Fermentation Optimization Of Compound Chinese Medicine And Its Effect On Mice Diarrhea Caused By Porcine Escherichia Coli

Infection with diarrhoeal Escherichia coli(DEC)in pigs can lead to edema disease(ED)in pigs,diarrhea in newborn piglets(ND),and diarrhea in weaned piglets(PWD),characterized by local or systemic inflammatory symptoms,severe diarrhea,and even sepsis.Diarrhea in piglets can lead to a large number of deaths in piglets,causing significant economic losses.In recent years,the occurrence of this disease tends to be acute,but the clinical efficacy of drug prevention and treatment is extremely poor.The widespread use of antibiotics has increased the resistance of Escherichia coli and increased the types of drug resistance,making prevention and control work extremely difficult,seriously restricting the healthy development of the pig breeding industry.Currently,a large number of studies have shown that many traditional Chinese medicines and microecological preparations have certain therapeutic effects on colibacillosis.Using microorganisms to ferment traditional Chinese medicines has the advantages of increasing the content of effective ingredients,enhancing therapeutic efficacy,and regulating the intestinal flora of animals.Therefore,the development of probiotic fermented Chinese medicine to prevent and treat piglet diarrhea caused by DEC is one of the ways to solve this problem.Based on the summary and practice of predecessors,this project will deeply explore the application of microecological preparations combined with traditional Chinese medicine,and develop safe,effective,economic,and quality controllable fermented traditional Chinese medicine preparations to prevent and treat piglet diarrhea.Firstly,probiotics are used to ferment the compound traditional Chinese medicine(Gegen Qinlian Tang,an ancient formula for relieving diarrhea,is made by adding or subtracting the reasonable middle decoction),and pig derived DEC is used to establish a mouse diarrhea model.The therapeutic and preventive effects of fermented compound Chinese medicine on mouse diarrhea caused by pig derived DEC are explored,providing scientific data support for the clinical application of fermented traditional Chinese medicine in preventing and treating piglet diarrhea caused by DEC.The specific content and results are as follows:1.This study carried out a meta-analysis of the prevalence of pathogenic Escherichia coli(ETEC,STEC)in pigs worldwide.626 relevant studies were retrieved from the four databases of CNKI,Science Direct,Web of Science and Pub Med,and 23 were selected for inclusion in the study.After extracting the relevant risk factors information,Cochran-Q test,I~2 test andχ~2Test and calculate the heterogeneity of the study,and use univariate regression analysis to verify the potential sources of heterogeneity and the impact of different risk factors on the prevalence of swine colibacillosis.The results show that the global prevalence rate of swine colibacillosis is13.31%,and there is an increasing trend after 2016,mainly caused by enterotoxin producing Escherichia coli(ETEC).2.In this study,three strains of lactic acid bacteria,six strains of Bacillus and six strains of yeast were tested for acid,bile salt,artificial gastrointestinal fluid tolerance,and cellulase,amylase,protease production capacity to screen fermentation bacteria.The results showed that Lactobacillus fermentans RF1(producing cellulase),Saccharomyces cerevisiae N3(producing amylase)and Bacillus subtilis YK(producing amylase and protease)had strong enzyme production ability and tolerance;Although the tolerance of Bacillus licheniformis YD is weak,its ability to produce amylase and protease is strong.Therefore,RF1,N3,YK and YD were selected as fermentation strains.3.This study takes the p H value after comprehensive fermentation,the number of viable bacteria per gram of fermented Chinese medicine,the content of flavonoids and polysaccharides per gram of fermented Chinese medicine as the evaluation criteria,and optimizes the six fermentation conditions of nitrogen source addition,carbon source addition,water content,bacteria amount,fermentation time,and fermentation temperature through single factor test,and optimizes the optimal water content,bacteria amount The best fermentation time and temperature were further optimized to obtain the best fermentation conditions.The results showed that the optimal fermentation conditions were 3%inoculation,40%water content and35℃for 2 days.4.This study established a diarrhea model by intraperitoneal injection of different doses of porcine DEC into mice,and established a blank group,a model group,a florfenicol group,an unfermented traditional Chinese medicine group(62.5 mg/m L),a high dose group(125 mg/m L),a medium dose group(62.5 mg/m L),and a low dose group(31.25 mg/m L)to explore the therapeutic and preventive effects of different doses of fermented compound Chinese medicine on porcine Escherichia coli.The results showed that the mice were injected with 200μL concentration is a dose of DEC of 1.0×10~8 CFU/m L,the challenged mice showed significant diarrhea symptoms,with a diarrhea rate of 100%and a mortality rate of 20%.A mouse diarrhea model can be successfully established;After 3 days of high-dose administration,all diarrhea ceased,and the mortality rate was the lowest except for the blank group(1 animal died);High dose treatment can significantly alleviate liver and spleen enlargement caused by Escherichia coli,and significantly reduce the proinflammatory cytokine(IL-1β,IL-6,IFN-γand TNF-α),Significantly increase the level of anti-inflammatory cytokine(IL-4,IL-10).Therefore,fermented compound Chinese medicine has a good therapeutic effect on diarrhea caused by Escherichia coli from pigs,but has no good preventive effect.To sum up,the global prevalence of porcine coliform disease was calculated by meta-analysis to be 13.31%;The solid fermentation system for fermented compound Chinese medicine fermentation by probiotics was established and optimized;On the basis of the successful construction of the porcine DEC diarrhea mouse model,it was found that fermented compound Chinese medicine could alleviate the liver and spleen swelling caused by Escherichia coli,and reduce the inflammatory cytokines(IL-1β,IL-6,IFN-γand TNF-α)Level and increase the level of anti-inflammatory cytokines(IL-4,IL-10)to control inflammation in mice,thus effectively treating diarrhea.