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.

Inactivation of Bacillus subtilis spores by a combination of high-pressure thermal treatment and potassium sorbate.

Combining High-pressure Thermal Treatment (HPTT) and Potassium Sorbate (PS) may have a stronger spore inactivation effect. Spores of Bacillus subtilis were subjected to HPTT at 600 MPa-65 °C/75 °C and a combination of HPTT and PS of 0.1% and 0.2% concentrations. After these treatments, different procedures and techniques were employed to investigate the spore's inactivation. The results revealed that 4.92 ± 0.05 log spores were inactivated after treatment at 600 MPa-75 °C, while 5.97 ± 0.09 log spores were inactivated when the HPTT treatment was combined with 0.2% PS. Changes in permeability of the spore's inner membrane were characterized by OD600 value and release rates of nucleic acids, protein, and dipicolinic acid (DPA). Compared with HPTT treatment at 600 MPa-75 °C, the OD600 value of spores decreased further by about 50% after treatment with a combination of HPTT and 0.2% PS. Additionally, the combined treatments resulted in a significant increase in the OD260 and OD280 values, as well as the DPA release. The spore size analysis indicated a significant decrease in the size of spores treated with a combination of HPTT at 600 MPa-75 °C and PS of 0.2% concentration. Furthermore, the flow cytometry analysis and confocal laser scanning microscopy (CLSM) analysis indicated that the inner membrane damage of spores was higher after combined treatments than that after HPTT treatment alone. A significant reduction was also found in the Na+/K+-ATPase activity after the combined treatments. Also, the FTIR analysis revealed that the combined treatments resulted in significant adverse changes in the spores' inner membrane, cell wall, cortex, and nucleic acid. Therefore, the combination of HPTT and PS has a stronger inactivation effect and can be suggested as a promising strategy for the inactivation of Bacillus subtilis spores.

High Pressure Thermal Sterilization and ε-Polylysine Synergistically Inactivate Bacillus subtilis Spores by Damaging the Inner Membrane.

ABSTRACT: This study was conducted to determine the sterilization effect of a combination of high pressure thermal sterilization (HPTS) and ε-polylysine (ε-PL) on Bacillus subtilis spores. The spores were treated with HPTS (550 MPa at 25, 65, and 75°C) and ε-PL at 0.1 and 0.3%. HPTS and ε-PL synergistically decreased the number of surviving spores and increased the release of the intracellular components in the spore suspension, with the maximal effects from treatment with 550 MPa at 75°C plus 0.3% ε-PL. Maximum fluidity and permeability of the cell inner membrane were observed with 550 MPa at 75°C plus 0.3% ε-PL. Changes in membrane lipids were detected from 3,000 to 2,800 cm-1 by Fourier transform infrared spectroscopy. The results provide new insights into the mechanism by which HPTS and ε-PL synergistically sterilize B. subtilis spores.

Glucose protects the cell membrane, Na+/K+-ATPase, nucleic acids, and proteins in Bacillus subtilis spores under high pressure thermal sterilization.

The extreme resistance of bacterial spores to sterilization makes them a major concern to the food industry and consumers. In this study, the effect of glucose on the inactivation of Bacillus subtilis spores by high pressure thermal sterilization (HPTS) was evaluated. The results showed that the protective effects of glucose increased with the increase in its concentration. Compared with the HPTS control (no addition of glucose), the activity of Na+/K+-ATPase was increased, the leakage of proteins and the release of 2,6-pyridine dicarboxylic acid (DPA) was decreased, and the vibrational strength of the functional group P = O was reduced by the addition of glucose. At the same time, glucose treatment increased the content of α-helix by 6%-22%, while decreased the random coil content by 5%-13% of the cellular protein. In conclusion, the addition of glucose protected the cell membrane, Na+/K+-ATPase, cellular nucleic acids and proteins of B. subtilis under HPTS treatment.