Mechanism Of Injury Escherichia Coli O157:H7 Caused By High Hydrostatic Pressure

High hydrostatic pressure (HHP) treatment is being investigated as a nonthermal processing technique to inhibit effectively Escherichia coli O157:H7, one of the most important food-borne pathogens. However, the injury of E. coli O157:H7 will be induced by HHP treatment, which will be underestimate or ingnored in the detection because of their lack of normal characteristics. To date, the injured mechanism of E. coli O157:H7 induced by HHP is lack of systematic researches, especially, those by proteomics. Moreover, the strains of the same species differ in resistance to HHP. Therefore, in the present study, E.coli CICC 21530, one of pressure resistant strains to HHP was selected to be treated with different pressures and its injury was investigated by the conventional plate count method and flow cytometry(FCM), Evaluation of cell structure changes, analysis of physiological characteristics, and analysis of functions and metabolic pathways of differentially expressed proteins by means of preteomics, would be contributed to reveal the mechanisms of injury of E. coli O157:H7 induced by HHP. The overall purpose was to provide a theory basis for HHP application in effective inhibition E. coli O157:H7 during the food manufacture and process. The contents and results are as follows:1. Detection of injured E. coli O157:H7 induced by HHP treatmentThe injury of E. coli O157:H7 CICC 21530 was investigated by the conventional plate count method and FCM after 200 MPa,400 MPa,500 MPa (25℃,5 min), respectively. The results of conventional plate count method showed the higher the pressure was, the stronger the inbibitory effect on E. coli O157:H7 was, but the lethal effect was not consistent with the injury effect and the most injure cells were caused after exposure to 400 MPa treatment. The result of FCM showed that the cytoplasmic membrane integrity was destroyed after HHP treatment, and the higher the pressure was, the more serious the damage of cytoplasmic membrane was, while no obvious change in esterase activity was observed. Compared with traditional plate count method, more normal cells were detected by FCM which indicated that the FCM could obtain more date and thus give better insights about pressure-induced changes on E.coli 0157:H7.2. Influence of HHP treatment on morphological structure of E.coli O157:H7E.coli O157:H7 was treated by 200 MPa,400 MPa,500 MPa (25℃,5 min), respectively. Changes of surface morphology and internal structure, variations of the DNA and ribosome thermal stability, and change of the total apparent enthalpy values of control and HHP-treated cells by meaning of scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC). SEM showed that some abnormal morphology, including rough and cracked cells, was observed after HHP treatment. The higher the pressure was, the more obvious the change of cell surface was, and cells size decreased significantly after exposure to 400 MPa and 500 MPa (P<0.05). TEM showed that HHP caused morphological changes in external and internal structures, such as the cells shape changes, plasmolysis and membrance rupture, light transparent area and so on. The higher the pressure was, the serious the damage of structure was. DSC showed that HHP destroyed the ribosome structure, and the disordering of the ribosome structure increased. There was no effect of HHP on DNA of E.coli O157:H7.3. Influence of HHP treatment on the cell membrane physiological property of E.coli O157:H7After treatment of 200 MPa,400 MPa,500 MPa (25℃,5 min), the membrane permeability, leakage of ions, activities of Na+K+-ATPase and Ca2+Mg2+-ATPase and the membrane fatty acid composition of E.coli O157:H7 were examined. The results showed that with the pressure increasing, membrane permeability increased, more leakages of K+, Mg2+, Ca2+were caused except for Na+. The activities of Na+K+-ATPase and Ca2+Mg2+-ATPase decreased, and the ratio of saturated vs. unsaturated fatty acids of cell membranes decreased after HHP treatment. The results indicated that the higher the pressure was, the more serious the damage of physiological properties of cell membrane was.4. Analysis of injury-related proteins of E.coli O157:H7 caused by HHP treatment by means of iTRAQ-based quantitative proteomicExperiment 1 indicated the most injure cells were caused after exposure to 400 MPa 5 min. Therefore, in this study, the differential expression proteins of the control and HHP-treated E. coli O157:H7 were analyzed by iTRAQ-based quantitative proteomics. The result showed that a total of 135 different proteins were identified, in which 4 were up-regulated and others were down-regulated. Geneontology analysis showed that there were 83 biological processes,50 molecular function,14 cell components were enriched, in which 11,7,4, respectively, was different significantly (P<0.05). Thirty-two KEGG pathways were enriched, in which 2 was different significantly (P<0.05). The results indicated that there was significant difference in ribosome synthesis and phosphotransferase system of E. coli O157:H7 after pressure treatment.