Studies On Intestinal Flora Of Monopterus Albus And Application Of Probiotics

In order to improve the utilization rate of the swamp eels to the compound feed and realize the replacement of wild ice fish with compound feed of swamp eels.This study explored the intestinal microflora of swamp eel in different states and the effects of compound probiotics on the growth performance and intestinal microflora of swamp eels.The main results are as following:1.Study on the structure of intestinal flora of swamp eels under different statesBy sampling swamp eels with different nutritional status(different fat sources,different protein levels),health status(healthy and sick)and gender.Analyzing the composition differences of the intestinal microflora of swamp eels under different status via high-throughput sequencing of bacterial 16 Sr DNA.The results showed that the core flora in the intestines of swamp eels was similar in structure at the phylum level,and the dominant phyla were Proteobacteria,Firmicutes,Fusobacteria and Bacteroidetes.At the genus level,the core intestinal flora of swamp eels are dominated by the Desulfovibrionaceae,Clostridium＿Sensu＿Stricto＿1,Cetobacterium,Aeromonas and Plesiomonas,and changes with the state of swamp eels.Cetobacterium were the dominant differential bacteria in the suitable protein group,healthy intestinal mucosa group and male eel group,suggesting that it was the core genus of intestinal tract of healthy swamp eels.Conclusion:(1)The intestinal microflora of swamp eels was significantly different under different conditions.(2)Cetobacterium were the core bacteria in the intestinal tract of healthy swamp eels.2.Isolation and screening of probioticsThe purpose of this experiment is to isolate and screen Bacillus which can produce a variety of digestive enzymes with strong activity and Lactobacillus with good performance of producing lactic acid.Selective medium was used to isolate and screen Lactobacillus and Bacillus.A total of 25 strains of Lactobacillus were screened.Among them,Lactobacillus plantarum L3,isolated from the intestinal contents of grass carp,had the strongest acid-producing capacity.A total of 10 strains of Bacillus were screened,all of which could produce amylase and protease.Among them,Bacillus subtilis MZ can also produce cellulase and has antibacterial effect on Aeromonas veronii AV-S2.The safety test results showed that both Lactobacillus plantarum L3 and Bacillus subtilis MZ were non-hemolytic and pathogenic,and could be used as candidate probiotics in subsequent trials.3.Effect evaluation of compound probiotics fermentation feedIn this experiment,three strains of Bacillus subtilis MZ,Lactobacillus plantarum L3 and Saccharomyces cereviscera J1 were used as mixed fermentation feed.There were 16 groups in the experiment,three in parallel in each group.The orthogonal experiment was designed for the three kinds of microorganism with basically the same number of viable bacteria in accordance with 3 factors and 4 levels.After the fermentation,the crude protein and crude fiber indexes were determined to determine the best ratio.Results:when the feed crude protein and crude fiber were used as the screening index,the optimum inoculation ratio of compound strains of bacillus subtilis MZ: Saccharomyces cereviscera J1: Lactobacillus plantarum L3 = 2:1:1.With the proportion of 2:1:1,the contents of crude protein,small peptide and total acid were significantly increased(p<0.05),and the contents of crude fiber,ether extract and ash were significantly decreased(p <0.05).4.Effects of compound probiotics on growth performance of swamp eelIn order to explore the effective use of compound probiotics in the production of swamp eels,direct spraying feed and fermented feed were used to evaluate the effects of feeding on the growth performance of swamp eels.A total of 450 swamp eels were randomly divided into 6 groups with 3 replicates per group and 25 eels per parallel group.The first 5 groups were treated with different proportion(0%、25%、50%、75%、100%)of fermented feed to replace basal feed,and the 6 group was treated with the compound probiotics sprayed with basal feed as bacterial liquid spraying group,which were labeled as F0,F25,F50,F75,F100 and MS,in turn.Swamp eels were fed for 60 days.The results showed that the direct spraying of compound probiotics and partly replace fermentation feed can promote the growth of swamp eel,improve eel liver,stomach and intestinal structure,improve the intestinal digestive enzyme activity and antioxidant capacity.However,the replacement of excessive fermented feed will slow down the growth of swamp eels,damage the tissue structure of the liver and gastrointestinal tract,and influence the activity of digestive enzymes.The suitable substitution ratio of fermented feed was 34.92～35.29%.5.Influence of compound probiotics on intestinal flora of swamp eelsBased on the experiment of compound probiotics culture,18 samples were randomly selected from F0,F25,F100 and MS groups,and their intestinal contents were collected.High-throughput sequencing of bacterial 16 Sr DNA was conducted to analyze and compare the intestinal flora of the four groups.The results showed that the composition of intestinal flora of each group at the taxonomic level of phyla,genera and species and PLS-DA analysis indicated that compound probiotics could change the structure of intestinal flora of swamp eels.The effects of the compound probiotics on the level of phyla were as follows: the abundance of Proteobacteria decreased and the abundance of Firmicutes increased.At the genera level,there were significant differences between the groups of Burkholderia-Caballeronia-Paraburkholderia,Chlamydiaceae and Staphylococcus(p<0.05).At the species level,there were extremely significant differences between the groups of Lactobacillus plantarum,Rhodococcus erythropolis,Ralstonia Picketti and Staphylococcus kloosil(p<0.01).The abundance of some functional bacteria in the intestinal microflora of swamp eels(such as Rhodococcus erythropolis and Ralstonia Picketti)can be increased,and the abundance of potential pathogenic bacteria such as Staphylococcus kloosil decreased,so as to improve the microflora structure when 25% fermented feed was used instead of basal feed.Spraying feed with live bacteria can make the exogenous beneficial Lactobacillus plantarum and Bacillus subtilis were detected in the intestinal flora of swamp eels.However,100%fermented feed may cause excessive exogenous Lactobacillus plantarum to enter the intestines of swamp eels,causing imbalance of intestinal flora of swamp eels.