Study On The Sublethal Injury Formation,Repair And Adhesion Change Of Escherichia Coli O157:H7 Sublethal Damage After Freezing Stress

Escherichia coli O157:H7 has a serious impact on public health and food safety and becomes one of the globally recognized and widely studied food-borne pathogens due to its low infectious dose,strong pathogenicity,high risk.Frozen treatment is widely used to ensure quality and extend the shelf life of food.But sublethally injured E.coli O157:H7 is found in frozen food,which is difficult to eliminate completely and can recover all physiological activity and toxicity under suitable conditions.This is a potential threat in food safety and attracts extensive attention of researchers.At present,E.coli O157:H7 monitored of is mostly viable bacteria state,and detection of sublethally injured E.coli O157:H7 has also become a hot and difficult point in food safety testing.Bacterial adhesion is the first step to invade and contaminate food.It is a prerequisite for the formation and development of biofilms and an important indicator of E.coli O157:H7 pathogenicity.Therefore,this study investigated the sublethal injury,biological characteristics and recovery conditions of E.coli O157:H7 under freezing stress,and further explored the adhesion change of E.coli O157:H7 during the sublethal injury and repair process.It provides new ideas to control E.coli O157:H7 for food processing industry and reduces the persistence of bacteria and the food contamination.The main findings are as follows:（1）The effects of different freezing liquids,thawing temperatures,repeated freeze-thaw cycles and different metal ions on sublethally injured E.coli O157:H7were investigated.The results showed that sublethally injured E.coli O157:H7 was induced by freezing stress,and the sublethal ratios of E.coli O157:H7 increased with increase of nutrient content in freezing solution and the decrease of thawing temperature,but the dead ratios decreased.The optimal preparation conditions of sublethally injured E.coli O157:H7 were that cells were frozen in TSB at-20℃for 16h and thawed at 25℃for 5 min,and the maximum sublethal ratio was 99.5%.Compared with untreated cells,the sublethally injured cells were more sensitive to acid（hydrochloric acid,acetic acid,lactic acid）and Na Cl.（2）The effects of different repair media,treatment temperatures and exogenous additives on recovery of sublethally injured E.coli O157:H7 were investigated.The results showed that the repair of injured cells needed nutrition,and the more abundant the nutrition,the higher the repair ratios.Injured cells were completely recovered by TSB at 37℃for 90 min.Mn²⁺,Zn²⁺and Fe²⁺（≥1 m M）promoted resuscitation,but Mg²⁺and Ca²⁺had no effects on the recovery.Cinnamaldehyde,thymol,carvacrol,gallic acid,rutin and chlorogenic acid effectively inhibited the repair of injured cells,and the lowest concentration completely inhibiting repair was 0.078 mg/m L,0.039mg/m L,0.010 mg/m L,0.156 mg/m L,0.078 mg/m L and 0.039 mg/m L,respectively.（3）The adhesion changes of E.coli O157:H7 during freezing treatment and recovery were investigated.The results showed that adhesion of E.coli O157:H7 on iron sheet and lettuce was significantly enhanced compared with untreated cells.The adhesion ability of E.coli O157:H7 increased with the extension of freezing treatment time,and gradually recovered to the untreated level during the repair process.The improvement of adhesion ability may be related to the increase of motility and the change of extracellular polymeric substances content and composition.During the freezing treatment,the motility of E.coli O157:H7 decreased significantly but rebounded and expression of flagella-related genes（fli A,flg C,flg D,mot A and che A）increased at 16 h.The motility was not restored during the repair process.The content of extracellular polymeric substances and the ratio of sugar/protein are positively correlated with adhesion,and the expression of extracellular polymeric substances-related genes（cps B,rcs C,rcs F and wca F）is all up-regulated during recovery.Freezing stress and repair treatment had no significant effect on the molecular structure and molecular weight of lipopolysaccharide,and the lipopolysaccharide-related genes（msb B and lpt D）were not significantly expressed.