# Lactic Acid Bacteria Co-Encapsulated with Lactobionic Acid:
## 3 Results and Discussion
### Viability over Time of the Probiotic Encapsulated in Dried Capsules  
The viability of the microorganism is a key factor in ensuring the probiotic effect on the health of the host. Actually, there is no standard recommendation on the bacterial concentration to consume to have a benefit; however, it is generally accepted that the microorganism, to be defined as a probiotic, must have a concentration of at least 10\({}^{6}\) CFU per g of product [37]. This bacterial concentration must be guaranteed at the time of consumption and, considering that during storage the vital count can decrease, an assessment of the viability over time is essential. We evaluated the impact on viability over time of: (1) the carbon source used as substrate by the microorganism (testing two different broths: AFB-G and AFB-L; (2) the coating (testing the least (0.1 M CaCl\({}_{2}\) for 10 min) and the most (0.5 M CaCl\({}_{2}\) for 10 min and chitosan) crosslinked capsules); (3) the drying technology used (freeze- or thermal-dried, considering that for both, a water content of about 0.1% on dry basis was measured immediately after drying, while a value of less than 2% was measured after 30 days). In Figure 2, the microbial concentration observed over time (days) is shown. It may be noted that there were no differences due to the carbon source used: the same trend was obtained on capsules with the same structure but revitalized in different broths, and this could be affirmed for both the freeze- and thermal-dried capsules. On the contrary, the resistance of the structure of the capsules seemed to be very important to guarantee stability: the capsules crosslinked in 0.1 M CaCl\({}_{2}\) for 10 min protected the microorganism less than the other types of capsules, with a decay in the microbial concentration of about 4 log\({}_{10}\) at the end of the time considered. The same reduction was indeed not present if the capsules were produced by crosslinking in 0.5 M CaCl\({}_{2}\) for 10 min and then coated with chitosan. Additionally, in this case the trend between the freeze- and thermal-dried capsules was very similar and no statistically significant differences were found. Furthermore, in the study by [PERSON] et al. [38], the structure of the capsule seemed to be an important factor in determining the probiotic viability over time: it was found that chitosan-coated alginate capsules were more capable than others (with skim milk, dextrin, or denatured whey protein) in protecting _L. plantarum_ during storage. The importance of chitosan coating for probiotic viability during storage was also shown by [PERSON] et al. [39]. Based on these data, only the microorganisms encapsulated in the most crosslinked capsules, then coated with chitosan, had a useful microbial concentration to be considered probiotics after 30 days of storage, while for the other capsules only half the storage time should be considered.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Capsules** & **A** & **B** & **C** & **D** \\ \hline Force (g) & 253.7 \(\pm\) 7.4 \({}^{\text{a}}\) & 283.2 \(\pm\) 68.3 \({}^{\text{a}}\) & 304.3 \(\pm\) 52.0 \({}^{\text{a}}\) & 476.9 \(\pm\) 38.7 \({}^{\text{b}}\) \\ \hline \hline \end{tabular}
\end{table}
Table 3: Bloom values (g) for the different capsules. Different letters in the same column indicate significant differences (\(p\) < 0.05).