Probiotic bacteria and bifidobacteria, in specific, constitute a significant part of the human gut microflora, while they have been widely incorporated into various fermented foods and dairy products . The consumption of bifidobacteria has been associated to certain health benefits conferred to the human host, including reduction of serum cholesterol levels, enhancement of immune function, diarrhea alleviation, decrease of lactose intolerance, modulation of the gut microflora and allergy alleviation . However, in order to exert their health benefits, probiotic bacteria should be able to survive during food processing and storage, as well as under the harsh conditions of the gastrointestinal (GI) system, in order to successfully colonize the colon . Due to their high sensitivity to various environmental factors, such as heat, high acidity, oxidative stress, freezing and moisture, probiotic bacteria are prone to cell wall deterioration, lipid oxidation or undesirable alterations of the cell membrane . Therefore, the protection of probiotics is necessary and for this purpose their encapsulation in suitable carriers has been proposed. Various methods are reported for the encapsulation of probiotic bacteria, including extrusion, emulsification, coacervation, spray-drying or freeze-drying . Εncapsulation of probiotic cells has been studied mainly through the application of the spray drying technology, using various materials such as alginate and chitosan , maltodextrin along with whey protein concentrate, skim milk powder or sodium caseinate and/or trehalose or D-glycose  or even hydrolyzed black waxy rice flour . In contrast, milder techniques such as emulsification have not been extensively examined.
Rodrigues et al. (2012) encapsulated probiotic bacteria in alginate beads through the extrusion method and studied its effect on their viability during storage at 5oC. Encapsulation had a beneficial effect, whereas the double coating with chitosan or dextran sulphate did not significantly enhance the viability of the cells. Extrusion method has, also, been extensively examined in a previous study , using a variety of encapsulating blends and providing satisfactory results in the protection of probiotic cells during storage or in vitro simulation of the GI tract. In the current study, the application of emulsification method for the encapsulation of the probiotic strain, Bifidobacterium animalis subsp. lactis (BB-12) was selected to be examined, as it involves mild conditions and presents low cost and high cellular retention .
The emulsification technique includes the dispersion of probiotic cells in a water-based polymer suspension (discontinuous phase), which is then added in an appropriate amount of oil (continuous phase) in order to form a water-in-oil emulsion; it is substantially based on the association and interactions between the discontinuous and continuous phase. The subsequent addition of a calcium chloride solution leads to the insolubilization of the water-soluble polymer and the formation of gels beads within the oil phase, thus encapsulating the probiotic bacteria. The beads produced by emulsification may be of a wide range of shapes and sizes, whereas their diameter may be sufficiently small, even below 300 µm . This technique also presents the potential for large-scale production, due to bulk beads’ formation in short time .
Biocompatible and non-toxic materials are investigated for the incorporation of encapsulated products in food matrices . In particular, for the encapsulation through emulsification the use of sodium alginate as encapsulating agent has already been reported , as it is inexpensive, nontoxic and compatible with most other materials . Moreover, alginate is widely used as encapsulating material due to its ability to develop nets under mild conditions . Other materials, such as guar gum, xanthan, locust bean, and carrageenan have been tested by Ding and Shah (2009) for their protective properties against acidic conditions or in the presence of bile salts. Carrageenan and xanthan gums were proved to be as effective as sodium alginate during probiotic cells’ exposure to harsh conditions. Adhikari et al. (2000) encapsulated bifidobacteria in carrageenan beads through emulsification in order to further incorporate them into yogurt, thus maintaining their bacterial load stable throughout the 30-days storage at 4oC. Τhe application of alginate alone is limited due to its instability in the presence of Ca2+ chelating agents and monovalent ions or harsh conditions . In order to improve the chemical and mechanical stability of alginate beads, the combination of alginate with other polymers has been proposed, such as gellan gum  or corn starch . However, research on the combination of sodium alginate with a variety of materials for the reinforcement of the beads is still limited. Moreover, probiotic cells have been successfully encapsulated in a blend of whey protein isolate and pullulan through emulsification, thus improving their viability during storage as well as under simulated gastrointestinal conditions . Emulsification can be further combined with spray-drying  or freeze-drying [22, 23], since extension of probiotics’ shelf life can be achieved by reducing the moisture levels . Although drying can significantly prolong the viability of probiotic cells, a such operation may, also, have a detrimental effect on probiotics’ viability. Thus, milder approaches are recommended so as to improve existing drying systems. The prebiotic substances are nondigestible food components that stimulate selectively the growth, activity, or both of probiotic bacteria . Another practice proposed by Raddatz et al. (2019) studied the additional use of prebiotics; they incorporated a variety of prebiotic substances, such as Hi-maize starch, inulin, and rice bran, into the encapsulating mixture in order to address the probiotics’ viability and achieved improved results. The incorporation of Hi-maize starch into the alginate systems has also been studied by others researchers [25–27].
In this work, the elaboration of integrated and complex encapsulating systems consisting of sodium alginate, other hydrocolloid materials (xanthan, carrageenan, pectin and cellulose nanocrystalline-CNC), milk and/or milk proteins, glycose and prebiotics (inulin) is investigated. Additionally, the incorporation of cryoprotectants (glycerol) or oxygen scavengers (L-cysteine-HCl) is examined. The occurring blends are evaluated and compared regarding their effectiveness, in terms of protecting BB-12 cells during refrigerated or frozen storage as well as their transition through a simulated gastrointestinal system.