The Composition And Enzyme-producing Function Of The Microflora In The Digestive Tract Of The Sea Cucumber Apostichopus Japonicus

In the present study，molecular biological technique was used to assess the thecomposition of the bacterial communities in the contents of the foregut and hindgut ofthe sea cucumber Apostichopus japonicus and in the ambient surface sediment, and theenzyme-producing function of the indigenous flora in the intestine of the sea cucumberwas surved by traditional culture method combined with molecular biologicaltechnique.The composition of the bacterial communities in the contents of the foregut andhindgut of the sea cucumber Apostichopus japonicus and in the ambient surfacesediment was surveyed by16S rDNA gene454-pyrosequencing. A total of188,623optimized reads and15,527operational taxonomic units (OTUs) were obtained from theten gut contents samples and four surface sediment samples. The sequences in thesediments, foregut contents, and hindgut contents were assigned to38.0±4.7,31.2±6.2and27.8±6.5phyla, respectively. The bacterial richness and Shannon diversity indexwere both higher in the ambient sediments than in the gut contents. Proteobacteria wasthe predominant phylum in both the gut contents and sediment samples. Thepredominant classes in the foregut, hindgut, and ambient sediment were Holophagaeand Gammaproteobacteria, Deltaproteobacteria and Gammaproteobacteria, andGammaproteobacteria and Deltaproteobacteria, respectively. The potential probiotics,including sequences related to Bacillus, lactic acid bacteria (Lactobacillus, Lactococcus,and Streptococcus) and Pseudomonas were detected in the gut of A. japonicus. Principlecomponent analysis and heatmap figure showed that the foregut, hindgut, and ambientsediment respectively harbored different characteristic bacterial communities. Selectivefeeding of A. japonicus may be the primary source of the different bacterial communities between the foregut contents and ambient sediments.The enzyme-producing microbes were isolated and assessed from the gut of A.japonicus, the study was made by traditional culture method combined with molecularbiological technique. A total of39strains were qualitatively screened on the basis oftheir extracelluar enzyme-producing anility. The39enzyme-producing strains wereclustered into8groups at the genetic similarity level of100%by analysis of therestriction patterns of amplified16S rDNA with MboI. Phylogenetic analysis shows that37strains belong to genus Bacillus and2are the members of genus Virgibacillus. Theenzyme-producing capability analysis results indicated that Bacillus is the mainenzyme-producing bacteria in the gut of A. japonicus, which can produce protease,amylase and cellulase, but Vigibacillus can only produce protease. The highenzyme-producing capability of the isolates suggested that the gut microbiota makegreat contributions to the digestive process of the sea cucumber A. japonicus, and theBacillus can be used as the candidate probiotics for A. japonicus aquaculture.The activities of protease, amylase and cellulase produced by isolatedenzyme-producing strains were determined. Bacteriostasis test was also carried out todetect the inhibition of39isolated enzyme-producing strains against the Vibrosplendidus by the filter paper method. The results showed that the activities of protease,amylase and cellulase produced by enzyme-producing microflora isolated from the gutof the sea cucumber were3.56-92.11U/mL、3.27-87.23U/mL and0.97-57.74U/mL,respectively. The IBSC2-8showed the highest protease activity（92.11U/mL）, thestrains IBSC2-8, IBSC2-6and IBSC2#-4showed the highest protease activity(92.11U/mL）, the highest amylase activity (87.23U/mL) and the highest cellulaseactivity (57.74U/mL), respectively. A total of10strains from39enzyme-producingstrains showed the ability to inhibit the growth of Vibro splendidus, and the IBSC2-10has the highest bacteriostasis effect (the inhibition zone diameter was37.6mm).