## 2 Materials and Methods
### Preparation of Probiotic Suspension  
Probiotics were pre-cultured twice with 5% (v/v) inoculum in MRS (5 mL) broth and incubated at 37 \({}^{\circ}\)C for 12 h. For strains of the genus _Bifidobacterium_, MRS broth (Difco, USA) was supplemented with L-cysteine-HCl (0.05%, w/v) and incubated under anaerobic conditions (GasPak(tm) anaerbiosis generator system, BD, USA). The probiotics for the encapsulation assays by spray drying were grown in MRS (200 mL) broth inoculated (5%, v/v) with the grown pre-culture and incubated at 37 \({}^{\circ}\)C for 12 \(\pm\) 2 h. Probiotics in the late-log phase were harvested by centrifugation (6000\(\times\)\(g\) for 10 min at 4 \({}^{\circ}\)C); the pellets were washed with sterile distilled water and, again, centrifuged as described above. Finally, the probiotics were re-suspended in sterile distilled water and added to the encapsulating solution. In this stage, the bacterial suspension presented a total viable count between 10\({}^{9}\) and 10\({}^{10}\) CFU/mL.  
Effect of Inulin, or Mucilage from Chia or Flax Seeds, and Inlet Air Temperature on the Survival of L. rhamns after Spray Drying  
The effect of CM, FM, or INL as an encapsulating agent was determined on _L. rhamns_ survival after spray drying. Bacterial suspensions fed to the spray dryer contained maltodextrin (MD, food grade, with a dextrose equivalent of 15, Prinal(r) and different concentrations (0, 0.3, 0.6% w/v) of CM, (0, 0.1, 0.2% w/v) FM, or INL (0, 5, 10% w/v) such that the total solids concentration was 15% (w/v). The effect of encapsulating agents on _L. rhamns_ survival was determined at three inlet air temperatures (90, 110, and 130 \({}^{\circ}\)C). A response surface methodology (RSM) matrix was applied as the experimental design. The experiments were organized according to a face-centered central composite design with two variables (encapsulating agent and drying temperature) and three replicated center-point experiments. A total of 11 experiments were performed for each encapsulating agent (Table 1). The survival after spray drying and viability of _L. rhamns_ during storage under the refrigerated condition (4 \({}^{\circ}\)C) was followed periodically for 40 days and then determined at 80 and 250 days.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Treatment**} & \multicolumn{4}{c}{**Encapsulating Solution**} & \multirow{2}{*}{**T (\({}^{\circ}\)C)**} \\ \cline{2-2} \cline{4-5}  & **EAC \({}^{\dagger}\) (\%, w/v)** & & **MD \({}^{\dagger}\) (\%, w/v)** & \\ \hline
**1** & INL & 0.0 & 15.0 & 90 \\
**2** & & & & 110 \\
**3** & & & & 130 \\
**4** & & 5.0 & 10.0 & 90 \\
**5** & & & & 110 \\ \hline \hline \end{tabular}
\end{table}
Table 1: A design central composite face-centered with two variables, encapsulating agent (chia seed mucilage, flaxseed mucilage, and inulin) content (EAC) and drying inlet air temperature (T).  
Effect of Chia Seed Mucilage on the Survival After Spray Drying and Viability during Storage of L. plantarum and Strains of the Genus Bifidobacterium  
This stage assessed the effect of the operation conditions (0.6% w/v of CM and 90 \({}^{\circ}\)C of inlet air temperature) that maximized the survival of _L. rhamn_ during the spray-drying process on survival after spray drying and viability during storage of _L. plantarum_, _B. infantis_, and _B. longum_. Probiotics for the spray-drying assays were prepared according to item 2.3. The viability of encapsulated bacteria during storage was determined periodically for 60 days at 4 and 25 \({}^{\circ}\)C.