Fate Of Vibrio Parahaemolyticus During Simulated Digestion Process

Vibrio parahaemolyticus,as a common food-borne pathogen,is one of the most important pathogens causing food poisoning in coastal areas in summer and autumn.It can cause fever,vomiting,diarrhea and abortion.Serious Vibrio parahaemolyticus can cause primary sepsis and even death,which poses a great threat to food safety and public health.Revealing the pathogenic mechanism of Vibrio parahaemolyticus has always been the focus of attention.It helps to reveal the underlying causes of human diseases and curbs the risk of Vibrio parahaemolyticus from the source.However,there is still a blank description of how Vibrio parahaemolyticus passes through the human digestive tract before it reaches the intestinal tract.The application of "gold" standard formula of simulated digestive juice and artificial simulated digestive tract model can help monitor the fate of Vibrio parahaemolyticus in human digestive tract,provide a new perspective,further explore the pathogenic mechanism of Vibrio parahaemolyticus,and play a great role in the diagnosis,treatment of related diseases and the development of new drugs.Therefore,in order to clearly understand the fate changes of Vibrio parahaemolyticus in human digestive tract,the following research work was carried out in this paper:(1)the fate changes of Vibrio parahaemolyticus in three simulated digestive juices;(2)the fate changes of Vibrio parahaemolyticus in artificial static digestive model;(3)the changes of gastric acid and escape of parahaemolysis.The main contents and results of this study are as follows: 1.The fate changes of Vibrio parahaemolyticus in three simulated digestive juicesThe survival rate,morphological changes,heterogeneity and gene expression of Vibrio parahaemolyticus from different sources in three simulated digestive juices in Shanghai were investigated and analyzed,which provided data basis for the study of pathogenic mechanism of Vibrio parahaemolyticus.The survival rate of 60 Vibrio parahaemolyticus strains(28 clinical strains,30 food-borne strains and 2 ATCC standard strains)from different sources in Shanghai was monitored by using Minekus standardized formula and traditional plate coating method.The heterogeneity of the strains in three simulated digestive juices was analyzed.The presence of Vibrio parahaemolyticus strains was observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The relative expression of virulence genes of Vibrio parahaemolyticus in three simulated digestive juices was further revealed by fluorescence quantitative PCR.The results showed that saliva had no significant effect on Vibrio parahaemolyticus and had no bactericidal effect;Vibrio parahaemolyticus could not survive in gastric juice(pH=2.0/3.0),and the microscopic structure of cells was seriously damaged.When the acidity and basicity of gastric juice increased to 4.0,the survival rate of bacteria was 54.70(+1.11%).The viability of pathogenic strains in gastric juice was higher than that of non-pathogenic strains,and the viability of clinical strains was higher.It was higher than that of environmental strains.The bactericidal effect of intestinal juice is lower than that of gastric juice.The results of gene expression showed that the expression of virulence-related genes in intestinal juice treatment was significantly higher than that in saliva and gastric juice treatment.In this study,the survival,morphological changes,heterogeneity and virulence gene expression of pathogenic and non-pathogenic Vibrio parahaemolyticus isolated from food and clinic in simulated digestive juice were compared systematically for the first time.The main bactericidal sites of Vibrio parahaemolyticus isolated from human digestive tract and the related changes of Vibrio parahaemolyticus in human digestive tract were preliminarily analyzed,which provided reliable data support 2.The fate change of Vibrio parahaemolyticus in artificial simulated static digestion modelOn the basis of Chapter 2,the survival rate,morphological changes,heterogeneity and gene expression of Vibrio parahaemolyticus from different sources in Shanghai during simulated digestion were validated by artificial static digestion model.Using Minekus' s continuous static digestion model in vitro and traditional plate coating method,the survival rate of 60 Vibrio parahaemolyticus strains(28 clinical strains,30 food-derived strains and 2 ATCC standard strains)from different sources in Shanghai were monitored.The heterogeneity of the strains was analyzed.The strains were observed by scanning electron microscopy and transmission electron microscopy.The relative expression of virulence genes in Vibrio parahaemolyticus during digestion was further revealed by fluorescence quantitative PCR.Saliva has no obvious influence on Vibrio parahaemolyticus in human digestion process and has no bactericidal effect;Vibrio parahaemolyticus has low survival rate in gastric juice and serious damage to the cell microstructure,which is basically consistent with the findings in chapter 2.The most effective bactericidal site for Vibrio parahaemolyticus is the human stomach,with pathogenicity(trh+trh+,tdh-,tdh-trh+)and non-pathogenicity(trh+trh+).The survival rates of sex(trh-tdh-)strains were also significantly different.The difference is that in the continuous digestion process,Vibrio parahaemolyticus has VBNC phenomenon.After passing through gastric juice,the intestinal pH value is about 7.0,and the body temperature of human body is 37 degrees C,which provides the best growth environment for Vibrio parahaemolyticus.Vibrio parahaemolyticus has "resuscitation" phenomenon in the intestinal fluid treatment stage,regardless of shape or quantity.The results of gene expression showed that the expression of virulence-related genes in intestinal juice treatment was significantly higher than that in saliva and gastric juice treatment.To sum up,this study used in vitro continuous digestion model to verify that some Vibrio parahaemolyticus can survive to the intestine through oral and gastric environment with high pH,resuscitate in the intestine,and express virulence-related genes.3.Changes of gastric acid and escape of paralysisIn order to study the changes of gastric pH and the escape of Vibrio parahaemolyticus with food intake,Vibrio parahaemolyticus with 107 CFU/g concentration was inoculated into salmon and Penaeus vannamei.The digestive simulation was carried out by in situ digestive system of human stomach in vitro.After 120 minutes,the food emptying rate,the change of gastric pH value and the survival of Vibrio parahaemolyticus from chylous were measured.Situation.The results showed that the gastric emptying lag time of salmon was 60 min,that of Penaeus vannamei was 90 min,and that of two kinds of chyme were not completely emptied when simulated digestion lasted 120 min.The pH value of stomach is 1.6 + 0.1,which increases sharply after 10 minutes of food intake.With the continuous secretion of gastric acid and digestion and decomposition of food,the pH value of stomach begins to decrease and remains around 5.41.Vibrio parahaemolyticus was not completely killed by gastric acid after 120 minutes of food digestion.The survival rate of Vibrio parahaemolyticus was 0.119(+0.025%)in shrimp and 0.007(+0.005%)in salmon.In conclusion,Vibrio parahaemolyticus enters the intestinal tract through gastric emptying following digestion and decomposition of food matrix,thus avoiding the killing of gastric acid and causing human diseases.In this study,a simulated dynamic human stomach in situ simulation system was used for the first time to analyze the correlation between food intake and the survival of pathogenic bacteria,which provided a new way of thinking for understanding the pathogenic mechanism of Vibrio parahaemolyticus and reliable basic data for the later construction of a real simulated digestion model..