The Fermentation Technique Of Four Strains Of Probiotics And It’s Effect In Apostichopus Japonicus Culture

This study focused on four strains of probiotics Aj080718lA15, Aj080719lA12,Aj080719lA16and Aj080718lA25, which were isolated from the intestine of healthy seacucumber Apostichopus japonicus. Based on the experimental study, the best fermentationformula and the fermentation conditions were determined for the four strains of beneficialbacteria. And the application effect of the probiotics in sea cucumber culture was accomplishedthrough a series of indoor experiments, mainly including the growth, immunity andmicroecological changes of sea cucumber’s intestine and culture environment. Then the safety ofthe probiotics was analyzed to common aquatic animals. On the basis of research work, twobeneficial probiotic products were established, including a single bacteria preparation andmulti bacteria preparation.The mainly research achievement of this thesis as below:1. The best fermentation conditions for the four strains of probiotics were determinedthrough the single factor experiment, while the optimal fermentation formula was studiedthrough the orthogonal experiment. As a result, a fermentation process in experimental scale wasestablished. These data included the followings: optimal fermentation time for32h, the bestfermentation temperature at28degree, the initial pH at7.5, inoculation amount for5%, rotationspeed at170rpm; The optimal fermentation formula for glucose2.5g/l, soybean meal powder15g/l, NaCl10g/l. In addition, the antagonistic action was founded between Aj080719lA12and other three strains.2. Based on the experimental application of the probiotics in sea cucumber culture, theinfluence of large scale and long term use of beneficial bacteria agents were evaluated in termsof growth, enzymatic activity, the microecological structure of intestine and the environment.The artificial infection using Vibrio splendidus was studied to understand the effect of usingprobiotics. Experimental results show that the single bacteria preparation and the multi bacteriapreparation could significantly improve the acid phosphatase (AKP), alkaline phosphatase (ACP)and superoxide dismutase (SOD) activity in sea cucumber (P <0.05), but no significant difference for Lysozyme vitality (LSZ); Observably improved the intestinal protease activity(P <0.05), the multi bacteria preparation could improve protease activity three times as comparedwith those fed a control diet, and could also improve sea cucumber growth speed, the highestspecific growth rate of the multi bacteria preparation almost2times to the control group; Certainconcentration of probiotics could significantly affect the microecological structure of intestineand the culture environment, in which the total number of heterotrophic bacteria increasedsignificantly, and much reduced the vibrio number; The artificial infection tests using Vibriosplendidus, illustrated that the relatively immune protective rate could reach75%.3. The safety of probiotics used for common marine aquaculture animals was evaluated. Theseanimals were sea cucumber, abalone, turbot, black fish, sea bass and shrimp. Soaking with singlestrain and multi bacteria agent for15d, the results showed that the safety concentration was106cfu/ml. It had no negative effect on growth, feeding and alive for the tested animals.This study determined the probiotic fermentation process in laboratory condition and willprovide the theory basis for large scale production. It also proved that the positive influence ofbeneficial bacteria for the growth, enzymatic immunity, disease resistance, protease vitality,microecological structure of intestinal and the culture environment. In addition, the safetyconcentration of probiotics was determined for the cultural application. These will provide abasic data and technical support for effective and safe use of probiotics in aquaculture. Inpractice, it has significance for widespread application of probiotics and healthy management insea cucumber culture.