Mechanism of Bacillus subtilis spore inactivation induced by moderate electric fields

Bacterial endospores are the key safety targets for inactivation within low-acid foods. Herein, we investigated the inactivation of Bacillus subtilis CGMCC 1.1087 spores (10(7) CFU/mL) in sterile distilled water using moderate electric fields (MEF, 300 V/cm) under various temperatures (< 30, 55, 65 and 75 degrees C). MEF treatment at below 30 degrees C resulted in 0.6-log reduction of spores, while treatments for 60 min without electric fields showed no inactivation. Inactivation induced by MEF in the same treatment time increased to 1.8-, 2.0- and 2.5-log as temperature increased to 55, 65 and 75 degrees C. Spores treated with MEF at < 30, 55, 65 and 75 degrees C or mild heat (55, 65 and 75 degrees C) scarcely lost heat resistance, suggesting that spores did not germinate during MEF or mild heat treatment. The viability of MEF-treated spores did not increase by addition of lysozyme (3 mu g/mL) in recovery plates, preincubation for 1 h in a 1:1 mixture of 60 mM Ca2+ and DPA, or lysozyme treatment in hypertonic medium. Confocal laser scanning microscopy photomicrographs showed that exposure to MEF induced a marked increase in the permeability of inner membrane and cortex. These findings suggested that damage of the cortex and inner membrane, rather than spore nutrient germinant receptors or cortex lyric enzymes, are possible reasons contributing to inactivation of B. subtilis spores by MEF. This study indicates that MEF at mild temperatures (55 to 75 degrees C) have the potential for spore inactivation. Industrial relevance: Literature in the past few years has shown that moderate electric fields (MEF), typically associated with ohmic heating, have nonthermal effects on bacterial spores, leading to accelerated inactivation. The current work extends the range of temperatures to those well below thermally lethal conditions, and shows that some spore inactivation occurs under MEF, even when temperatures are sublethal. Little or no germination is observed, and spore inner membranes are increasingly compromised over time. The elucidation of such nonthermal effects would be significant to the food industry as it seeks increasingly nonthermal methods for inactivation of spores.