Diversity Analysis Of Intestinal Microbial Of Grass Carp And Bighead Carp And The Isolation And Identification Of Their Candidate Probiotics Bacteria

Due to the large-scale intensive aquaculture and the use of antibiotics and various drugs in the aquaculture industry,the intestinal flora of fish has been extensively disturbed.The abundance of intestinal flora of cultured fishes is significantly lower than that of natural living fishs.The diversity of intestinal flora in fish involves in its health.The investigation regarding the intestinal flora of fishes is for the overall understanding of the health status of cultured fish.In addition,the development and application of probiotics can reduce the use of antibiotics and drugs,which is conducive to the healthy and sustainable development of the aquaculture industry.In this study,grass carp and bighead carp were selected as the research objects that were derived from a natural pond in Conghua,Guangzhou,China.The diversity of intestinal flora of grass carp and bighead carp was studied using 16 S rRNA amplification sequencing technology,and the abundance and differences of dominant phyla,beneficial bacteria and potential pathogenic microorganisms in the intestinal tract were analyzed.Furthermore,the beneficial bacteria in intestinal tract grass carp and bighead carp were isolated,analyzed,and identified.In order to isolate the candidate probiotics with enzyme production as well as antibacterial function,the probiotic properties of candidate strains were evaluated by the indexes of hydrolase production and antibacterial function.The main results are as follows:(1)Based on the sequencing and diversity analysis of 16 S rRNA amplifiers of intestinal flora of grass carp and bighead carp,Proteobacteria,Firmicutes,and Bacteroidota were the dominant intestinal flora of grass carp.The dominant flora of bighead carp intestines are Firmicutes and Proteobacteria.At the class level,the intestinal flora of grass carp and bighead carp consisted of Gammaproteobacteria,Bacteroidia,Bacilli,Clostridia,Alphaproteobacteria,and Verrucomicrobiae,meanwhile the differences between grass carp group and bighead carp group was significant at all class levels(p<0.05).At the genus level,the beneficial bacteria in grass carp and bighead carp included Bacteroides,Bacillus,Cetobacterium,Prevotella,Lactococcus,Bifidobacterium,Lactiplantibacillus,and Lactiplantibacillus.However,the potential pathogens contained Kluyvera,Aeromonas,Escherichia＿Shigella,Arcobacter,Staplococcus,Fusobacterium,Paeniclostridium,Clostridium＿sensu＿stricto＿1,Legionella,Sarcina,and Luteolibacter.The results showed that the intestinal microbial diversity of grass carp was more abundant.(2)38 strains of Bacillus were isolated from the intestinal contents of grass carp and bighead carp by microbial plate method.32 strains produced cellulase,and all strains could produce protease and amylase.After the enzyme activity of quantitative analysis,determination of antibacterial ability,and strain adhesion ability related index determination,the strains named YB6,YB42,WB6,and WB16 were obtained.Indeed,these strains exhibited a high enzymatic activity on protease,amylase,and cellulose,and displayed a good antibacterial ability.In addition,they performed good properties in hydrophobicity,selfaggregation,and co-aggregation experiments,which indicated that they have potential abilities of developing probiotics for feeding.It was identified as Bacillus velezensis strain YB6,Bacillus siamensis strain YB42,Bacillus sp.strain WB6,and Bacillus velezensis strain WB16 by 16 S rRNA sequence alignment and phylogenetic tree analysis.(3)Six strains of Clostridium butyricum were isolated from bighead carp intestines by microbial plate method.The optimal strains YC5 and YC7 were obtained after the enzyme and acid production analysis.YC5 and YC7 produced butyric acid with yields of 9.66 m M and 5.23 m M,respectively.YC5 could produce acetic acid and amylase,YC7 could produce acetic acid and cellulase.The acetic acid yields of YC5 and YC7 were 17.73 m M and 14.54 m M,respectively.It was identified as Clostridun butyricum by 16 S rRNA sequence alignment and phylogenetic tree analysis,both of the two strains.In conclusion: In this study,four strains of bacillus with hydrolase producing and bacteriostatic functions were obtained by isolation and functional analysis,and they had good adhesion performance.At the same time,two strains of Clostridium butyricum producing acetic acid and butyricum had amylase and cellulase activities,respectively.The six candidate strains had the potential of probiotics.