Post-high-pressure temperature and time - Overlooked parameters in high pressure treatment of bacterial spores.

High pressure (HP) processing has high potential for bacterial spore inactivation with minimal thermal input. To advance HP germination and subsequent inactivation of spores, this study explored the physiological state of HP-treated spores using flow cytometry (FCM). Bacillus subtilis spores were treated at 550 MPa and 60 °C (very HP (vHP)) in buffer, incubated after the HP treatment, and stained for FCM analysis with SYTO16 indicating germination and propidium iodide (PI) indicating membrane damage. FCM subpopulations were analyzed depending on the HP dwell time (≤20 min), post-HP temperature (ice, 37 °C, 60 °C) and time (≤4 h), germination-relevant cortex-lytic enzymes (CLEs) and small-acid-soluble-proteins-(SASP)-degrading enzymes by using deletion strains. The effect of post-HP temperatures (ice, 37 °C) was additionally studied for moderate HP (150 MPa, 38 °C, 10 min). Post-HP incubation conditions strongly influenced the prevalence of five observed FCM subpopulations. Post-HP incubation on ice did not or only slowly shifted SYTO16-positive spores to higher SYTO16 levels. At 37 °C post-HP, this shift accelerated, and a shift to high PI intensities occurred depending on the HP dwell time. At 60 °C post-HP, the main shift was from SYTO16-positive to PI-positive subpopulations. The enzymes CwlJ and SleB, which are CLEs, seemed both necessary for PI or SYTO16 uptake, and to have different sensitivities to 550 MPa and 60 °C. Different extents of SASP degradation might explain the existence of two SYTO16-positive subpopulations. Shifts to higher SYTO16 intensities during post-HP incubation on ice or at 37 °C might rely on the activity and recovery of CLEs, SASP-degrading enzymes or their associated proteins from reversible HP-induced structural changes. These enzymes seemingly become active only during decompression or after vHP treatments (550 MPa, 60 °C). Based on our results, we provide a refined model of HP germination-inactivation of B. subtilis spores and an optimized FCM method for quantification of the safety-relevant subpopulation, i.e., vHP (550 MPa, 60 °C) superdormant spores. This study contributes to the development of mild spore inactivation processes by shedding light on overlooked parameters: post-HP incubation conditions. Post-HP conditions significantly influenced the physiological state of spores, likely due to varying enzymatic activity. This finding may explain inconsistencies in previous research and shows the importance of reporting post-HP conditions in future research. Furthermore, the addition of post-HP conditions as HP process parameter may open up new possibilities to optimize HP-based inactivation of spores for potential industrial applications in the food industry.

Isolation, stability, and characteristics of high-pressure superdormant Bacillus subtilis spores.

Bacterial spores are a major challenge in industrial decontamination processes owing to their extreme resistance. High-pressure (HP) of 150 MPa at 37 °C can trigger the germination of spores, making them lose their extreme resistance. Once their resistance is lost, germinated spores can easily be inactivated by a mild decontamination step. The implementation of this gentle germination-inactivation strategy is hindered by the presence of a subpopulation of so-called high-pressure superdormant (HPSD) spores, which resist germination or germinate only very slowly in response to HP. It is essential to understand the properties of HPSD spores and the underlying causes of superdormancy to tackle superdormant spores and further develop germination-inactivation strategies involving HP. This study investigated factors influencing the prevalence of HPSD spores and successfully isolated them by combining buoyant density centrifugation and fluorescence-activated cell sorting, which allowed further characterisation of HPSD spores for the first time. The prevalence of HPSD spores was shown to be strongly dependent on the HP dwell time, with increasing treatment times reducing their prevalence. Spore mutants lacking major germinant receptors further showed a highly increased prevalence of HPSD spores; 93% of the spores remained dormant even after a prolonged HP dwell time of 40 min. In contrast to nutrient germination, sublethal heat treatment of 75 °C for 30 min prior to pressure treatment did not induce spore activation and increase germination. The isolated HPSD spores did not show visible structural differences compared to the initial dormant spores when investigated with transmission electron microscopy. Re-sporulated HPSD spores showed similar germination capacity compared to the initial dormant spores, indicating that HPSD spores are most likely not genetically different from the rest of the population. Moreover, the majority of HPSD spores germinated when exposed a second time to the same germination treatment; however, the germination capacity was lower than that of the initial population. The fact that the majority of spores lost superdormancy when exposed a second time to the same trigger makes it unlikely that there is one factor that determines whether a spore germinates with a certain HP treatment or not. Instead, it seems possible that there are other reversible or cumulative causes. This study investigated the factors influencing spore HP superdormancy to improve the understanding of HPSD spores with regard to their stability, germination capacity, and potential underlying causes of spore HP superdormancy. This knowledge will contribute to the development of HP-based germination-inactivation strategies for gentle but effective spore control.