大肠杆菌O157:H7的冷冻损伤及解冻存活

Survival of Frozen Escherichia coli O157:H7 after Thawing

为探讨冷冻后残存的大肠杆菌O157:H7(Escherichia coli O157:H7)在解冻后的存活情况,本研究首先比较4株E.coli O157:H7冷冻后的死亡和损伤情况,进而采用无营养的磷酸盐缓冲液作为基质研究冷冻后不同解冻方式对E.coli O157:H7存活的影响。结果表明:4株E.coli O157:H7-20℃冷冻24、48、72 h后均发生了一定程度的死亡和损伤,冷冻时间越长细菌致死和致伤程度越明显,且存在菌株差异,冷冻72 h时菌株CICC21530的损伤率最高,为87.70%。采用混合菌株进行解冻实验,4株E.coli O157:H7磷酸盐缓冲液菌液冷冻后立即置于20、30、37℃解冻,细菌发生了进一步的死亡,解冻温度越高死亡越明显,3个温度组在解冻48 h时菌落数均显著低于冷冻72 h时菌落数(P<0.05)。进一步探讨缓慢解冻方式对菌体存活的影响,菌液冷冻后先置于4℃一定时间(0、2、6、12 h),再置于37℃不同时间(5、10、30 min)观察菌株存活情况,结果表明4℃缓慢解冻时间越长,越有利于细菌的存活,4℃、12 h/37℃、5~30 min解冻方式下改良山梨醇麦康凯琼脂上菌落数仍显著低于胰蛋白胨大豆琼脂上的菌落数(P<0.05),表明仍有损伤菌的存在。本实验提示采用缓慢解冻反而有利于残存菌的存活,冷冻食品风险评估时应重视残存菌尤其是损伤菌的检测和控制。

The objective of this study was to investigate the survival of frozen Escherichia coli O157:H7during thawing.The injury and death of four E.coli O157:H7strains doe to freezing were compared and the effect of different thawing methods on the survival of frozen E.coli O157:H7in nutrient-free phosphate buffer saline(PBS)solution was determined.The results showed that after being frozen at?20℃for24,48or72h,cell injury and even death occurred in all the four strains and the severity was time dependent and varied among strains.After being frozen for72h,strain CICC21530showed the highest injury rate(87.70%).When a mixture of four strains was frozen in PBS and then immediately thawed at20,30or37℃,cell death was aggravated with increasing thawing temperature.The colony counts of72h frozen E.coli cells were significantly reduced at48h of thawing at each temperature(P<0.05).Additionally,to explore the effect of slow thawing on the survival of E.coli O157:H7,the frozen strains were thawed slowly at4℃for different durations(0,2,6,and12h),and then kept at37℃for5,10,and30min,respectively.The results showed that slow thawing for a longer time was more beneficial to bacterial survival.Frozen E.coli were thawed at4℃for12h/37℃for5–30min and then cultured on sorbitol macconkey agar(SMAC).As a result,higher bacterial counts were found compared with they were cultured on tryptose soya agar(TSA),indicating that the injured cells existed.This study suggests that slow thawing is conductive to bacteria surviving freeze and more attention needs to paid to the detection and control of surviving bacteria,especially injured bacteria during the risk assessment of frozen food.