Synergistic inactivation and mechanism of thermally enhanced photodynamic treatment with germinant against Bacillus altitudinis and Bacillus safensis spores

Bacillus species form highly heat-resistant spores, causing food spoilage and economic losses in the food industry. This study employed a “germinate and inactivate” strategy to effectively inactivate Bacillus altitudinis and Bacillus safensis spores. We first prepared curcumin-thymol nanoparticles (CT NPs) to mediate photodynamic inactivation (PDI) of vegetative cells under white light. Subsequently, optimal synergistic parameters were determined. Combining 5 mM L-aspartic (Asp) and 50 μM CT NPs with 65 °C thermal treatment under white light (TL-Asp) for 120 min achieved log reductions of 3.75 for B. altitudinis spores and 4.05 for B. safensis spores. Multiparameter analysis, including pyridine-2,6-dicarboxylic acid (DPA) release, nucleic acid and protein leakage, Na + /K + -ATPase activity, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and heat resistance, provided mechanistic insights into spore inactivation. Specifically, germinant reduced spore resistance, while 65 °C thermal treatment enhanced photodynamic penetration into spore inner membrane (IM) and core, thereby exacerbating IM damage and nucleic acid leakage, leading to synergistic inactivation. Additionally, 65 °C heat synergistically promoted core hydration by increasing membrane fluidity, which accelerated spore germination. These findings suggest that TL-Asp treatments hold potential for controlling spores in food industry applications.