The stable superdormant Bacillus spores under 500 MPa: Isolation, characterization and development of strategy to reduce their abundance under high-pressure processing.

High hydrostatic pressure (HHP) can trigger bacterial spores to germinate and reduce their resistance, thereby facilitating the subsequent inactivation of germinated spores through mild treatments such as pasteurization. This approach is known as the "germinate to eradicate" strategy. However, the presence of superdormant (SD) spores that fail to germinate under HHP-termed high-pressure superdormant (HPSD) spores-poses a significant challenge to the efficacy of this method. We have previously found that a subset of HPSD spores isolated after 500 MPa treatment enter an irreversible germination commitment state and undergo spontaneous germination. However, there are still some stable HPSD spores that remain in deep superdormant state after 500 MPa induction treatment, hindering the effective implementation of the "germinate to eradicate" strategy. In this study, stable HPSD spores of Bacillus subtilis were isolated after 500 MPa treatment at 20 °C for 5 min and subsequent post-incubation (37 °C/12 h) to allow completion of spontaneous germination. These spores were characterized for germination capacity and resistance profiles. Compared with conventionally defined HPSD spores isolated immediately after 500 MPa treatment, the stable HPSD spores exhibited more pronounced germination defects in response to various stimuli, including L-alanine, the AGFK mixture, dodecylamine, and high-pressure treatments at 200 MPa and 500 MPa. Furthermore, they demonstrated reduced resistance to heat, hydrochloric acid, sodium hypochlorite, and formaldehyde, with decimal reduction times (D-values) decreased by 1.78, 2.42, 1.82, and 2.58 fold, respectively. In contrast, their resistance to UV254 nm irradiation was enhanced, showing a 2.18-fold increase in D-value. Additionally, we optimized the HHP treatment parameters along with post-incubation temperature and duration to minimize the quantity of stable HPSD spores of both B. subtilis and B. cereus, whether suspended in sterile double-distilled water or in food matrices such as skimmed milk and vegetable juice. The findings of this study enhance our understanding of HPSD spore behavior and contribute to the development of effective spore inactivation strategies for industrial HHP applications.