Effects of heat combining with Nisin treatment on the sterilization of Bacillus subtilis spores

Abstract: Bacterial spores are the most difficult microorganisms to be inactivated, which can cause food spoilage and poisoning in food production. High-temperature treatment above 100 ℃ is often used to inactivate bacterial spores in food, but the heat treatment at high temperature inevitably affects the nutrition and sensory quality of products. In this study, a lower-temperature heat treatment combined with Nisin on bacterial spores was proposed to explore the inactivation effects and mechanism, in order to find a feasible way to inactivate bacterial spores with lower-temperature. A plate-counting method, fluorescence polarization method, spectrophotometry and flow cytometry were used to determine the survival rate, fluorescence polarization degree, the absorbance value and inner membrane permeability of the spore samples. The results showed that: 1) The 80 ℃ treatment or Nisin alone cannot inactivate bacterial spores, but the 80℃ heat treatment combined with Nisin can effectively inactivate bacterial spores. After treated at 80 ℃ with 500 mg/L Nisin for 20 min, the survival concentration of bacterial spores decreased by about 1.4 lg (CFU/mL). 2) The fluorescent probe DPH was used to mark the inner membrane of bacterial spores, and further to analyze the fluidity of bacterial spores in the inner membrane. The results showed that the heat treatment combined with Nisin can significantly improve the fluidity of the inner membrane. Nisin can directly affect the degree of fluorescence polarization in the inner membrane. With higher Nisin concentration, the influence of Nisin on the degree of fluorescence polarization of inner membrane was stronger, whereas, the influence was smaller in the heat treatment at 80 ℃. The effects of 500 mg/L Nisin on the bacterial spores were much stronger than that of heat at 80 ℃. 3) The content release of spore was closely related to the inner membrane permeability of bacterial spores, and the absorbance value of spore suspension was often used to estimate the release of spore's content. The higher release degree of spore's content caused more significant decrease of the absorbance value of spore suspension. After 80℃ thermal treatment combined with Nisin, the absorbance value of spore suspension was significantly lower than that of thermal or Nisin treatment alone. After the treatment with 80 ℃ heat and 500 mg/L Nisin for 20 min, the absorbance value of spore suspension was the lowest. 4) The spores treated with heat combined with Nisin were stained with PI (Propidium Iodide), whereas, the changes of inner membrane permeability were studied by flow cytometry. The 80℃ thermal treatment combined with Nisin can significantly increase the permeability of inner membrane. Compared with heat or Nisin used alone, the permeability of inner membrane increased significantly after thermal treatment combined with Nisin. The higher Nisin concentration caused the higher permeability of inner membrane. It was found that Nisin had significant effects on the fluidity and permeability of inner membrane for the spore, where the 80 ℃ thermal treatment combined with different concentrations of Nisin had synergistic effect to improve the permeability of inner membrane. With the increase in the permeability of inner membrane, water was easier to enter the core of bacterial spores, which reduced the heat resistance of bacterial spores. The 80 ℃ thermal treatment combined with 500 mg/L Nisin can effectively inactivate bacterial spores, while the addition of Nisin greatly reduced the temperature required for inactivation of bacterial spore. The findings can contribute to reduce the damages of thermal sterilization on the nutritional and sensory quality in food production.