【作者】 孙海涛；

【导师】 吉红；

【作者基本信息】 西北农林科技大学 ， 水生生物学， 2011， 硕士

【摘要】 鱼类消化酶受年龄、食性、食物组成及环境因素等的影响。本文研究了0～53日龄匙吻鲟消化酶的发育模式,匙吻鲟、鳙和杂交鲟消化酶活力、特性及酶谱的差异,池塘与网箱养殖匙吻鲟消化酶活力及活力分布的不同,饲料蛋白水平对匙吻鲟消化酶活力的影响以及温度、pH对匙吻鲟蛋白酶和淀粉酶活力的影响。初步了解匙吻鲟的消化生理特点,以期为了解匙吻鲟营养需求和研制匙吻鲟专用配合饲料提供基础资料。本研究获得以下结果:1、碱性蛋白酶和酸性蛋白酶分别在出膜后3d(3DAH)和刚出膜时(0DAH)检测出活力;试验期间碱性蛋白酶活力高于酸性蛋白酶;在12DAH～40DAH期间α-淀粉酶活力相对稳定;发育早期脂肪酶活力较高;磷酸酶活力在20DAH开始显著增加,且碱性磷酸酶活力高于酸性磷酸酶。结果表明,蛋白酶,α-淀粉酶和磷酸酶随个体发育活力增加;碱性蛋白酶在个体发育早期对蛋白质的消化具有重要作用。养殖环境和食物发生改变时,酸性蛋白酶、α-淀粉酶、碱性磷酸酶和酸性磷酸酶活力在生长减慢时增加,生长加快时降低;脂肪酶活力在环境和食物发生变化时维持稳定。2、匙吻鲟总蛋白酶活力在pH 2.3～3达到最大,随pH增大活力显著下降,但在pH 7.0处出现另一个峰值。鳙总蛋白酶活力在pH 4.0～4.5、pH 8.0～8.5和pH 11.0处表现出三个峰值,而杂交鲟分别在pH2.5～3.0和pH 8.0～8.5处出现两个峰值。匙吻鲟酸性蛋白酶和脂肪酶活力显著高于鳙和杂交鲟,而碱性蛋白酶和α-淀粉酶活力显著低于鳙和杂交鲟。TLCK对匙吻鲟碱性蛋白酶活力的抑制率低于TPCK,而TLCK对鳙和杂交鲟碱性蛋白酶的抑制率均高于TPCK。匙吻鲟和杂交鲟存在一些相同或相似的条带;匙吻鲟与鳙的酶谱特征表现出较大的差异。3、池塘组碱性蛋白酶、α-淀粉酶和脂肪酶活力均显著低于网箱组;两组间酸性蛋白酶活力差异不显著;酸性蛋白酶活力在食道、胃和瓣肠中检测出,且食道和胃中的活力高于瓣肠;碱性蛋白酶在十二指肠和瓣肠中检测出,十二指肠中的活力高于瓣肠;淀粉酶活力在四个部位中都检测到,十二指肠中淀粉酶活力最大;脂肪酶只在胃中检测出活力。食道和胃中酸性蛋白酶活力在两组中差异不显著,而池塘组瓣肠酸性蛋白酶活力显著高于网箱组;池塘组十二指肠和瓣肠中碱性蛋白酶活力均显著低于网箱组;池塘组食道和十二指肠中淀粉酶活力显著低于网箱组,胃和瓣肠中淀粉酶活力两组间差异不显著;池塘组胃中脂肪酶活力显著低于网箱组。4、酸性蛋白酶和肝胰脏碱性蛋白酶活力随饲料蛋白水平的增加而增加,而肠道碱性蛋白酶活力随饲料蛋白水平的增加而降低;消化道α-淀粉酶活力随饲料蛋白水平的增加而降低,而肝胰脏α-淀粉酶活力表现出相反的趋势;饲料蛋白水平对消化道和肝胰脏脂肪酶活力影响不显著,各组脂肪酶活力差异不显著。结果表明,饲料蛋白水平对匙吻鲟肌肉粗蛋白含量,酸性蛋白酶,碱性蛋白酶和α-淀粉酶都会产生显著影响;饲料蛋白质水平为39.10%比较适宜。5、匙吻鲟酸性蛋白酶的最适温度为35～40℃,最适pH为2～3;碱性蛋白酶的最适温度为35℃,最适pH为7以上;淀粉酶活力受温度的影响不显著,在pH 5.5和pH8.0时出现峰值。更多还原

【Abstract】 A series of biochemical determination and electrophoretic observations have been conducted to analyze development, characterization, distribution and zymogram of protease,α-amylase and lipase in paddlefish Polyodon spathula. The research included study on development of digestive enzymes in paddlefish from 0 to 53 day after hatching, comparing activrty and characterization of protease, amylase and lipase in paddlefish with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus♀×Acipenser schrenki Brandt♂), a comparative study of a comparative study of enzymatic activity, distribution and characterization between pond-reared (with commercial feed) and cage-reared (with natural live food) paddlefish, effects of diet protein level on digestive enzyme activity of paddlefish and effects on tempareture and pH value on protease and amylase on paddlefish. The important results were in the followings:1. The alkaline protease activity was detected at 3 DAH and acid protease activity was quantifiable from eggs. Alkaline protease activity was consistently higher than acid protease activity.α-amylases activity was relatively stable during incubation period in workshop (12DAH～40DAH). High lipase activity indicated the importance of lipid utilization. Phosphatases activity significantly increased from 20DAH and alkaline phosphatases activity was higher than acid phosphatases activity. It is suggested that protease,α-amylase and phosphatase activities were increased with the ontogenesis of paddlefish and alkaline protease was important to paddlefish larval. Acid protease,α-amylase, acid and alkaline phosphatase activities were increased with growth decreasing and decreased with growth increasing when fish transferred to pond.2. Higher proteolytic activity was observed in the pH range 2.5–3.0 and at a pH of 7.0 for paddlefish; proteolytic activity of bighead carp had three peak in pH 4.0～4.5, pH 8.0～8.5, pH 11.0 and hybrid sturgeon indicated higher proteolytic activity in pH2.5～3.0 and pH 8.0～8.5. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp; while alkaline protease and amylase activity of paddlefish was significantly lower than those of bighead carp and hubrid sturgeon. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone (TPCK) was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone (TLCK). The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis further showed that there were certain types of enzymes, especiallyα-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon.3. There was no significant difference observed for acid protease activity between pond and cage groups, whereas the activity of alkaline protease,α-amylase and lipase in the pond group were significantly lower than those in the cage group. Acid protease activity was detected in the esophagus, stomach and intestine, and alkaline protease demonstrated detectable activity in the duodenum and intestine; Theα-amylase showed activity across the entire digestive tract, whereas lipase activity was only detected in the stomach. The activity of acid protease in the intestine of the pond group was significantly higher than that in the cage group; however, there was no difference in enzyme activity of esophagus and stomach between both groups. The alkaline protease activity of pond group was significantly lower than that of cage group in both duodenum and intestine . In addition, theα-amylase activity of pond group was significantly lower than that of cage in the esophageal and duodenal sections; however, there was no difference in the activity in the stomach and intestine. Lipase demonstrated higher activity in the stomach of the pond group than that of the cage group.4、Both acid protease activity and alkaline protease activity of hepatopancreas were significantly increased with the increase of diet protein level, while alkaline protease activity of intestine was decreased with increase of diet protein level. The activity ofα-amylases in digestive tract was decreased with increase of diet protein level, whileα-amylases activity in hepatopancreas showed an opposite trend. There was no significant difference on lipase activity in all tested tissues among dietary protein treatments. The results indicated that there were significant effects of diet protein level on activities of acid protease, alkaline protease andα-amylase. The test indicated that 39.10% protein content suited the protein demands of paddlefish juvenile.5、The optimum temperature for acid protease and alkaline protease were 35～40℃and 35℃, respectively. Effect of temperature on amylase was not significant and there was no optimum temperature for amylase. The optimum pH value for acid protease, alkaline protease and amylase were2～3, >7.0, 5.5 and 8.0, respectively.更多还原