高压热处理下芽孢皮层肽聚糖支架水解机理研究

Bacterial spores are the most difficult microorganisms to be inactivated. High-pressure thermal sterilization (HPTS) can inactivate the spores and cause smaller damages on food qualities. However, there has been no major breakthrough in the study on spore inactivation mechanism by HPTS for a long time. HPTS can induce the hydrolysis of peptidoglycan support of spore's cortex, which is a critical reason for spore inactivation by HPTS. However, the hydrolysis principle of cortex peptidoglycan under HPTS is still not clarified. This project uses cortex-lytic enzymes and cortex peptidoglycan isolated from Bacillus subtilis spores as entry points, and treats the enzymes and peptidoglycan with HPTS respectively. The effects of HPTS on the activity of cortex-lytic enzymes are discovered at different research levels of primary structure, secondary structure, senior structure and ultrastructure by the methods of electrophoresis, circular dichroism spectrum, Fourier transform infrared spectroscopy, spectrofluorimetry, ultraviolet absorption spectrum scanning, measurement of surface sulfydryl content and atomic force microscope. Meanwhile, the hydrolysate of cortex peptidoglycan is analyzed by reverse-phase HPLC and matrix-assisted laser desorption ionization-time of flight MS, and the effects of HPTS on the non-enzymatic hydrolysis of cortex peptidoglycan are clarified. Finally, the hydrolysis principle of peptidoglycan support of bacterial spore's cortex under HPTS is discovered, which will improve the illumination of spore inactivation mechanism by HPTS, and impel effectively the application of HPTS in food industry.

芽孢是最难杀灭的微生物，高压热杀菌技术（HPTS）能有效杀灭芽孢，同时对食品品质的影响较小。然而，对HPTS杀灭芽孢机理的研究长期没有大的突破。HPTS会引起芽孢皮层肽聚糖支架水解，这是其造成芽孢死亡的一个关键原因，但HPTS诱发皮层肽聚糖水解的机理尚不明确。本项目以从枯草杆菌芽孢中分离的皮层裂解酶和皮层肽聚糖为切入点,对其进行HPTS处理，采用电泳法、圆二色光谱法、傅里叶变换红外光谱法、荧光光谱分析法、紫外吸收光谱扫描法、表面巯基含量测定法及原子力显微镜分别从一级、二级、高级及超微结构等不同研究层次上揭示HPTS对皮层裂解酶活性的影响。同时，采用反相高效液相色谱法、基质辅助激光解吸电离飞行时间质谱法分析皮层肽聚糖的水解产物，阐明HPTS对皮层肽聚糖支架非酶水解的作用。进而揭示HPTS导致芽孢皮层肽聚糖支架水解的机理，推进对HPTS杀灭芽孢机理的阐明，有力地推动HPTS在食品工业中的应用。

芽孢是食品杀菌的关键目标。高压热杀菌技术（HPTS）能有效杀灭芽孢，同时对食品品质的影响较小，然而对HPTS杀灭芽孢机理的研究长期没有大的突破。HPTS会引起芽孢皮层肽聚糖支架水解，这是其造成芽孢死亡的一个关键原因，但HPTS诱发皮层肽聚糖水解的机理尚不明确。本项目以HPTS导致芽孢萌发而死亡，或HPTS导致芽孢皮层肽聚糖非酶水解为科学假说，开展了以下研究：超高压、热和各种化学物质对芽孢萌发的影响；芽孢皮层肽聚糖的提取、结构与组分分析、水解产物分析；超高压、热、乙醇及HPTS对芽孢皮层肽聚糖的作用；天然芽孢皮层裂解酶的提取或皮层裂解酶基因克隆与重组表达，皮层裂解酶的分离纯化；HPTS对枯草杆菌芽孢皮层裂解酶的作用。研究发现超高压、热和各种化学物质会对芽孢萌发产生影响，但是这些处理对芽孢的作用与HPTS对芽孢的作用有根本不同。芽孢皮层肽聚糖与营养体细胞壁肽聚糖的特征氨基酸种类和含量大不相同，这表明芽孢皮层肽聚糖与营养体细胞壁肽聚糖在结构上有重大不同，这种结构和氨基酸含量上的不同很可能与芽孢皮层的耐压性有关。使用RP-HPLC法能有效分离皮层肽聚糖的各种水解产物，但由于缺乏标准品，无法对水解产物进行定性。使用MALDI-TOF MS能分离HPTS处理后芽孢皮层肽聚糖样品中的各种物质，但由于没有相应的质谱库和大分子标准品，所以也无法确定肽聚糖是否发生了非酶水解。转而针对皮层肽聚糖小分子特征水解产物NAG和NAM，采用HPLC法分析HPTS处理后的皮层肽聚糖样品，使用Aminex HPX-87H糖柱成功分离鉴定了NAG和NAM标准品，并使用该柱检测出HPTS处理后皮层肽聚糖样品上清液中存在NAM，证明HPTS造成了芽孢皮层肽聚糖的非酶水解。成功将皮层裂解酶CwlJ基因克隆并重组表达了该酶，对重组表达的皮层裂解酶或芽孢中天然的皮层裂解酶进行分离纯化并成功地保持了酶活。研究发现HPTS不但没能激活皮层裂解酶，反而将其钝化。综合以上研究可知，HPTS处理下芽孢皮层肽聚糖支架的水解机理为非酶水解，这推进了对HPTS杀灭芽孢机理的阐明，有力地推动HPTS在食品工业中的应用。