# Lactic Acid Bacteria Co-Encapsulated with Lactobionic Acid:
## 2 Materials and Methods
### Simulated In Vitro Digestion of Rehydrated Freeze-Dried and Thermal-Dried Capsules  
The in vitro digestion test was performed only with the aim to test the survival of _L. paracasei_, since LBA is resistant to human digestive enzymes [5]. For this purpose, the viability of the microorganisms was tested at several times during the digestion trial and calculated per gram of capsule. In addition, a sample of free bacteria, unencapsulated, was tested also as control, with an initial concentration of 7 \(\mathrm{log_{10}}\) CFU/mL. The three digestive conditions (salivary, gastric, and intestinal phases) were prepared according to [PERSON] et al. [32] with some modifications. Simulated salivary fluid (SSF) was prepared by adding 1.12 g/L KCl, 0.50 g/L KH\({}_{2}\)PO\({}_{4}\), 1.14 g/L NaHCO\({}_{3}\), 0.031 g/L MgCl\({}_{2}\)(H\({}_{2}\)O)\({}_{6}\), and 0.0058 g/L (NH\({}_{4}\))\({}_{2}\)CO\({}_{3}\). Simulated gastric fluid (SGF) was prepared by mixing 0.51 g/L KCl, 0.12 g/L KH\({}_{2}\)PO\({}_{4}\), 2.10 g/L NaHCO\({}_{3}\), 2.76 g/L NaCl, 0.020 g/L \begin{table}
\begin{tabular}{c c c} \hline \hline
**Capsules** & **Carbon Source** & **Coating** \\ \hline I & AFB-G & 0.1M 10 min (A from Table 1) \\ \hline II & AFB-G & 0.5M 10 min + chitosan (D from Table 1) \\ \hline III & AFB-L & 0.1M 10 min (A from Table 1) \\ \hline IV & AFB-L & 0.5M 10 min + chitosan (D from Table 1) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Capsule formulations used to test probiotic viability over time, to study the impact from the carbon source used and from the crosslinking of the coating. MgCl\({}_{2}\)(H\({}_{2}\)O)\({}_{6}\), and 0.048 g/L (NH\({}_{4}\))\({}_{2}\)CO\({}_{3}\). Simulated intestinal fluid (SIF) was prepared by adding 0.51 g/L KCl, 0.11 g/L KH\({}_{2}\)PO\({}_{4}\), 7.14 g/L NaHCO\({}_{3}\), 2.24 g/L NaCl, and 0.067 g/L MgCl\({}_{2}\)(H\({}_{2}\)O)\({}_{6}\) (all reagents from Sigma-Aldrich). Before the in vitro digestion test, the thermal-dried and freeze-dried capsules were rehydrated in two different ways: in a saline solution (0.9% NaCl, 1:100 (_w/v_) at 37 \({}^{\circ}\)C for 15 min) and in 120 g of natural yogurt (Danone, Danone Group SA, France) (at 4 \({}^{\circ}\)C for 24 h). For the salivary simulation, 25 mL of SSF were mixed with CaCl\({}_{2}\) (Sigma-Aldrich) and \(\alpha\)-amylase (1333 U/mg protein, CAS 900-90-2, Sigma-Aldrich) to obtain a concentration in the final volume of 0.083 g/L and 75 U/mL, respectively. Capsules rehydrated in saline solution were recovered and added directly while those rehydrated in yogurt were added with 5 g of yogurt. The oral mixture was incubated for 3 min at 37 \({}^{\circ}\)C. Afterwards, 25 mL of SGF were added to the mixture. Porcine pepsin (2500 U/mg protein, CAS 9001-75-6, Sigma-Aldrich) and CaCl\({}_{2}\) were also added to this mixture to achieve a final concentration of 2000 U/mL and 0.0083 g/L. Then, the mixture was well homogenized, the pH was adjusted to 2.0 with HCl 5 M (Sigma-Aldrich) and incubated at 37 \({}^{\circ}\)C for 2 h. Finally, porcine pancreatin (0.1% (_w/v_), CAS 9049-47-6, Sigma-Aldrich), bovine chymotrypsin (3% (_w/v_), CAS 9004-07-3, Sigma-Aldrich), amyloglucidase (260 U/mL, CAS 9032-08-0), with a concentration in the final volume of 1.12 U/mL, [33]), and CaCl\({}_{2}\) (0.033 g/L) were mixed with 50 mL of SIF. This solution was added to the previous mixture and was well homogenized, the pH adjusted to 6.5 with NaOH 5 M (Sigma-Aldrich), and incubated at 37 \({}^{\circ}\)C for 2 h.  
The bacterial viability was assessed by taking samples at different times: one after the simulated salivary phase and one after each 30 min in the simulated gastric and intestinal phases (9 samples in total for the entire simulated digestion trial). For the sampling, a number of capsules were collected and weighed under sterile conditions. The capsules were homogenized and dissolved with a sterile sodium citrate solution (1% (_w/w_), pH 6.0, Sigma-Aldrich) in a 1:10 proportion (capsule weight/sodium citrate volume).