| ObjectiveWe had selected the potential probiotic Bacillus which have effect on immune enzymes and digestive enzymes of sea cucumber Apostichopus japonicus through preliminary identification and screening, and constructed complex-probiotic which can improve sea cucumber immunity and digestion, In order to provide theoretical basis for the sea cucumber aquaculture development.MethodsBy the methods of spread plate method and streaked plate,we isolated probiotics strains from the intestine gut of sea cucumber and aquaculture ponds, At the same time,we identificated the strains through teh dyeing, morphological featured and 16 S r DNA sequence analysis. determined the optimal growth conditions of the stains by spectrophotometer method.The safety test of five isolates showed that no probable pathogenic for sea cucumber in the acute toxic test.Fresh bacterial cells of five stains were fed to sea cucumbers, We determined the enzyme activity of acid phosphatase(ACP),alkaline phosphatase(AKP) and total superoxide dismutase(T-SOD), phenol oxidase activity(PO) of coelomic fluid and intestinal protease,amylase and lipase activity in sea cucumber, in order to screened the strains which can stimulate on sea cucumber.Used the orthogonal experiment to explored the better proportion of composite strains.Resultswe isolated five probiotics strains from the intestine gut of sea cucumber and sediments of aquaculture ponds from liaoning surrounding, and numbered B1,B2,B4,B6 and B7. we determined thefive strains were bacillus genus through observing the colony morphology, physiological and biochemical characteristics,glen dyeing and 16 Sr DNA homologya nalysis. B1 belonged to Bacillus Vallismortis, B2 belonged to Bacillus subtilis, B4 belonged to Bacillus sp., B6 belonged to Bacillus methylotrophicus, B7 belonged to Bacillus aryabhattai.The rusult of characteristics of growth and enzyme production showed that the optimal growth tempreture of B1,B2 was 20~35℃,The B6 and B7 strain were growing well at temperature in the 20~35℃. Five strains could grow at a wide range of p H,B1,B4 and B6 stain could grow well at p H in the 6.0~9.0, the optimal p H of B2,B7 was 6.0~8.0. The optimal growth salinity of B1,B4 and B7 range from 3% to 7.5%,salinity is within the scope of 3%~5%,the B2 and B6 could grow well.Enzyme production characteristics analysis results showed that the 5 strains all can product protease, amylase. As well as B1 and B7 can product lipase.Through the acute toxicity test on sea cucumber,which concluded the five stains were safe at the final concentration of five strains under 1500mg/L.Feeding different strains to sea cucumbers in order to investigate the stimulating effect of the strains on sea cucumbers. We could draw a conclusion that B1, B2, B4, B6 and B7 all have significantly positive stimulation on acid phosphatase(ACP), alkaline phosphatase(AKP), total superoxide dismutase(T-SOD) and phenol oxidase(PO) in coelomic fluid of sea cucumber.The adding amount of 0.1% and 0.2% of B1 strains significantly increased the activity of acid phosphatase(ACP), alkaline phosphatase(AKP), total superoxide dismutase(T-SOD) and phenol oxidase(PO)(p<0.05), but 0.3% B1 strains had extremely significant effect only on AKP and PO activity(p < 0.01); compared to control group, B2 had significantly effect on ACP, AKP, SOD and PO activity under the condition of adding dose of 0.3%(p<0.05); after 7 days, The B4,B6 and B7 strains had significantly higher ACP, AKP, SOD and PO activity than control group under the feeding ammount of 0.2%(p<0.05).By observaing index of digestive enzymes in the gut of sea cucumber B1,B2,B4,B6 and B7 can remarkably improve the activity of sea cucumber gut protein activity(p<0.05), especially B6(p<0.01). Protease activity is control group 10 times fed with 0.1% of B6 strains.B4,B2 and B6 strains can significantly improve the activity of amylase, B1,B7 had no significant stimulation on amylase activity of sea cucumber gut(p﹥0.05). B1, B4, B6 and B7 significantly improved intestinal lipase activity(p < 0.05), B2 had no significant effect on lipase activity(p﹥0.05). Comprehensive from immune enzymes and the digestive enzymes results, to obtain the five strains all had positive stimulation, the suitable additions of B1 is 0.1% of the sea cucunmbers’ weight,B2 is 0.3%,and B4,B6 and B7 is 0.2%.According to the results of feeding experiment, we selected five factors 4 levels orthogonal experiment. Using process analysis, comprehensive balance method to analysis the orthogonal experiment results, we determined the best proportion of 5 strains was : A4B2C1D3E4, The B1 strain at dose of 0.09 g, B2 add a dose of 0.09 g, B4 add a dose of 0.03 g, B6 strain is 0.03 g, the end of B7 content is 0.06 g.ConclusionsThe five stains were safe and had no toxic effect on sea cucumber at the final concentration of five strains under 1500mg/L. And all have significantly positive stimulation on immune enzymes and the digestive enzymes activities.The better additions of B1 is 0.1% of the sea cucunmbers’ weight,B2 is 0.3%,and B4,B6 and B7 is 0.2%.According to the results of orthogonal experiment, we obtained the best proportion of 5 strains was : A4B2C1D3E4, The B1 strain at dose of 0.09 g, B2 was 0.09 g, B4 was 0.03 g, B6 strain was 0.03 g, the end of B7 was 0.06 g.Key words... |
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