Application of Bifidobacterium spp in beverages and dairy food products: an overview of survival during refrigerated storage

Probiotic is a preparation of viable microorganisms that are added to the diet of humans to control the growth of undesirable or less desirable microorganisms in the gastrointestinal tract that rich in the flora of more than 500 different bacterial species some of which have important health functions (Shori, 2016; Zendeboodi et al., 2020). A probiotic strain must maintain viability and functionality during manufacturing food products and subsequent storage without loss of the sensory properties of these foods (Shori et al., 2016; Muniandy et al., 2017; Champagne et al., 2018).


Introduction
Probiotic is a preparation of viable microorganisms that are added to the diet of humans to control the growth of undesirable or less desirable microorganisms in the gastrointestinal tract that rich in the flora of more than 500 different bacterial species some of which have important health functions (Shori, 2016;Zendeboodi et al., 2020). A probiotic strain must maintain viability and functionality during manufacturing food products and subsequent storage without loss of the sensory properties of these foods Muniandy et al., 2017;Champagne et al., 2018).
Factors such as diet, antibiotics, and stress can influence normal bifidobacteria concentrations in the human digestive system. Thus, incorporation of bifidobacteria has been shown to enhance the therapeutic value of foods and the survival of bifidobacteria in foods is varying depending on the food matrices (Adhikari et al., 2000;Shori, 2015a). Several factors may affect the viability of Bifidobacterium spp. in food and beverage products including the time, pH and temperature of incubation and storage, ratio and strains in the inoculum, availability of nutrients, presence of undesirable microflora and their enzymes, the presence of hydrogen peroxide and dissolved oxygen, the concentration of metabolites (Donkor et al., 2006;Pereira et al., 2011;Shori, 2013a;Baba et al., 2014). Throughout the shelf life, it is highly desirable that the viable counts of Bifidobacterium spp in the final product to be at least 10 6 -10 7 cfu/ml to offering health benefits to the consumers (Shori, 2013b). Therefore, the objective of this study is to review the applications of Bifidobacterium spp in food and beverage products and the effect of different food matrices on their survival during the storage.

Probiotic bacteria
Probiotics can be defined as " [...] live microbial, dietary supplements or food ingredients that have a beneficial effect on the host by influencing the composition and or metabolic activity of the flora of the gastrointestinal tract [...]" (Fuller, 1989, p.366). Probiotics are present in fermented food products and known as lactic acid bacteria (LAB). These bacteria consist of several genera including Lactobacillus, Bifidobacterium, Streptococcus, and Enterococcus which have different beneficial effects on the host and has been used for decades in the food and natural sources to promote good health of human (Shori & Baba, 2013;Shori et al., 2020a, b).

Characteristics of Bifidobacterium spp as probiotics
Bifidobacteria are probiotic commonly found throughout the colon of both humans and animals which are considered normal residents of the gastrointestinal tract (Nielsen et al., 2003). They are once classified under the genus lactobacillus but they are found phylogenetically distinct from LAB and classified under phylum Actinobacteria. Out of 25 species, only ten are commercially used in conjunction with L. acidophilus as probiotics. For example, B. bifidum, B. infants, and B. lactis. Bifidobacteria usually appear in pairs with a V or Y-like shaped. These microbes are anaerobic, non-pathogenic, gram-positive, non-spore forming, pleomorphic rod, and catalase-negative (Nielsen et al., 2003;Zacarías et al., 2020). Unlike LAB, bifidobacteria have high G+C content (55% to 67 mol%) and plasmids are rarely found in the cytoplasm, except for B. longum. They grow optimally within temperature 37 °C to 41 °C and pH optima for growth ranges from pH 6.5 to pH7.0 (Sonomoto & Yokota, 2011). Bifidobacteria are nutritionally fastidious. Bifidobacterium spp. of human origin can utilize fructose, galactose, and lactose. They are the main saccharolytic bacteria found in the human colon, and able to utilize non-digestible oligosaccharides in the colon to produce acetic acid as well as lactic acid in the ratio of 3:2 through a unique fructose-6-phosphate phosphoketolase pathway (Toscano et al., 2015). Different species and/or strains of bifidobacteria may exhibit different beneficial health effects, such as regulation of intestinal microbial homeostasis, repression of procarcinogenic enzymatic activities within the microbiota, production of vitamins, and the bioconversion of a number of dietary compounds into bioactive molecules (Toscano et al., 2015;Chugh & Kamal-Eldin, 2020). In addition, bifidobacteria enhance several immune functions i.e. the activation of macrophages and lymphocytes, antibody production, mitogenic response in the spleen, and Peyer's patches and function of natural killer cells (Marin et al., 1997).

Survival of Bifidobacterium spp in beverages
Beverages containing probiotics are a new and promising approach for therapeutic products (Shori, 2012). The success of probiotic beverages is often limited by the nature of the ingredients, the contamination, and the low viability of strains during storage (Shori et al., 2019;Jaimez-Ordaz et al., 2019;Bruno et al., 2020). One of the most commercial probiotics available in the beverages market is a species of Bifidobacterium. Several researchers have been studied the viability of Bifidobacterium spp. in dairy and non-dairy beverages (Table 1). For example, fermented whey-based goat milk beverage was prepared using S. thermophilus TA-40, B. animalis BB-12 and L. rhamnosus Lr-32 (Buriti et al., 2014). The viability of B. animalis BB-12 in wheybased goat milk beverages in the presence of guava or soursop pulps reduced (1 log cycle; p < 0.05) during 21 days of storage (Table 1). Allgeyer et al. (2010) found that the soluble corn fiber or inulin (5 g) did not affect the viability of B. lactis Bb-12 in yogurt-like drink during one month of storage. An earlier study showed that the inclusion of inulin (3 g/100 mL) and/or okara flour (5 g/100 mL) with ABT-4 culture (L. acidophilus La-5, B. animalis Bb-12, and S. thermophilus) in soymilk during fermentation have no influence on the viability of B. animalis during 28 days of storage (Bedani et al., 2013; Table 1). However, B. animalis B94 showed 1 log cycle increased in fermented soymilk after 28 days of storage (Donkor et al., 2007; Table 1). Kun et al. (2008) found that Bifidobacterium strains (B. lactis Bb-12, B. bifidum B7.1 and B3.2) showed significant (p < 0.05) growth in pure carrot juice without nutrient supplementation during fermentation (Table 1). However, another study reported that B. lactis 420 and B. lactis Bb-12 have declined rapidly in carrot juice (without nutrient supplementation) after 7 days of refrigerated storage and they were untraceable after 2 months (Tamminen et al., 2013). In non-fermented probiotic milk/ carrot juice drink with L. acidophilus LA5, L. plantarum and L. rhamnosus GG, B. lactis Bb-12, showed about 1 log cfu/ml reduction during 3 weeks of storage (Daneshi et al., 2013).

B. lactis
Bb-12 has demonstrated good survival in orange juices during 6 weeks (6.9 log cfu/mL) whereas showed significantly (p < 0.05) decreased in pineapple juices to 3.0 log cfu/ml (Sheehan et al., 2007). Furthermore, the study found that the commercial probiotic (B. lactis Bb-12) cannot survive in cranberry juice (Sheehan et al., 2007). B. animalis B94 maintained a level of counts > 6.5 log cfu/ml after 72 hours of incubation in green tea extract (López de Lacey et al., 2014). Beverages could have positive health properties in the gut once the populations of Bifidobacterium spp. were beyond the minimum recommended level 7 log cfu/g for health benefits (Salva et al., 2011;Shori, 2015b).

Survival of Bifidobacterium spp. in yogurt
Yogurt is a semi-solid fermented milk product (Mahmood et al., 2019;Shori, 2020). It defined as a coagulated milk product that results from lactic acid bacteria i.e. Lactobacillus bulgaricus and Streptococcus thermophilus (Shori & Baba, 2012;Coskun & Karabulut Dirican, 2019). Probiotics such as Bifidobacterium spp. are added to enhance the fermentation process for production bio-yogurt and can survive under the acidic conditions in the fermented milk (Donkor et al., 2006;Altuntas & Korukluoglu, 2019). However, these bacteria showed slow growth in milk and low survival rate during storage (El-Dieb et al., 2012). Therefore, active components such as plant extracts, milk proteins, inulin, and lactulose have been added to increase the growth and viability of Bifidobacterium spp. in yogurt (Table 2). Kailasapathy et al. (2008) reported that the amount of 5 or 10 g/100 g added fruit mixes (mango, mixed berry, passion fruit, and strawberry) in yogurt showed no effect on B. animalis ssp. lactis LAFTIs B94 growth. Similarly, inclusion of flavouring agents such as strawberry (0.08 & 0.16; w/w), vanilla (0.07 & 0.14; w/w), peach (0.08 & 0.16; w/w) and banana (0.1 & 0.2; w/w) essences did not affect the viability of bifidobacteria in yogurt (Vinderola et al., 2002). The presence of pectins and fructooligosaccharides in either apple or banana fibers showed an increase in the numbers of B. animalis subsp. lactis BL04 by 1 log cfu/ml in skim milk yogurt compared to the absence (Espírito Santo et al., 2012). A reduction in the viability of B. animalis Bb-12 was shown in stirred fruit yogurt made from goat's milk during one month of storage but it still in the recommended level of 7 log cfu/g for health benefits (Ranadheera et al., 2012). Another study reported that supplementation of whey protein concentrate (WPC; 3%-5%) in fermented goat milk sustained a high population of B. lactis Bb-12 even after 21 days of refrigerated storage (Martín-Diana et al., 2003; Table 2). The high growth of B. lactis from 7.41 log cfu/ml to 10.3 log cfu/ml was significantly associated with the presence of lactulose in milk during fermentation (Oliveira et al., 2011a). Likewise, Oliveira et al. (2011b) found that the growth of B. lactis in fermented skim milk dramatically stimulated (p < 0.05) by 40 mg/g inulin during 7 days of storage at 4 °C ( Table 2). Counts of 7.52 cfu/ml were recorded for B. longum in fermented milk with S. thermophilus TA040, L. delbrueckii ssp. bulgaricus LB340 and L. acidophilus  Table 2).
La14 after one day of storage (Cruz et al., 2012). This viable cell count of B. longum has decreased about 1 log cfu/ml after 30 days of storage (Table 2). Recently found that green tea infusion (5%, 10%, or 15%) has no influence on the viability of B. animalis ssp. lactis BB-12 in yogurt during 3 weeks of storage (Najgebauer-Lejko, 2014). However, green tea sustained

Survival of Bifidobacterium spp. in cheese
Cheese is one of the best carriers for probiotics (Pivetta et al., 2020). The development of probiotic cheeses can be very strain-dependent as many of the probiotic strains showed insufficient performance in the cheese environment (Shori et al., 2018;Silva et al., 2018;Prezzi et al., 2020). Several studies have reported the applications of Bifidobacterium spp. in different types of cheeses (Table 3). Tharmaraj & Shah, (2004) studied the changes in the population of B. animalis in cheese-based dips with different bacterial combinations over 10 weeks of storage. The authors found the best combination of probiotic bacteria can be used when combined B. animalis with L. acidophilus and L. paracasei subsp. paracasei, together (inoculation at 9 cfu log/g). B. animalis showed a high level of the population required for health benefits through 10 weeks of storage. Cheddar cheeses inoculated with B. longum 1941 or B. animalis subsp. lactis B94 used as an adjunct with starter lactococci showed high viability of Bifidobacterium spp. cells during 24 weeks ripening period (Ong & Shah, 2009; Table 3). Despite, B. lactis Bb-12 can survive in a high level of cheddar cheese (≥10 8 cfu/g) during six months of ripening, the increase was associated with a high level of moisture content (40%) which considered slightly above the legal limit permitted for Cheddar cheese (Mc Brearty et al., 2001). B. longum 15708 showed slow growth in salted cheddar cheese with about 3 log cfu/g loss of their viable cells after 3 days of storage (Fritzen-Freire et al., 2010a). A previous study demonstrated high survival of B. lactis Bb-12 in cheddar cheese followed by Bifidobacterium sp. DR10 and B. lactis B94 respectively with viable cell counts above 7 log cfu/g (Phillips et al., 2006; Table 3). B. lactis registered high cell counts (7.5 log cfu/g) in semi-hard cheese incubated with L. acidophilus and L. paracasei for 60 days (Bergamini et al., 2009). However, inulin or oligofructose (10%) had no significant effect on the survival of B. animalis subsp. lactis in petit-suisse cheese during 30 days of storage (Cardarelli et al., 2008; Table 3). Likewise, the presence of fructooligosaccharides or a mixture of inulin and fructooligosaccharides (50:50) in the synbiotic cheeses has not affected the viability of B. lactis B94 during 60 days of ripening period .
The viability of B. animalis Bb-12 in Minas Frescal cheese did not effect by the presence of lactic acid (Fritzen-Freire et al., 2010b). This may explain the high viability of Bifidobacterium in Minas cheeses. The viable cell counts of B. animalis Bb-12 added to Minas fresh cheese with L. acidophilus La-5 and S. thermophilus increased significantly (p < 0.05) during 21 days of storage (Buriti et al., 2007; Table 3). B. animalis subsp. lactis showed the highest counts (~8.43 log cfu/ml) when combined with S. thermophilus in Minas frescal cheese whey as compared with other probiotics such as L. acidophilus and L. rhamnosus (Almeida et al., 2008).

Survival of Bifidobacterium spp in ice cream
Ice cream is a frozen dairy product made from a combination of served ingredients other than milk (Shori et al., 2018). The effectiveness of probiotics ice cream consumption on consumer's health is related to bacterial viability. The incorporation of Bifidobacterium spp. has been studied in ice cream (Table 4).
It is very important to maintain the stability of Bifidobacterium spp. in ice cream during storage. Silva et al. (2015) found that B. animalis had the ability to maintain satisfactory viability in goat's milk ice cream during 120 days of frozen storage (Table 4). Akın et al. (2007) reported that the addition of 2% of inulin in ice cream containing 10% w/w of fermented milk increased the viability of B. lactis from 10 5 cfu/g to 10 6 cfu/g compared to the absence. Akalin & Erisir (2008) found that the presence of 4% inulin or oligofructose did not influence the viability of B. animalis BB-12 in low-fat ice cream stored at -18 °C for 90 days. Another study reported that non-fermented soymilk ice cream improved the viability of B. lactis during 30 days of storage at -20 °C (Aboulfazli et al., 2016; Table 4). Bifidobacterium species (B. lactis BBDB2 and B. lactis Bb-12) showed initial populations of 10 7 -10 8 cfu/g in frozen soy dessert (Heenan et al., 2004). Besides, both probiotics species maintained sufficient viability of 10 7 cfu/g after 28 weeks of storage at -20 °C. Fermented acerola (Malpighia emarginata) ice cream has demonstrated a good carrier of Bifidobacterium where B. lactis and B. longum showed viability of 8.65 log cfu/g and 7.51 log cfu/g respectively over 15 days of storage (Favaro-Trindade et al., 2006).
The viable cell counts of B. animalis  in synbiotic ice cream showed a decrease by 2.9 log after 6 months of storage (Homayouni et al., 2008;Table 4). However, microencapsulated of B. animalis showed a reduction by only 0.7 log and maintained B. animalis viability between 10 8 and 10 9 cfu/g overall shelf. This indicated that encapsulation can significantly maintain the high viability of probiotic bacteria in ice cream over storage (Shori, 2017). A previous study revealed that encapsulated B. bifidum 231 in ice cream increased significantly (p < 0.05) the viability of probiotic (8.06 log cfu/g) compared to non-encapsulated bacteria (6.33 log cfu/g) after 90 days of storage (Sahitya et al., 2013). In addition, B. bifidum 231 has been improved after coencapsulated with 3% Fructooligosaccharides during storage at -20 °C (Sahitya et al., 2013). Similar behavior has been shown by B. Lactis Bb-12 with 30% rise in their viability in ice cream microencapsulated with calcium alginate and whey protein after 6 months of storage (Karthikeyan et al., 2013).

Conclusion
The utilization of beverages and dairy foods as Bifidobacterium spp. carrier is representing potential advantages and appreciated alternatives for the food industry. In order to improve the survival rates of Bifidobacterium spp. in beverages and dairy foods during refrigerated storage, ingredients with active components such as plant phytochemicals, milk proteins, inulin, and lactulose could be a promising approach for controlled Bifidobacterium spp. viability. Furthermore, microencapsulation is good processing to maintain the high viability of Bifidobacterium spp. Encapsulation materials are known as safe ingredients and can be used in food applications. Therefore, there is a big interest in the improvement of the physical properties and mechanical stability of the polymers used in probiotics encapsulation, to ensure a high population of probiotics not only in food during storage but also after gastrointestinal digestion.   (Kalicka et al., 2019)