【作者】 叶林超；

【导师】 叶均安； 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 硕士

【摘要】 本研究旨在研制一种能促进缢蛏生长并能降低缢蛏养殖环境污染的蛋白酶解物。首先筛选出最佳的酶解条件、酶解物形式,确定最佳的无机氮源形式以及最佳氮、磷源添加浓度。然后以缢蛏的一种饵料来源——小球藻为研究对象,通过细胞密度、叶绿素、蛋白质含量等生长生化指标,研究蛋白酶解物对小球藻生长的影响,并与多种动植物蛋白的酶解物进行比较。并拆分蛋白酶解物的组成成分,深入研究其作用机理。最后应用到缢蛏养殖,通过壳长、个体重、养殖塘底泥有机物含量、氮含量等指标,研究蛋白酶解物对缢蛏生长及养殖环境的影响。试验一、首先以普通豆粕为研究对象,通过对酶解时间、液固比等条件的摸索,筛选出最佳的酶解条件。试验表明,最佳酶解条件为：液固比15：1,酶解时间为8h,温度为50℃,pH为7.2,酶与底物浓度比为6：1(mg／g),此时,水解度和三氯乙酸溶解度分别达到最大值11.68%和26.96%。酶解时间超过8h或者液固比超过15：1,普通豆粕酶解物的水解程度明显减小。然后通过酶解蛋白酶解物、豆粕、棉粕、菜粕和花生粕等多种蛋白酶解物,以水解度、三氯乙酸溶解度和悬浮性等指标,筛选出最佳的蛋白酶解物。研究结果表明,蛋白酶解物在人工海水中的有机物沉降比例最小(21.58%),显著低于鱼浆等其它各蛋白饲料酶解物(p<0.05),表明其在海水中的悬浮性最好。蛋白酶解物的水解程度显著大于其它各蛋白饲料酶解物(p<0.05),表明其酶解后的产物更充分,更能促进海洋生物吸收和利用；试验结果还表明,棉粕酶解后的悬浮性和酶解效果相对其它植物粕类蛋白的酶解物更佳。试验二、首先,通过细胞密度、光密度值、干重、叶绿素含量以及可溶性蛋白含量等生长生化指标,对碳酸氢铵、硫酸铵、硝酸钠、尿素、氯化铵和磷酸二氢铵等不同氮源的筛选,选出小球藻生长最佳的氮源。结果表明,碳酸氢铵可更好地促进小球藻生长(p<0.01),硝酸钠和尿素次之,硫酸铵、氯化铵和磷酸二氢铵的效果不显著。然后以磷酸二氢钠为磷源,通过正交组合,研究不同浓度的氮和N／P比对小球藻生长的影响,确定最佳的N、P源组合。在培养液中添加N质量浓度分别为0、50、100mg/l的碳酸氢铵和P质量浓度为O、3、6mg/l的磷酸二氢钠,进行双因素正交试验。结果显示,添加N50mg/l、P3mg／l时小球藻的细胞密度、光密度值(OD)和单位体积叶绿素等生长指标均显著高于其它各处理组(P<0.05)。然后缩小浓度梯度进一步试验,分别添加N浓度为40、50、60mg／l和P浓度为2、3、4mg／l,结果表明,NH4HCO3作为N源,NaH2PO4作为P源时,小球藻生长的最适N、P浓度分别为50mg／l、3mg／l,N／P比为16.7。最后再与蛋白酶解物进行3x3正交试验,筛选出对小球藻生长最佳的氮、磷源与蛋白酶解物之间的组合。结果表明,同时添加N50mg／l、P4mg／l、蛋白酶解物12ml／L时,能显著促进小球藻生长、提高叶绿色和藻体蛋白质的合成(P<0.05)。试验三、通过分解蛋白酶解物的组成成分(酶制剂和微量元素制剂),采用单因素试验设计,分别对各种成分的酶解物进行分析,以细胞密度、光密度值、干重、叶绿素含量以及可溶性蛋白含量等指标,研究蛋白酶解物对小球藻生长的影响机理。结果表明,培养液中同时添加动物蛋白加酶与微量元素的降解物以及N、P源对小球藻生长效果要明显好于没有添加N、P源组(P<0.01),说明无机N、P的添加对小球藻的生长有较好的促进作用；结果还表明,添加微量元素组或酶制剂组相对对照组均能显著促进藻类生长(P<0.01),但添加蛋白酶解物对小球藻的生长效果是最佳的,细胞密度、叶绿素水平,蛋白质含量都相对最高(P＜0.05)。试验四、以缢蛏为研究对象,在各试验塘分别添加蛋白酶解物、鱼浆和鸡粪进行比较,以缢蛏的壳长、个体重、总重以及塘底泥的含氮量、有机物含量等指标来反应不同添加物对缢蛏生长性能和养殖环境的影响,同时分析添加蛋白酶解物后的养殖经济效益。研究结果表明,添加蛋白酶解物的各个处理组缢蛏平均总重达到18.5千克,分别比鸡粪组高3.1%,比鱼浆组高8.8%。从经济效益上看,蛋白酶解物组的缢蛏每500克单价达到7.5-8.0元,比鸡粪组的平均单价高出了33.3%-66.7%。从对环境的影响看,鸡粪组和鱼浆组有机物含量分别比蛋白酶解物组高28.5%和9.8%；氮含量分别比蛋白酶解物组高94.6%和16.1%。由此可见,使用蛋白酶解物可显著促进缢蛏的快速生长,增加经济收益,并能降低蛏涂及养殖环境的污染。更多还原

【Abstract】 This study was conducted to develop a kind of animal protein enzymatic hydrolysis that could improve growth of Sinonovacula constricta, and reduce the pollution of culture environment. First, the best enzymatic hydrolyzed condition and best form of enzymatic hydrolysis were selected. The best form of inorganic nitrogen and concentration of nitrogen and phosphorus sources were definited. Then the effects of animal protein enzymatic hydrolysis on growth of Chlorella vulgaris were studied, by parameters such as cell density、chlorophyll content and protein content. The reaction mechanism was also studied. Then the animal protein enzymatic hydrolysis was used in culture of Sinonovacula constricta to study the effects on its growth and environment by the index of carapace length、body weight、nitrogen content and organic matter content of soil from the bottom pond.In Expt.1, the best enzymatic hydrolyzed condition of soybean was selected by indicators such as hydrolyzed time、ratio of liquid and solid. Results showed that the degree of hydrolyzed reached 11.68%, and trichloroacetic acid degree of dissolved reached 26.96%, when the ratio (15:1)、hydrolyzed time(8h)、temperature(50℃)、pH(7.2)。Then the best form of enzymatic hydrolysis was selected by indicators such as degree of hydrolyzed、trichloroacetic acid degree of dissolved and suspension quality from five kinds of hydrolysis such as soybean、cotton、vegetable、peanuts dregs and animal protein enzymatic hydrolysis. Results showed that the suspension quality in sea water of animal protein enzymatic hydrolysis was smallest (p< 0.05),and its degree of hydrolyzed was better than any other hydrolysis. It could promote growth of marine life significantly (p<0.01)In Expt.2, the best kind of nitrogen resource for growth of Chlorella was selected by indicators of cell density、light density、dry matter、chlorophyll content and protein content from six different. nitrogen sources such as NH4HCO3, (NH4)2SO4, NaNO3, NH2CONH2、NH4Cl, and NH4H2PO4.. Results showed that NH4HCO3 could promote growth of Chlorella significantly compared with the other nitrogen sources (p< 0.01).Then the best combination of nitrogen and phosphorus sources was established by orthogonal design(2x2) adding 0、50、100mg/l NH4HCO3 and 0、3、6mg/l NaH2PO4. Results showed that the group (N50mg/l、P3mg/l) was best for growth of Chlorella. Then shorten the concentration gradient,and added 40、50、60mg/l NH4HCO3 and 2、3、4mg/l NaH2PO4. Results showed that the best combination was N50mg/l、P3mg/l. The ratio N/P was 16.7. Then added animal protein enzymatic hydrolysis to design a orthogonal experiment(3x3). Results showed that the best combination to promote growth and improve synthesis protein significantly was N (50mg/l)、P (4mg/l)、animal protein enzymatic hydrolysis (12ml/L) (P<0.05)In Expt.3, the mechanism of animal protein enzymatic hydrolysis effecting growth of Chlorella was studied by analyzing component of animal protein enzymatic hydrolysis by indicators of cell density、light density、dry matter、chlorophyll content and protein content. Results showed that adding nitrogen and phosphorus sources could improve growth of Chlorella significantly than not added (P<0.01).Results also showed that trace elements group and enzyme group had obviously effects on growth of Chlorella compared with the control, and adding them together was best to improve biomass、chlorophyll content and protein content of Chlorella significantly (P<0.05).In Expt.4, effects of animal protein enzymatic hydrolysis on the growth and culture environment of Sinonovacula constricta were studied compared with fish starch and chicken excrement, by indicators such as length of shell、height of body、nitrogen concentration and organism content of bottom pond. Results showed that the mean weight of Sinonovacula constricta of animal protein enzymatic hydrolysis group reached 18.5kg, more than chicken excrement group (3.1%), fish starch group(8.8%) respectively. The price of Sinonovacula constricta reached 7.5-8.0 rmb/kg from animal protein enzymatic hydrolysis group which higher than chicken excrement group by degree 33.3-66.7%. Results also showed that the organic matter concentrations of soil from bottom pond of chicken excrement group and fish starch group were higher than animal protein enzymatic hydrolysis group by degree 28.5% and 9.8% respectively, while nitrogen concentration higher by degree 94.6% and 16.1% respectively. In conclusion, animal protein enzymatic hydrolysis could improve growth of Sinonovacula constricta、increase financial gain and decrease the pollution significantly.更多还原