High pressure CO2 reduces the wet heat resistance of Bacillus subtilis spores by perturbing the inner membrane

Spores of wild-type Bacillus subtilis PS533 were treated by wet heat at 75 °C for 30 min, and high pressure CO2 (HPCD) at 6.5 MPa and 30 °C or 75 °C for 30 min. The spores were analyzed for wet heat resistance (85 °C, 90 °C, 95 °C) and typical germination events including DPA release and cortex hydrolysis, inner membrane permeability, and germination triggered by nutrient (L-valine and AGFK) or non-nutrient (dodecylamine and high pressure at 150 MPa or 550 MPa) germinants. The results showed that (i) HPCD-treated spores exhibited reduced wet heat resistance compared to the untreated or wet heat-treated spores; (ii) HPCD-treated spores did not undergo typical germination events such as DPA release or cortex hydrolysis compared to normally germinated spores; (iii) HPCD-treated spores released more metal ions and exhibited decreased ability to maintain DPA, indicating that the permeability of inner membrane of HPCD-treated spores was increased; (iv) HPCD-treated spores exhibited reduced germination rate when triggered by L-valine or 150 MPa, but increased germination rate when triggered by dodecylamine or 550 MPa, suggesting that the fluidity of the inner membrane of HPCD-treated spores might be increased. These results indicated that HPCD could reduce the wet heat resistance of spores, and this resistance decrease was probably due to the modification of the inner membrane caused by HPCD. Industrial relevance The extremely high wet heat resistance of spores makes them a significant problem in the thermal processing of foods. Thus, it of great interest to develop a process to reduce the wet heat resistance of spores. In this work, we found that HPCD can significantly reduce the wet heat resistance of B. subtilis spores, and this was achieved by perturbing the inner membrane of spores. These results can improve our understanding of the inactivation mechanism of spores by HPCD, and also provide an alternative approach for spore inactivation in foods.