Screening Of Hypoglycemic Probiotics From Canine And Its Effect On Type 1 Diabates In Mice

Canine diabetes mellitus is a secretory disease characterized by persistent hyperglycemia caused by a combination of various factors.Exogenous insulin is used as the main treatment in clinical practice.However,insulin is expensive and the preservation conditions are strict,which limits the treatment of canine diabetes mellitus.Studies have shown that diabetes is closely related to the imbalance of intestinal microbiota.Probiotics can play an important role in the disturbance of sugar metabolism by improving the intestinal microbiota.In addition,homologous probiotics isolated from animals themselves are more adaptable to the environment and can rapidly colonize and play an important role.Therefore,the purpose of this study was to analyze and compare the differences in the abundance of intestinal flora between diabetic dogs and healthy dogs,so as to provide a theoretical basis for the correlation between diabetes and intestinal microflora.In addition,canine probiotics with good probiotics activity and hypoglycemic function were isolated and screened from the feces of healthy dogs,and the feasibility of hypoglycemic probiotics was evaluated in the streptozotocin induced mouse type 1 diabetes model,so as to provide reference for the prevention and treatment of canine diabetes.Specific research results are as follows:1)Comparative analysis of fecal bacteria abundance between diabetic dogs and normal dogsAlpha analysis showed that ACE index,Chao 1 index and Shannon index in diabetes group were significantly lower than those in control group.Beta analysis showed that the samples of the control group showed a clustered distribution,while the samples of the diabetes group showed a diffuse distribution.The analysis of bacterial flora structure showed that Firmicutes,Fusobacteriota and Bacteroidota were the main dominant bacteria in the diabetic group and the control group at the phylum level.Lachnospiraceae,Fusobacterium,Escherichia＿Shigella,Bacteroides and Faecalibacte were the main dominant bacteria in the diabetic group and the control group at the gene level.Matestats analysis showed that at the phylum level,the relative abundance of Bacteroidota in the diabetic group was higher than that in the control group.At the genus level,the relative abundance of unclassified＿Erysipelotrichales,Peptococcus,Alloprevotella,Phascolarctobacterium,Negativibacillus,Muribaculaceae,Parabacteroides and Faecalibacterium in the diabetic group was higher than those in the control group,but the relative abundance of unclassified＿Lachnospiraceae and Blautia in the diabetic group was lower than those in the control group.2)Isolation,identification and screening of canine hypoglycemic probioticsTen strains of Lactobacillus were isolated from the feces of healthy dogs,They included 7 strains of Ligilactobacillus animalis,2 strains of Limosilactobacillus reuteri and 1 strain of Enterococcus faecium.The results ofα-glucosidase inhibition test showed that all 10 strains of Lactobacillus had inhibitory effects onα-glucosidase,among which2 strains of Lactobacillus reuteri(A3 and E7)and 2 strains of Lactobacillus animalis(B12 and D14)had higher inhibitory rates.Therefore,these four strains of canine Lactobacillus were selected to study their probiotics.3)The probiotic activity of canine hypoglycemic probioticsThe growth curve showed that 4 strains of canine Lactobacillus reached the plateau after 10 h growth.All the four strains had a certain degree of tolerance to adverse environment,and the survival rate was about 20%in the environment with p H=2 and bile salt concentration of 0.4%.They showed good growth activity at 45℃and 55℃,but could not tolerate high temperature at 65℃.Antibiotic sensitivity test showed that 4strains were sensitive or moderately sensitive to gentamicin,neomycin,erythromycin and other antibiotics.Inhibition test showed that the four strains could effectively inhibit the growth of Enterotoxigenic e.coli,Salmonella enteritidis,Listeria monocytogenes and Staphcoccus aureus.Hemolysis test showed that there was no hemolysis ring around the colonies of 4 strains on the blood plate.The antioxidant tests showed that the reducing capacity of the four strains was weak,but the clearance rate of DPPH was strong.Hydrophobic and self-agglutination tests showed that the four strains had different degrees of adhesion ability,and E7 had the strongest adhesion ability.According to the test results,strain E7 was selected for subsequent animal experiments after comprehensive analysis.4)Effect of canine hypoglycemic probiotics on mice type 1 diabetes modelThe mice were randomly divided into 5 groups:control group(C),modeling group(M),probiotic prevention group(P),probiotic treatment group(LT)and insulin treatment group(ST),with 5 mice in each group.Probiotics prevention group was given Lactobacillus reuteri E7(1×10~9 CFU/m L,0.2 m L)for 28 days,and other groups were given the same amount of saline.The modeling method was intraperitoneal injection of streptomycin(STZ)for five consecutive days.The random blood glucose was detected72 h after the last day of injection,when the level was higher than 16.7 mmol/L,the modeling was considered successful.The results of animal experiments showed that,compared with the model group,probiotics could alleviate the diabetes of mice,reduce the content of blood sugar and blood lipid,improve glucose tolerance,protect pancreatic tissue,and have no damage to liver and jejunum,indicating that probiotics have a good preventive effect on STZ-induced type 1 diabetes model of mice.Compared with the modeling group and insulin treatment group,feeding probiotics after modeling had no obvious effect on the alleviation of diabetes in mice,which could not effectively reduce the levels of blood sugar and blood lipid and had poor glucose tolerance and serious pancreatic tissue damage.Compared with the control group,Alpha analysis showed that ACE index and Chao1 index were significantly decreased in the modeling group.The ACE index and Chao 1index of probiotic prevention group had no significant difference from the control group,but significantly increased compared with the molding group.The ACE index and Chao 1index of probiotic treatment group and insulin treatment group were significantly reduced compared with the control group,but had no significant difference compared with the modeling group.Beta analysis showed that the distance between the samples of the control group and the probiotic prevention group was close,while the distance between the samples of the modeling group was far,indicating that diabetes caused intestinal flora disorder,and prophylactic feeding of probiotics could reduce the difference of intestinal flora between normal mice and diabetic mice.The analysis of flora structure showed that Firmicutes was the dominant species in the five groups of mice at phylum level.unclassified＿Lachnospiraceae was the dominant species in the five groups of mice at genus level.LEf Se analysis showed that the dominant bacteria in the control group were Lachnospirales,Firmicutes,Enterobacter and Clostridia,the dominant bacteria in the modeling group were Desulfobacterota,the dominant bacteria in the probiotic prevention group were Lachnospiraceae,Coriobacteriales and Eggerthellaceae,and the dominant bacteria in the probiotic treatment group were Muribaculaceae and Bacteroides.the dominant bacteria in the insulin treatment group were Alloprevotella.In conclusion,there are differences in fecal bacteria abundance between diabetic dogs and healthy dogs.Hypoglycemic probiotics isolated and screened from the feces of healthy dogs have a good preventive effect on STZ-induced mouse type 1 diabetes model.