*3.3. Growth in Mutual Biofilm Increases the Survivability of the LAB during Acid Stress Following Freeze-Drying*

It was further investigated whether the mutual biofilm could also protect the cells of LAB during freeze-drying, which is considered as the most common technique for drying and storage of probiotic bacteria for a long time [10,36]. Since, after consumption, the LAB cells are usually exposed to additional stress (acidic stress during the passage in the gastrointestinal tract), the survivability of the bio-coated cells during their exposure to acid stress was tested, following freeze-drying. To mimic the acid stress conditions, the bio-coated cells were freeze-lyophilized and exposed to pH 2. It was found that the bio-coated *L. plantrum* cells showed increased survivability of around 0.45 log·CFU/mL, compared to the control, following freeze-drying (Figure 5). In the case of the bio-coated *L. rhamnosus* cells, an increase of about 0.49 log·CFU/mL was observed, while, in the case of *P. acidilactici*, there was no significant change in the number of viable cells following freeze-drying (Figure 5). Concerning acid stress tolerance, the freeze-dried LAB cells were exposed to low pH (pH 2) for 1–3 h. As shown in Figure 5, the encapsulated LAB consistently demonstrated increased survival (up to around a one-log increase) following their exposure to this stress, especially after three hours of exposure to the low pH.

**Figure 5.** Bio-coating increases the survivability of the LAB during acid stress following freeze-drying. Mono- and dual-species cultures of *B. subtilis* and the LAB cells were generated in MMRS medium during bacterial growth for 8 h in 37 ◦C, 50 rpm. Survival rates of LAB cells were determined based on CFU counts following freeze-drying and an exposure to low pH (HCl 1 M, pH = 2) for 1–3 h. \* *p* < 0.05 for comparison of the control and tested samples. Error bars represent standard deviation (SD). BFD—before freeze-drying; AFD—after freeze-drying; AC1—freeze-dried and exposed for 1 h to acid conditions; AC3—freeze-dried and exposed for 3 h to acid conditions. The survival rates are shown for (**A**) *L. plantarum*, (**B**) *P. acidilactici*, and (**C**) *L. rhamnosus.*
