Sublethal Injury And Recovery Of Salmonella In Food Matrix

In recent years,with the improvement of economy level and acceleration rhythm of life,frozen food becomes more and more popular in our daily life because of its convenience.However,there is food safety problem that the detection rate of Salmonella in frozen food is very high.In frozen food,Salmonella has three states: lethal,sublethal and not damaged.Sublethally injured Salmonella which has not been completely killed can be self-repaired and return to normal under suitable environment,which may lead to a secondary contamination of food.Sufficient attention must be given to this situation.The effect of low tempreture(-20 ℃)on sublethal injury of Salmonella typhimurium,a well characterized strain,was investigated in this study.The recovery of sublthally injured cells was also studied.Besides,the molecular detection method of viable Salmonella in frozen meat was also studied.The main research contents were as follows :(1)Sublethal injury of Salmonella cells by low temperature at-20 ℃ were determined by using LIVE / DEAD Baclight kit and culture-based plate counting.The external morphology of Salmonella before and after freezing was observed by scanning electron microscopy(SEM).It was found that freezing caused significant changes in cell structure.In the frozen period,the total number of cells decreased with time in standard medium or in foodstuffs such as pork,beef,milk and egg,and the sublethal level was maintained at a high level of 90% or higher.It was found that sublethal rate of Salmonella cultured at 37 ℃ in logarithmic period following cold shocking at 15 ℃ for 2 h decreased from 90% to 60% in frozen food such pork,beef,milk and egg.(2)Sublethally injured cells were significantly more susceptible to the tempreture of 50 ℃,acid and lysozyme compared to the untreated cells.The effect of media,temperature,media pH and inhibitors of specific metabolic processes,Mg cations on the recovery of the sublethally injured cells was also investigated.Sublethally injured cells can be resuscitated not only in standard medium such as TSB and phosphate buffer(PBS)for 1 h and 2 h,but also in food matrixes,for emample,pork,beef,egg and milk for 3.5 h,2.5 h,2 h and 1.5 h respectively.The recovery rate was positively related to temperature,the degree and exposure of the nutriment.The recovery process was inhibited when pH value was lower than 5.0.As pH value raised to 5.0-8.0,the recovery rate of the sublethally injured cells increased with the increase of the pH.The addition of inhibitors such as sodium azide,chloramphenicol,rifampicin,penicillin and cerulenin showed that repair of most sublethally injured cells required energy and RNA synthesis,but was not dependent on protein,peptidoglican and fat synthesis.Moreover,Mg cations facilitated the recovery of sublethally injured cells.(3)Rapid detection of viable Salmonella in frozen meat products by SD-PMA-qPCR.In order to solve the problem that PMA-qPCR technology is prone to false-positive for the detection of viable bacteria of frozen samples,we introduced and optimized the SD(sodium deoxycholate)pretreatment process: incubated sample for 20 min with 0.1%SD.SD can reduce the membrane stability of injured cells,help PMA permeates through injured cells,which can inhibit the amplification of DNA in PCR.The accuracy of the SD-PMA-qPCR detection method was verified by the experimental results of frozen pork meat.The accuracy was not affected by the presence of non-target bacteria,and the sensitivity was stable at the limit of 102 cfu/g.