Correlation Between Heat Resistance and Biofilm Formation of Bacillus Strains' Spores Isolated From Raw Cow's Milk

Bacillus spp. pose a critical challenge to the dairy industry due to their ability to form highly heat-resistant endospores and robust biofilms on processing surfaces, leading to recontamination and food safety risks. This study aimed to investigate the correlation between the heat resistance of spores and the biofilm formation capacity of seven Bacillus strains isolated from raw cow's milk in the Mekong Delta, Vietnam.

Thermal resistance (D-values) was determined by treating spores at 80, 85, 90, and 95 °C for intervals of 12, 15, 20, 25, and 30 minutes. Biofilm formation was quantified using the crystal violet assay in 96-well plates via OD₅₇₀ measurements. Additionally, phylogenetic relationships were elucidated through 16S rRNA gene sequencing and compared with GenBank sequences to identify the isolates.

A significant positive correlation was observed between biofilm density and spore heat resistance, with an R-squared (R²) value of = 0.881 (p = 0.00174). Strains with higher OD₅₇₀ values exhibited significantly greater D₉₅ values. Notably, strain TGS11.1 showed the highest thermal resistance (D₉₅ = 11.37 minutes) and the strongest biofilm-forming ability (OD₅₇₀ = 0.3196). Phylogenetic analysis indicated a high degree of genetic similarity between these isolates and Bacillus cereus.

These findings reveal a “double risk” from indigenous Bacillus spp. strains, where the ability to persist via biofilms and survive thermal processing through resistant spores occurs simultaneously. This synergy complicates sterilization protocols and suggests that biofilm-forming capacity could potentially serve as an indicator for thermal persistence in dairy processing environments.

The study highlights a clear link between biofilm formation and spore heat resistance in Bacillus spp. These results underscore the necessity for more stringent interventions and offer practical guidance for optimizing sterilization practices to ensure the safety of dairy products.