| In the advantage of promoting the growth and immunity, probiotics are widely used in aquaculture. Shrimp paste is fermented by shrimp or its in vivo microbial, the presence of various probiotics, such as Lactobacillus,yeast and bacillus. There is no report in screening probiotics which used for Penaeus monodon breeding from fermented shrimp, so the traditional shrimp paste used as material in this study, and we found candidate probiotics which have the supurior capability of enzyme producing and pathogenic antagonistic with the mathod of plate streaking and identified as Bacillus, then mixed the candidate probiotics with feedstuff to feed Penaeus monodon and determined the growth, digestive enzyme activity, non-specific immunity and impacts on water of the probiotic Bacillus.Firstly. the research isolate the bacteria from the traditional shrimp, through separation and purification, hemolytic activity, enzyme activity and antagonistic activity assay, results are as follows: the 306 acid-forming strains was isolated from Hainan Hui shrimp sauce and Haikou shrimp paste by the BCP medium. 36 of them were gamma hemolysis strains by the determination of hemolytic activity, that is 11.8% in total. Determined the enzyme activities which including protease, amylase and lipase activities and the pathogenic antagonistic which including seawater aquatic pathogens Vibrio Harveyi, Vibrio anguillarum, Vibrio alginolyticus and freshwater aquatic pathogenic bacteria Aeromonas hydrophila and Streptococcus agalactiae. In the 36 gamma hemolysis strains, it reflects prominent protease activity and amylase activity, A27, A3 5 and B47 were productive strains and significant difference with other strains (P < 0.05). It indicated supurior capability of enzyme activities and antagonistic of A20 strain, and significant difference (P < 0.05) with other strains in producing amylase and in antagonistic of 4 species of aquatic animal pathogens. It indicated supurior capability of antagonistic of B3 strain which antagonises 5 species of aquatic animal pathogens and 4 of them was significant difference (P < 0.05) with other strains. All, B15 and B89 reflects enzyme activities and antagonistic which includ one pathogenic bacteria antagonistic at least and significant difference (P <0.05) with other strains. So we identified the eight strains as Bacillus by 16S rRNA.Secendly, 8 bacillus strains were evaluated through tolerance experiment of bile salt, acid and simulated gastrointestine, the test of adhesion ability and drug sensitivity test, results are as follows: the A20 and A27 strain were dominant strains.All, A20, A27, B47 and B89 indicated supurior capability in tolerance experiment of acid, bile salt and simulated gastrointestine, the lag time of A20 and A27 strain was significant difference (P < 0.05) in the 60 min and 120 min in the experiment of strong acid, and it the same with the experiment of simulated gastric juice in 3-hour.The lag time of A20 strain was significant difference (P < 0.05) in the densities of 0.1%,0.3%, 0.5% in the experiment of bile salt. It was significant difference (P <0.05) with other strains in the experiment of simulated intestinal juice in 4-hour.The auto-aggregation ratio of A20 and A27 strain was significant (P < 0.05) superior then other strains in the experiment of adhesion test, moreover, the auto-aggregation ratio of A20 and A27 strain sustained in a high level and reached its highest and over then 98% in 24-hour, and the co-aggregation ratio of 5 species of aquatic animal pathogens was sustained higher level of 70% and significant difference (P < 0.05) in treament of A20 and A27 strain. The A20 and A27 strain was the most two sensitive strians in the experiment of antibiotic tolerance.Thirdly, Feeding the Penaeus monodon with feedstuff which mixed the living A20 and A27 strain with 2.5×108 CFU/g, evaluating the effects of two Bacillus strains on growth non-specific immunity and digestive enzyme activities in Penaeus monodon, Compare with control group, experimental group more remarkable with the A20 and A27 strain mixed separately feedstuff in feeding Penaeus monodon 6-week later,and details as follows: A, the chemical oxygen demand (COD),concentration of ammonia nitrogen and nitrite of the experimental group was significant rare (P < 0.05)in water, and A27 group was extremly significant difference (P < 0.01). The contents of toxics was less in the experimental group compared with control, and A27 strain reflect supurior capability of water purification. B, the body length, body weight,fatness, weight gain rate and specific growth rate of Penaeus monodon was more remarkable in the experimental group, the weight of the group A20 were significant higher than the control group (P <0.01) and weight gain rate and specific growth rate was significant higher (P < 0.05), the growth performance of group A20 was remarkable in comparing with group A27. C, the superoxide dismutase (SOD) and catalase (POD) was significant highly (P < 0.05) in the experimental group, and the acid phosphatase (ACP) was extremly significant difference (P < 0.01). The lysozyme(LSZ) of group A20 was significant highly(P < 0.05),and it supurior in the nonspecific index of group A20. D, the trypsin activity and amylase.activity in the experimental group were significant remarkable (P < 0.01).The selected gamma hemolysis Bacillus A20 and A27 strain could enhance the digestive enzyme activity, growth and nonspecific immunity of Penaeus monodon and purified water based on its supurior capability of tolerance, adhesiveness, antibiotic sensitive, enzyme activities, acid production and pathogenic antagonistic. the Bacillus strain A20 was significantly promote the growth, immunity and digestive enzyme activity of Penaeus monodon, and the Bacillus strain A27 was significantly improve water. |
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