New insights into different structural damages of Bacillus subtilis spores under dry heat, saturated steam, and wet heat.

Spore inactivation remains a major challenge in food sterilization. In this study, the structural damage and inactivation of Bacillus subtilis spores were systematically compared under dry heat, saturated steam, and wet heat treatments. Increasing moisture content significantly intensified heat-induced damage to the spore coat and inner membrane and promoted cortex hydrolysis. RHS values increased by 39.68 % after dry heat at 240 °C, whereas increases of 42.98 % and 47.09 % were observed after saturated steam and wet heat at 121 °C, respectively. Elevated moisture also enhanced the exposure of free -SH groups and the fluorescence intensity of ANS. Flow cytometry revealed significant coat protein damage, with positive staining areas increasing to 69.12 %, 77.84 %, and 98.92 % following dry heat (240 °C), saturated steam (121 °C), and wet heat (121 °C), respectively. Confocal laser scanning microscopy confirmed these results and further demonstrated cortex hydrolysis across all treatments. FTIR spectroscopy showed distinct changes in proteins and membrane phospholipids; α-helix content decreased by 9.08 %, 9.12 %, and 9.64 % after dry heat (240 °C), saturated steam (121 °C), and wet heat (121 °C), respectively, while a 7 cm-1 red shift was observed in the 2961 cm-1 band after wet heat. Scanning electron microscopy and particle size analyses further supported that moisture substantially affected spore morphology and structural integrity during heat exposure. These findings highlight moisture as a critical determinant of the extent of spore structural damage during thermal sterilization.