Sublethal Injury And Recovery Of Escherichia Coli O157:H7 Induced By High Hydrostatic Pressure

High hydrostatic pressure（HHP）is a non-thermal sterilization method,which can effectively inactivate Escherichia coli O157:H7.However,with HHP treatment,Escherichia coli can be induced into sublethal state,which will repair quickly under favorable conditions.Therefore,sublethal cells is the potential risk source of human food poisoning.At present,studies on the formation and repair of Escherichia coli sublethal cells induced by HHP have not attracted much attention,so this research will carry out more experiments on this aspect,so as to find out the possible mechanism of the formation and repair of sublethal cells.This is of great significance for avoiding the risk of sublethal microorganisms and ensuring food safety.The main results as follows:（1）Construction of E.coli O157:H7 sublethal injury cell model,and detection of its physiological indicators.400 MPa 5 min was selected as optimal induction conditions,under this condition,the proportion of sublethal cells was 99.44% and all the surviving cells were determined to be sublethal cells.It was found that HHP induced sublethal cell formation could lead to cell membrane damage,membrane breakage,increased permeability,membrane potential depolarization,as well as leakage of intracellular nucleic acid,protein,Mg²⁺ and K⁺.For intracellular macromolecules,the secondary structure of mycoprotein and the content of α-helix andβ-folding both changed.On the other hand,catalase responded to oxidative stress（enzyme activity increased）,while superoxide dismutase and ATPase activity decreased,lost their original functions.（2）Analysis of the recovery process of sublethal cells and detection of their physiological indicators.As fast as 2 h,was needed to repair sublethal cells in liquid medium,the repair speed TSB（Tryptic Soy Broth）>BPW（Buffered Peptone Water）>PBS（Phosphate Buffer Saline）>min A（a kind of basic medium）.It was shown that the deficiency of Mg²⁺,PO₄^3-,casein hydrolysates and glucose did not change the time to complete repair（5 h）,but it affected cell proliferation.3 days were needed to complete recovery of sublethal cells on solid media.The results showed that,intracellular protein content remained low for 8 h of repair（≤0.231 mg/m L）,ATPase activity was not elevated,while catalase and superoxide dismutase activities gradually recovered,ATP concentrations were also rising.These results revealed that the mechanism of macromolecular formation and activation such as protein was inhibited during resuscitation,yet preferred triggered ATP generation pathway to ensure the energy supply of subsequent life activities.（3）The morphology and structure of sublethal and repair cells were observed.It was found that the damage of the surface morphology of E.coli was mainly reflected in the shrinkage of cell membrane and the appearance of pores.As pressure increased,the membrane eventually became fragmented.During repair process,the holes on the cell membrane were filled first,then the inner of cells was repaired until them returned to a full,round rod,like untreated cells.（4）By means of transcriptome analysis,the possible mechanism of formation and resuscitation of Escherichia coli O157:H7 sublethal state was proposed.The membrane,ribosome and other cellular components were significantly affected when sublethal cells were formed,while the amino acid synthesis pathway and enzyme activity were inhibited.During repair process,differential genes in ribosomal metabolic pathway,intracellular components and synthesis and metabolism of nitrogenous compounds were expressed in large quantities.The above results indicated that the formation of sublethal cells would affect the macromolecule synthesis,however,cellular resuscitation could weaken these effects.