## 3 Results and Discussion
### Effect of Immobilization and Freeze-drying on Cell Viability during Storage
#### 3.2.1 Storage at RT  
Storage of freeze-dried cultures is strongly recommended at temperatures 1-4 \({}^{\circ}\)C, as storage at RT or higher results in rapid cell death [41]. However, since refrigerated storage and maintaining an unbroken cold chain implies an increase in cost, storage at RT is advantageous [42]. The effect of freeze-drying, cell immobilization, and storage time on cell concentration and survival rates is presented in Table 2a-c. All factors (culture form, cell condition, and storage time) significantly affected cell viability (_p_ < 0.05). Levels of wet free cells ranged <7 log cfu/mL after 40 days of storage, for all strains. Counts of freeze-dried free cells ranged >7 log cfu/mL after 40 days of storage, but dropped <7 log cfu/mL after 90 days of storage, which is the minimum recommended concentration for conferring a beneficial effect, as already reported [33]. \begin{table}
\begin{tabular}{l l c c c c} \hline \hline \multirow{2}{*}{**Microorganisms**} & \multicolumn{3}{c}{**Immobilized Cells on Natural Food Carriers**} & \multirow{2}{*}{**Free Cells**} \\ \cline{3-3} \cline{5-6}  & & **Zea flakes** & & **Pistachio** & **Raisin** \\ \hline \multirow{4}{*}{_L. plantarum_ B282} & W & 8.6 \(\pm\) 0.2 & 8.4 \(\pm\) 0.1 & 8.5 \(\pm\) 0.1 & 9.6 \(\pm\) 0.1 \\  & FD & 8.3 \(\pm\) 0.1 & 8.5 \(\pm\) 0.1 & 8.0 \(\pm\) 0.1 & 10.8 \(\pm\) 0.1 \\  & FD (ww) & 8.0 \(\pm\) 0.1 \({}^{\star}\) & 8.3 \(\pm\) 0.1 & 7.4 \(\pm\) 0.1 \({}^{\star}\) & 8.4 \(\pm\) 0.1 \({}^{\star}\) \\  & \% survival & 92.6 \(\pm\) 0.3 \({}^{\star\star}\) & 98.8 \(\pm\) 0.2 \({}^{\star\star}\) & 86.8 \(\pm\) 0.6 & 87.8 \(\pm\) 0.8 \\  & W & 9.0 \(\pm\) 0.1 & 9.0 \(\pm\) 0.1 & 8.8 \(\pm\) 0.1 & 9.7 \(\pm\) 0.1 \\  & FD & 9.0 \(\pm\) 0.1 & 9.0 \(\pm\) 0.1 & 8.0 \(\pm\) 0.2 & 11.4 \(\pm\) 0.1 \\  & FD (ww) & 8.3 \(\pm\) 0.1 \({}^{\star}\) & 8.5 \(\pm\) 0.1 \({}^{\star}\) & 7.4 \(\pm\) 0.1 \({}^{\star}\) & 9.1 \(\pm\) 0.1 \({}^{\star}\) \\  & \% survival & 92.3 \(\pm\) 0.4 & 94.3 \(\pm\) 0.5 & 83.5 \(\pm\) 1.6 \({}^{\star\star}\) & 93.7 \(\pm\) 0.8 \\  & W & 8.3 \(\pm\) 0.1 & 8.2 \(\pm\) 0.1 & 8.2 \(\pm\) 0.1 & 9.3 \(\pm\) 0.1 \\  & FD & 8.5 \(\pm\) 0.1 & 8.4 \(\pm\) 0.1 & 7.8 \(\pm\) 0.2 & 11.4 \(\pm\) 0.1 \\  & FD (ww) & 8.2 \(\pm\) 0.1 & 8.2 \(\pm\) 0.1 & 7.4 \(\pm\) 0.1 \({}^{\star}\) & 8.8 \(\pm\) 0.1 \({}^{\star}\) \\  & \% survival & 98.2 \(\pm\) 0.4 \({}^{\star\star}\) & 99.0 \(\pm\) 0.5 \({}^{\star\star}\) & 89.9 \(\pm\) 0.4 \({}^{\star\star}\) & 94.4 \(\pm\) 0.1 \\ \hline \hline \end{tabular}  
* We: Wet cultures after growth in MRS medium, FD: freeze-dried cultures (before rehydration), FD (ww): freeze-dried cultures after rehydration. Free cells were used as control samples. Data are expressed as mean values (average) \(\pm\) standard error of the mean (STDE). Statistically significant differences are marked \({}^{\star}\): \(p\) < 0.05 compared to wet cells, \({}^{\star\star}\): \(p\) < 0.05 compared to free cells.  
\end{table} 
Table 1: Effect of freeze-drying and cell immobilization on cell levels and survival rates. Regarding wet immobilized cultures, in all cases, levels < 7 logcfu/g were recorded, except for immobilized _P. acidilactici_ SK cells on pistachio, which retained cell levels \(\geq\) 7 logcfu/g after 40 days of storage. However, the levels decreased <7 logcfu/g at 90 days of storage. During storage at RT, levels of freeze-dried immobilized _P. acidilactici_ SK cells on zea flakes and pistachios remained high throughout the storage period and increased survival rate was observed compared to wet cells (_p_ < 0.05). Specifically, after 180 days of storage, cell  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \multicolumn{8}{c}{**(a) Storage at RT—L.