Detection And Elimination Of The Foodborne Bacteria In VBNC State And Its Application To Food Sample

Rice flour-based food are natural media for many spoilage and pathogenic bacteria because of their rich nutrients and sufficient moisture,Staphylococcus aureus,Salmonella,Lactobacillus and E.coli O157 are the common contaminated bacteria.At present,the“national standard method”is often adopted to detect the bacteria in food sample with complex procedure and time-consuming.Furthermore,it has been demonstrated that bacteria could be induced into viable but non-culturable?VBNC?state under some conditions,while the bacteria in VBNC state remain its pathogenicity and corruption ability and can not be detected by the“national gold standard”method,resulting in potential food safety problems.Therefore,it is of great significance to take effective measures to eliminate its formation in food sample.In addition,current studies related to the requirement of formation of VBNC state mainly focus on a certain condition.Therefore,understanding the key factors for these bacteria entry into VBNC state is of great significance to eliminate its contamination.In this study,we firstly established a novel method for rapid detection of the main bacteria and its VBNC state in rice flour-based food.And the we investigated the effects of environmental conditions on the formation of VBNC cells of S.aureus and E.coli O157 under low temperature,and the key conditions for its formation were also obtained.Finally,the inhibition effects of some measures on the formation of VBNC state were explored,and validation in food samples.1.The PSR assay was established and optimized by designing primers of the conservative region of rfbE.The optimal concentration of betaine is 0.7 M,and under the ration of calcein and Mn²⁺at 1:4 or 1:8,it is helpful for us to judge the result of PSR reaction according to the color change.The amplification products could be visually detected through adding the fluorescence indicator until 40 min by designing the accelerate primers..The results of the specificity and sensitivity analysis show that the primers targeting rfbE are specific highly,the detection limit is 11.2 pg/?L and 10 times higher than that of conventional PCR.2.Primers were designed based on the conserved regions of S.aureus,Salmonella,and L.caesi.The specificity and sensitivity of the established PSR assay was evaluated.The four mentioned bacteria was inoculated in the crystal cake and its DNA was extracted with appropriate method respectively,the rapid and highly sensitive detection of bacteria in rice flour-based could be achieved by color change and electrophoresis of the established PSR assay.The detection limit of artificially inoculated crystal cake by PSR assay was found to be 10³CFU/mL,10⁴ CFU/mL,10⁵ CFU/mL,10⁴ CFU/mL.The sensitivity of PSR for bacteria detection in food system was not affected by food components.3.S.aureus ATCC 25923 and E.coli O157 ATCC43895,the common contaminated bacteria,were selected as the target.Under the condition of freezing oligotrophic environment,they could be induced into VBNC state.With 5?g/mL PMA treatment of dead and VBNC state of S.aureus and E.coli O157,PSR assay was successfully distinguished bacteria in VBNC state or membrane-damaged.The color of reaction system changed from orange to green when the DNA template of VBNC cells added,while the template DNA of the dead strain remained orange.Meanwhile,the sensitivity of PMA-PSR assay was evaluated.The detection limit of PSR assay for detection of E.coli O157 and S.aureus in VBNC state was found to be 10³CFU/mL and 10⁴ CFU/mL,which is similar with that of the bacteria in food samples.4.The effects of environmental conditions,including nutritional status,acidity and salt concentration,on the formation of VBNC state of S.aureus ATCC25923 and E.coli ATCC43895 were studied.The key conditions for S.aureus entry into VBNC state as follows:?adequately nutrition and strongly acidic environment?stored at 4???nutrition deficiency and weak acidic environment?stored at 4?or-20??iii oligotrophic refrigeration condition.The key conditions for E.coli O157 entry into VBNC state as follows:eutrophic and high salt environment?adequately nutrition and weak acidic environment iii oligotrophic refrigeration condition.5.The influence of environmental conditions on the formation of VBNC of S.aureus was as follows:nutritional status>acidity>salt concentration.The effect of external environmental conditions on the formation of VBNC state of E.coli O157 was acidity>nutrient state>salt concentration.Environmental factors present different effects on the formation of VBNC state of different bacteria.Therefore,the control of VBNC formation depends on the characteristics of bacteria and their resistance to the environment.6.Adding 1.0%?v/v?HAc into the medium to control the formation of VBNC state of S.aureus is relatively ineffective when there are some nutrients,while the nutritional status presents high performance in the survival S.aureus.Therefore,the best way to control and eliminate the S.aureus in VBNC state is nutrient-free supply and acidic environment.When nutrient concentration is higher than 50%,adding 1.0%?v/v?HAc can directly kill E.coli O157and eliminate the formation of VBNC state.Under the condition of weak acid and high salt,the formation of E.coli O157 VBNC could be eliminated by changing the nutritional status.In food sample,S.aureus and E.coli O157 will directly die by adding 1.0%?v/v?HAc in the food sample with the concentration of nutrients was 25%,50%,100%,respectively.Therefore,the formation of VBNC state of S.aureus and E.coli O157 could be eliminated by this method in rice flour-based food...