*3.2. Growth in Mutual Biofilm Increases the Survivability of the LAB during Desiccation*

It was hypothesized that the growth in the mutual biofilm system could provide a relative protection of LAB during the desiccation process, which might indicate about the relative improvement in survivability of the bio-coated cells through industrial processing and storage conditions. Therefore, the LAB cells grown in the mutual biofilm were exposed to desiccation conditions for either 20 or 40 h. The LAB cells grown in mono-species culture were used as a control sample. It was found that *L. plantarum* cells grown in mutual biofilm showed increased survival (relatively to mono-species culture) of around 1.12 log·CFU/mL and 1.52 log·CFU/mL, following 20 and 40 h of desiccation, respectively. Surprisingly, *L. rhamnosus* cells grown in the mutual biofilm demonstrated an even more significant increase in survivability, of around 3.12 log·CFU/mL, during 20 h of desiccation. Even more profoundly, the bio-coated cells of *L. rhamnosus* demonstrated around a five-log increase in their survivability after 40 h of desiccation. Concerning the cells of *P. acidilactici*, the bio-coated cells showed a relatively moderate increased survival following 20 and 40 h of desiccation of around 0.71 log·CFU/mL and 2.09 log·CFU/mL, respectively (Figure 3).

To reinforce our assumption about the ECM protection of the LAB cells, the dual- and mono-species biofilms were visualized using SEM imaging, after desiccation treatment. It was found that *L. plantarum* cells grown in the presence of *B. subtilis* were surrounded with the coating substance(s), which could be interpreted as the biofilm matrix (Figure 4).

**Figure 3.** Growth in dual-species biofilm increases survivability of LAB during exposure to drying process. Mono and dual-species cultures of *B. subtilis* and LAB cells were generated in MMRS medium during bacterial growth for 8 h in 37 ◦C, 50 rpm. Survival rates of the LAB cells (grown in presence or absence of biofilm forming *B. subtilis*) were determined based on CFU counts following desiccation conditions (40% relative humidity (RH)) for 20–40 h. Error bars represent standard deviation (SD). \* *p* < 0.05 comparison of the control and tested samples. (**A**) *L. plantarum* survival rates, (**B**) *P. acidilactici* survival rates, and (**C**) *L. rhamnosus* survival rates.

**Figure 4.** Bio-coating of *L. plantarum* cells by extracellular polymeric substance(s) produced by *B. subtilis*. Mono and dual-species cultures of *B. subtilis* and *L. plantarum* cells were generated in MMRS medium during bacterial growth for 8 h in 37 ◦C, 50 rpm. (**A**) SEM images of the mono-species culture of *L. plantarum* and (**B**) dual-species culture of *L. plantarum* with *B. subtilis* following desiccation conditions (40% RH) for 20 h. Images were taken at a magnification of 20,000× with a JEOL, JSM 7800F, Japan.
