**4. Viability of probiotics in food products**

Improving the viability of probiotic bacteria in different food products (especially fermented products) until the time of consumption has been the subject of hundreds of studies. Viability of probiotic microorganisms, namely, the number of viable and active cells per g or mL of probiotic food products at the moment of consumption is the most critical value of these products, because determines their medicinal efficacy (Khorbekandi et al., 2011; Tamime et al., 2005). Therefore, in order to maintain consumer confidence in probiotic products, it is important to ensure a high survival rate of the bacteria during the production of product as well as over the product shelf life (Saxelin et al., 1999). Although there is no world-wide agreement on the minimum of viable probiotic cells per gram or milliliter of probiotic product, generally, the concentrations of 106 and 107-108 cfu mL-1 (cfu g-1), respectively, have been accepted as the minimum and satisfactory levels. It has also stated that probiotic products should be consumed regularly with an approximate amount of 100 g d-1 in order to deliver about 109 viable cells into the intestine (Karimi et al., 2011; Mohammadi et al., 2011; Vinderola et al., 2000a).

In order to have a positive effect in the intestinal tract some specific requirements regarding food products should be fulfilled. First, probiotics need to resist the manufacturing process; second, they should remain viable during the storage period in the commercial products until the end of the shelf-life. Many factors influence the viability of probiotic microorganisms in food products during production and storage periods. The main mentioned factors are: pH, titrable acidity, molecular oxygen, redox potential, hydrogen peroxide, bacteriocins, short chain fatty acids, flavoring agents, microbial competitions, packaging materials and packaging conditions, rate and proportion of inoculation, stepwise/stage-wise fermentation, micro-encapsulation, milk solid non-fat content, supplementation of milk with nutrients, heat treatment of milk, incubation temperature, storage temperature, carbonation, addition of salt, sugar and sweeteners, cooling rate of the product and scale of production. Figure 2 implies main factors affecting viability of probiotics in food products. These factors are discussed below:

Fig. 2. Main factors affecting the viability of probiotic food products and during delivery through gastrointestinal tract.

#### **4.1 Strains of probiotic bacteria**

126 New Advances in the Basic and Clinical Gastroenterology

Improving the viability of probiotic bacteria in different food products (especially fermented products) until the time of consumption has been the subject of hundreds of studies. Viability of probiotic microorganisms, namely, the number of viable and active cells per g or mL of probiotic food products at the moment of consumption is the most critical value of these products, because determines their medicinal efficacy (Khorbekandi et al., 2011; Tamime et al., 2005). Therefore, in order to maintain consumer confidence in probiotic products, it is important to ensure a high survival rate of the bacteria during the production of product as well as over the product shelf life (Saxelin et al., 1999). Although there is no world-wide agreement on the minimum of viable probiotic cells per gram or milliliter of probiotic product, generally, the concentrations of 106 and 107-108 cfu mL-1 (cfu g-1), respectively, have been accepted as the minimum and satisfactory levels. It has also stated that probiotic products should be consumed regularly with an approximate amount of 100 g d-1 in order to deliver about 109 viable cells into the intestine (Karimi et al., 2011;

In order to have a positive effect in the intestinal tract some specific requirements regarding food products should be fulfilled. First, probiotics need to resist the manufacturing process; second, they should remain viable during the storage period in the commercial products until the end of the shelf-life. Many factors influence the viability of probiotic microorganisms in food products during production and storage periods. The main mentioned factors are: pH, titrable acidity, molecular oxygen, redox potential, hydrogen peroxide, bacteriocins, short chain fatty acids, flavoring agents, microbial competitions, packaging materials and packaging conditions, rate and proportion of inoculation, stepwise/stage-wise fermentation, micro-encapsulation, milk solid non-fat content, supplementation of milk with nutrients, heat treatment of milk, incubation temperature, storage temperature, carbonation, addition of salt, sugar and sweeteners, cooling rate of the product and scale of production. Figure 2 implies main factors affecting viability of

Fig. 2. Main factors affecting the viability of probiotic food products and during delivery

**4. Viability of probiotics in food products** 

Mohammadi et al., 2011; Vinderola et al., 2000a).

probiotics in food products. These factors are discussed below:

through gastrointestinal tract.

Care must be taken in selecting the most appropriate strain for a particular food application. Indeed, the first step in incorporating a probiotic into a food is identifying compatibilities between the attributes of the selected strains and the food production steps, food matrix and storage conditions. Selection of probiotic strains used in food products should be according to both the criteria of compatibility with and resistance to the product and *in vivo* conditions in order to increase the viability of the probiotic bacterial strains (Korbekandi et al., 2011). The tolerance of probiotics both to the product and to the internal conditions of the living consumer is strain-dependent (strain-specific). Suitable probiotic strains are those enable to maintain their survival and stability during commercial production of products as well as during the storage period (Godward et al., 2000; Talwalker and Kailasapathy, 2004). Furthermore, high viable survival rate during delivery through the gastrointestinal tract is necessary to allow enough live cell arrival to the human intestine. Therefore, selection of resistant probiotic strains against production, storage and gastrointestinal tract condition is of prime importance. Researchers have indicated that the survival of bacteria against harsh conditions in food products such as pH, titrable acidity, oxygen toxicity, freezing and low temperatures and storage temperatures are species- and strain-specific (Godward et al., 2000; Kailasapathy and Sultana, 2003; Ravula and Shah, 1998; Takahashi et al., 2007; Tamim et al., 2005).

Selected probiotic strains should also results in adequate sensory characteristics of final product. Some studies have shown that flavor is the first indicator with respect to the choice of a food, followed by considerations with respect to health (Mohammadi and mortazavian, 2011; Tuorila and Cardello, 2002). Consumers are not interested in consuming a functional food if the added ingredients confer disagreeable flavors on the product, even if this results in advantages with respect to their health. Therefore, a pleasant aroma and taste profiles are of importance in the formulation of probiotic functional foods and is strain-dependent. The metabolism of the probiotic cultures can result in the production of components that may contribute negatively to the taste and aroma of the product, such as acetic acid produced by *Bifidobacterium* spp. during fermentation and over storage period. Figure 3 shows main criteria for selection of probiotic strains in food products.

#### **4.2 pH and titrable acidity**

pH and titrable acidity of probiotic products considerably affect cell survival of probiotic microorganisms (Mortazavian et al., 2010). Low pH is of the most important factor that restricts the growth and stability of probiotic bacteria in fermented products. Hydrogen ions damage probiotic cells via disrupting mass transfer through the cell membranes and acidic starvation of the cells (Mortazavianand and Sohrabvandi, 2006). Very low pH ranges in fermented milks might cause an increase in the concentration of undissociated organic acids in them and, as a result, enhances the bacteriocidal effect of these acids. The aforementioned effect of organic acids arises from their lipophilic nature. They can be transferred through the microbial cells and dissociate within them, changing the intracellular pH. Also, organic acids might bind to various intracellular compounds. Both of these phenomena disturb cell metabolism (Korbekandi et al., 2011).

The optimum pH for growth of *Lactobacillus acidophilus* is 5.5-6.0, but for bifidobacteria this range is 6.0 –7.0 (De Vuyst, 2000). In food products, lactobacilli are able to grow and survive

Delivery of Probiotic Microorganisms into Gastrointestinal Tract by Food Products 129

molecular oxygen is detrimental to probiotic growth and survival. However, the degree of oxygen sensitivity varies considerably between different species and strains (Kawasaki et al., 2006). In general, lactobacilli, which are mostly microaerophilic, are more tolerant of oxygen than bifidobacteria, to the point where oxygen levels are rarely an important consideration in maintaining the survival of lactobacilli (Lee and Salminen, 2009). Oxygen content and redox potential have been shown to be important factors for the viability of bifidobacteria especially during the storage period. Oxygen affects probiotic cultures in three ways. Firstly, it is directly toxic to some cells; secondly, in the presence of oxygen, certain cultures, especially *L. delbrueckii* ssp. *bulgaricus* produces peroxide (especially in dairy products), which is toxic to probiotic cells particularly *L. acidophilus*; and thirdly, free radicals produced from the oxidation of components (e.g., fats) are toxic to probiotic cells

Several methods have been used to decrease oxygen content. The most important ones are accomplishing fermentation under vacuum (for fermented products), using vacuum packaging, using packaging materials with low permeability to oxygen, adding antioxidants and oxygen scavengers to milk (such as ascorbic acid), and controlling the production process in such a way that minimum dissolved oxygen entered into product (Dave and Shah 1997; Korbekandi et al 2011; Shah, 2000; Talwalkar and Kailasapathy, 2003; Talwalkar et al.,

The compatibility of probiotics with other ingredients within food formulations can have a significant impact on bacterial survival. Interactions between probiotics and other ingredients can be protective, neutral, or detrimental to probiotic stability (Lee and Salminen, 2009; Mattila-Sandholm et al., 2002). The main affective food ingredients are

Food additives used in the food industry could significantly affect the growth and viability of probiotic bacteria (e.g. *L. acidophilus*, *L. casei*, *L. paracasei*, *L. rhamnosus* and bifidobacteria) and starter cultures (e.g. *S. thermophilus*, *L. delbrueckii* ssp. *Bulgaricus*, *Lactococcus lactis* and *Saccharomyces* spp.) used for fermented and nonfermented products (Vinderola et al., 2002). These additives include salts (NaCl and KCl), sugars (sucrose and lactose), sweeteners (acesulfame and aspartame), aroma compounds (diacetyl, acetaldehyde and acetoin), natural colorings for fermented milks (red, yellow and orange colorings), flavoring agents (strawberry, vanilla, peach and banana essences), flavoring–coloring agents (strawberry, vanilla and peach), nisin (a polypeptide-type antibiotic produced by *L. lactis* which is active against spore- forming bacteria and could be used as a natural preservative in addition to lactic acid), natamycin, lysozyme and nitrate (Vinderola et al., 2002). Elevated levels of ingredients can inhibit probiotics during storage (Arihara et al., 1998; Boylston et al., 2004;

Probiotic lactobacilli and, in particular, bifidobacteria grow poorly in milk due to lack of non-protein nitrogen (free amino acids and small peptides) and some vitamins, as well as to

(Korbekandi et al., 2011; Tamim et al., 2005).

Kourkoutas et al., 2006; Lee and Salminen, 2009).

**4.4.2 Growth and protective factors** 

2004).

**4.4 Food ingredients** 

mentioned as follows:

**4.4.1 Food additives** 

Fig. 3. Main criteria for selection of probiotic strains in food products.

in fermented milks and yogurts with pH values between 3.7 and 4.3 (Boylston, 2004). Bifidobacteria tend to be less acid tolerant, with most species surviving poorly in fermented products at pH levels below 4.6 (Boylston, 2004; Lee and Salminen, 2009; Ross et al., 2005). The tolerance of *Bifidobacterium* spp. to acidic conditions is strain-specific. The best survivability of bifidobacteria have been observed in *B. longum* in the presence of acidic conditions and bile salts, and for *B. lactis* (*B. animalis* ssp. *lactis*) in fermented milks conditions (korbekandi et al., 201; Tamim et al., 2005).

Survival in low pH beverages such as fruit juices (pH 3.5-4.5) possesses a significant challenge to probiotic survival. Researchers have reported that cell viability depends of the strains used, the characteristics of the substrate, the oxygen content and the final acidity and the concentration of lactic acid and acetic acid of the product (Shah, 2001). According to Sheehan et al., (2007), when adding *Lactobacillus* and *Bifidobacterium* orange, pineapple and cranberry juice, extensive differences regarding their acid resistance were observed. All of the strains screened survived for longer in orange and pineapple juice compared to cranberry. *L. casei*, *L. rhamnosus* and *L. paracasei* display a great robustness surviving at levels above 7.0 log cfu/ml in orange juice and above 6.0 log cfu ml-1 in pineapple juice for at least 12 weeks (Rivera-Espinoza and Gallardo-Navarro, 2010).

#### **4.3 Molecular oxygen**

Lactobacilli are aerotolerant or anaerobic, and strictly fermentative, while bifidobacteria are strictly anaerobic and saccharoclastic (Holzapfel et al., 2001; De Vuyst, 2000). Therefore, molecular oxygen is detrimental to probiotic growth and survival. However, the degree of oxygen sensitivity varies considerably between different species and strains (Kawasaki et al., 2006). In general, lactobacilli, which are mostly microaerophilic, are more tolerant of oxygen than bifidobacteria, to the point where oxygen levels are rarely an important consideration in maintaining the survival of lactobacilli (Lee and Salminen, 2009). Oxygen content and redox potential have been shown to be important factors for the viability of bifidobacteria especially during the storage period. Oxygen affects probiotic cultures in three ways. Firstly, it is directly toxic to some cells; secondly, in the presence of oxygen, certain cultures, especially *L. delbrueckii* ssp. *bulgaricus* produces peroxide (especially in dairy products), which is toxic to probiotic cells particularly *L. acidophilus*; and thirdly, free radicals produced from the oxidation of components (e.g., fats) are toxic to probiotic cells (Korbekandi et al., 2011; Tamim et al., 2005).

Several methods have been used to decrease oxygen content. The most important ones are accomplishing fermentation under vacuum (for fermented products), using vacuum packaging, using packaging materials with low permeability to oxygen, adding antioxidants and oxygen scavengers to milk (such as ascorbic acid), and controlling the production process in such a way that minimum dissolved oxygen entered into product (Dave and Shah 1997; Korbekandi et al 2011; Shah, 2000; Talwalkar and Kailasapathy, 2003; Talwalkar et al., 2004).

#### **4.4 Food ingredients**

128 New Advances in the Basic and Clinical Gastroenterology

Fig. 3. Main criteria for selection of probiotic strains in food products.

conditions (korbekandi et al., 201; Tamim et al., 2005).

12 weeks (Rivera-Espinoza and Gallardo-Navarro, 2010).

**4.3 Molecular oxygen** 

in fermented milks and yogurts with pH values between 3.7 and 4.3 (Boylston, 2004). Bifidobacteria tend to be less acid tolerant, with most species surviving poorly in fermented products at pH levels below 4.6 (Boylston, 2004; Lee and Salminen, 2009; Ross et al., 2005). The tolerance of *Bifidobacterium* spp. to acidic conditions is strain-specific. The best survivability of bifidobacteria have been observed in *B. longum* in the presence of acidic conditions and bile salts, and for *B. lactis* (*B. animalis* ssp. *lactis*) in fermented milks

Survival in low pH beverages such as fruit juices (pH 3.5-4.5) possesses a significant challenge to probiotic survival. Researchers have reported that cell viability depends of the strains used, the characteristics of the substrate, the oxygen content and the final acidity and the concentration of lactic acid and acetic acid of the product (Shah, 2001). According to Sheehan et al., (2007), when adding *Lactobacillus* and *Bifidobacterium* orange, pineapple and cranberry juice, extensive differences regarding their acid resistance were observed. All of the strains screened survived for longer in orange and pineapple juice compared to cranberry. *L. casei*, *L. rhamnosus* and *L. paracasei* display a great robustness surviving at levels above 7.0 log cfu/ml in orange juice and above 6.0 log cfu ml-1 in pineapple juice for at least

Lactobacilli are aerotolerant or anaerobic, and strictly fermentative, while bifidobacteria are strictly anaerobic and saccharoclastic (Holzapfel et al., 2001; De Vuyst, 2000). Therefore, The compatibility of probiotics with other ingredients within food formulations can have a significant impact on bacterial survival. Interactions between probiotics and other ingredients can be protective, neutral, or detrimental to probiotic stability (Lee and Salminen, 2009; Mattila-Sandholm et al., 2002). The main affective food ingredients are mentioned as follows:

#### **4.4.1 Food additives**

Food additives used in the food industry could significantly affect the growth and viability of probiotic bacteria (e.g. *L. acidophilus*, *L. casei*, *L. paracasei*, *L. rhamnosus* and bifidobacteria) and starter cultures (e.g. *S. thermophilus*, *L. delbrueckii* ssp. *Bulgaricus*, *Lactococcus lactis* and *Saccharomyces* spp.) used for fermented and nonfermented products (Vinderola et al., 2002). These additives include salts (NaCl and KCl), sugars (sucrose and lactose), sweeteners (acesulfame and aspartame), aroma compounds (diacetyl, acetaldehyde and acetoin), natural colorings for fermented milks (red, yellow and orange colorings), flavoring agents (strawberry, vanilla, peach and banana essences), flavoring–coloring agents (strawberry, vanilla and peach), nisin (a polypeptide-type antibiotic produced by *L. lactis* which is active against spore- forming bacteria and could be used as a natural preservative in addition to lactic acid), natamycin, lysozyme and nitrate (Vinderola et al., 2002). Elevated levels of ingredients can inhibit probiotics during storage (Arihara et al., 1998; Boylston et al., 2004; Kourkoutas et al., 2006; Lee and Salminen, 2009).

#### **4.4.2 Growth and protective factors**

Probiotic lactobacilli and, in particular, bifidobacteria grow poorly in milk due to lack of non-protein nitrogen (free amino acids and small peptides) and some vitamins, as well as to

Delivery of Probiotic Microorganisms into Gastrointestinal Tract by Food Products 131

The temperature at which probiotic organisms grow is important in food applications where fermentation is required. Also, storage temperature exhibits considerably important role. Fermentation temperature is one of the most important factors affecting the qualitative parameters of probiotic fermented milks, including the viability of probiotic microorganisms and fermentation time (incubation time). The optimum temperature for growth of most probiotics is between 37°C and 43°C (Boylston et al., 2004; Doleyres and Lacroix 2005; Lee and Salminen, 2009). Although the growth of *L. acidophilus* may occur at temperatures as high as 45 °C, the optimum growth occurs within 40-42°C. The optimum growth temperature for bifidobacteria is 37-41°C (Korbekandi et al., 2011). Species of bifidobacteria isolated from the human intestinal tract such as *B. longum* (infantis), *B. breve*, *B. bifidum*, and *B. adolescentis* have optimum growth temperatures in the range of 36–38°C, whereas *B. animalis* ssp. *lactis* can grow at higher temperatures of 41–43°C (Crittenden, 2004; Doleyres

Temperature is also a critical factor influencing probiotic survival during storage period. Probiotic food products usually, should be stored at a refrigerated temperature, preferably 4-5ºC. The storage temperature of probiotic food products affects the viability of the probiotics via effects of temperature on the cells survival, the type and concentration of metabolites formed between starter bacteria and probiotics in fermented products. Mortazavian et al. (2007a) found that storage in ABY-type culture (*L. acidophilus*, *B. lactis* and yogurt bacteria) at 2ºC for 20 days resulted in the highest viability of *L. acidophilus* LA-5*,* whereas, for *Bifidobacterium lactis* BB-12, the highest viability was obtained when yogurt was stored at 8ºC (Mortazavian et al., 2007b). Low resistance of bifidobacteria cells to low refrigeration temperatures (2°C or less) has been proven (Kailasapathy and Rybka, 1997; Korbekandi et al., 2011). In general, in ABY-type culture, storage of the product at 4-5ºC appears to result in greatest viability of both probiotics, i.e., *L. acidophilus* and bifidobacteria (Mortazavian et al., 2008). During processing, temperatures above 45–50°C will be detrimental to probiotic survival. The higher the temperature, the shorter the time period of exposure required to severely decrease the numbers of viable bacteria, ranging from hours or minutes at 45–55°C to seconds at higher temperatures. It is obvious that probiotics should be added downstream of heating/cooking/pasteurization processes in food manufacture (Lee and Salminen, 2009). Freezing temperatures can also affect viability of probiotics. This is discussed in the next

Probiotics can survive well over long shelf lives in products such as frozen yogurts and ice cream. During the freezing process, the cells of probiotics can be lethally injured by damaging their cell walls or their cell membranes caused by mechanical stresses of the ice crystals formed in the external medium or inside the cells, by temperature decrease chock to the cells and cold injuries, by condensation of solutes (those are detrimental to probiotic cells) in the extracellular/intracellular media, or by dehydration of the cells. All mentioned factors cause reduction or interruption of vital metabolic activities of the cells that are necessary for their live (Akin et al., 2007; Davies and Obafemi, 1985; Gill 2006; Jay et al., 2005). The size of the ice crystals increases with decrease in freezing rate and larger

**4.5 Temperature** 

section.

and Lacroix 2005; Lee and Salminen, 2009).

**4.6 Freezing and thawing operations** 

their slow activity of β-galactosidase (Korbekandi et al., 2011). A good and common method to compensate for slow growth is to fortify milk with different growth factors (consumed by probiotics as nutrients) and/or growth promoters (which improve viability of probiotics but not as a direct nutrient) such as casein, whey protein hydrolysates, *L*-cysteine, yeast extract, glucose, vitamins, minerals and antioxidant. These supplements have significant positive effects on the survival of probiotic microorganisms (Mohammadi et al., 2011).

The addition of *L*-cysteine, whey protein concentrate, acid casein hydrolysate and tryptone improved the viability of *L. acidophilus* and bifidobacteria by providing growth factors as these probiotic bacteria lack proteolytic activity (Dave and Shah, 1998). Protein derivatives promote probiotic survival due to several reasons; namely, their nutritional value for the cells, reducing redox potential of the media as well as increasing buffering capacity of the media (which results in a smaller decrease in pH) (Dave and Shah, 1998; Mortazavian et al., 2010). It should be pointed out that effects of milk proteins on the viability of probiotics depends on various factors such as the type of the strains used, specifications of the milk protein derivative added, inoculation conditions and formulation of the product. It has been reported that casein and whey protein hydrolysate enhanced the acidification rate of *S. thermophilus* and reduced the growth rate of probiotic organisms (*L. acidophilus* La-5 and *L. rhamnosus* Lr-35) in fermented milks during the manufacturing stages, although the survival of the latter bacteria was improved after storage (Lucas et al., 2004).

Prebiotics are non–viable and non-digestible (or minimally digestible) food ingredients which are metabolized selectively by beneficial intestinal bacteria and enhance their growth and/or activity. They are mostly sugar-like compounds (oligosaccharides) comprising between two and ten monomers that largely resist digestion by pancreatic and brush border enzymes. The term "synbiotic" is used to describe products that contain both probiotics and prebiotics (Nobakhti et al., 2009). These compounds (such as fructooligosaccharides and galactooligosaccharides) can have suitable effect on retention of probiotics viability (especially bifidobacteria) in food products as well as in gastrointestinal tract (Gibson et al., 2004; Mizota, 1996; Mohammadi et al., 2011; Rycroft, 2001).

The food matrix, itself, can be protective. An example is cheese, where the anaerobic environment, high fat content and buffering capacity of the matrix helps to protect the probiotic cells both in the product and during intestinal transit (Boylston et al., 2004; Lee and Salminen, 2009). In contrast to liquid foods, the solid matrices in food products, such as the gel structure in yogurt or cheese, support probiotic cells by reducing their exposure to detrimental factors (e.g., hydrogen ions and organic acids) (Karimi et al., 2011; Mohammadi and Mortazavian, 2011). These matrices can act as a barrier (by physically and chemically binding hydrogen ions and organic acids (Korbekandi et al., 2011). Increasing the buffering capacity of milk would stimulate growth and activity of probiotics in fermented milks. It leads to higher viability of probiotics in dairy fermented products as well as in the gastrointestinal tract due to the maintenance of pH at higher values. Also, the pH of the products with higher buffering capacity declines slowly during refrigerated storage and results in a greater survival of probiotic cells. Moreover, by absorbing hydrogen ions into the dry matter of product matrix (such as proteins), the amounts of undissociated organic acids are increased, resulting in the reduction of bacteriocidic effect of these compounds on probiotics (Mortazavian et al., 2011; korbekandi et al., 2011; Heydari 2011).

#### **4.5 Temperature**

130 New Advances in the Basic and Clinical Gastroenterology

their slow activity of β-galactosidase (Korbekandi et al., 2011). A good and common method to compensate for slow growth is to fortify milk with different growth factors (consumed by probiotics as nutrients) and/or growth promoters (which improve viability of probiotics but not as a direct nutrient) such as casein, whey protein hydrolysates, *L*-cysteine, yeast extract, glucose, vitamins, minerals and antioxidant. These supplements have significant positive

The addition of *L*-cysteine, whey protein concentrate, acid casein hydrolysate and tryptone improved the viability of *L. acidophilus* and bifidobacteria by providing growth factors as these probiotic bacteria lack proteolytic activity (Dave and Shah, 1998). Protein derivatives promote probiotic survival due to several reasons; namely, their nutritional value for the cells, reducing redox potential of the media as well as increasing buffering capacity of the media (which results in a smaller decrease in pH) (Dave and Shah, 1998; Mortazavian et al., 2010). It should be pointed out that effects of milk proteins on the viability of probiotics depends on various factors such as the type of the strains used, specifications of the milk protein derivative added, inoculation conditions and formulation of the product. It has been reported that casein and whey protein hydrolysate enhanced the acidification rate of *S. thermophilus* and reduced the growth rate of probiotic organisms (*L. acidophilus* La-5 and *L. rhamnosus* Lr-35) in fermented milks during the manufacturing stages, although the survival

Prebiotics are non–viable and non-digestible (or minimally digestible) food ingredients which are metabolized selectively by beneficial intestinal bacteria and enhance their growth and/or activity. They are mostly sugar-like compounds (oligosaccharides) comprising between two and ten monomers that largely resist digestion by pancreatic and brush border enzymes. The term "synbiotic" is used to describe products that contain both probiotics and prebiotics (Nobakhti et al., 2009). These compounds (such as fructooligosaccharides and galactooligosaccharides) can have suitable effect on retention of probiotics viability (especially bifidobacteria) in food products as well as in gastrointestinal tract (Gibson et al.,

The food matrix, itself, can be protective. An example is cheese, where the anaerobic environment, high fat content and buffering capacity of the matrix helps to protect the probiotic cells both in the product and during intestinal transit (Boylston et al., 2004; Lee and Salminen, 2009). In contrast to liquid foods, the solid matrices in food products, such as the gel structure in yogurt or cheese, support probiotic cells by reducing their exposure to detrimental factors (e.g., hydrogen ions and organic acids) (Karimi et al., 2011; Mohammadi and Mortazavian, 2011). These matrices can act as a barrier (by physically and chemically binding hydrogen ions and organic acids (Korbekandi et al., 2011). Increasing the buffering capacity of milk would stimulate growth and activity of probiotics in fermented milks. It leads to higher viability of probiotics in dairy fermented products as well as in the gastrointestinal tract due to the maintenance of pH at higher values. Also, the pH of the products with higher buffering capacity declines slowly during refrigerated storage and results in a greater survival of probiotic cells. Moreover, by absorbing hydrogen ions into the dry matter of product matrix (such as proteins), the amounts of undissociated organic acids are increased, resulting in the reduction of bacteriocidic effect of these compounds on probiotics (Mortazavian et al., 2011;

effects on the survival of probiotic microorganisms (Mohammadi et al., 2011).

of the latter bacteria was improved after storage (Lucas et al., 2004).

2004; Mizota, 1996; Mohammadi et al., 2011; Rycroft, 2001).

korbekandi et al., 2011; Heydari 2011).

The temperature at which probiotic organisms grow is important in food applications where fermentation is required. Also, storage temperature exhibits considerably important role. Fermentation temperature is one of the most important factors affecting the qualitative parameters of probiotic fermented milks, including the viability of probiotic microorganisms and fermentation time (incubation time). The optimum temperature for growth of most probiotics is between 37°C and 43°C (Boylston et al., 2004; Doleyres and Lacroix 2005; Lee and Salminen, 2009). Although the growth of *L. acidophilus* may occur at temperatures as high as 45 °C, the optimum growth occurs within 40-42°C. The optimum growth temperature for bifidobacteria is 37-41°C (Korbekandi et al., 2011). Species of bifidobacteria isolated from the human intestinal tract such as *B. longum* (infantis), *B. breve*, *B. bifidum*, and *B. adolescentis* have optimum growth temperatures in the range of 36–38°C, whereas *B. animalis* ssp. *lactis* can grow at higher temperatures of 41–43°C (Crittenden, 2004; Doleyres and Lacroix 2005; Lee and Salminen, 2009).

Temperature is also a critical factor influencing probiotic survival during storage period. Probiotic food products usually, should be stored at a refrigerated temperature, preferably 4-5ºC. The storage temperature of probiotic food products affects the viability of the probiotics via effects of temperature on the cells survival, the type and concentration of metabolites formed between starter bacteria and probiotics in fermented products. Mortazavian et al. (2007a) found that storage in ABY-type culture (*L. acidophilus*, *B. lactis* and yogurt bacteria) at 2ºC for 20 days resulted in the highest viability of *L. acidophilus* LA-5*,* whereas, for *Bifidobacterium lactis* BB-12, the highest viability was obtained when yogurt was stored at 8ºC (Mortazavian et al., 2007b). Low resistance of bifidobacteria cells to low refrigeration temperatures (2°C or less) has been proven (Kailasapathy and Rybka, 1997; Korbekandi et al., 2011). In general, in ABY-type culture, storage of the product at 4-5ºC appears to result in greatest viability of both probiotics, i.e., *L. acidophilus* and bifidobacteria (Mortazavian et al., 2008). During processing, temperatures above 45–50°C will be detrimental to probiotic survival. The higher the temperature, the shorter the time period of exposure required to severely decrease the numbers of viable bacteria, ranging from hours or minutes at 45–55°C to seconds at higher temperatures. It is obvious that probiotics should be added downstream of heating/cooking/pasteurization processes in food manufacture (Lee and Salminen, 2009). Freezing temperatures can also affect viability of probiotics. This is discussed in the next section.

#### **4.6 Freezing and thawing operations**

Probiotics can survive well over long shelf lives in products such as frozen yogurts and ice cream. During the freezing process, the cells of probiotics can be lethally injured by damaging their cell walls or their cell membranes caused by mechanical stresses of the ice crystals formed in the external medium or inside the cells, by temperature decrease chock to the cells and cold injuries, by condensation of solutes (those are detrimental to probiotic cells) in the extracellular/intracellular media, or by dehydration of the cells. All mentioned factors cause reduction or interruption of vital metabolic activities of the cells that are necessary for their live (Akin et al., 2007; Davies and Obafemi, 1985; Gill 2006; Jay et al., 2005). The size of the ice crystals increases with decrease in freezing rate and larger

Delivery of Probiotic Microorganisms into Gastrointestinal Tract by Food Products 133

This process has been recently used as an efficient method for improving the viability of probiotic bacteria in fermented milk drinks, fermented frozen dairy desserts, ice cream and juices (Adhikari et al., 2000; Krasaekoopt et al., 2004; 2005; Kailasapathy, 2006; Mohammadi et al., 2011), and simulated gastrointestinal tract (Hansen et al., 2002; Korbekandi et al., 2011; Lee and Heo, 2000; Krasaekoopt et al., 2004; Mortazavian et al., 2008; Sultana et al., 2000; Wenrong and Griffiths, 2000). Encapsulated probiotic organisms, when incorporated into fermented frozen dairy desserts, showed an improved viability of >105 cfu g-1 in the product compared to counts of <103 cfu g-1 when non-encapsulated organisms were used (Mortazavian et al., 2010 ; Shah and Ravula 2004). Studies suggest that, micro-encapsulation of free probiotic cells can increase their viability by ≥2 log cycles in fermented milks during a refrigerated storage period. As mentioned earlier, in fermented milk drinks with pH values of less than 4.2, free cells of *L. acidophilus* LA-5 lost their viability to less than 106 cfu mL-1 after 1 week; and in the case of *Bifidobacterium lactis* BB-12, a similar loss occurred after 2 weeks of storage. For encapsulated cells, viable population of *L. acidophilus* and bifidobacteria remained higher than 105 and 106 cfu mL-1 after 42 days of refrigerated storage, and counts of free probiotic free cells were not detected and 102 cfu mL-1,

The packaging of probiotic food products influences the oxygen permeability into the product, and as a result, affects the viability of bifidobacteria, *L. acidophilus* and other probiotic species during the storage period. Several aspects of food packaging materials including the type of the packaging materials (Glass and plastic) their thickness, and the application of active/intelligent packaging systems could influence survival of probiotic bacteria (Korbekandi et al., 2011). In general, two important points are worth mentioning. Firstly, apart from the packaging materials, the temperature and relative humidity of the atmosphere are the key factors affecting oxygen permeability. Secondly, besides the efficiency of packaging, the economic aspect should also be taken into account (the price of packaging materials as well as the price of packaging machines) because they can

The gastrointestinal tract (GIT) with its diverse and concentrated microbial population (at birth 1014 cfu g-1 and between 400 and 500 species) is one of the key organs of the human body, and it is in fact an ecosystem of highest complexity that mediates numerous interactions with the chemical (and nutritional) environment. The gastrointestinal tract starts in mouth, travels through the stomach, intestines and ends at the anus. In each section of the gastrointestinal tract, different types and quantities of microbes are found. The average adult carries about four pounds of microbes in their intestinal tract (Tannis, 2008). Nonetheless, diversity at a division level is among the lowest (Bäckhead et al., 2005) and the lactobacilli and bifidobacteria comprise less than 5% of the total microbiota (Lay et al., 2005;

Probiotics targeting the intestine clearly encounter the greatest hurdles in order to be delivered to their targeted site. The main factors to be considered that influence the viability

significantly influence the final price of products and their sale volumes.

**5. Viability of probiotics in food products during delivery through** 

respectively (Mortazavian et al., 2008).

**gastrointestinal tract** 

Lee and Salminen, 2009).

**4.9 Packaging materials and conditions** 

intracellular ice crystals causes greater damage to the cells (Gill, 2006; Jay et al., 2005). Therefore, rapid freezing after inoculating with the probiotic microorganisms contributes to the good maintenance of the populations of these microorganisms in the product (Mohammadi et al., 2011).

Probiotic cells are subjected to some chemical stresses during melting (freeze-thaw) of the frozen products which can cause mortality to them. On one hand, the cells are exposed to osmotic effects (Jay et al., 2005). On the other hand, the high concentrations of detrimental factors such as hydrogen ions, organic acids, oxygen and other poisoning components to probiotic cells in melting media, associated with freezing concentration, are the factors having a great effect on viability loss of probiotics. pH has been found to exhibit a crucial role in this regard.
