Studies On Selection Of Probiotics From The Misgurnus Anguillicaudatus And Their Application

In this study, we investigated the bacterial diversity of the intestine from juvenile and adult loach( Misgurnus anguillicaudatus). A strain of potential probiotic Bacillus subtilis was screened in vitro based on isolates from the intestinal flora of loach, being able to produce a variety of enzymes and having strong antagonistic activity against pathogen. Effects of dietary Bacillus subtilis on growth performance, digestive enzymes and nonspecific immune of juvenile loach were evaliated to provide a reference for its application in formulated diet of loach. The main results are as follows:First, high throughput sequencing was used to analyze and compare the bacterial community in the intestine of juvenile and adult wild loach. Genomic DNA was extracted from intestine samples of domesticated juvenile fish, wild juvenile fish and wild adult fish. and a gene segment from the V3-V4 portion of the 16 S rRNA gene was amplified and sequenced using an Illumina Miseq sequencer.In phylum level, Proteobacteria was the most dominant in the intestine of domesticated and wild juvenile loach, accounting for 91.3% and 94.4% respectively, followed by Firmicutes accounting for 8.5% and 4.0%, respectively. Proteobacteria was the most dominant in the intestine of the wild adult loach accounting for 79.6%, followed by Fusobacteria accounting for 18.4%. In genus level, there were fourteen genera bacteria presented in the intestine of domesticated juvenile, Aeromonas and Acinetobacter accounted for 84.4%. Seventeen genera bacteria presented in the intestine of wild juvenile, Aeromonas and Acinetobacter accounted for 90.2%. While twenty-four genera bacteria prensented, Aeromonas, Acinetobacter and Cetobacterium accounted for 89.3%.Second, ninety-three bacterial strains isolated from the intestine of juvenile and adult loach were tested in vitro for extracellular protease, amylase, and lipase produced and antibacterial activity against common pathogen of aquatic animals(Aeromonas hydrophila, Aeromonas salraonicida, Vibrio parahaemolyticus and Shewanella putrefaciens). Six strains producing three enzymes and two strains producing antagonistic activities were screened. Hemolytic assays showed that strains N1-08, N2-08 and N2-26 did not secreted hemolysin. Strain N1-08 both producing antagonistic and enzyme activity was selected to safety test, evaluated by intraperitoneal injection of 200 μl bacterial suspension into juvenile loach. Results showed that strain N1-08 was harmless to M.anguillicaudatus. The 16 S rRNA sequence analysis indicated that strain N1-08 comprised sequences more related to Bacillus subtilis.Third, the effects of dietary potential probiotics N1-08 on growth, digestive enzymes and nonspecific immune of the juvenile loach was evaluated. Fish in control group(C) was fed the basal diet, those in treatment groups were fed diets containing strain N1-08 at 107 cells/g(Ⅰ) and 109 cells/g(Ⅱ), respectively. The results showed that there were no significant differences in growth performance of fish between treatment groups and control(P<0.05). Strain N1-08 enhanced the hepatopancreas and intestinal digestive enzymes activities(trypsin activity, amylase activity and lipase activity) and the nonspecific immune activity(serum lysozyme activity, and serum superoxide dismutase activity) of the juvenile loach.