*3.1. Formation of Mutual Probiotic Biofilm of B. subtilis with Lactic Acid Bacteria (LAB)*

The starting point of this investigation was generating the dual-species biofilms for the different LAB strains together with robust ECM-producing bacterium *B. subtilis*. Thus, the bacterial cells were incubated in the biofilm-promoting MMRS medium, which promotes increased biofilm formation by *B. subtilis* through the KinD-Spo0A pathway [33]. To visualize the mutual biofilms, a transcriptional fusion of the *tapA* promoter to the *cfp* gene (encoding cyan fluorescent protein) was used [35]. The observed upregulation in the CFP expression, during the mutual growth of *B. subtilis* with three different species of the probiotic LAB, indicates that the *tapA* operon was activated and there that there was notable matrix production by *B. subtilis* (Figure 1). This finding was quite noticeable following a comparison of morphological changes that occurred during LAB growth in the presence of *B. subtilis* (Figure S1, Supplementary Materials). In this regard, the LAB cells could not form any biofilm bundles during their growth as a mono-species culture (Figure S1, Supplementary Materials), whereas a notable incorporation of those cells was observed into biofilm bundles produces by *B. subtilis* cells (Figure 1).

**Figure 1.** Formation of dual-species biofilm bundles of *B. subtilis* with LAB. Bacterial biofilms were generated during co-culture growth of *B. subtilis* cells with either *Lactobacillus plantarum*, *Pediococcus acidilactici*, or *L. rhamnosus* cells in modified MRS (MMRS) medium at 37 ◦C for 8 h. The biofilm samples were prepared as described in Section 2 and analyzed using a confocal laser scanning microscope (CSLM, Leica, Germany). *B. subtilis* cells express CFP under the control of the *tapA* operon, which is responsible for the matrix production. LAB cells are not stained. Scale bar = 10 μm.

To confirm that there are no antagonistic interactions between the LAB and *B. subtilis* cells, the bacterial growth was analyzed in this mutual growth system. Consequently, there was no significant inhibition in either of the bacterial species following their mutual growth (Figure 2), meaning that the LAB and *B. subtilis* cells can grow together without interference through generating the mutual probiotic biofilm. In addition, the growth in dual-species biofilm did not change the medium acidification rate by the LAB cells (Figure S2, Supplementary Materials).

**Figure 2.** Co-culture growth of *B. subtilis* with the LAB bacteria does not influence growth rate. Growth curve analysis of LAB cells in presence or absence of *B. subtilis*. The blue line represents the growth rate of a single-species culture of LAB, whereas the orange line represents the growth rate of (**A**) *L. plantarum,* (**B**) *P. acidilactici*, and (**C**) *L. rhamnosus* in co-culture with *B. subtilis*. (**D**) Growth curve of *B. subtilis* cells in the presence of LAB species compared to single culture. The cells were analyzed during growth for 8 h in 37 ◦C, 150 rpm.
