Proteolytic Activity of Bacillus subtilis upon κ-Casein Undermines Its "Caries-Safe" Effect

Milk is believed to be a relatively "caries-safe" food. This belief relies on the fact that caseins, which constitute around 80% of milk's protein content, were found to inhibit the adhesion of Streptococcus mutans to enamel and, therefore, decrease biofilm formation. While S. mutans is considered a leading cause of dental disorders, Bacillus subtilis is a non-pathogenic foodborne bacterium, frequently contaminating milk and its products. This study aimed to investigate the effects of dairy-associated foodborne bacteria such as B. subtilis on biofilm formation by S. mutans in the presence of casein proteins. Our results indicate that there is a significant decrease in total biofilm formation by S. mutans exposed to a casein protein mixture in a mono-species culture, whereas, in the co-culture with B. subtilis, an inhibitory effect of the caseins mixture on S. mutans biofilm formation was observed. Proteolytic activity analysis suggested that B. subtilis is capable of breaking down milk proteins, especially κ-casein, which enables biofilm formation by S. mutans in the presence of milk caseins. Therefore, these findings may challenge the assumption that milk is "caries-safe", especially in a complex microbial environment.