Sustainable Food Processing Laboratory, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zurich, Zurich, Switzerland.
Sustainable Food Processing Laboratory, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zurich, Zurich, Switzerland.
Int J Food Microbiol. 2023 Oct 2;402:110279. doi: 10.1016/j.ijfoodmicro.2023.110279. Epub 2023 Jun 13.
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.
高压（HP）处理具有在最小热输入下实现细菌孢子灭活的巨大潜力。为了推进 HP 发芽和随后的孢子灭活，本研究使用流式细胞术（FCM）探索了 HP 处理孢子的生理状态。枯草芽孢杆菌孢子在缓冲液中于 550 MPa 和 60°C（超高 HP（vHP））下处理，处理后孵育，并使用 SYTO16 进行 FCM 分析以指示发芽和碘化丙啶（PI）以指示细胞膜损伤。根据 HP 驻留时间（≤20 分钟）、HP 后温度（冰、37°C、60°C）和时间（≤4 小时），用缺失菌株分析与 HP 发芽相关的皮层裂解酶（CLE）和小酸溶性蛋白（SASP）-降解酶的 FCM 亚群。还研究了 HP 后温度（冰、37°C）对中压（150 MPa、38°C、10 分钟）的影响。HP 后孵育条件强烈影响五个观察到的 FCM 亚群的流行率。HP 后在冰上孵育不能或仅缓慢地将 SYTO16 阳性孢子转移到更高的 SYTO16 水平。在 37°C 下孵育时，这种转移加速，并且根据 HP 驻留时间发生向高 PI 强度的转移。在 HP 后 60°C，主要的转移是从 SYTO16 阳性到 PI 阳性亚群。CLE 似乎都需要 CwlJ 和 SleB 酶来摄取 PI 或 SYTO16，并且对 550 MPa 和 60°C 的敏感性不同。不同程度的 SASP 降解可能解释了两个 SYTO16 阳性亚群的存在。在 HP 后冰上孵育或在 37°C 孵育时向更高的 SYTO16 强度的转移可能依赖于 CLE、SASP 降解酶或其相关蛋白的活性和从可逆 HP 诱导的结构变化中的恢复。这些酶似乎仅在减压或 vHP 处理（550 MPa、60°C）后才变得活跃。基于我们的结果，我们提供了一个经过改进的枯草芽孢杆菌孢子 HP 发芽-失活动力学模型，以及一种用于量化与安全性相关的亚群（即 vHP（550 MPa、60°C）超休眠孢子）的优化 FCM 方法。本研究通过揭示被忽视的参数：HP 后孵育条件，为温和孢子失活动力学的发展做出了贡献。HP 后条件显著影响孢子的生理状态，可能是由于酶活性的变化。这一发现可以解释先前研究中的不一致，并表明在未来的研究中报告 HP 后条件的重要性。此外，将 HP 后条件作为 HP 过程参数添加可能为在食品工业中潜在的工业应用中优化基于 HP 的孢子失活动力学开辟新的可能性。
