Thermosonication suppresses heat-stress-induced biofilm enhancement in Bacillus: Metabolomics insights into reduced contamination risk compared with pasteurization

Conventional pasteurization inactivates microorganisms but can also enhance the biofilm-forming ability of some Bacillus species. This undesirable consequence increases the risk of contamination. This study explored thermosonication (TS) as an alternative and identified its unique mechanism using non-targeted metabolomics and targeted gene expression analysis. Pasteurization triggers “programmed biofilm synthesis” in strains such as BC01 and BS01. It depletes key metabolites such as L-glutamine, histidine, and arginine, and upregulates biofilm genes ( spo0A , tasA , epsA ) to promote matrix production. In contrast, TS causes a “metabolic crisis” by disrupting the tricarboxylic acid cycle, leading to the accumulation of intermediates (citrate, α-ketoglutarate) and severe energy deficit. A significant upregulation of the universal stress regulator sigB marks this. As a result, bacteria stop biofilm synthesis and shift focus to survival and repair. Strain-specific accumulation of stress-protectant metabolites occurs, including betaine and phosphate in BC01 and sucrose and 5′-ribose-5′-phosphate in BS01. Inhibition of biofilm-promoting pathways means TS reduces biofilm formation more than pasteurization. Overall, TS offers dual bactericidal and biofilm-inhibitory effects by targeted metabolic disruption. This dual mechanism provides a strategy to lower biofilm risks, improve dairy safety, and extend shelf life. • Thermosonication curbs pasteurization-boosted Bacillus biofilms. • TS replaces biofilm-dependent heat resistance in Bacillus via SigB adaptation. • TS disrupts the TCA cycle, causing an energy deficit that weakens biofilms.