The reader is referred to Reineke and Mathys (2020) for an extensive review of different spore inactivation pathways by emerging technologies.Ī more gentle approach to control spores aims on not directly inactivating the dormant spores but rather using a so called germination-inactivation strategy ( Collado et al., 2004 Abee et al., 2011 Lovdal et al., 2011 Zhang and Mathys, 2019).
![spore stain spore stain](https://lh6.googleusercontent.com/--D0m8JwO87s/To3dVy5tPUI/AAAAAAAAAAw/EWpZLhlGRH0/s640/blogger-image-1667167475.jpg)
Therefore, effective and gentle alternative non-thermal spore control strategies are of high interest ( Storz and Hengge, 2010 Sevenich and Mathys, 2018 Zhang et al., 2018). However, such procedures often cause unwanted losses in food quality. For food products where absence of spores is essential, intensive wet heat treatment is usually applied to directly inactivate spores ( Storz and Hengge, 2010 Georget et al., 2013). Depending on food composition and storage conditions, surviving spores can eventually germinate and grow out, and cause food quality and/or safety problems. These spores are extremely resistant to heat, dehydration, and chemical or physical stresses, and thus a major challenge in food decontamination processes ( Setlow, 2006, 2007 Setlow and Johnson, 2007 Patrignani and Lanciotti, 2016 Zhang et al., 2018). These bacteria can form spores when the environmental conditions become unfavorable for their survival. As a consequence, they inevitably enter the food chain, and potentially cause food spoilage and food-borne illnesses, leading to economic losses and increased public health risks ( Setlow and Johnson, 2007 Reineke et al., 2013b Wells-Bennik et al., 2016). Spore-forming bacteria, mainly represented by the genera Bacillus and Clostridium, are ubiquitous in nature. Introduction Isostatic High Pressure Processing as a Basis for Mild Spore Control Strategies This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states. HP treatment at 150 MPa and 37☌ did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. Moreover, the kinetic transitions between different physiological states were characterized. Of particular interest was the physiological state of the third sub-population, which was previously referred to as “unknown”. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80☌, 10 min) and cultivability in a nutrient-rich environment.
![spore stain spore stain](https://image4.slideserve.com/521079/spore-stain-l.jpg)
Bacillus subtilis spores were treated with HP at 150 MPa and 37☌, stained with SYTO16 and PI, and analyzed via FCM. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies.
![spore stain spore stain](https://d3i71xaburhd42.cloudfront.net/4c52e7412c4cf987f74c5458738a453df2260fd7/4-Figure1-1.png)
2Cytometry Facility, University of Zurich, Zurich, Switzerland.1Sustainable Food Processing Laboratory, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zürich, Zurich, Switzerland.Off 1, Stephan Benke 2 and Alexander Mathys 1*