Identification And Application Of Two Strains Of Potential Probiotics Associate With Aquaculture Of Sea Cucumber(Apostichopus Japonicus)

1The identification and safety inspection of two probiotics forApostichopus japonicusBy the means of morphological, physiological and biochemical tests, and16S rDNAsequence analysis, two bacterial strains （BC-1, BC-2） isolated from sea cucumberculture pond were identified. The bacterial strain BC-1and BC-2were identified asBacillus cereus. The effects of temperature, salinity and pH on growth of strains weretested. The results show that, The suitable growth temperature of the strain BC-1was18³9℃, and the optimum growth temperature was24℃; the suitable growthtemperature of the strain BC-2was18³9℃, the optimum growth temperature was27℃. The suitable growth salinity range of the strain BC-1was0³0; the suitablegrowth salinity range of the strain BC-2was0³5. The suitable growth pH valuerange of the strains BC-1and BC-2were5.5⁹.0. The optimum growth pH for BC-1was7.5and the BC-2was6.5. Water purification study found that content ofharmful materials in breeding wastewater, such as ammonia nitrogen and nitritenitrogen, was significantly reduced by addition of the two strains （p<0.05）.Meanwhile, COD level in water was also decreased. Safety of these two strains wasevaluated by muscle injection and extraction. Results showed that these strains had nosignificantly negative influence on Apostichopus japonicus growth under appropriateadditive concentrations. Therefore, they could be used as further research materialsfor breeding effect study.2Effects of addition of Bacillus cereus on the growth, digestive andnon-specific immune enzymes activities of Apostichopus japonicus Effects of Bacillus cereus on the growth, activities of digestive and non-specificimmune enzymes in Apostichopus japonicus were studied over56days. The additivedoses of living bacillus cells in feed were0,10⁷,10⁹and10¹¹cfu/kg, respectively.Results showed that the growth of A. japonicus enhanced significantly （p<0.05）,while the coefficient of variation in body weight decreased significantly （p<0.05）when B. cereus was added in feed at the dose of10⁷and10⁹cfu/kg. Intestinal proteaseactivity exhibited a positive correlation with Bacillus concentration in feed whileamylase activity showed a negative correlation （p<0.05）. The activities ofnon-specific immune enzymes were affected by various addtive doses of Bacillus infeed. With the increase of Bacillus concentration in feed, superoxide dismutase （SOD）activity in coelomic fluid of A japonicus enhanced significantly （p<0.05）. And theactivities of acid phosphatase （ACP）, alkaline phosphatase （AKP） and lysozyme （LSZ）in A japonicus when Bacillus was added in feed at the dose of10⁹cfu/kg significantlyenhance （p<0.05）. According to the results in the present study, it could be concludedthat the optimal additive concentration of B. cereus BC-2in feed was10⁹cfu/kg, atwhich the growth rate, activities of digestive enzyme and the capacity of non-specificimmunity in A. japonicus could be significantly improved.3Effects of a probiotic on the growth, environmental microorganism，digestive and non-specific immune enzymes activities of ApostichopusjaponicusTwo feeding ways of mixing with bait and splashing water out were chosen toevaluate the effect of a Bacillus cereus on Apostichopus japonicus growth, digestiveand non-specific immune enzymes activities and environmental microbial amount.The additive concentrations of living bacillus in two feeding ways were10⁷、10⁹、10¹¹cfu/Kg in mixture group and10⁶、10⁸、10¹⁰cfu/m³in splashing group. In thecontrol, basic bait was added. The whole experiment was finished in56days. Strangebacterium quantity in water was determined every two weeks. Growth ofApostichopus japonicas was measured both in the mid-stage and at terminated period. Activities of digestive enzymes in the digestion gut and non-specific immuneenzymes in coelomic fluid were assayed at the end of the experiment. Results were asfollows:（1） Strange bacterium quantity in splashing group kept lower lever comparedwith the control, while mixture group showed significant higher level （p<0.05）. Thedivergence narrowed with time going on until almost disappeared.（2）The growth ofApostichopus japonicus was promoted and the weight coefficient of variation wasdecreased obviously with bacterium BC-1concentration of10⁹cfu/Kg in mixturegroup and10⁶cfu/m³in splashing group.（3）When the additive concentrations of BC-1were10⁹or10¹¹cfu/Kg in mixture group, amylase and protease activities in intestinalwere enhanced prominently. The lipase and protease activities were higher undermixing feed concentration of10⁷cfu/Kg and splashing concentration of10⁸cfu/m³compared with the control （p<0.05）.（4）Under the two feed ways, bacterium BC-1showed obvious stimulative effect on immune enzymes activities in coelom. Themixture group with bacterium concentrations of10⁹、10¹¹cfu/Kg exhibited higherSOD、AKP、ACP and lysozyme activities than that in the control. When the splashingconcentration was10¹⁰cfu/m³, except SOD, other immune enzymes were higher thanthat in the control. Considering all the results above, it could be conclude that thegrowth rate, digestive and non-specific immune enzymes activities could be improvedsignificantly when the mixing concentration was10⁹cfu/Kg and splashingconcentration was10⁸cfu/m³.