Figure 6.

Supplementation of protease inhibitors eliminates proteolytic effect of B. subtilis cells in the presence of casein proteins. S. mutans and B. subtilis cells were grown in BHI supplemented with 3% lactose with various concentrations of casein proteins mixture (0%, 0.5%, 1%, or 1.5%) with addition of a proteinase inhibitor cocktail. After 24 h of incubation at 37 °C in 95% air/5% CO2, the formed biofilms were washed twice. The formed biofilms were stained in Syto 9 (green) labeling live bacteria and PI (red) labeling dead bacteria. The graph displays data of fluorescent integrity of the formed biofilms as calculated by imageJ. The protease inhibitor cocktail canceled the proteolytic effect of B. subtilis in the dual-species culture, and there was a significant decrease in the formed biofilm (in all tested concentrations of casein proteins). The pictures are representative of two biological repeats, each preformed in duplicate. * p < 0.05 compared to control (0% casein).