Lactobacillus Plantarum ZDY2013 Antagonize Food-borne Pathogen Bacillus Cereus

Lactic acid bacteria have been widely used in dairy products,traditional food and health food,and their interaction with gut microbiota is the hot topic in the world.At present,the selection of high-efficiency dominant strains is the key to support the formation of probiotics industry and market,and whether lactic acid bacteria can become dominant bacteria during the process of interaction with pathogen is an important step to set it as a probiotics.This paper aims to explore the biological function and preliminary mechanism of Lactobacillus plantarum ZDY2013 to antagonize the foodborne pathogenic Bacillus cereus.The paper is divided into six chapters.In chapter one,the harm of food-borne pathogenic bacteria,the toxicity of B.cereus,the function of lactic acid bacteria and the beneficial properties of L.plantarum were summarized,and the interaction mechanism between lactic acid bacteria and food-borne pathogenic bacteria was overviewed.In chapter two,a specific method for toxin-producing B.cereus identification was established in complex food substrates.The complex amplification system was constructed to selectively identify the vomiting toxin or enterotoxin producing B.cereus with the specific primers target the relevant toxin gene,the species gene and the amplification internal standard.PMA was used to cross-link the cell membrane damaged B.cereus in the sample,then genomic DNA was extracted.The results showed that the optimized complex amplification system could specifically identify the emetic or enterotoxin-producing B.cereus,and was applicable to the analysis of complex food substrates such as starch and dairy products.The complex bacteria in the food matrix did not affect the signal acquisition of target DNA by this method,and the DNA signal of the dead B.cereus could be effectively eliminated.The lowest level of emetic toxin producing B.cereus could be identified in the complex food matrix is10~3 cfu/g,while the low concentration of enterotoxin producing B.cereus(10~1 cfu/g)could be specifically identified after a short enrichment process.In chapter three,to study how the enterotoxin B.cereus was antagonized by the probiotic L.plantarum ZDY2013 from multiple perspectives.The relationship between the enterotoxin producing B.cereus ATCC14579 and HN001 inhibition and the pH of L.plantarum culture was determined by in vitro test.How the dominant bacteria formed was tested by the model of joint milk fermentation with L.plantarum and enterotoxin-p roducing B.cereus.The Caco-2 model was used to study how L.plantarum prevents enterotoxin-producing B.cereus adhesion through different colonization methods.The activity of extracellular lactate dehydrogenase was used to indicate the toxicity of toxins secreted by B.cereus in culture,and if the L.plantarum against the genes over-expression related to the cell stress from cell culture of B.cereus.The results showed that the growth of enterotoxin-producing B.cereus was inhibited due to the organic acids producted by the metabolism of L.plantarum.Milk joint fermentation by the two kinds of strains can continuously reduce the pH value,and the number of B.cereus was continuously decreased after 24 hours,while no effect on the number of was obtained from the decreased pH value.B.cereus colonization can be inhibited by L.plantarum on Caco-2 cells with three different models,e.g.exclusion,competition and replacement,and the competition strategy was the best model.The supernatant from enterotoxin-producing B.cereus had the toxicity to destroy the membrane integrity of epithelial cell and induced the release of lactate dehydrogenase,and the toxicity decreases with the decrease of concentration of supernatant.The presence of L.plantarum protected epithelial cells against genes over-expression induced by the supernatant of enterotoxin-producing B.cereus.In chapter four,we tested how the L.plantarum fermented milk affects the damage from enterotoxin-producing B.cereus in mice.Mice were randomly assigned to four groups,the mice in the first group were given enterotoxin-producing B.cereus first and PBS solution subsequently;The mice in the second group were given L.plantarum fermented milk first and enterotoxin-producing B.cereus subsequently;The mice in the third group were given first and subsequently;The mice in the fourth group were given PBS solution for the two periods.The direct effects of the L.plantarum fermented milk and the enterotoxin-producing B.cereus on mice were determined by monitoring the change of body weight index.The levels of inflammatory factors and the changes of physiological and biochemical indexes in the serum of mice were investigated as well to test the interference from the enterotoxin-producing B.cereus,and whether the L.plantarum fermented milk had an improvement effect on them.The balance of gut microbiota of mice was monitored by high-throughput sequencing to analyze whether the enterotoxin-producing B.cereus disturbed it,and whether the milk fermented by L.plantarum had benefit to the health of intestinal flora.The results showed that the enterotoxin-producing B.cereus reduced the body weight growth rate of mice and also disturbed the related inflammatory factors levels.The L.plantarum fermented milk could prevent the enterotoxin-producing B.cereus from promoting the increase of platelet count in whole blood and uric acid in serum,and improve the injury degree of the cecum pathological tissues.The enterotoxin-producing B.cereus changed the abundance of some taxa,but without significantly change the overall structure of the intestinal flora in mice,while the L.plantarum fermented milk ameliorated this change.In chapter five,we further tested the characteristics of metabolites exopolysaccharides from L.plantarum against enterotoxin produced B.cereus.Exopolysaccharides were extracted and purified from L.plantarum,and the molecular weight and monosaccharide component characteristics related to biological activity were studied.The structure of exopolysaccharides were modified with sulfonation,and the functional groups were characterized by FT-IR.The free radicals scavenging abilities of sulfated EPS were measured to verify the change of biological activity.The cytotoxicity of different types of enterotoxin from B.cereus(one,two or three kinds of enterotoxin)was measured on the small intestinal epithelial cell model,and the protective effect for the cells with the presence of exopolysaccharides or its derivatives was tested.The results showed that the yield of exopolysaccharides from L.plantarum was 429.4±30.0 mg/L and the purity was 96.06%.The elution curve of size exclusion chromatography showed that exopolysaccharides only had a symmetrical sharp peak with a molecular weight of 5.17×10~4 Da.Gas chromatography showed that exopolysaccharides were composed of xylose and galactose,and galactose accounted for 98.3%of the total content.After the derivatization of exopolysaccharides,the antioxidant activity was significantly increased,and the ability to prevent damage from different types of enterotoxin of B.cereus on intestinal epithelial cells was also significantly enhanced.In chapter six,the biological functions and preliminary mechanisms of the L.plantarum against the foodborne pathogenic B.cereus from multiple perspectives and levels were summarized,and some related suggestions were reminded for the further improvement.Together,this paper systematically explored the interaction between the probiotic L.plantarum and food-borne pathogenic bacteria B.cereus from different perspectives,and explained that L.plantarum could antagonize the production of toxin-producing B.cereus from the aspects of in vitro,e.g.food,cells,mice and metabolites.Therefore,this study can deepen the understanding of the interaction between lactic acid bacteria and pathogenic microorganisms,and also provide a theoretical basis for the development and application of L.plantarum and its metabolites in the food industry in the future.