The Diversity Of Intestinal Microflora And The Bioactivities Of Culturable Bacteria Associated With Sea Cumumber Apostichopus Japonicus At The Feeding Stage

Sea cucumbers have high economic value and nutritional value.Sea cucumber Apostichopus japonicus in the Yellow Sea and Bohai Sea is of good quality.Sea cucumber is a benthic filter-feeding invertebrate.Due to its poor feeding ability and slow movement,the microorganism in sea cucumbers play a great role on the digestion,absorption,and immune defense of sea cucumbers.The composition and diversity of the intestinal microflora of sea cucumbers are closely related to the health of sea cucumbers and the breeding environment.The feeding amount of sea cucumbers varies greatly before and after the special period of summer dormancy.With the changes of feeding amount,the composition and diversity of the intestinal microflora of sea cucumbers will also change.This study is based on high-throughput sequencing technology to analyze the intestinal flora structure of pond cultured sea cucumbers at different feeding stages.At the same time,pure culture method is used to screen the enzyme-producing activity and antibacterial activity of the isolated strains.This study will provide the research foundation for the development and utilization of probiotics.Sea cucumbers Apostichopus japonicus were collected from the Weihai Nanhai New District,Shandong Province..The composition and diversity at the different feeding stages were studied by high-throughput sequencing.The pure culture method was used to isolate culturable bacteria from the intestines of sea cucumbers.The phylogenetic analysis of the isolated bacterial strains was determined by 16 S r RNA sequencing.The enzyme activities of the strains were determined,including protease,amylase,cellulase and alginase.The antimicrobial activities of the crude extracts of the fermentation products of the isolated bacteria were measured against seven indicator strains by the filter paper method.A total of 36 phyla of bacteria were detected in the intestinal samples of Apostichopus japonicus at different feeding stages,of which the Proteobacteria accounted for the highest proportion,followed by the Bacteroides and Actinomycetes.Among the 782 genera of bacteria detected,Halioglobus accounted for the highest proportion.The number of types of intestinal bacteria and the proportion of each type of bacteria have changed significantly during different feeding stages.A total of 27 strains of bacteria were isolated from the intestinal samples of sea cucumbers.16 S r RNA sequence analysis showed that Proteobacteria and Firmicutes were the majority of bacteria,among which Bacillus was the most.The results of enzyme activity screening showed that 6 strains of bacteria had the activity of producing 4 kinds of digestive enzymes,and the alginase activity of one strain(HSN-04)was significantly higher than that of other strains.The results of antibacterial activity showed that 27 bacterial strains had inhibitory effect on at least one indicator bacteria,and the strain HSJ-05 also had antibacterial activity against Vibrio anguillarum,Staphylococcus aureus and Bacillus subtilis.The results show that the changes of the intestinal flora of sea cucumbers are closely related to the feeding stages.The bacteria isolated from the intestines can promote digestion and absorption and have antimicrobial activities,which can have a good effect on the growth of sea cucumbers and adaption to the environment.