In this study, a method of Bacillus subtilis spore inactivation under high pressure (P, 200 MPa) combined with moderate temperature (T, 80 °C) and the addition of antimicrobial peptide LK (10² μg/mL) was investigated. Spores presented cortex hydrolysis and inner membrane (IM) damage with an 8.16 log reduction in response to treatment with PT-LK, as observed by phase-contrast and inverted fluorescence microscopy and flow cytometry (FCM) analysis. Furthermore, a tandem mass tag (TMT) quantitative proteomics approach was utilized because Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis data were used. After treatment with PT-LK, 17,017 polypeptides and 3166 proteins were detected from B. subtilis spores. Among them, 78 proteins showed significant differences in abundance between the PT-LK-treated and control groups, with 49 proteins being upregulated and 29 proteins being downregulated in the PT-LK-treated group. Genetic information processing, metabolism, cellular process, and environmental information processing were the main mechanisms of PT-LK-mediated spore inactivation.

Killing spores is an important challenge for the development of the food industry. After germination, the resistance of spores disappears and they are more easily killed, which is currently the main strategy for their destruction. Therefore, study of the mechanism of spore germination is of great significance for improving methods of spore inactivation. Previous studies have shown that the hydration of the spore core region, accompanied by the disappearance of bacterial spore resistance, is a key step in the germination pathway of bacterial spores. However, the specific mechanism of this process has been studied very little. In this study, Bacillus subtilis PY79 was used as a model strain, and its single water glycerol channel protein (GlpF) was regarded as a starting point to explore the mechanism of water transport during spore germination. First, we constructed glpF mutants and overexpression strains and discovered that the deletion of glpF did not affect the growth of bacterial vegetative cells and spores. Further germination experiments on the spores of the glpF-deficient strain through detecting calcium dipicolinate and absorbance of spores showed that the germination rate of the mutant strain spores increased, while increasing the water activity did not affect the results caused by glpF deletion. Meanwhile, overexpressed glpF affected the permeability of the spore coat. Finally, when treating spores with ultra-high pressure, the spores lacking glpF were more likely to be inactivated. The above results have suggested that the glpF gene plays an important role in spore germination.

High-pressure homogenization has been extensively studied for its excellent homogenization effect and the prospect of continuous liquid food production, but its sterilization ability still needs to be improved. In this study, we replaced the homogenization valve with two opposing diamond nozzles (0.05 mm inner diameter) so that the fluid collided at high velocity, corresponding to high-pressure micro-fluidization (HPM). Moreover, HPM treatment significantly inactivated Staphylococcus aureus ~7 log in the liquid with no detectable sub-lethal state at a pressure of 400 MPa and a discharge temperature of 50 °C. The sterilization effect of HPM on S. aureus subsp. aureus was attributed to a significantly disrupted cell structure and increased membrane permeability, which led to the leakage of intracellular proteins, resulting in bacterial death. At the same time, HPM treatment was able to significantly reduce the ability of S. aureus subsp. aureus to form biofilms, which, in turn, reduced its virulence. Finally, compared to the simulated system, more effective sterilization was observed in apple juice, with its color and pH remaining unchanged, which suggested that HPM can be used to process other liquid foods.

In the food industry, food spoilage caused by spores is a pressing scientific challenge that needs to be addressed urgently, and spore germination is a key approach to solving this problem. Studies have shown that peptidoglycan-induced spore germination represents a novel mechanism of action, which can bind to the PASTA domain of the serine/threonine kinase PrkC. However, the signaling mechanism of peptidoglycan-induced spore germination remains unclear. This study focuses on Bacillus subtilis, using pull-down experiments to screen for proteins interacting with PrkC. There are 80 interaction proteins of PrkC that were identified in the spore. GO analysis reveals that PrkC-interacting proteins in the spore are mainly involved in metabolic processes, cell part and catalysis. KEGG results indicate that PrkC-interacting proteins in the spore are mainly involved in RNA degradation, quorum sensing, oxidative phosphorylation, etc. Additionally, proteins are categorized into six groups by function based on events that may be associated with post-germination triggered by peptidoglycan-induced activation of the PrkC signaling pathway, including “stimulate translation initiation” and “ATP synthesis and energy metabolism”. The experimental results provide a theoretical basis for further elucidating the signaling mechanism of PrkC, revealing the signaling pathway of peptidoglycan-induced spore germination, and identifying targeted inducers and repressors of spore germination.