Cortex degradation plays a critical role in modulating the pressure resistance of Bacillus spores and their inactivation under high-pressure treatments

High hydrostatic pressure (HHP) is an emerging non-thermal technology capable of inducing spore germination and inactivation. However, the structural determinants of pressure resistance in germinated spores remain poorly understood. This study investigated the role of cortex degradation and other spore structures in the effectiveness of a germination-inactivation strategy using combined HHP treatments (200 MPa–500 MPa, or 500 MPa–500 MPa). Our results showed that HHP (100 to 500 MPa) induced germination and heat resistance loss in Bacillus subtilis and B. cereus spores. Cortex degradation induced by HHP, primarily mediated by CwlJ, contributed to the loss of pressure resistance and enhanced subsequent 500 MPa inactivation of B. subtilis spores. Incubation at 37 °C following 500 MPa treatment promoted further cortex degradation and enhanced inactivation during subsequent 500 MPa treatment, but this effect was abolished in spores with defective germinant receptors. Furthermore, spore coat defect lowered pressure resistance and improved inactivation in the combined 200 MPa and 500 MPa treatment, whereas deletion of small acid-soluble proteins had no effect. Finally, structural analyses supported that cortex degradation plays a crucial role in inner membrane damage and spore inactivation via HHP. These findings highlight cortex degradation as a key intermediate step in pressure-mediated spore inactivation and provide valuable insights for designing efficient non-thermal sterilization strategies.