Study On The Metabolism And Proliferation Of Foodborne Microorganisms And The Formation Of VBNC State In Rice And Flour-based Products

Rice,wheat and corn are main foods in China which are rich in nutrients.However,they are easily contaminated by food-borne microorganisms during food processing,storage and sales.According to investigations,Escherichia coli O157(E.coli O157),Salmonella,Staphylococcus aureus(S.aureus)and Pediococcus lactis(P.lactis)are the common food-borne microbes in rice and flour-based food.At present,the main detection method for these microbes is the traditional culture plate in the national standard which is time-consuming and complex.It is worth noting that in recent years,many studies report that these four microorganisms can enter into viable But Non-culturable(VBNC)which brings challenges to the standard detection method.Also,it was confirmed that bacteria in VBNC state can cause food spoilage and produce toxins threatening food safety and human health.In addition,few research studied the proliferation and metabolism of bacteria during the formation of VBNC state.Therefore,this study established a rapid cross-priming amplification(Cross Priming Amplification,CPA)technology to identify the VBNC cells in rice-flour based products.Also,the proliferation and metabolism of the four microorganisms in planktonic and biofilm cells and the formation of the VBNC state during the storage were explored.The main research contents and conclusions were as follows:(1)The established CPA system was optimized.The optimal final concentration ratio of calcein and Mn²⁺was 1:4,the Mg²⁺was 8 m M,while the betaine was 0.4 M.The determined reaction temperature and time were 63?and 60 minutes.The experiment further designed CPA detection reaction systems based on the conservative genes of E.coli(rfb E),Salmonella(inv A),S.aureus(fem A)and P.lactis(phe S)to verify the sensitivity and specificity.Then applied to the detection of artificial contaminated food samples.The detection limits of CPA reaction applied in food systems were 10³ CFU/m L,10⁴ CFU/m L,10² CFU/m L and 10⁴ CFU/m L,respectively,which were 100-1000 times higher than PCR.Finally,using the optimized CPA reaction system,combined with the PMA dye at a final concentration of 10?g/m L to treat the VBNC bacteria samples.The PMA-CPA detection and verification of VBNC status bacteria were realized successfully.(2)Taking planktonic and 8 h,24 h,72 h biofilm state of four microbes as research object,the proliferation and metabolism of microorganisms during low-temperature storage in rice and flour-based products were studied.The results showed that the speed of entering the VBNC state had a certain relationship with the physiological characteristics of the strain itself,cell state,nutritional conditions and temperature.The Gram-negative bacteria were more likely to enter the VBNC state.Only E.coli and Salmonella can enter VBNC status in Mifen at-20?.The speed trends of Salmonella and Escherichia coli entering VBNC were 72 h>24 h>planktonic>8 h and planktonic>8h>24h,respectively.(3)The PMA-qPCR combined with plate counting method was used to monitor the change of VBNC status bacteria during low-temperature storage.The results indicated that the process of reducing the culturable number of bacteria was accompanied by the accumulation of VBNC status bacteria.This accumulation did not cause changes in food quality,means that VBNC state cannot be evaluated by sensory evaluation.(4)The related gene expression of the four microorganisms during storage was explored.The results found that the virulence genes of planktonic and biofilm cells of E.coli,Salmonella can express continuously and stably at low temperature.However,continuous expression can be achieved in a short period of time,but ceased with the prolonged storage time.For P.lactis,dna K gene had a greater supporting effect on the short-term growth of lactic acid bacteria under low temperature conditions,due to the expression of dna K under low temperature stress was significantly up-regulated.(5)Though the virulence genes of VBNC cells of E.coli and Salmonella were negatively expressed in the Mifen group at-20?,their expression ability did not disappear with the extension of storage time.The virulence expression ability of Salmonella VBNC cells in the biofilms was stronger than that in the planktonic state.In summary,the continuous expression of virulence of VBNC status and their accumulation in food may cause greater hidden dangers to food safety and public health.In summary,this study established a detection assays for viable microbes in food systems and studied the law of metabolism and proliferation of foodborne microorganisms in rice and flour-based products.The amounts of detectable bacteria determined by the traditional plate counting method are below the detection limit which does not mean that the food is absolutely safe.The bacteria might exist in VBNC state which is more difficult to identify.The expression of toxin gene and accumulation of toxins in food have a huge potential safety hazard to food safety and public health.