Study On The Lactic Acid-sublethal Injury And Recovery Of Escherichia Coli

Escherichia coli is of vital importance to our daily life,it exists widely in layer of soil,water,cattle,poultry,flies and a variety of food,such as meat,milk and milk-made products,aquatic products,bean products,vegetable and fermented food.When E.coli resists to some adverse environment,such as acid,alkali,hot,low temperature,desciccation,and antimcrobial peptides,it's possible to form a unique physiological characteristic—sublethal injured state.Sublethal injured E.coli is difficult to be sterilizd thoroughly,causing great potential risk of food safety.It has caused great concern and become research focus in recent years.And lactic acid has been widely applied in food field,beacause of showed strong bacteriostasis activity and coordination with other preservatives.In addition,lactic acid is also commonly existed in fermented foods,including cheese,yogurt,mayonnaise,are produced by lactic acid bacteria.Otherwise,lactic acid bacteria,as for a kind of probiotics,can secrete some lactic acid to repress the growth of the gram-negative bacteria such as E.coli in the gut.The environment woud lead to lactic acid-sublethal injured E.coli.At present,however,the monitoring on E.coli are all in the state of viable.The research on the sublethal injury statement of E.coli becomes the hottest,but the most difficult.So,mastering the biological characteristics of E.coli in acid stress,understanding the recovery mechanisms of lactic acidsublethal injured E.coli,can provide new insight into controlling contamination in the food processing industry,and play a positive role in developing more reliable detecting technology.In this article,the research object is E.coli,which is commonly in food and the gut.First,studied on the optimum lactic acid-sublethal injury and resuscitate conditions of it,and investigated the relationship of the sublethal injured rates of E.coli and the permeability levels.Then,determined the oxidative stress,including levels of ROS and total glutathione,the SOD and CAT activity.Compared the rates of repair of defect types of oxidative stress genes of E.coli with wild strain,screen the regulatory genes ofrecovery in relieve oxidative stress approach.The main results as follows:(1)The factors,including lactic acid at low-p H values,temperature,oxygen flow volume,which can effect injury of E.coli were studied.The results showed that lactic acid lactic acid at low-p H values had a significant affect,when pH?5.50,lactic acid can't produce injured E.coli,wheras lactic acid at pH <3.00,E.coli can die directly,lactic acid at p H vary 3.00 from 5.40,the extent of injured E.coli increases,when pH decline.The best parameters were as follows: lactic acid at pH 4.20,temperature at37?,125 r/min.The injured rate of E.coli treated with the best parameters was 99.15%.The content material penetrated to the outside and the membrane permeability increased at the same time.The permeability levels correlated with the sublethal injured rates of microbes.The cell membrane permeability increased by 92.91%,at the optimum sublethal injury condition.(2)Knocked out some single genes such as rpoS,rssB,uvrY,phoQ,arcA can effect the acid resistance of E.coli were studied.The results showed that the lack of rpoS,rssB,uvrY,phoQ can decrease the acid resistance of E.coli.At 60 min,the injured rates of ?rpoS,?rssB,?uvrY,?phoQ were 99.66%,99.10%,99.62%,100%,respectively.However,the lack of arcA gene enhances its ability,the injured rates of?arcA was 97.98% at 60 min.(3)Many factors such as temperature,nutrition,additives can effect the recovery of lactic acid-sublethal injured E.coli.Among them,the best repair temperature was37 ?,the injured E.coli was unable to resuscitate completly at 4 ?.Damaged E.coli demand certain nutritional condition,but the rate of recovery was faster,when nutrition was richer.Moreover,Zn2 + and Mn2 +,Fe2 +,and CAT had significant affect of the repair of injured E.coli.Even,the minimum effective concentrations of Zn2 + and Mn2+,Fe2 + were 1 mmol/L,1 mmol/L and 1 mmol/L respectively.When the injured E.coli was succed to recovery,its cell membrane was also fixed.(4)Measured many index,including levels of ROS and total glutathione,the SOD and CAT activity of E.coli in the process of lactic acid-sublethal injury and recovery.Research showed that E.coli would occur oxidative stress in the process of lactic acid-sublethal injury.When the injured bacteria was the maximum degree of injury,the levels of ROS was 4.59 times of the normal bacteria.If E.coli can repair completely after injury,the levels of ROS would drop to normal levels.CAT can remove ROS directly and improve the acitivity of endogenous CAT to relieve the oxidative stress,and Zn2 +,Mn2 +,Fe2 + can improve the acitvity of endogenous SOD torelieve the oxida-tive stress.(5)Knocked out some single genes,which are related to oxidative stress,such as sod A/B/C,kat G/E,gsh A/B can effect the acid resistance of E.coli were studied.The results showed that the lack of sod A/B/C,katG/E,gshA/B can decrease the acid resistance of E.coli.At 60 min,the injured rates of ?sod A,?sod B,?sodC,?kat G,?kat E,?gshA,?gshB were 99.28%,99.16%,99.47%,99.50%,99.54%,99.16%,99.13%,respectectively.Otherwise Cu/ZnSOD,MnSOD were more effective than FeSOD in relieve ROS.Conclusion: This study found that,E.coli resisted to lactic acid environment,it would occur lactic acid-sublethal injury,then injury brought to oxidative stress and produced a great deal of ROS,altered the membrane permeability.The best parameters were as follows: lactic acid at pH 4.20,temperature at 37?,125r/min.The injured rate of E.coli treated with the best paramenters was 99.15%.Some mental ions had significant affect of the repair of injured E.coli.Particularly,the minimum concentrations(1 mmol/L)of Zn2 + and Mn2 +,Fe2 +,had a great significant affect of the recovery.rpoS was indispensable to the acid resistance of E.coli,because of it was a total regulatory gene.However,the expression of arcA can inhibit the acid resistance.SOD and CAT,which were as main antioxidant enzymes in E.coli,played an irreplaceable role in alleviating oxidative stress and improving acid resistance.