Preparation Of Microcapsules Of Bacillus Subtilis And B. Cereus And Effects On Immunological Function Of Chinese Mitten Crab Eriocheir Sinensis

The results of the preparation technology of alginate microcapsules of Bacillus subtilis and B. cereus and theirs effects on immunological function of Chinese mitten crab Eriocheir sinensis are first reported in the paper. The results have important reference value to study on the preparation form of feed probiotics of aquatic animals and immunostimulants of E. sinensis. The experiment is composing of two parts as follows:Experiment one: The sodium alginate which has good biology consistent was used as shell-material and the B. subtilis or B. cereus was used as core-material, then preparing microcapsulation with emulsification method. The optimal technological conditions of making the microcapsule were determined through single factor test and orthogonal experiment of L9 (34). The results were as follows: the concentration of sodium alginate was 2%, the emulsification time was 15 min, the stirring speed was 600 r/min, the proportion of B. subtilis or B. cereus and sodium alginate was 1:6. This method can prepare microcapsules which have good enteric solubility and acid resistance, embedding rate was 59.91% and 58.33%, respectively.Experiment two: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of alginate microcapsules of living B. subtilis, B. cereus, B. subtilis and B. cereus (1:1) (about 2.2×1011 cfu/g powder) dosed at 0, 1 or 5 g/kg dry diet for 1 week, respectively. The total haemocyte count (THC), different haemocyte count (DHC), respiratory burst (release of superoxide anion) of haemocytes, the simulated PO activity by Bacillus (POS) and total phenoloxidase (POT) in haemocyte lysate supernatant (HLS), phenoloxidase (PO), peroxidase (POD), lysozyme (LSZ), acid phosphatase (ACP), alkaline phosphatase (ALP) activities in serum were examined at 0,1,2,3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results indicated that experimental groups of feeding microcapsules of Bacillus showed all higher haemocyte count (THC), hyaline cells (HC), higher respiratory burst (release of superoxide anion) of haemocytes, while boosted up POS and POT activities in haemocyte lysate supernatant (HLS), phenoloxidase (PO), peroxidase (POD), lysozyme (LSZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in serum. The activities of these immune parameters of test groups were significantly higher than control group at many sampling time points (P<0.05). The stimulate effects of the 5 g/kg group is more superiority than the 1 g/kg group at many sampling time points. Crab of test groups were the more resistant. It was concluded that E. sinensis that were fed with diets concluding microcapsules of Bacillus showed increased immune ability as well as resistance to A. hydrophila infection.