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

Abstract

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.

Keywords: Bacillus; Flow cytometry; Germination; Mild inactivation; Superdormant.