## 3 Results and Discussion
### Effect of Immobilization and Freeze-drying on Cell Viability during Storage
#### 3.2.2 Storage at 4 \({}^{\circ}\)C  
Likewise, the effect of storage at refrigerated temperature (4 \({}^{\circ}\)C) on the levels of wet and freeze-dried cultures and survival rates were also studied (Table 3a-c). All factors (culture form and condition and storage) significantly affected cell viability (\(p<0.05\)) in all cases, with the exception of freeze-dried immobilized _P. acidilactici_ SK cells on zea flakes and pistachios, the concentration of which was not affected by the storage period (\(p>0.05\)). Specifically, the levels of immobilized _P. acidilactici_ SK cells on zea flakes and pistachios ranged 8.1 and 8.5 logcfu/g (98.2 and 99.7% survival rates) after 180 days of storage, values similar (\(p>0.05\)) to the initial levels, (in the 1 st day of storage). In the remaining cases, storage time affected cell viability negatively (\(p<0.05\)). The greatest reduction of cell viability was observed in wet free cells after 40 days of storage (levels \(<\) 7 logcfu/mL) with survival rates ranging in significantly (\(p<0.05\)) lower values compared to wet immobilized cultures. At 90 days, wet free cultures were detected \(\leq\)1.3 logcfu/mL. Freeze-dried free _L. plantarum_ B282 cells ranged \(<\)7 logcfu/mL, after 90 days of storage, but levels \(>\)7 logcfu/mL for _P. acidilactici_ SK and _L. plantarum_ F4 after 180 days of storage were recorded, indicating a strain-specific survival, as reported elsewhere [47]. On the other hand, immobilization had a significant effect on the viability of wet cells. In particular, wet immobilized _L. plantarum_ B282 and _L. plantarum_ F4 cells in zea flakes ranged at levels \(>\) 7 logcfu/g up to day 90 of storage, while similar loads were recorded at immobilized cells on pistachios up to day 180. Wet immobilized _P. acidilactici_ SK cells levels on raisins ranged 7.2 logcfu/g up to 40 days of storage, while for _L. plantarum_ B282 and _L. plantarum_ F4 strains, the limit of 7 logcfu/g was not achieved at the same timepoint. A similar study showed that cell loads of wet immobilized _L. casei_ CSL3 cells in guava or kiwi ranged \(<\)6 logcfu/g after 43 days of refrigerated storage [48]. Levels of freeze-dried immobilized _L. plantarum_ B282, _P. acidilactici_ SK, and _L. plantarum_ F4 cells on zea flakes and freeze-dried immobilized _P. acidilactici_ SK and _L. plantarum_ F4 cells on pistachios ranged \(>\)7 logcfu/g at the 180 th day of storage and were significantly higher (\(p<0.05\)) compared to freeze-dried free cells. Similarly, loads of freeze-dried immobilized _L. plantarum_ B282 cells on pistachios were recorded \(>\)7 logcfu/g up to 90 days of storage, whilst at the same timepoint freeze-dried free cell levels were significantly (\(p<0.05\)) lower. Regarding freeze-dried _P. acidilactici_ SK cells immobilized on zea flakes, the levels ranged to similar values to freeze-dried free cells, but a higher (\(p<0.05\)) survival rate was observed (92% for cells immobilized on zea flakes and 88.8% for free cells) at 180 days of storage. In contrast, levels of freeze-dried immobilized cultures on raisins did not exceed 7 logcfu/g at 40 days of storage. Raisins are well-known for their nutritional value, but they are rich in polyphenols that exert antimicrobial activity [45]. The above results showed that Immobilization on pistachios and zea flakes contributed to maintenance of high cell viability for up to 6 months (Table 3a-c), higher than the recommended levels (>7 logcfu/g) when stored at 4 \({}^{\circ}\)C. Immobilization on natural food carriers (such as zea flakes and pistachios) can be used to preserve the viability of probiotic products during storage at 4 \({}^{\circ}\)C, in high doses. This technology should allow probiotics to be incorporated into food systems and allow manufacturers to place assurances on the viability and quantity of probiotics in the final products.