Abstract
Purpose:
The purpose of this study was to establish a sterilization method that effectively inactivates Bacillus subtilis spores while retaining food quality through the combination of microwave (MW) heating with rosemary extract-induced germination, which reduces the treatment temperature and pressure required for sterilization.
Methods:
Spores were subjected to MW heating, and heat-stress responses were evaluated by gene expression analysis, while spore morphology was observed by fluorescence microscopy with staining of the cells with PI and DAPI. The influence of MW frequency (2.45 GHz vs. 27 MHz) and power on germination and sterilization, as well as comparisons with the conventional sterilization method of autoclaving, were considered. Rosemary extraction was conducted in solvents with different polarities and analyzed by liquid chromatography-mass spectroscopy (LC-MS), which led to the major compounds rosmanol, ursolic acid, and betulinic acid.
Results:
MW treatment resulted in significant structural damage to spores, reducing turbidity and increasing membrane permeability. Gene expression analysis revealed that MW preferentially induced the stress-related protein genes (dnaK, dnaJ, and clpP), which indicated that damage targeted the proteins. Rosmanol increased the hydrolytic enzyme gene cwlJ expression to comparable levels of L-alanine, indicating that rosemary extract and rosmanol followed the same germination induction pathway of L-alanine. These results suggested MW heating combined with rosemary-induced germination as a promising sterilization technique to preserve food quality while effectively targeting spore-forming bacteria.
