Fermentation Optimization Of Bacillus Subtilis And Its Application In The Breeding Of Sea Cucumber

Apostichopus japonicus, also called sea cucumber, is one of the most economicaquaculture species. As its high edible nutrition and medical value, it is known as thefirst position of "eight treasure marine products ".In recent years, as the industrialaquaculture developing rapidly, diseases of sea cucumber become increasingly serious,which has greatly influenced the economic benefits. Proper use of antibiotics in seacucumber breeding could inhibit the happening of disease to a certain extent,extensive use of antibiotics can be commonly seen, thus appeared terrible negativeeffects. Adding probiotics in the fodder or aquaculture water instead of antibiotics hasbeen a new research direction. Bacillus subtilis is one of the most frequently usedprobiotics in aquaculture, its impacts include: water quality purification, secretingexogenous enzymes to promote the body’s digestion and absorption of nutrients,improving the activity of body’s non-specific immune enzyme, and improving themicro ecological environment of animal gut, etc. In this study, the liquid fermentationconditions of B. subtilis (YSFRI403) were optimized and its application effects in thebreeding of sea cucumber were analyzed.Part one: The optimization of culture medium and fermentation conditions of B.subtilis (YSFRI403).By single-factor test and multi-factor orthogonal experiment，the optimummedium and its fermentation condition of B. subtilis was studied. The optimummedium was: indian meal10g/L, potato starch10g/L, soybean powder15g/L,Na2HPO43g/L, NaH2PO43g/L,30.8mg/kg MnSO40.5g/L. The best fermentationcondition of B. subtilis was37℃, initial pH7.5, rotational speed140r/min, inoculateamount7%, and liquid volume20%. The number of active bacteria was significantlyincreased up to2.72×1011CFU/ml under the optimum condition.The fermentation raw material in this study is inexpensive, better in economic benefits, thus having a good application in industrial fermentation production.Part two: The application effects of B. subtilis in the breeding of sea cucumber.To detect the probiotic function to Apostichopus japonicus, the water quality,specific growth rate, activity of non-specific immune enzyme, digestive enzymes,intestinal flora and other parameters of sea cucumber was analyzed by biochemicalmethods after adding different quantity of B. subtilis. The results showed that addingB. subtilis could significantly reduce the nitrate and nitrite in the aquaculture water,and the highest removal rate of nitrate and nitrite was42.28%and22.33%respectively. The SGR of sea cucumber obviously improved and the activity ofnonspecific immune enzymes was higher than that of the control group significantly.However, the quantity of B. subtilis did not have much effect on ammonia nitrogenand COD content in water. When the content of B. subtilis was6ppm, the SGRincreased40.74%; the impact on the activity of amylase in sea cucumber organizationincreased30.27%; the activity of peroxidase, alkaline phosphatase, lysozyme andsuperoxide dismutase increased by135%、27%、396%、192%respectively. Theactivity of protease improved53.93%when the content of B. subtilis was5ppm.Adding B. subtilis in aquaculture water could also affect the quantity change of seacucumber intestinal flora, with the increase of adding amount of B. subtilis, thenumber of B. subtilis in sea cucumber intestine increased significantly (P <0.05), andthe number of heterotrophic bacteria decreased significantly (P <0.05), the best effectappeared when the added quantity is6-9ppm. This research suggests that addingappropriate content of B subtilis can improve the water quality of sea cucumber andthe activity of body’s digestive enzymes and nonspecific immune enzyme, so as topromote the growth of sea cucumber, which provides scientific basis for theapplication of the B subtilis.