## 3 Results and Discussion
### Effect of Immobilization and Freeze-drying on Cell Viability during Storage
#### 3.2.2 Storage at 4 \({}^{\circ}\)C
casei_ CSL3 on soybean were similar to initial levels, when submitted to cold storage for 30 days [49]. Oat flakes were proposed as a suitable support for the immobilization of _L. casei_ CSL3 cells, as they maintained high cell viability during storage at 4 \({}^{\circ}\)C for 60 days [50]. Survival of probiotic cells in high levels up to the end of the product expiration date is a crucial factor for the food industry [51]. Incorporation of freeze-dried free or immobilized _L. casei_ 393 cells on oat flakes, raisins, and apple pieces on yoghurt and subsequently evaluation of its survival during storage resulted in elevated viability of immobilized cells compared to free after 60 days of storage, expanding the products usual shelf-life up to 30 days. The positive contribution of cell immobilization in cell survival was also documented in immobilized _L. casei_ 01 cells on dark or white chocolate, resulting in maintenance of cell viability after storage at 4 \({}^{\circ}\)C for 60 days and in higher cell survival after exposure to simulated gastric environment compared to free cells [52]. Co-encapsulation of _L. paracasei_ LBC81 and ELBAL cells with a calcium alginate matrix coated with gelatin improved cell survival during 35 days of storage at low temperature, while free cells' viability decreased to 28 and 35%, respectively [43]. Moreover, the levels of encapsulated _L. paraplantarum_ FT-259 in casein/pectin microparticles decreased by 0.41 and 1.26 log after 60 and 90 days of storage at 8 \({}^{\circ}\)C, respectively, with microbial counts remaining over 6.24 logcfu/g during the entire study period and retained high viability percentages throughout storage (over 83.2%) [40]. Of note, [PERSON] et al. [53] reported that encapsulation of _L. acidophilus_ probiotic cells on alginate/fish gelatin possesses protective effects, as it increased their viability by 2-3 logcfu/g during baking and 7 days of storage at 4 \({}^{\circ}\)C.  
Studies evaluating the effect of storage on the viability of probiotic strains support that low storage temperatures prolong cell survival, compared to high storage temperatures [54,55], in agreement with the results of this study. Regarding the decrease in cell viability, it is estimated that during storage, cells increase the saturated fatty acids of their membrane, due to lipid oxidation. Lipid oxidation, which is favored at high storage temperatures, is accompanied by free radical formation, and subsequently, DNA and cell membrane damage [56]. For industrial purposes, both stability and viability of the microbial probiotic cultures consist of the most important parameters influencing manufacturing and marketing [57]. Production of probiotic supplements has to be combined with viable cell counts as shelf life of the product is crucial [58]. Results of the present study clearly demonstrate that the presumptive probiotic wild-type strains were able to survive during storage in immobilized form, highlighting that pistachios and zea flakes could be good candidates as vehicles of probiotics. The strains showed better survival ability on zea and pistachio rather than on raisins, confirming that food formulation affects the viability of probiotics during storage [59]. Furthermore, the viability of probiotic cultures during storage seems to be strain-rather than species-specific [47]. Heat, oxygen, and acid tolerance in a strain-specific manner, can be taken into account to choose strains that could achieve a high survival during storage. In general, the use of stress-resistant strains can be useful for improving stability in industrial processes [60].