*2.7. Analysis of Survival Rates Following Transition within In Vitro Digestion System*

To analyze the survivability of the LAB during the passage in the gastrointestinal tract, the freeze-dried samples were resuspended in 5 mL of DW. Afterward, the samples were monitored for four hours through an in vitro digestion system using the method described previously [35].

#### *2.8. Determining the E*ff*ect of Conditioning Supernatant (CSN) on S. aureus Biofilm Formation*

Cells of *B. subtilis* and *Lactobacillus plantarum* were grown either in monoculture or co-culture using MMRS medium as described above for 24 h; *B. subtilis* cells were also grown in LB medium. Cultures were then centrifuged at 13,000 rpm for 5 min, and the supernatants were sterilized by passing through a 0.45-μm filter (Merck, Rockland, MA, USA). For analysis of the CSN effect on biofilm formation by pathogenic bacteria, the *S. aureus* starter culture was generated through its growth into 10 mL of TSB medium overnight at 37 ◦C and 150 rpm. The, *S. aureus* biofilm was grown into 1 mL TSB medium (within a 24-well culture plate) supplemented by the CSN (10% v/v) harvested from the above probiotic cultures. The TSB medium without supernatant was used as control. The plate was incubated for 24 h at 37 ◦C.

#### *2.9. Biofilm Quantitation Assay*

Crystal violet staining was performed similarly as described previously [34]. Briefly, following 24 h of incubation at 37 ◦C, unattached cells were removed by washing the well plates two times using DW. Then, 1% crystal violet (CV) solution was added to the wells. Following 2 min of incubation, the excess CV was removed by washing with DW. Afterward, the fixed CV was released by 33% acetic acid washing. Then, 100 μL of each sample was transferred to a new well plate for the absorbance detection at 570 nm. To confirm the CV results, the CFU quantitation was performed for surface-attached cells. Following 24 h of incubation at 37 ◦C, unattached cells were removed by washing the well plate two times using DW. Then, 1 mL of DW was added to each well, and the cells attached to the surface were scratched out using sterilized swab; the bacterial suspensions were serially diluted and underwent CFU counting on LB agar plates.
