To Select Strains With Higher Enzyme Activity From The Distinguishment Of A. Japonicus And Its Application In Feed

Bacillus amyloliquefaciens X3-3, Bacillus methylotrophicus X4-1 and Bacillus megaterium X4-5 were selected from A. Japonicus intestinal using Gram staining, phenotypic properties of norphology, physiology and biochemical and 16S rDNA identification. Two of these, Bacillus amyloliquefaciens X3-3 and Bacillus methylotrophicus X4-1 with higher digestive enzymes. Bacillus megaterium X4-5 can purify water obviously. The results showed that Bacillus imyloliquefaciens X3-3 with the best growth conditions at 34°C-37°C, 8.0 pH and 30 salinity and Bacillus methylotrophicus X4-1 with best growth conditions and optimum conditions of producing digestive enzyme at 37°C, 7.0-8.0 pH and 30 salinity by some single factor experiments. In the respect of water quality of aquaculture, ammonia density in water with ndophenol blue photometric method and the nitrite content of which was determined by N-(l-naphthyl)-ethylenediamine dihydrochloride spectrophotometric method. The result show hat Bacillus megaterium X4-5 has potential application value in improving water quality of aquaculture.Several security indicators could be used as the basis for choosing the three strains as a beneficial bacteria, such as, the effects of strains adhering to cells in a Stichopus japonica gut, he acute toxicity to zebra fish and antagonistic ability of the different pathogenic bacteria. The esults of toxicity studies using zebra fish as laboratory animal model have shown that the surviving rate had not obvious difference from that in control group (P>0.05) and all survival ates were higher than 90%. The adhere rate of this three strains on sea cucumber digestive cells was higher than 85%. And the adhere rate of this three strains on sea cucumber digestive cells was prerequisites for bacterial colonization. In the respect of regulating balance of intestinal nicroflora of stichopus japonicus, all these strains were tested to have strong antagonism to )athogens, such as pseudomonas and Vibrio parahaemolyticus.The results showed that the activity of digestive and SGR values of the trial echinoders ncreased obviously (P<0.05) and reached new heights within non-distinctive differences (P> 0.05) from 30d to 60d due to intake of single strains microbial preparation of Bacillus imyloliquefaciens X3-3 or 2 in 1 compound bacteria preparation of Bacillus amyloliquefaciens X3-3 and Bacillus methylotrophicus X4-1 or Bacillus megaterium X4-5, that the activities of ease alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), superoxide dismutas (SOD) and peroxidase (POD) which involve in the animal humoral immune effectors raised apparently after feeding diets acquired with compound agent made by Bacillus amyloliguefaciens X3-3 and Bacillus methylotrophicus X4-1 and Bacillus megaterium X4-5 was superior to that of two bacterial strains, and all of this compared with single bacterial strain only except Bacillus amyloliquefaciens X3-3, the difference was significant(P<0.05).The results ndicated that this living bacteria composed of Bacillus amyloliquefaciens X3-3, Bacillus methylotrophicus X4-1 and Bacillus megaterium X4-5 could play a good role for improvement ofnonspecific immunity and growth promoting effects on Apostichopus japonicus and was suitable as feedstuff additive.It has great value of development and application.