteristics, including antagonistic activity against spoilage strains and antibiotic resistance. The researchers determined that all strains of *L. plantarum* had good acidifying activity; however they showed some differences in antimicrobial, proteolytic and enzymatic activities. Başyiğit Klç et al. (2011b) investigated the technological properties of twenty *L. plantarum* strains to evaluate their potential usage as starter cultures in the dairy industry. During two months in cold storage, there were no signicant changes in the number of bacteria or the pH of the skim milk inoculated with *L. plantarum* strains. The authors suggested that *L. plantarum* AC3-10 and AB6-25 can be used in industrial yogurt manufacture, based on their technological properties such as proteolitic

Floros et al. (2012) tested 19 facultatively heterofermentative lactobacilli from Feta, Kasseri, and Graviera cheeses for potential probiotic strains. Data from this study revealed that isolates B1, G16, G22, E22, E35, and H30 from Feta; PB2.2 from Kasseri; and 631 from Graviera have promising probiotic properties *in vitro*. β-galactosidase, low proteolytic and coagulation activities, and antibacterial activities make them promising candidates as adjunct cultures for the food industry. In another study, yoghurt was produced using a mixture of potential probiotic *L. plantarum* AB6-25, AC18-82, AK4-11 and a commercial starter culture. The yoghurt was divided into four experimental batches to which were added 0.25%, 0.5%, 1%, and 1.5% β-glucan. The survivability of these potential probiotic strains and the physico-chemical properties of the yoghurts were analyzed during a 21-day storage period. The highest *L. plantarum* count was found in the yoghurt containing 0.25% βglucan. The study found the best physico-chemical properties to be in the 0.25% and 0.5% βglucan containing yoghurts. Therefore, the researchers suggested using 0.25% and 0.5% βglucan in yoghurts produced using these potential probiotic bacteria and commercial starter

Wang et al. (2010) identified and established the functional and technological characteristics of potential probiotic *Lactobacillus* strains isolated from two sources: the faeces of breast-fed infants and traditional Taiwanese pickled cabbages. The authors selected the strains *L. reuteri* F03, *L. paracasei* F08 and *L. plantarum* C06 for producing probiotic fermented milk, due to their acid and bile tolerance and ability to adhere to Caco-2 cells. The milks were fermented with these 3 strains separately, and rats were fed a daily dose of 108 CFU/day for 14 days. After the consumption of the *Lactobacillus*-fermented milk, the rats showed increased faecal lactobacilli counts, while the counts of coliform and *C. perfringens* were signicantly decreased. On the other hand, Başyiğit Klç et al. (2010) investigated the effects of a probiotic culture mix (*L. fermentum*, *L. plantarum* and *E. faecium*) and alfa-tocopherol administration on the microbial flora in rat GIT and faeces during a 14-day feeding period. The results indicated that the probiotic culture and alfa-tocopherol administration had no significant effects on the microbial flora of the rat intestinal tract during the 14 days of intake. Minelli et al. (2004) reported that in rats administered milk fermented with L. casei, the faecal *E. coli* counts remained stable, but *Clostridia* counts decreased significantly. Yang et al. (2005) also reported decreased faecal coliform counts as one of advantages of *Lactobacillus* and *Bidobacterium* proliferation in the rat

activity, acidifying ability, and production of flavour compounds.

culture (Başyiğit Klç, 2012).

Although the number of cells required to produce therapeutic benets is not known and might vary as a function of the strain and the health effect desired, in general a minimum level of more than 106 viable probiotic bacteria per mililitre or gram of food product is accepted (Ouwehand & Salminen, 1998). The study of new probiotic strains for their technological relevance and use in food products is important for trade and industry. The search for strains which show resistance to biological barriers of the human GIT, and which possess physiological characteristics compatible with probiotic properties among LAB isolated from food, may eventually lead to the discovery of new probiotic strains for functional food products (Bude-Ugarte et al., 2006).

Studies of fermented food products as a source of new isolates are rapidly accumulating. For example, a mixture of human-derived probiotic strains was tested in the manufacture of ice cream; some of the ice cream was sweetened with sucrose and some was sweetened with aspartame (Başyiğit et al. 2006). The results showed that neither frozen conditions during the storage period nor the type of sweeteners used had any undesired effect on the survival of the probiotic cultures. Georgieva et al. (2009) studied technologically relevant properties of eight candidate probiotic *L. plantarum* strains isolated from cheeses. Researchers tested their capacity to survive over extended shelf-times at refrigerated temperatures and their growth viability in the presence of preservatives widely used in food processing. The researchers determined that the cultures' acidifying and coagulating abilities and enzyme activity make them appropriate for diverse food applications, but especially for dairy products. In another study, the survival of the probiotic strains *L. fermentum* (AB5-18 and AK4-120) and *L. plantarum* (AB16-65 and AC18-82), all derived from human faeces, was investigated in Turkish Beyaz cheese production (Başyiğit Klç et al., 2009). The researchers determined the viability of probiotic bacteria in Turkish Beyaz cheese during 4 months of ripening and the bacteria's effect on chemical properties of the cheese. The results of the study revealed that the test probiotic culture mix was successful for cheese production and did not adversely affect cheese quality during ripening.

Essid et al. (2009) characterized 17 strains of *L. plantarum* isolated from traditional Tunisian salted meat products to select the most suitable for use as starters for fermenting meat. Critical characteristics included acidication and enzymatic activities responsible for nal sensory properties; also important were safety characteristics, including antagonistic activity against spoilage strains and antibiotic resistance. The researchers determined that all strains of *L. plantarum* had good acidifying activity; however they showed some differences in antimicrobial, proteolytic and enzymatic activities. Başyiğit Klç et al. (2011b) investigated the technological properties of twenty *L. plantarum* strains to evaluate their potential usage as starter cultures in the dairy industry. During two months in cold storage, there were no signicant changes in the number of bacteria or the pH of the skim milk inoculated with *L. plantarum* strains. The authors suggested that *L. plantarum* AC3-10 and AB6-25 can be used in industrial yogurt manufacture, based on their technological properties such as proteolitic activity, acidifying ability, and production of flavour compounds.

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

functional food products (Bude-Ugarte et al., 2006).

did not adversely affect cheese quality during ripening.

2000; Lacroix & Yildirim, 2007).

consequences. A number of dairy products containing probiotic bacteria are currently on the market. Fermented milk and cheeses have been described as the most suitable carriers, because they enhance the transit tolerance of bacteria (Saarela et al., 2000; Lourens-Hattingh & Viljoen, 2001). Some strains of *Lactobacillus* and *Bidobacterium* have been shown to tolerate acidic stress when ingested with milk products (Mater et al., 2005). Lactobacilli (e.g. *L. acidophilus*, *L. casei* subsp. *casei*, *L. gasseri*, *L*. *paracasei*, *L*. *reuteri* and *L. rhamnosus*) and bifidobacteria (e.g. *Bifidobacterium adolescentis*, *B*. *bifidum*, *B. breve*, *B. infantis* and *B. longum*) constitute a significant proportion of probiotic lactic acid bacterium cultures used in the dairy industry (Wood & Holzapfel, 1995; Klein et al., 1998). It is also important to determine the technological features of the strains because they could greatly affect food quality. Further, probiotic starter cultures need to be tested for large-scale production feasibility in regard to acidification, proteolysis, and aroma formation. They must accomplish this without losing viability and functionality or creating unpleasant flavor or texture (De Vuyst,

Although the number of cells required to produce therapeutic benets is not known and might vary as a function of the strain and the health effect desired, in general a minimum level of more than 106 viable probiotic bacteria per mililitre or gram of food product is accepted (Ouwehand & Salminen, 1998). The study of new probiotic strains for their technological relevance and use in food products is important for trade and industry. The search for strains which show resistance to biological barriers of the human GIT, and which possess physiological characteristics compatible with probiotic properties among LAB isolated from food, may eventually lead to the discovery of new probiotic strains for

Studies of fermented food products as a source of new isolates are rapidly accumulating. For example, a mixture of human-derived probiotic strains was tested in the manufacture of ice cream; some of the ice cream was sweetened with sucrose and some was sweetened with aspartame (Başyiğit et al. 2006). The results showed that neither frozen conditions during the storage period nor the type of sweeteners used had any undesired effect on the survival of the probiotic cultures. Georgieva et al. (2009) studied technologically relevant properties of eight candidate probiotic *L. plantarum* strains isolated from cheeses. Researchers tested their capacity to survive over extended shelf-times at refrigerated temperatures and their growth viability in the presence of preservatives widely used in food processing. The researchers determined that the cultures' acidifying and coagulating abilities and enzyme activity make them appropriate for diverse food applications, but especially for dairy products. In another study, the survival of the probiotic strains *L. fermentum* (AB5-18 and AK4-120) and *L. plantarum* (AB16-65 and AC18-82), all derived from human faeces, was investigated in Turkish Beyaz cheese production (Başyiğit Klç et al., 2009). The researchers determined the viability of probiotic bacteria in Turkish Beyaz cheese during 4 months of ripening and the bacteria's effect on chemical properties of the cheese. The results of the study revealed that the test probiotic culture mix was successful for cheese production and

Essid et al. (2009) characterized 17 strains of *L. plantarum* isolated from traditional Tunisian salted meat products to select the most suitable for use as starters for fermenting meat. Floros et al. (2012) tested 19 facultatively heterofermentative lactobacilli from Feta, Kasseri, and Graviera cheeses for potential probiotic strains. Data from this study revealed that isolates B1, G16, G22, E22, E35, and H30 from Feta; PB2.2 from Kasseri; and 631 from Graviera have promising probiotic properties *in vitro*. β-galactosidase, low proteolytic and coagulation activities, and antibacterial activities make them promising candidates as adjunct cultures for the food industry. In another study, yoghurt was produced using a mixture of potential probiotic *L. plantarum* AB6-25, AC18-82, AK4-11 and a commercial starter culture. The yoghurt was divided into four experimental batches to which were added 0.25%, 0.5%, 1%, and 1.5% β-glucan. The survivability of these potential probiotic strains and the physico-chemical properties of the yoghurts were analyzed during a 21-day storage period. The highest *L. plantarum* count was found in the yoghurt containing 0.25% βglucan. The study found the best physico-chemical properties to be in the 0.25% and 0.5% βglucan containing yoghurts. Therefore, the researchers suggested using 0.25% and 0.5% βglucan in yoghurts produced using these potential probiotic bacteria and commercial starter culture (Başyiğit Klç, 2012).

Wang et al. (2010) identified and established the functional and technological characteristics of potential probiotic *Lactobacillus* strains isolated from two sources: the faeces of breast-fed infants and traditional Taiwanese pickled cabbages. The authors selected the strains *L. reuteri* F03, *L. paracasei* F08 and *L. plantarum* C06 for producing probiotic fermented milk, due to their acid and bile tolerance and ability to adhere to Caco-2 cells. The milks were fermented with these 3 strains separately, and rats were fed a daily dose of 108 CFU/day for 14 days. After the consumption of the *Lactobacillus*-fermented milk, the rats showed increased faecal lactobacilli counts, while the counts of coliform and *C. perfringens* were signicantly decreased. On the other hand, Başyiğit Klç et al. (2010) investigated the effects of a probiotic culture mix (*L. fermentum*, *L. plantarum* and *E. faecium*) and alfa-tocopherol administration on the microbial flora in rat GIT and faeces during a 14-day feeding period. The results indicated that the probiotic culture and alfa-tocopherol administration had no significant effects on the microbial flora of the rat intestinal tract during the 14 days of intake. Minelli et al. (2004) reported that in rats administered milk fermented with L. casei, the faecal *E. coli* counts remained stable, but *Clostridia* counts decreased significantly. Yang et al. (2005) also reported decreased faecal coliform counts as one of advantages of *Lactobacillus* and *Bidobacterium* proliferation in the rat

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

Highlights in Probiotic Research 251

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,

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

Chandramouli et al., 2004).

assimilate cholesterol.
