Evaluation of the membrane damage mechanism of thymol against Bacillus cereus and its application in the preservation of skim milk

The goal of this study was to elucidate the mechanism by which thymol caused membrane damages in Bacillus cereus cells and the feasibility of its application in the preservation of skim milk. The minimum inhibitory concentration (MIC) of thymol was determined using the broth microdilution method. The membrane potentials, intracellular adenosine triphosphate (ATP) concentrations, and pH gradients across the membrane were characterized using fluorescence spectroscopy. The degree of membrane damage and membrane structural integrity were observed under an electron microscope. The quantity of B. cereus in skim milk at different temperatures and concentrations of thymol was calculated using the plate counting method. As a result, the MIC of thymol against B. cereus BNCC 103930 was 0.625 mg/mL. Thymol significantly induced B. cereus cell dysfunction, membrane potential hyperpolarization, pH gradient dissipation, the blockade of ATP synthesis, and intracellular ATP depletion. Confocal laser scanning microscopy (CLSM) and field emission gun scanning electron microscope (FEGSEM) assays revealed pronounced membrane damages of B. cereus after thymol administration. Of note, low thymol concentrations, together with low-temperature storage, have the potential to control B. cereus in skim milk. These results indicated that thymol could be used as a food additive preventing B. cereus food poisoning.