Synergistic inactivation and mechanism of thermosonication treatment combined with germinants and licorice extracts against Paraclostridium bifermentans.

Spore contamination is a critical factor that contributes to food spoilage and economic losses in the food industry. In this study, we employed a "germination-inactivation-inhibition" strategy to effectively inactivate Paraclostridium bifermentans spores. We systematically screened and optimized the germinants, thermosonication conditions, and inhibitors to determine the most effective combination for spore inactivation. We found that the optimal conditions were germinant "A" GFNa-60 (90 mmol/L L-alanine, 10 mmol/L D-glucose, 10 mmol/L D-fructose, and 60 mmol/L NaCl), thermosonication (40 KHz, 480 W) at 80 °C for 60 min, and licorice extract (6.25 mg/mL) as an inhibitor. This combination was highly effective in deactivating P. bifermentans spores, resulting in a reduction of approximately 3.59 log CFU/mL. Detailed analyses, including particle size analysis, fluorescence microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), provided insights into the mechanisms underlying spore inactivation. Specifically, germinants decreased spore resistance, thermosonication induced spore surface expansion and perforation, and licorice extract facilitated spore dispersion while exacerbating thermosonication-induced inner membrane damage and nucleic acid leakage, leading to synergistic spore inactivation. Additionally, licorice extract continued to inhibit the growth and reproduction of the remaining spores. A spore inactivation rate of 99.97 % was achieved. These findings offer valuable insights into improved sterilization practices in the food industry, particularly for the management of spore contamination. The proposed "germination-inactivation-inhibition" strategy demonstrates potential as an effective approach for controlling spores in industrial applications.