Biofilm formation assay

The biofilm formation assay was adapted from Ouarabi et al. [61]. Initially, suspensions (10⁸ CFU mL^-1) of L. plantarum strains were obtained as previously described. An aliquot (10 μL) of each Lactobacillus strain was inoculated separately in trypticase soy broth (TSB, Acumedia) (200 μL) supplemented with peptone (20 g mL^-1) in a 96-well polystyrene plate and then incubated overnight. After incubation, plates were washed twice with sterile saline to remove non-adherent cells. Cells were fixed with 96% ethanol (200 μL) and incubated for 15 min at room temperature (25°C). Plates were emptied and then filled with violet crystals (200 μL, 0.1%) and incubated for 15 min at room temperature (25°C). The plates were then washed twice with sterile saline, and the wells were resuspended in 96% ethanol (200 μL); absorbance (650 nm) was immediately measured and used as an indication of biofilm formation. Sterile medium was included as a negative control to ensure that the influence on biofilm formation was not attributed to a non-specific binding effect to crystal violet. Based on the optical densities of the isolates (OD_I) and the negative control (OD_C), the formation of biofilms by Lactobacillus strains was classified according to their adherence: non-adherent, OD_I ≤ OD_C; weakly adherent, OD_C < OD_I ≤ (2 × OD_C), moderately adherent: (2 × OD_C) < OD_I ≤ (4 × OD_C); strongly adherent: (4 × OD_C) < OD_I.