| At present,most probiotics commonly used in aquaculture come from the intestines of livestock or their living environment,the adhesion and colonization of these probiotics in the intestines of fish is poor,resulting in their unsatisfactory probiotic effects on fish.Therefore,screening fish-derived probiotics from fish intestines is one of the main ways to solve this problem.This research takes Rhynchocypris lagowskii(Dybowski,1869),a characteristic economic fish in Jilin Province as the research object,probiotics with the ability of producing protease,amylase and lipase were isolated and screened from the intestinal tract of its,the bacteria with high enzymatic capacity were screened by enzyme-producing capacity test,in vitro bacteriostatic test and antibiotic sensitivity test.The probiotics of bacteria were further evaluated by detecting their high temperature resistance,acid resistance,gastric juice resistance,intestinal fluid resistance,bile salt resistance and bacterial strain safety.The adhesion ability and adhesion mechanism of potential probiotics were studied by in vitro adhesion model.The results showed that:Test 1: In this study,214 strains of bacteria were isolated from the intestinal tract of Rhynchocypris lagowskii.Among them,protease producing bacteria were 86 strains,accounting for 40.19%.amylase producing bacteriac were 53 strains,accounting for 24.77%.Lipase producing bacteria were 47 strains,accounting for 21.96%.The bacteria of produced three enzymes simultaneously were T28 strains,accounting for 13.08%.The antagonistic ability of 15 strains of bacteria with strong enzyme-producing ability against common pathogens such as Staphylococcus aureus,Aeromonas hydrophila,Escherichia coli,Aeromonas veronii,Edwardsiella and Aeromonas sobria was determined by AGAR perforation diffusion method.Then,10 strains of bacteria with strong antagonistic ability were screened out according to the antagonistic test results,and further determined the sensitivity of these 10 strains to 17 antibiotics.Finally,8 strains of bacteria with strong ability to produce enzymes,antagonize pathogenic bacteria and less resistance factors were screened as potential probiotics.Name them respectively as: LSG1-1,LSG2-1,LSG2-3-2,LSG2-5,LSG2-8,LSG3-3,LSG3-7 and LSG3-8.Test 2: 8 strains of potential probiotics were identified by combining with the morphological,physiological and biochemical characteristics of bacteria and 16 S r RNA sequence analysis.The results showed that strains LSG1-1,LSG2-1,LSG2-3-2,LSG2-5,LSG2-8,LSG3-3,LSG3-7 and LSG3-8 had the highest similarity with Bacillus licheniformis,Bacillus subtilis,Bacillus methylotrophicus,Bacillus velezensis,and Bacillus amyloliquefaciens,Bacillus amyloliquefaciens,Bacillus tequilensis,Bacillus aryabhattai and Bacillus mojavensis,respectively.After further analysis of phylogenetic tree,8 strains were identified as Bacillus licheniformis,Bacillus subtilis,Bacillus methylotrophicus,Bacillus velezensis,and Bacillus amyloliquefaciens,Bacillus amyloliquefaciens,Bacillus tequilensis,Bacillus aryabhattai and Bacillus mojavensis,respectively.Test 3: The probiotics of bacteria were tested in vitro by simulating the intestinal digestion environment of fish.The tolerance of bacteria were evaluated by high temperature resistance test,acid resistance test,bile salt resistance test,gastric juice resistance test,intestinal juice resistance test and safety test.The results showed that all strains had strong tolerance.LSG2-1and LSG2-8 had significantly higher tolerance to high temperature than other strains(P<0.05),while LSG2-3 and LSG3-6 had significantly lower tolerance to high temperature(P< 0.05).LSG3-7 had the highest acid resistance(P<0.05),while LSG2-5 had the lowest acid resistance(P<0.05),LSG3-7 had the highest tolerance to gastric juice(P<0.05),while LSG2-1 and LSG3-8 had the lowest tolerance to gastric juice(P<0.05).LSG2-5 had a higher tolerance to intestinal fluid(P<0.05),while LSG3-8 had a significantly lower tolerance to intestinal fluid(P<0.05).LSG3-6 had a strong tolerance to bile salt(P<0.05),while LSG2-3-2 had a significantly poor tolerance to bile salt(P<0.05).The results of safety test showed that each strain had a good safety against Rhynchocypris lagowskii.Test 4: An in vitro intestinal mucin protein model was established by using the intestinal mucin protein of Rhynchocypris lagowskii.The adhesion of 8 potential probiotics to the intestinal mucin protein of Rhynchocypris lagowskii was detected by using the fluorescent marker hoechst3325 in the vitro model.The results showed that: LSG1-1 adhesion rate was 10%,LSG2-1 adhesion rate was 16%,LSG2-3 adhesion rate was 18%,LSG2-5 adhesion rate was35%,LSG2-8 adhesion rate was 36%,LSG3-6 adhesion rate was 24%,LSG3-7 adhesion rate was 34%,and LSG3-8 adhesion rate was 33%.Then sodium periodate,protease K and trypsin were used to modify the surface proteins of the bacteria and the mucin proteins of the intestinal tract of Rhynchocypris lagowskii,and the main properties of adhesion receptors on the surface lectin and intestinal mucin were analyzed.The results showed that: The adhesion receptors of LSG1-1,LSG2-3,LSG2-5,LSG2-8 and LSG3-6 in mucin were protein substances,while the adhesion receptors of LSG2-1,LSG3-7 and LSG3-8 in mucin were glycoprotein substances.Finally,the adhesion inhibition of 8 strains of potential probiotics against Aeromonas hydrophila and Aeromonas Vickers was investigated by means of competition,rejection and substitution tests.The results showed that:In the competitive test,LSG2-5,LSG2-8 and LSG3-6 hadsignificantly higher adhesion inhibition against Aeromonas hydrophila than other strains(P<0.05),while LSG1-1 and LSG2-1 had significantly higher adhesion inhibition against Aeromonas Vickers than other strains(P<0.05).In the rejection test,LSG2-5,LSG2-8 and LSG3-6 had significantly higher adhesion inhibition against Aeromonas hydrophila than other strains(P<0.05),and LSG3-8 and LSG2-8 had significantly higher adhesion inhibition against Aeromonas hydrophila than other strains(P<0.05).In the substitution test,LSG2-5,LSG2-8 and LSG3-6 had significantly higher adhesion inhibition against Aeromonas hydrophila than other strains(P<0.05),and LSG3-7 and LSG2-8 had significantly higher adhesion inhibition against Aeromonas Vickers than other strains(P<0.05).Finally,8 strains of Bacillus were screened out,all of which had good probiotics.Among them,LSG3-3(Bacillus tequilensis),LSG3-7(Bacillus aryabhattai),and LSG3-8(Bacillus mojavensis)were screened out in fish for the first time,showing good application prospects.This study provides a reference and scientific basis for the development and application of probiotics preparation of Rhynchocypris lagowskii. |
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