Experimental setup

In the production of probiotic food one of the important factors is the matrix of the food substrate. It acts as a medium to achieve the growth of microbes to at least 9 log cfu/g or ml (FAO/WHO, 2001), which is considered necessary to confer health benefits to the host (Santo et al., 2011). Characterisation of specific probiotic strains, food matrix and dietary content interaction with the probiotics are the research areas for food technologists and industrialists (Isolauri, 2007). Composition of food substrate such as fat content, type of proteins, carbohydrates and pH can affect probiotic growth and survival.

Charalampopoulos et al. (2003) suggested that the nature of food matrix could affect the stability of the probiotic microorganisms during gastrointestinal transit. Dairy and non-dairy substrates are considered as a vehicle for delivering probiotic bacteria to the human GIT and base for the development of probiotic foods.

Dairy matrices are an extremely promising source for the development of probiotic foods (Yeo et al., 2011). Various food products have been developed as carriers for probiotics, mainly of dairy origin because consumers commonly associate them with fermented dairy products and perceive health benefits in the presence of probiotic cultures (Sanders, 2000).

The base for the production of dairy fermented products is milk, which has a typical composition of 87.4% water, 4.7% lactose, 3.8% fat, 3.3% protein (80% casein and 20%

whey protein), 0.2% citrate and 0.6% minerals, with pH in the range 6.5–6.7 (De Sukumar, 2007).

Most probiotics proliferate well in a dairy-based matrix due to the lactose-hydrolysing enzyme and proteolytic system involved in casein utilisation, which provides probiotic cells with a carbon source and essential amino acids for growth. Metabolism of these nutrients produced organic compounds that are essential for the development of flavour, preservation and appearance of the products (Yeo et al., 2011). Some additives like prebiotics, pulses and cereal flours are used to speed up the acidification process and survivability of probiotics, as some lactobacilli are unable to consume lactose as a carbon source.

According to Rogelj (2000), dairy-fermented products such as yogurt, probiotic beverages and cheese-containing lactic acid bacteria and their constituents such as omega-3 fatty acid, phytosterols, isoflavones, conjugated linoleic acid, minerals and vitamins have a prominent position in the development of functional foods. In some cases, fermented milk products are fermented by monocultures of probiotic bacteria, but usually supporting cultures are applied to speed up the acidification process and provide the desired texture and flavour (Schmid et al., 2006). Many lactobacilli and bifidobacteria survive in fermented milk products for 4–8 weeks in refrigerated storage. Probiotic dairy products, which contain health promoting lactic acid bacteria (LAB) in addition to traditionally used starter LAB, are good examples of successful fermented functional foods. Today, numerous commercial dairy-based beverages incorporate various strains of probiotic bacteria that are available for human consumption.

Increasing demand for new foods and tastes initiated development of non-dairy probiotic products that are part of the day-to-day normal diet to maintain the minimum therapeutic level (Lavermicocca, 2006). The application of probiotic microbial strains for fermentation of cereals and legumes is a rational approach for the development of functional foods.

Cereals contain high levels of carbohydrates, which act as a source of carbon and energy for microbes during fermentation. Most of the carbohydrates in cereals are present as starch and only available for microbes after amylolytic hydrolysis. Endogenous cereal enzymes, malt or selected enzymes can be used to break down the starch to simple fermentable sugars (i.e., maltose and glucose), which can be utilised by probiotics as a carbon source (Salovaara and Simonson, 2004).

Pediococcus spp. VA403 (Pintado et al., 1999), Lactobacillus manihotivorans (Ohkouchi and Inoue, 2006) and Lactobacillus plantarum (Thomsen and Guyot, 2007) are known as LAB, which have the ability to breakdown the starch and utilise it as a carbon source to produce lactic acid. Cereal-based products‘ ability to support the growth of probiotics is mainly due to their high concentration of fibres such as xylooligosaccharides, xylan and arabinoxylan, which may act as a growth substrate for probiotics. Besides carbohydrates, cereals also contain relatively high levels of minerals, vitamins, sterols, and other growth factors, which support the growth of microbes, including the LAB. Whole grains are also a source of many phytochemicals, including phytoestrogens, phenolic compounds, antioxidants and phytic acid (Katina et al., 2007), which provide additional functionality to probiotic foods.

The nutritional quality of grains is sometimes inferior to that of milk because of its lower protein content, deficiency of certain essential amino acids, low starch availability, antinutrients (phytic acid and tannins) and the coarse nature of the grains (Blandino et al., 2003). Fermentation has been postulated to decrease the level of starch as well as some non-digestible poly- and oligosaccharides, improve protein quality and increase the level of amino acids and group B vitamins. Fermentation also provides optimum pH conditions for enzymatic degradation of phytate and release minerals such as manganese (which is an important growth factor of probiotic), iron, zinc and calcium (Blandino et al., 2003).

Strains of Lactobacillus have been recognised as complex microorganisms that require fermentable carbohydrates, amino acids, vitamin B, nucleic acids and minerals to grow.

Charalapompoulos et al. (2003), conducted experiments with different cereals to determine the main parameters required for the growth of probiotic microorganisms, such as composition and processing of cereal grains, substrate formulation, growth capability and productivity of the starter culture, stability of the probiotic strain during storage, organoleptic properties and nutritional value of the final product. Different cereals were found to provide different conditions to support the growth of probiotics (Charalapompoulos et al., 2003). It has been reported that Lactobacillus reuteri, Lactobacillus acidophilus and Bifidobacterium bifidum grow well in oat-based substrates (Martenson et al., 2002). Yosa, a new oat-based fermented food similar to flavoured

yogurt or porridge, is considered as a health food due to its oat fibre, lactobacilli and bifidobacteria (Blandino et al., 2003).

Helland et al. (2004b), studied the growth ability of probiotics in a corn-based fermented substrate and observed that maize fermentation induces fruity flavours in traditional Mexican foods, which could have good worldwide acceptance. Nyanzi et al. (2010), evaluated the sensory attributes of a maize beverage fermented by four species of probiotics and reported that the beverages fermented by L. acidophilus or L. rhamnosus were well accepted by trained and untrained panels.

Soy is an excellent raw material for the development of non-dairy probiotic foods to overcome the limitations associated with dairy products. The benefits of soy have drawn much attention recently and numerous soy products have been evaluated as possible probiotic vehicles. Experiments revealed that soy milk is a good food matrix for probiotics such as Lactobacillus spp., L. casei, L. helveticus, L. fermenti, L. fermentum, L. reuteri and L. acidophilus (Wang et al., 2006).

Soy-based fermented foods may provide additional benefits for the consumer due to their various functional properties: they are hypolipidaemic, anticholesterolaemic and antiatherogenic and have reduced allergenicity (Lopez-Lazaro and Akiyama, 2002).

According to Champagne etal. (2005), development of a fermented soy product containing probiotics requires strain selection for the ability to grow in the substrate, as well as the ability to compete or even establish a synergy between strains. Donkor et al. (2005), reported that the protein in fermented soy milk could encourage the growth of many probiotic strains such as L. acidophilus, L. casei and S. thermophilus.

Scientific research has shown that probiotic-containing soy-fermented beverages have good sensory acceptance for potential consumers (Shimakama et al., 2003). Hauly et al.

(2005), reported that soy yoghurt supplemented with fructooligosaccharide had an acceptance index above 70%. The texture and taste of soy yoghurt are essential attributes for product acceptability (Donkor et al., 2007). Gel formation of soy milk proteins is a key process step in the manufacture of a non-dairy fermented product like yoghurt. The rheological properties of set gels determine the texture, organoleptic properties and shelf

life of the product (Lee and Lucey, 2006; Cayot et al., 2008). Soy milk has a low acidification rate and slow growth of probiotic bacteria, which take longer to complete fermentation and produce undesirable changes in the product that are not acceptable to the consumer (Donkor et al., 2007).

Addition of certain additives like prebiotics (inulin and fructooligosaccharide) and whey protein concentrate improves the textural and sensory characteristics of fermented soy yoghurt (Hauly et al., 2005; Donkor et al., 2007). Soy is the most studied matrix for the formulation of probiotic food, but other substrates like peanut have also been explored for the development of probiotic food (Mustafa et al., 2009).

Fruits and vegetables are a rich source of minerals, vitamins, dietary fibres and antioxidants (Yoon et al., 2004). Therefore, there has been increasing interest in the application of vegetable and fruit juices as alternative carriers of probiotics. A number of studies found that probiotic strains have the capability to grow in fruit and vegetable matrices (Rivera-Espinoza and Gallardo-Navarro, 2010). Researchers also observed significant differences in the acid resistance of lactobacilli and bifidobacteria in orange, pineapple, cranberry, bitter gourd, carrot and other juices. According to Sheehan etal.

(2007), Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus paracasei survived longer in orange and pineapple juice than in cranberry juice. They survived at levels above 7.0 and 6.0 log cfu/ml in orange juice and pineapple juice for at least 12 weeks at refrigerated storage temperature.

Sheehan etal. (2007), reported that fruit juices appear as a more complex system for the development of probiotic foods, due to the more acidic pH of the products. Thus, the selection of probiotic strains that are more resistant to acidic environments is crucial in the development of a probiotic juice (Yeo et al., 2011). Microencapsulation has been shown to provide protection to acid-sensitive probiotics. Ding and Shah (2008), studied the effect of microencapsulation on the viability of probiotic bacteria in orange and apple juices and reported that encapsulated probiotic bacteria was found to survive over 6 weeks of cold storage with counts of more than 10⁵cfu/ml or g, while free probiotic cells lost their viability within 5 weeks. The addition of prebiotics can also improve the viability and stability of the probiotics (Vergara et al., 2010). Kyung etal. (2005), developed a probiotic

red beet beverage using Lactobacillus acidophilus and Lactobacillus plantarum and reported that both strains reduced the pH of the juice from an initial value of 6.3 to less than 4.5 after 48 h of fermentation, due to their ability to produce a greater amount of lactic acid.