高压热杀菌结合酸对枯草杆菌芽孢的杀灭作用

Effects of high-pressure thermal sterilization combining with acid on the sterilization of Bacillus subtilis spores

高压热杀菌(high-pressure thermal sterilization,HPTS)能够有效的杀灭食品中最难杀死的芽孢,研究表明,HPTS结合酸处理能取得更好的杀灭效果。为明确HPTS结合酸杀灭芽孢的机理,该研究调整芽孢悬浮液的pH值为1、4、7,于550 MPa结合25、65、75℃处理20 min。采用平板计数法、紫外分光光度法、流式细胞术和傅里叶红外变换光谱对HPTS结合酸处理后枯草杆菌芽孢的杀灭率、紫外吸收泄漏量、内膜通透性及芽孢成分变化进行研究。结果表明,随着pH值降低,HPTS结合酸对芽孢的杀灭作用明显增强,芽孢最多被杀灭了6.25 lgCFU/mL;紫外吸收泄漏量及内膜通透性都显著增加;同时芽孢内膜磷脂相态发生转变,膜脂流动性增加;蛋白质二级结构变化,导致蛋白变性;核酸物质变性,皮层肽聚糖和细胞壁结构改变。综上,酸处理是辅助HPTS杀灭芽孢的有效手段,两者具有良好的协同杀菌作用。

High-pressure thermal sterilization(HPTS)is effective in inactivating the most difficult-to-inactivate bacterial spores in foods,and studies have shown that HPTS combined with acid treatment can achieve better inactivating effect.To clarify the mechanism of inactivating bacterial spores by HPTS combined with acid,the pH of the bacterium suspension was adjusted to 1,4 and 7,and the suspension was treated at 550 MPa combined with 25,65 and 75℃for 20 min in this study.The killing effect,UV absorption leakage,inner membrane permeability and changes in spore composition of Bacillus subtilis after HPTS combined with acid treatment were investigated by plate counting,UV spectrophotometry,flow cytometry and Fourier infrared transform spectroscopy.It was found that:(1)Under normal temperature and pressure,the pH value alone could not cause the inactivation of bacterial spores.The inactivating effect of HPTS combining with acid on bacterial spores increased effectively with the decreasing of pH,and bacterial spores were inactivated by up to 6.25 lgCFU/mL.(2)The UV absorption leakage reflects the changes in the permeability of the inner membrane of bacterial spores.OD_(260) and OD_(280) values increased significantly after HPTS combined with different pH treatments,the UV absorption leakage of bacterial spores increased,indicating that HPTS combined with different pH caused some degree of damage to the permeability barrier of bacterial spores.As the pH decreased,the amount of UV-absorbing material leaked from bacterial spores after 550 MPa combined with 25℃/65℃/75℃treatment increased significantly,indicating that acid would further damage the permeation barrier of bacterial spores,allowing more nucleic acid and protein-based UV absorbers to leak out.(3)Flow cytometry is commonly used to detect damage to bacterial cell membranes.The bacterial spores were stained with the fluorescent dye PI,which releases red fluorescence after embedding double-stranded DNA.Compared to the action of HPTS alone,the proportion of positive areas after treatment with HPTS combined with pH 4 and pH 7 was essentially similar,whereas the proportion of positive areas after treatment with HPTS combined with pH 1 was significantly greater,indicating that HPTS combined with pH 1 had a stronger damaging effect on the inner membrane of bacterial spores than HPTS alone.It can be assumed that acid increased the permeability of the inner membrane of bacterial spores and improved the inactivating effect.(4)Fourier transform infrared spectroscopy is now widely used for the comprehensive detection of the physiological state of microorganisms.By applying the second derivative to the original infrared spectrum,changes in the lipid phase state of bacterial cell membranes and substances such as proteins and nucleic acids can be specifically analyzed.The wavelength range for infrared detection in this experiment was 4000-500 cm^(-1).The 3000-2800 cm^(-1)band reflects the phase changes of the membrane lipids.The infrared spectroscopy(IR)absorption peaks at the original 2960 cm^(-1)and 2919 cm^(-1)of bacterial spores treated with HPTS combined with acid shifted and the IR absorption peaks changed more strongly,indicating that the phospholipid phase state of the inner membrane of bacterial spores shifted,the phospholipid molecules of its inner membrane changed from a gel state to a liquid crystal state,and the membrane lipid mobility increased.The 1700-1600 cm^(-1)band reflects changes in protein secondary structure in the protein amide I band.The IR absorption peak of 1674 cm^(-1)representing theβ-folded structure of the protein shifted to 1676 cm^(-1)for bacterial spores treated with HPTS combined with acid,meanwhile,the IR absorption peaks of 1652 cm^(-1)and 1644 cm^(-1)representing theα-helical structure of the protein shifted to 1650 cm^(-1)and 1646 cm^(-1),indicating that changes in the secondary structure of proteins resulted in protein denaturation.The 1300-900 cm^(-1)band reflects changes in nucleic acid backbone vibrations.The bacterial spores treated with HPTS combined with acid showed a shift in the position of the IR absorption peaks at 1080 cm^(-1)and 1220 cm^(-1)towards the lower wave numbers and a weakening of the intensity of the absorption peaks.This indicates that the nucleic acid material within bacterial spores is denatured and the peptidoglycan layer and cell wall structure are significantly altered,possibly causing bacterial spores’cortex to rupture and thus leading to bacterial spores’death.The experimental results show that acid treatment is an effective means to assist HPTS in inactivating bacterial spores,with good synergistic bactericidal effect.