# Lactic Acid Bacteria Co-Encapsulated with Lactobionic Acid:
## 3 Results and Discussion
### Simulated In Vitro Digestion of Rehydrated Freeze-Dried and Thermal-Dried Capsules  
To evaluate the capacity of the alginate capsules to protect _L. paracasei_ survival, freeze-dried and thermal-dried capsules rehydrated in saline solution and yogurt were subjected to a simulated in vitro digestion test. As a control, unencapsulated _L. paracasei_ (7 log\({}_{10}\) CFU/mL) was used. Results are shown in Figure 4.  
In the simulated salivary phase, the probiotic concentration was equal to or greater than 6 log\({}_{10}\) CFU/g capsules or mL (in the case of control). The differences observed may be due to the initial concentration of microorganisms in each type of rehydrated capsule and not to the effect of salts and \(\alpha\)-amylase present in the environment, as the differences were not related to the type of capsule (freeze- or thermal-dried) or the type of hydration (saline solution or yogurt). The main changes were observed in gastric simulation. After 30 min in the stomach simulation, no survival was observed in the case of the control. It is known that the acidic environment (pH \(<\) 2.0) as well as the presence of pepsin can cause the death of the microorganisms [43]. Significant differences (\(p\) \(<\) 0.05) were found in terms of the way the capsules were rehydrated but not between freeze- or thermal-dried capsules. Higher survival was obtained in capsules rehydrated in yogurt than in those rehydrated in saline solution, with a difference of 57.75%. After the simulated gastric phase, there was a small reduction in the microbial load in the simulated intestinal phase. At the final point, the mean survival of capsules rehydrated in saline solution was 2.3 \(\pm\) 0.1 log\({}_{10}\) CFU/g capsules, while for those rehydrated in yogurt, it was 4.8 \(\pm\) 0.1 log\({}_{10}\) CFU/g capsules. Therefore, the way in which the capsules were rehydrated influenced the survival of the probiotic throughout the simulated in vitro digestion test in both types of capsules (freeze-dried and thermal-dried). Yogurt, with its rich nutritional composition, could play a dual role in the protection of microorganisms: first, by acting as a more complex matrix to digest, compared to saline solution, with more \"engaged\" digestive enzymes (the same results were obtained by [PERSON] et al. (2013) [44], where different food carriers of probiotics were tested during in vitro digestion, and higher survival was  
Figure 4: Variation of the concentration of _L. paracasei_ (log\({}_{10}\) CFU/g of capsule and log\({}_{10}\) CFU/mL in the case of control sample) throughout the in vitro digestion test. F-S refers to freeze-dried capsules rehydrated in saline solution, T-S to thermal-dried capsules rehydrated in saline solution, F-Y to freeze-dried capsules rehydrated in yogurt, and T-Y to thermal-dried capsules rehydrated in yogurt. Experiments were carried out in triplicate and reported results correspond to the mean value. Different letters indicate significant differences (\(p\) \(<\) 0.05). observed in complex and/or nutrient-rich ones) and second, by feeding microorganisms with nutrients: during rehydration in yogurt, capsules can absorb water enriched with nonrighing molecules such as sugars, which can promote viability. For probiotic microorganisms to be effective, they must withstand the conditions of the digestive tract to reach the GIT. Although there is no scientific consensus on the probiotic concentration needed to obtain a beneficial effect, several authors suggest that this concentration would be between 6 and 9 log\({}_{10}\) CFU/day [10,45]. Regarding LBA, it was not measured during the simulated in vitro digestion test, as it is resistant to human digestive enzymes (non-digestible fibre), and it is capable of reaching the large intestine intact [5,46]. In the case of yogurt, the recommended daily intake is between 200 and 250 g [47], meaning two portions per day (120 g/portion).