Combined treatment of ε-polylysine and heat damages protective structures and spore inner membranes to inactivate Bacillus subtilis spores.

The sterilizing effect of a combination of heat (80, 90, and 100 °C) and ε-polylysine (ε-PL, 0.25 and 1 g/L) treatments on Bacillus subtilis spores was investigated and compared with that of conventional heat sterilization. The inactivation rate of spores and changes in their protective structure were evaluated using different methods and techniques. Changes in cell membrane's fatty acids, cell walls, proteins and nucleic acids were also analyzed. The results showed that the combined heat and ε-PL treatments significantly (p < 0.05) inactivated the Bacillus subtilis spores compared with the single heat treatment. Besides, the inactivation of spores was enhanced as the temperature and ε-PL concentration of combined treatments increased. The inactivation rate was found to be 2.18 log after heating at 90 °C for 60 min combined with the addition of 1 g/L ε-PL. Additionally, the electrical conductivity of spores' suspension and the positive region of flow cytometry significantly (p < 0.05) increased depending on temperature and ε-PL concentration of a combination treatment, indicating significant damage in the cell membranes and increased permeability. Significant changes in the spore morphology were also observed by the microscopy analysis after a combination treatment. Furthermore, the Fourier transform infrared spectra indicated a phase change in the inner membrane and alteration in the structure of peptidoglycan layer, as well as protein and nucleic acids denaturation after combined treatments. Therefore, the combined heat and ε-PL treatments can be suggested as sterilizing alternative to conventional heat sterilization in the food industry.