超高压对金黄色葡萄球菌的杀菌机理研究

The high hydrostatic pressure (HHP) sterilization technology is an alternative food low-temperature (room-temperature) sterilization technology. However, the mechanisms of HHP-induced microbial inactivation have not been clearly explained. The effects of HHP on the structure, membrane proteins, membrane fluidity, membrane integrity, absorption and utilization of the nutritional substance, metabolic activity and biological energy conversion will be investigated with the foodborne pathogenic Staphylococcus aureus as test microorganism in this the project. The new methods to determine the integrity of cell membrane and the absorption of the nutrient will be developed. The relationships between the detachment of membrane proteins from cell membrane, the changes of membrane fluidity and membrane integrity, and the corresponding death rates of this microorganism subjected to HHP will be constructed, and the mechanisms of cell membrane damage will be clarified. Before and after cell membrane physical injuries due to HHP, the differences of the changes for the physiological and biochemical indices such as the absorption of the nutritional substance, metabolic activity, and biological energy conversion and the mutual relationships between the indices will be defined, and the mechanisms of the physiological and biochemical injuries will be revealed. The relationships between the changes for the physiological and biochemical indices described above and the corresponding death rates of this microorganism due to HHP will be verified. Based on the investigation descried above, the primary target for pressure-induced inactivation and the inactivation mechanisms of this microorganism by HHP will be elucidated, which would establish theoretical and technological bases for the application of HHP-sterilization technology in the food industry.

超高压杀菌技术是一项崭新的食品低温(常温)杀菌技术，其确切杀菌机理尚未完全阐明。本项目以食源致病性金黄色葡萄球菌为试材，在已有工作的基础上，进一步研究超高压对其结构、细胞膜蛋白、膜的流动性和完整性、营养吸收和利用、代谢活力和生物能量转化等的影响；建立测定细胞膜完整性、营养的吸收和利用的新方法；建立超高压作用下菌体细胞膜蛋白脱落、膜脂流动性和完整性的变化与菌体数致死率之间的联系，揭示超高压对细胞膜损伤的机制；确立超高压作用下细胞膜在损伤前与损伤后菌体对营养的吸收利用、代谢活力和能量转化等生理生化活性的变化规律的差异及相互联系，揭示超高压对菌体生理生化活性伤害的机制；探明超高压作用下细胞膜在损伤前与损伤后菌体的这些生理、生化指标的变化与菌体数致死率之间的关系，明确在超高压作用下菌体失活的关键部位，阐明超高压对金黄色葡萄球菌的杀菌机理，为超高压杀菌技术在食品工业中的应用奠定理论和技术基础。

以金黄色葡萄球菌为试材，建立超高压对其失活曲线。当压力超过300MPa作用10min，在培养基上菌落数低于检测限。以荧光偏振法测定超高压对菌体细胞膜流动性、荧光偏振度和微粘度的变化。不同的压力对细胞膜的荧光偏振度和微粘度的影响不同，300~400MPa作用10~40min细胞膜荧光偏振度及微粘度达到稳定。.用紫外分光光度法和原子纸吸收法测定了经超高压作用后细胞内紫外吸收物与金属离子的泄漏和激光共聚焦显微镜观察其结构的变化。150~300MPa作用10min，细胞内紫外吸收物没有泄漏到胞外，350MPa作用10min细胞内紫外吸收物泄漏量达到最大；350MPa作用10min菌体几乎全部失活，紫外吸泄漏达到最大，此时的细胞膜才受到超高压的损伤。150~300MP作用10min时K+和Mg2+没有泄漏到胞外，350MP作用后泄漏达到最大。部分细胞在培养基上虽不能生长，但其膜还完好，表明超高压作用下细胞膜损伤与失活之间的对应关系不好。.经高压处理，部分菌体在培养基上不能存活，但细胞膜完好，荧光染色剂未进入细胞内，核未被染色。采用细胞内外pH达到平衡所用的时间的一半t1/2来表示超高压作用对菌体细胞膜H+通透性的影响，经300MPa作用10min后维持细胞质内pH的屏障被破坏，表明H＋对在压力的作用下通透性比细胞内紫外吸收物和荧光染料的通透性好。.以放射性探针标记菌体细胞膜，测定经超高压作用后细胞膜电位的变化；建立了荧光染料法测定胞内pH值，用荧光探针测定经超高压作用后细胞内pH值的变化；用比色法测定了超高压对F1F0ATP酶活性的影响。150~300MPa作用10min在培养基上随着压力的增大菌落数显著降低，电子显微镜下观察细胞膜结构完整，细胞内紫外吸收物与金属离子没有泄漏到细胞外，F1F0ATP酶活性降低显著，膜电势、跨膜H+浓度梯度和质子动力势基本保持不变；300MPa作用10minF0F1ATP酶活性降低到0，质子回路不通，电子传递链质子泵打出细胞膜外的质子无法重新回到细胞内，质子动力势达到最大值，偶联磷酸化过程中断，呼吸作用不能继续下去，细胞死亡。超高压作用下菌体的灭活与F1F0ATP酶活性的降低的显著相关。说明菌体细胞膜上参与与呼吸链有关的酶和电子载体较F0F1ATP酶耐压。F0F1ATP酶对超高压敏感，超高压作用下F0F1ATP酶的失活是其超高压灭活的重要原因。