流式细胞术在超高压诱导大肠杆菌O157:H7亚致死研究中的应用  

Flow Cytometric Assessment of Sublethal Injury of Escherichia coli O157:H7 Cells Caused by High Hydrostatic Pressure

作　　者：孔晓雪 韩衍青[2] 付勇 姬赛赛 王娴静 禹金龙 江芸[1] 

机构地区：[1]南京师范大学金陵女子学院食品科学系,江苏南京210097 [2]江苏省生产力促进中心,江苏南京210042

出　　处：《食品科学》2018年第3期135-141,共7页Food Science

基　　金：国家自然科学基金面上项目(31371861,31671915);江苏省"六大人才高峰"高层次人才项目(NY-080)

摘　　要：超高压作为一种应用日趋广泛的食品非热杀菌技术,对食源性大肠杆菌O157:H7有显著的致死作用,但对其致死效应缺乏快速有效的评价手段,对其致死机制也需进一步证实。本研究运用流式细胞术结合传统培养方法,评价了超高压对大肠杆菌O157:H7的致死效应,并通过扫描和透射电子显微镜技术观察分析了超高压处理后菌体细胞微观结构的变化,初步探讨了超高压对大肠杆菌O157:H7的致死机制。结果表明:双染色流式细胞术检测方法能够快速准确地对超高压处理后不同生理状态的大肠杆菌O157:H7细胞进行分析和分选,且具有较高灵敏度和精确度;400 MPa的超高压处理对大肠杆菌O157:H7有明显的损伤和致死效果,菌体细胞出现严重变形,部分细胞的细胞膜出现破裂;500 MPa处理时,致死效应更加明显,菌体细胞壁和细胞质膜严重分离,光透明区出现大量辐射丝状物。菌体细胞膜完整性丧失很可能是超高压诱导大肠杆菌O157:H7致死的主要原因,即使经过500 MPa的超高压处理,依然有不少大肠杆菌O157:H7菌体细胞处于亚致死状态,这部分亚致死状态的细菌是食源性疾病爆发的潜在隐患。As a widely used non-thermal decontamination technology in the food processing industry, high hydrostatic pressure processing has a significant lethal effect on foodborne Escherichia coli O157：H7. However, effective methods for detecting viable cells after high hydrostatic pressure treatment are still lacking, and the inactivation mechanism of pressurized Escherichia coli O157：H7 needs to be confirmed. In this study, the living state of high hydrostatic pressuretreated Escherichia coli O157：H7 cells was evaluated by flow cytometric analysis and the conventional culture technique. The microstructure of pressurized bacterial cells was observed by scanning and transmission electron microscopy. The inactivation mechanism of high-pressure-treated cells was further explored. Results showed that double-staining flow cytometry was a rapid and effective method to sort Escherichia coli O157：H7 cells in different physiological states with high sensitivity and accuracy. Significant damage and sub-lethal injury of Escherichia coli O157：H7 cells were observed after treatment at 400 MPa, and the bacterial cells were seriously deformed with partial rupture of the cell membrane at this pressure level. When the pressure was increased up to 500 MPa, the bacterial cell wall and membrane were separated severely from each other with lots of radial filaments appearing in the light transparent area. It is worth noting that there is a large part of sub-lethally injured pathogenic cells surviving even after high hydrostatic pressure treatment at 500 MPa, which may be a potential risk factor for food-borne disease outbreaks.

关 键 词：大肠杆菌O157:H7 超高压 流式细胞术 亚致死 电子显微镜 

分 类 号：TS201.3[轻工技术与工程—食品科学]