| Vibrio parahaemolyticus (Vp) is a main pathogenic bacteria causing foodborne gastrointestinal disease due to consumption of seafood. With the development of the aquatic products circulation, Vp caused food poisoning is not only result from the seafood, but also due to the cross-contaminated food by improper seafood processing and storage, therefore the control of Vp load in seafood is very important. At present, the measures for reducing Vp in seafood are relaying and depuration, thermal treatment, high-pressure, radiation and so on, however, none of these methods are effective giving consideration to both the effect of sterilization and maintain the original flavor of seafood. Many scholars have proposed organic acid can be used in food preservation and storage in recent years, so the combination of organic acids and other methods has become the research focus of control bacterial load in food.The formation of bacterial acid tolerant cell is the key problem of treating bacteria by mild acid, but the mechanism of tolerant Vp induced by acidic condition is not clear. In this study, the method based on citric acid and temperature treatment was established to reduce Vp in seafood and the synergetic effect of mild acid and heat on bacteria survival were studied. This method not only reduces the carrying load of Vp, but also maintains the freshness of the seafood. Morever, We studied the acid tolerance response of Vp produced in the weak acid condition, and analyzed the expression pattern of regulation factors in the acid tolerant cells. The study provided the theoretical and practical basis for establishing the efficient technique for reducing Vp load in seafood.1.Decreasing effect of organic acid and temperature on Vibrio parahaemolyticus in seafoodThe conditions for reducing Vp load in the artifical system were optimized by response surface analysis method. After the single factor analysis of Vp sterilization effect and evaluation of temperature, time, pH factors and their interactions by Box-Benhnken design (BBD), the best parameters for reducing Vp were selected as55.5℃,25s and pH4.96. In this condition, the predicted value of Vp death was97.36%, the verification of the value on artificially contaminated salmon samples was97.07%and there was no significant difference in fish myofibrillar protein Ca2+-ATPase activity between the treated and untreated samples.2. Vibrio parahaemolyticus acid tolerance induction and expression pattern of δ factorsSix strains of Vp were choosed to study the effects of acid induced tolerance by different adaptive pH and adaptive time, and it was found that Vp acid tolerance was related to strains, adaptation pH, and adaptation time. Vp acid tolerance inducing conditions in this study were environment adaption at pH5.5for1hour and environment stress at pH4.0for1hour. Under this condition, we analyed the survival rate of6strains of Vp and detected the expression levels of cadA, toxR genes and ten potential8factors by relative quantitative method. The results showed that the expression of cadA, VPA1690, VP2358genes in6strains were up-regulated and VP2553, VPA1555, VP2953, VP2210upregulated in the strains which can be detected in viable bacteria, and the upregulation of toxR was found to lead strains to acid tolerance response.The survival rate of the pandemic strain RIMD2210663at series of adaptive pH for1hour following by the environmental stress at pH4.0for1hour and the expression levels of cadA, toxR and ten potential δ factors were tested. Results showed that the selected genes were induced by low pH, and the pH had more effect on the expression of cadA, VP2358, VP2578than those of other genes. VP2553may play an important role on survival of Vp under acid stress and toxR plays an important role for the acid tolerance. Different pH has a significantly different effect on expression of cadA, toxR and δ factors. Conditions at both pH4.5and pH6.0can induce the expression of acid tolerance related genes, and the expression level induced at pH4.5was higher, but acid tolerance response was not effectively formed. The main reason is that pH4.5adaptation stage make most of the Vp died, although at pH6.0, VP can survive in the adaptation phase, but the cell acid tolerance ability is not strong enough to resist lethal acid environment.3.The role of VP2358in acid tolerance of Vibrio parahaemolyticusAccording to the analysis results of the expression of δ factors in acid tolerance response, we furtherly explored the regulation of cadA expression by studying the effect of VP2358on RIMD2210663acid tolerance response. The mutant of RIMD2210663⊿VP2358was constructed by suicide plasmid pDS132mediated homologous recombination method. The results showed that the mutant acid tolerance induction dropped when adapted at pH4.5, pH5.0, pH5.5, pH6.0and pH7.5for1hour following by environment stress at pH4.0and37℃treatment for1hour, which showed that VP2358influenced the acid tolerance of Vp, but it may not be the unique influencing factor. Further study showed that the expression levels of acid tolerance related genes changed in the strain which was mutant for VP2358. The expression levels of cadA, rpoS and toxR were0.7,2.8,1.7times of wild strain after environment adaption and stress treatment. This implies that VP2358is mainly related with the expression of cadA, and that VP2358-cadA gene regulation system may play a major role in RIMD2210663acid tolerance, but the detailed mechanism needs further study. Our results provided information and data for studying mechanism of Vp acid tolerance and its regulatory factors.The survival rate of RIMD2210663⊿VP2358strain was significantly lower than that of the wild strain after the treatment of pH4.96,55.5℃for25s, however that had no significant difference between the mutant and wild strain in the treatment of55.5℃for25s or pH4.96for25s respectively, indicating that VP2358had significant effects on the survival of RIMD2210663in acid combined with heat treatment. The further study on the expression regularity and its mechanism of environmental factors inducing VP2358will contributes to the optimization of acid-heat synergic sterilization parameters and clarify the survival mechanism of Vp under acid-heat stress. |
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