Study On The Existence Of Acid Tolerance Response Of Salmonella During Simulated Chilled Beef Storage And The Mechanism Based On Two-component Regulatory System

The nutritional and mild acidic environment of beef and the use of acidic bactericides in the production process may induce the production of the acid tolerance response(ATR)in Salmonella.ATR can significantly enhance the acid tolerance and virulence of bacteria,and produce cross-protection effect,leading to the useless of hurdle factors and endangering the health of consumers.Physiological changes in carcass and the aging of beef have a potential impact on the production of ATR in Salmonella,but the specific mechanism is still unclear.In the current study,the existence and change of acid tolerance of Salmonella during simulated chilled beef storage was explored.At the same time,the specific regulatory mechanism of Salmonella ATR was explored based on two-component regulatory system(TCS)regulating amino acid metabolism.The purpose is to evaluate the threat of ATR to chilled beef,and provide a theoretical reference for enterprises to prevent and control the bacterial ATR in the industrial chain.In this study,the meat medium(ME)was used to simulate the chilled storage environment of beef,the acid tolerance and survivability of Salmonella was measured within 25 days.RT-q PCR was used to compare the relative expression levels of ATR-related genes in normal Salmonella(14028)and PhoP/PhoQ or Pmr A/Pmr B mutation strains(?phoP and?pmrA),the apparent acid tolerance of each strain was also measured.The effect of TCS on ATR and its pathways were explored,as well as the the expression of decarboxylase-encoding genes in different ionic conditions that simulating the biochemical environment of beef.The main findings are as follows:By using the ?Red recombination system,the DNA fragment with 50 bp homologous sequence was successfully recombined with the target gene,and TCS-mutation strains?phoP and?pmrA that can be stably passaged were obtained.By measuring the acid tolerance of Salmonella during chilled storage,it was found that the acid induction process significantly improve the acid resistance of Salmonella(P<0.05),and this high acid tolerance can maintain for at least 25 days.In the mild acidic ME,chilled storage can significantly reduce the survivability and the acid tolerance of Salmonella,and even inhibit the production of ATR,confirming the necessity of strictly observing the low temperature operation procedures in the production process.After phoP was knocked out,the relative expression levels of adi A,adi Y,cad A and cad B related to arginine and lysine metabolism were reduced and the expression of rpo S related to?factor was increased,indicating that the strong acid tolerance of Salmonella is related to the regulataion of PhoP/PhoQ on arginine and lysine metabolism pathways and the feedback regulation of?factor.By analyzing the differences of the relative expression of decarboxylase-realted genes in14028,?phoP and?pmrA,and the amino acid adding experiments,it was found that the addition of amino acid in acid-challenge media significantly enhanced the acid tolerance of Salmonella(P<0.05).At the same time,adding amino acid to the?phoP and?pmrA groups also improved the acid tolerance,but still lower than control.These results showed that the mechanism of Salmonella ATR production is multi-faceted:1)PhoP/PhoQ and Pmr A/Pmr B recognized the acid signal and positively regulates the ATR of Salmonella through the regulation of arginine and lysine metabolism.2)In addition to PhoP/PhoQ and Pmr A/Pmr B,there are other TCSs or amino acid receptors to help bacteria tolerate acid.3)In addition to amino acid metabolism,there are still other pathways or global regulatory factors(such as?factor)that are conducive to bacterial acid tolerance,and some factors are related to TCS.By comparing the relative expression levels of adi A and cad A of normal Salmonella and mutant Salmonella under different concentrations of ion signal combinations,it was found that H⁺and Ca²⁺interacted with the relative expression level of adi A,while the acidic environment and Ca²⁺concentration in the beef production process were in the promotion expression range,which has a significant improvement effect on the expression of adi A(P<0.05).And there was no interaction between H⁺and Ca²⁺on the relative expression of cad A,which suggested the different regulatory mechanisms of arginine and lysine metabolism in Salmonella.