Isolation,Identification And Beneficial Effects Evaluation Of Lactobacillus Reuteri From Rabbit

The Lactobacillus reuteri is a kind of lactobacillus which exists widely in animal intestine.It has strong adhesion to intestinal mucosa and can adjust the distribution of intestinal flora,characteristics of antagonistic colonization of harmful bacteria and inhibiting growth of pathogenic bacteria in intestinal tract.In addition,it is often used as a feed additive because of its good resistance to adverse circumstance.This study isolated and identified Lactobacillus reuteri from rabbit intestinal contents,and studied its biological characteristics,stress resistance,adhesiveness in vitro and implantability in vivo,in order to obtain the rabbit Lactobacillus reuteri with good probiotics,resistance to adverse events and strong ability of colonization,so as to provide theoretical basis and necessary materials for the effective prevention and treatment of rabbit intestinal diseases and the development of rabbit oral vaccine.In this study,the intestinal contents of 8-week-old healthy rabbits were isolated and cultured,and the morphological,biochemical and 16 S r DNA sequences of the isolated bacteria were identified.The results was 10 strains of lactic acid bacteria were isolated and 4 strains of Lactobacillus reuteri(named R-L.reu(21)-1,R-L.reu(21)-2,R-L.reu(21)-3,R-L.reu(21)-4)were obtained.The growth curves and acid-producing ability of four strains of Lactobacillus reuteri were determined.It was found that all the four strains of Lactobacillus reuteri isolated in this study were in accordance with the growth and reproduction law of the bacteria,the PH of the medium decreased to 4.20 after 24 hours of culture,and the rate of acid production was related to the rate of proliferation of lactic acid bacteria.The in vitro bacteriostatic test showed that 4 strains of Lactobacillus reuteri had different degree of bacteriostatic effect on ETEC K88 and salmonella enterica,the inhibitory effect of R-L.reu(21)-4 on E.Coli K88 and salmonella enterica was the most obvious.The results of antibiotic sensitivity test showed that 4 strains of Lactobacillus reuteri were sensitive to chloramphenicol,rifampicin and erythromycin.The stress resistance of four strains of Lactobacillus reuteri was analyzed by acid tolerance test,bile salt tolerance test,artificial gastroenteric fluid tolerance test and high temperature tolerance test.The results of acid resistance test showed that all the four strains could maintain good growth at p H 3.0,survival rate was 97-99%.When p H decreased to 2.0,the growth of some bacteria was blocked,but R-L.reu(21)-4 had the strongest resistance to low p H,which was significantly higher of the survival rate than the other three strains(P<0.05),it was still above 98%.The results of bile salt tolerance test showed that all the four strains could survive well in 0.1% bile salt condition,with the survival rate ranging from 95%-97%(P>0.05),but the number of viable bacteria of RL.reu(21)-1,R-L.reu(21)-2 and R-L.reu(21)-4 in 0.3% bile salt concentration was significantly higher than that of R-L.reu(21)-3(P<0.05).The results of tolerance test of artificial gastric and intestinal fluid showed that the survival rate of the four strains was 90%-93% for 2 h in artificial gastric fluid(P>0.05).After entering the intestinal fluid,the survival rate of the four strains decreased with the increase of incubation time,and the survival rate was still between 68%-71%after 8h of incubation(P>0.05).These results indicated that the strains had certain tolerance to the simulated artificial gastric juice and intestinal juice.The results of high temperature tolerance test showed that the survival rate of 4 strains decreased with the increase of temperature and the extension of incubation time.The survival rate of the four strains was 87%-91% at 50℃ for 15 min(P>0.05),and the survival rate was still above 78% at 60℃ for 15 min(P>0.05).According to the above experimental results,all the Lactobacillus reuteri isolated in this study had good stress resistance,among which R-L.reu(21)-4 strain had the best stress resistance.The adhesion ability of lactic acid bacteria is closely related to its colonization ability.The yield of exopolysaccharides,surface hydrophobicity and self-polymerization ability are commonly used to evaluate the adhesion ability of lactic acid bacteria.n order to determine the adhesive ability of the isolated strains,the exopolysaccharide yield,surface hydrophobicity,self-polymerization ability and co-polymerization ability were measured,and the adhesive test in vitro was carried out.The results showed that the exopolysaccharides produced were higher,and their surface hydrophobicity was strongerby R-L.reu(21)-1 and R-L.reu(21)-4 of the 4 strains isolated.The results of in vitro adhesion test showed that strain R-L.reu(21)-4 had the highest in vitro adhesion rate,which was consistent with the above results.In this study,strain R-L.reu(21)-4 was selected for in vivo colonization test to determine the colonization ability of Lactobacillus reuteri isolated from rabbits.To further explore whether there are differences in colonization ability among different strains of lactobacillus,therefore,Lactobacillus reuteri P-L.reu(18)-1 from swine was selected as the control group.The results showed that strains P-L.reu(18)-1 and R-L.reu(21)-4 could be detected in the intestine of rabbits on the 7th day after oral administration of lactic acid bacteria,but the colonization of R-L.reu(21)-4 was much higher than that of P-L.reu(18)-1 and only R-L.reu(21)-4could be detected on the 14 th day.The results showed that both R-L.reu(21)-4 and P-L.reu(18)-1strains could be colonized in rabbit intestine,but the homology R-L.reu(21)-4 strain was the best.The safety of R-L.reu(21)-4 and P-L.reu(18)-1 strains to young rabbits was evaluated by animal experiment,and the results showed that R-L.reu(21)-4 and P-L.reu(18)-1 strains had good biological safety to young rabbits.In summary,We were isolated 4 strains of lactobacillus reuter from rabbits,which had typical characteristics of lactic acid bacteria.They were certain resistance to gastrointestinal environment.The bacteriocin is the main substance of Lactobacillus reuteri.The strain R-L.reu(21)-4 could be successfully colonized in rabbits.The colonization ability of strain R-L.reu(21)-4 in rabbits is significantly higher than that of Lactobacillus reuteri from pigs,and it is not pathogenic to rabbits.The results of this study can provide theoretical support and important materials for the effective prevention and treatment of rabbit intestinal diseases and the development of rabbitderived Lactobacillus reuteri preparations.