Research On The Preparation Of Immobilized Enzyme And Its Application In Sea Cucumber Bait

This thesis focuses on the application research of improving sea cucumber breeding baits digestion and absorption by adopting enzyme engineering technology. At present, the digestive enzymes are widespread applied in the animal feed, which can promote digestion and absorption of nutrients, improve the growth rate and feed conversion rate of animals, and improve the animal health, and it also can cut down nitrogen and phosphate emissions of breeding, and protect breeding ecological environment. Therefore, the feeding enzyme preparation plays dual role in increasing economic benefits and protect the ecological environment in the modern aquaculture industry, and the relevant research on digestive enzyme is more and more concerned by people.In this work, based on the growing environment characteristics of sea cucumber, the main digestive enzymesα-amylase and acidic pepsin were embedding immobilized, which exists in sea cucumber intestinal canal as research object, and then them were used to improve the digestive absorption of sea cucumber baits.Their enzymology character and immobilized condition optimizing are studied, and then, the related properties of immobilized enzyme and its application on sea cucumber feed are researched. The main research contents and conclusions are as follows:(1) Free enzymology character study. It involves the optimum reaction temperature and pH. Results show that: when the fungusα-amylase liquid is diluted 1,000 times, the optimum value of temperature and pH for reaction are 60℃and 5.3. When the acidic pepsin powder is diluted 1,500 times, the optimum value of temperature and pH for reaction are 58℃and 2.6.(2) Immobilized preparation conditions optimization research. Through single factor and orthogonal test, the influencing factors of immobilized preparation conditions is discussed, such as sodium alginate concentration, calcium chloride concentration, plus enzyme quantity, immobilized time. Then, the optimal preparation process is obtained. The results of study proved the activity of the immobilizedα-amylase is the highest when the related parameters are as follows: 2.5% sodium alginate, 15% enzyme amount, 2% CaCl₂ and immobilized time 1 h. And the activity of immobilized acidic stomach protease is the best when the related parameters are as follows: 2.5% sodium alginate, 2.5% CaCl₂, 150% enzyme amount and immobilized time 1h. (3) The related properties of immobilizedα-amylase are studied. The results show that: the optimum value of temperature and pH for reaction are 58℃and 5.4, the continuous operating stability of immobilized enzyme is better, the optimum value of mechanical strength is when the size of gel diameter between 2.0 to 2.5 mm, and the optimum serving temperature is 4℃.(4) Research on the application of immobilized enzyme for hydrolyzing sea cucumber feed. The effects of hydrolysis time, enzymatic hydrolysis temperature, immobilized enzyme additive amount on baits zymolysing rate are studied. It turned out that: the optimum value ofα-amylase hydrolysis time, immobilized enzyme additive amount and enzymatic hydrolysis temperature are between 25 to 30 min, 40mg/ml, and 30℃.The optimum concentration of feeding is 5%. And the optimum value of acid pepsin hydrolysis time, immobilized enzyme additive amount and enzymatic hydrolysis temperature are between 25 to 30min, 20mg/g, and 30℃. At this time, its continuous operation is better.