Study On Fermentation Products Of Microbial From Koumiss On Pathogenic Escherichia Coli

The aim of this research was to study the structure of bacteriostatic protein produced by fermented koumiss microorganism and its inhibitory effect on bovine pathogenic E.coli O₈ in vitro and infection of intestinal mucosal barrier and intestinal flora of pathogenic E.coli O₈ in mice,and the inhibitory mechanism of bacteriostat produced by fermented milk microorganisms on bovine pathogenic E.coli O₈.Provide theoretical basis.This study mainly includes the following three experiments:Experiment 1:Using ammonium sulfate precipitation method,ultrasonic wave concussion method and microporous filtration method to prepare bacteriostatic protein extract,determine bacteriostatic action and antibacterial spectrum of bacteriostin protein,separate bacteriostatic active component after Sephadex LH-20 chromatography column of antibacterial protein,and identify 17 kinds of amino acid content and molecular weight.The experimental results showed that the extracted antibacterial protein had a wide antibacterial spectrum.The minimum inhibitory concentration?MIC?and minimum bactericidal concentration?MBC?for pathogenic E.coli O8 were 89.13 and 178.25 g/mL respectively.The molecular weight of the isolated active group was mainly below 1000Da,and only 10%of the protein molecular weight was above 1000Da.The bacteriostatic active components contain a variety of small molecular proteins or peptides.The total amount of17 amino acids was 455.52 g/kg,the highest content of proline was 286.64 g/kg,accounting for 64.36%of the total amino acid content,followed by aspartic acid and cysteine,101.92 g/kg and 35.56 g/kg respectively,accounting for 22.88%and 7.98%of the total amino acid content,and the 3 amino acids accounted for more than 95%of the total amino acid content.Experiment 2:E.coli O₈ with high pathogenicity and lethality was screened as an experimental strain to study the bacteriostatic mechanism of bacteriostatic protein against pathogenic E.coli O8 in vitro.The results showed that bacteriostatic protein could destroy the permeability of E.coli O₈ cell membrane and the permeability of cell wall,and change the surface hydrophobicity of E.coli O₈ cells,thus releasing the alkaline phosphatase?AKP?,large molecular protein and beta galactosidase in the cell membrane of the pathogenic bacteria to the culture medium.Inhibitory effect of bacteriostin on pathogenic E.coli O₈ decreased with the decrease of concentration,and the growth of E.coli O₈ could be inhibited in 48 h when the concentration of bacteriostin was higher than that of 1/2MIC.Experiment 3:The bacteriostasis mechanism of koumiss microorganism fermentation product on pathogenicity E.coli O₈ in vivo.The experiment was conducted to study the experimental mice first and then to study the pathogenicity test.The purpose of this study was to study the protective effect of bacteriostin on infected E.coli O₈ mice.This experiment mainly includes the following aspects:?1?Establishment of a mouse model to determine the 80%minimum lethal dose?MLD?of pathogenic E.coli O₈ in mice.Test set blank control group,negative control group,positive control group,CFS high dose group,CFS medium dose group,CFS low dose group,Nisin group,7 groups,continuous perfusion of 7th day,4th day intraperitoneal injection of 80%MLD bacteria suspension in the gastric perfusion,every day to collect the feces of each group of mice,cryopreservation and reserve.?2?The effect of fermented product on the immune regulation of mice was significantly lower than that in the blank control group.The content of IL-2,IL-4,IL-10,Ig A,Ig G,INF-?,T3,T4 and INS in the negative control group decreased significantly?P<0.05?.CFS high dose group can regulate the stability of anti inflammatory factors,proinflammatory factors,immunoglobulin and stress hormones in infected E.coli O₈ in mice,maintain the balance of biochemical indexes in the serum of mice and improve the resistance of mice.?3?The effect of koumiss microbial fermentation products on the ileum mucosal barrier in mice was significantly lower than that in the blank control group?P<0.05?,and there was no significant difference between the CFS high dose group and the positive control group?P>0.05?,and the TJ related protein of the ileum?OCCLUDIN,CLAUDIN-1,Z0-1?.The content of m RNA gene expression and protein content decreased significantly?P<0.05?,and the content of serum DAO,D-LA and LPS increased significantly?P<0.05?.Compared with the blank control group,the content of sIg A,IL-2,IL-10,IFN-?and TGF-?1 in the ileum of mice decreased significantly?P<0.05?:there was no significant difference in the content of sIgA,IL-2,IL-10,IFN-gamma and TGF-beta in the ileum of CFS high dose group and positive control group?P>0.05?,and there was no significant difference in the content of IL-1 beta,IL-6,IL-8 and TNF-??P>0.05?.As a result,E.coli O₈ can destroy the intestinal mucosal chemical barrier,physical barrier and immune barrier,and high dose CFS can effectively improve and repair the intestinal mucosal barrier damage caused by E.coli O₈.?4?The effect of koumiss microorganism fermentation product on the intestinal mucosal barrier of mice was sequenced by Illumina Hi Seq sequencing platform,and the species annotation and abundance analysis were carried out to reveal the species composition of the samples.Mainly through Acidobacteria?Firmicutes?Bacteroidetes?Deferribacteres?Proteobacteria?Verrucomicrobia?Tenericutes?Actinobacteria?Saccharibac Teria and Cyanobacteria,10 different phylum,carry out analysis of species relative abundance at door level.Compared with the blank control group,the OTUs of the intestinal flora in the 1st day,4th day feces and the cecum content of 7th day in the mice fed with saline was increased,the number of Firmicutes in the group structure increased,but the number of Bacteroidetes decreased,and the increase of Deferribacteres significantly.After the gavage of ciprofloxacin,Firmicutes decreased significantly,and the 4th day intraperitoneal injection of E.coli O₈ decreased to the lowest level,but in the7th day cecum content,Firmicutes increased significantly to the normal level,and the number of Proteobacteria and Verrucomicrobia increased significantly.After intragastric Nisin,the number of Firmicutes decreased to the same as ciprofloxacin,after 4th day intraperitoneal injection of E.coli O₈,the amount increased,but was still lower than that in the saline group.With the number of Verrucomicrobia,Firmicutes was more than the normal saline group in the 7th day cecum content,and the amount of Verrucomicrobia decreased with the ciprofloxacin 4th day.The number of Saccharibacteria has also increased.After taking high dose of CFS,the number of Firmicutes decreased slightly,while the number of Bacteroidetes increased slightly,and the proportion of Cyanobacteria increased.It can be seen that the most cecum in mice is Bacteroidetes and Firmicutes.The different ways of treating mice make Bacteroidetes and Firmicutes in a relatively stable range.When the mice infected with E.coli O₈,Firmicutes begins to decrease and Bacteroidetes increases.The early perfusion of ciprofloxacin in the stomach makes the decrease of Firmicutes in the intestinal tract of mice and the increasing trend of Bacteroidetes.The trend is more significant when E.coli O₈ is intraperitoneally injected.Firmicutes can significantly increase the level of normal mice at 7th day,indicating that ciprofloxacin hydrochloride can destroy the stability of intestinal microflora structure in normal mice,but when the intestinal tract is invaded by pathogenic bacteria,the intestinal flora can be regulated to the normal state.Ciprofloxacin hydrochloride can be used to treat colibacillosis,but it has damage to the normal organism and does not have the function of preventing disease.The intestinal flora of mice was maintained at a relatively stable level at high dose of CFS.After intraperitoneal injection of E.coli O₈,the content of Bacteroidetes and Firmicutes was slightly fluctuated,but it remained consistent with the normal saline group,indicating that CFS could maintain steady state changes between the various intestinal flora of mice and inhibit the E.coli O₈ good results.