Spontaneous germination of superdormant Bacillus subtilis spores under high hydrostatic pressure: Influencing factors and underlying mechanism

High hydrostatic pressure (HHP) as an advanced non-thermal food processing technology, can induce bacterial spores to germinate and reduce their resistance, and these germinated spores can be effectively inactivated by subsequent mild treatments such as pasteurization. This method for spore inactivation is known as the "germinate to eradicate" strategy. However, the presence of superdormant (SD) spores, which do not germinate under HHP and are referred to as high-pressure superdormant (HPSD) spores, poses a challenge to the successful implementation of this strategy. Hence, gaining insights into the characteristics of HPSD spores is crucial for their elimination. In this study, we reported that HPSD Bacillus subtilis spores isolated after 500 MPa/15-35 degrees C/3-5 min treatment (500 MPa-SD) showed spontaneous germination when incubated in sterile distilled water at 4 degrees C-37 degrees C, indicating the instability of these SD spores. Notably, elevating the 500 MPa treatment temperature from 15 degrees C to 35 degrees C, or increasing the incubation temperature from 4 degrees C to 37 degrees C, could enhance the efficiency of spontaneous germination of 500 MPa-SD spores. Additionally, we discovered that the germinant receptor GerA was crucial for the occurrence of spontaneous germination of 500 MPa-SD spores. Meanwhile, the 500 MPa-SD spores with GerA showed a substantial decrease in potassium ions and reduced heat resistance. These observations suggested that the 500 MPa-SD spores, at least a proportion of them, had an activated GerA, entering commitment state that led to irreversible spontaneous germination. Industrial relevance: The existence of SD spores greatly limits the successful application of HHP technology in low-acid foods processing. The new insights gained from this work are valuable for understanding the properties of the HPSD spores and could potentially pave the way for the development of innovative methods to effectively inactivate bacterial spores in industrial HHP food processing systems.