| The intestinal flora plays an important role in maintaining the health of the body,and is closely related to the nutrition digestion,immune metabolism and biological antagonism.But in recent years,the overuse of antibiotics has led to the disorder of intestinal flora,which has a great impact on the health of the body.Lactic acid bacteria in the human gut is important bacteria can regulate the intestinal flora disturbance by inhibiting the proliferation of pathogenic bacteria,improve the intestinal micro environment effect.The separated and screened with a probiotic function of lactic acid bacteria,study the ability of mixed fermented milk in vitro for intestinal pathogens,by high-throughput Illumina sequencing technology of mixed lactic acid bacteria fermented milk on antibiotic induced intestinal dysbacteriosis regulation.The main research contents are as follows:(1)Lactic acid bacteria(LAB)were isolated from the feces of Yunnan Bama longevity population and Xinjiang traditional fermented dairy products of Guangxi,and the ability of resisting acid and bile salt,the ability of adhering cells,the ability of inhibiting bacteria and drug resistance were studied.The results showed that 45 strains of lactic acid bacteria,strain J,La,Xn and Yd of 4 strains of LAB resistant and acid resistant high bile salt tolerance,pH in artificial gastric juice 3 in cultured 3H survival rates were 43.38%,65.89%,20.32%,57.75%,in the bile salt concentration of 0.30%medium to culture 24h the survival rates were 45.96%,53.33%,22.33%,31.05%;4 strains of LAB on the cell adhesion rate of IEC-6 was greater than 8/cell;inhibition on Escherichia coli,Salmonella and Clostridium difficile 3 intestinal pathogenic bacteria were highly antibacterial,bacteriostasis circle diameter was greater than 20mm;the physiological and biochemical 16S and rRNA identification,Xn strains was Lactobacillus fermentum,La and Yd strains were Lactobacillus plantarum,J strains were Lactobacilus rhamnosus.(2)Research on fermentation characteristics of LAB and hybrid strains,diacid,protein and hydrogen peroxide treatment to determine its ability to inhibit intestinal pathogens were made by fermentation supernatant of single strains and mixed strains,and the preparation conditions of surface optimization of mixed LAB fermented milk production response.The results show that the acidity of mixed LAB in fermented after 10h was 120.07°T,higher than that of single strain fermentation;4 strains fermentation supernatant after the acid of 3 kinds of pathogenic bacteria had no inhibitory effect;fermentation supernatant after removal of protein,L.plantarum La has influence on Salmonella inhibition had no significant effects on Escherichia coli inhibitory effect of Bacillus strain L.rhamnosus J(P>0.05),had no inhibitory effect on Escherichia coli and Salmonella effect(P>0.05),L.fermentum Xn of Clostridium difficile significantly inhibited(P<0.05),L.rhamnosus J the bacteriostatic effect of L.plantarumYd and L.plantarum La of Clostridium difficile decreased but not significantly(P>0.05);fermentation the supernatant after inhibition of hydrogen peroxide removal,4 strains of Escherichia coli changed significantly(P<0.05),L.fermentum Xn,L.plantarum Yd of Salmonella and Clostridium difficile inhibition effect significantly(P<0.05),L.plantarum La strains of Salmonella significantly(P<0.05)but had no significant effects on Clostridium difficile(P>0.05),fungi static effect of strain L.rhamnosus J of Salmonella and Clostridium difficile were not significant(P>0.05).After the fermentation of lactic acid bacteria,the antibacterial circle diameter of Escherichia coli was 30.81mm,and the diameter of inhibition circle for Salmonella was 26.22mm,and the diameter of inhibition circle was 23.53mm.The optimum conditions of mixed LAB fermented milk preparation is:sugar content is 6.06%,3.88%inoculation,fermentation temperature is 39?.this model of fermented milk bacteria number was 9.50 lg(CFU/mL),the actual measured is 9.44 lg(CFU/mL),the diameter of inhibition zone of Escherichia coli was 31.75 mm and the diameter of inhibition zone of Salmonella was 26.34mm,the diameter of inhibition zone of Clostridium difficile was 24.51mm,were higher than that of single strain fermentation.(3)Through the establishment of lincomycin hydrochloride intestinal dysbacteriosis model intervention using mixed LAB and fermented milk,the ratio of determination of mouse intestinal Bifidobacteria and E,scherichia coli(B/E)and body weight,daily food intake,serum endotoxin content,spleen index,serum globulin content and immune globulin(IgG)content.The results show that the lincomycin hydrochloride(300mg/mL)3d orally,intestinal Bifidobacterium and Lactobacillu.s were 7.54 1g CFU ·g-1,5.86 1g CFU g-1,were significantly lower than the control group(P<0.05),and the number of enterococci was significantly higher than the control group(P<0.05).At the same time,the B/E value of intestinal tract in the model group was significantly lower than that of the control group(P<0.05),and was not recovered in 7d.Mixed LAB after 7d intervention,the bacterial suspension and fermented milk group increased the weight of mice was higher than the control group,daily food intake is also higher than the model group;the serum endotoxin level of the bacterial suspension intervention group,intervention group,bacteria fermented milk and fermented milk suspension prevention group prevention group mice were significantly lower than the model grroup(P<0.05)were 0.3725 EU· L-1,0.3631 EU·L-1,0.3758 EU·L-1,0.3831 EU·L-1;spleen index of bacterial suspension liquid and fermented milk intervention group the intervention group mice were 2.48 mg·g-1,2.45 mg·g-1,and the bacterial suspension prevention group and prevention group were fermented milk 2.37 mg·g-1,2.16 mg· g-1,and they were significantly higher than model group(P<0.05);serum globulin and IgG content of mice in model group were 19.82 g·L-1,0.78 g· L-1,and the bacterial suspension intervention group and intervention group,fermented milk bacteria suspension fermented milk prevention group and prevention group The serum protein content were 25.75 g·L-1,27.54 g·L-1,22.47 g·L-1,24.06 g·L-1,IgG content were 0.92 g·L-1 and 0.98 g·L-1,0.85 g·L-1,0.91 g·L-1,compared with the model group were significantly increased(P<0.05).(4)To regulate the intestinal flora of mice using Illumina high-throughput sequencing of mixed LAB fermented milk,and to observe the colon tissue of mice after HE staining Mouse intestinal flora composition at the division level,mainly for the Firmicutes,Bacteroidetes and Proteobacteria;control group of mice intestinal Firmicutes content was 32.68%,antibiotics reduced the content of gastrointestinal tract in mice,Firmicutes was 3.87%,the bacterial suspension prevention group and fermented milk the prevention group mice in Firmicutes were respectively 30.44%,33.90%,mixed LAB and fermented milk intervention,Firmicutes were restored to the level of the control group were 35.30%,32.39%;control group of mice intestinalbacteriodetes content was 53.58%,antibiotic intervention increased the content.Bacteroidetes was 54.44%,bacterial suspension prevention group and prevention group of fermented milk in mouse intestinal bacteriodetes content were 51.05%and 49.15%,mixed LAB and fermented milk intervention decreased the content of Bacteroidetes,respectively 50.37%,51.33%;the content of bacteria in the intestines of mice group deformation control blank 7.24%,antibiotic intervention increases the content of Proteobacteria was 36.58%,the bacterial suspension prevention group and prevention group of fermented milk in mouse intestinal Proteobacteria were respectively 12.47%,12.73%,mixed LAB suspension and fermentation milk intervention group in mouse intestinal Proteobacteria were respectively 7.76%,10.29%. |
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