【作者】 刘文斌；

【导师】 王恬； 夏德全；

【作者基本信息】 南京农业大学 ， 水产养殖学， 2005， 博士

【摘要】 植物蛋白酶解后会产生活性肽类，活性肽不仅有营养作用，而且能促进动物生长、提高免疫机能、增强抗病能力等。本研究选用饼粕植物蛋白作为酶解底物，以几种内切蛋白酶类对饼粕植物蛋白进行酶解，对其蛋白酶解产物生物效价进行分析，并将其酶解产物添加到饲料中喂养鱼类，探讨蛋白酶解产物对鱼类生长发育的影响及其影响机制，为植物蛋白酶解产物开发与应用提供理论依据。1、酶解豆粕蛋白适宜酶类及其酶解条件筛选通过正交试验设计（L₉（3⁴）），分别对木瓜蛋白酶、胰蛋白酶和As1.398枯草蛋白酶等三种酶解大豆粕的水解条件中的PH、温度、时间、酶浓度进行了适宜范围的筛选。试验共分27组，酶解结束后分别对27组酶解产物SDS-PAGE电泳分析和水解度测定，并以水解度为衡量指标，对三种酶解大豆粕的水解条件筛选结果表明：木瓜蛋白酶水解大豆粕的最佳条件为：PH7.0、温度60℃、时间5h、酶浓度5％，在此条件下，大豆粕的水解度可达到3.67％；胰蛋白酶水解大豆粕的最佳条件为：PH7、温度50℃、时间7h、酶浓度5％，在此条件下，大豆粕的水解度可达到5.53％；As1.398蛋白酶水解大豆粕的最佳条件为：PH 6.5、温度50℃、时间7h、酶浓度5％，在此条件下，可使大豆粕的水解度达到8.73％。因此，在这三种酶种以As1.398枯草蛋白酶对豆粕酶解能力最强。2、常见几种饼粕蛋白酶解产物的超滤与电泳比较分析用木瓜蛋白酶、胰蛋白酶、Asp1.398枯草蛋白酶酶解豆粕、棉粕、菜粕和花生粕，采用超滤法结合SDS-PAGE电泳法对其酶解产物进行分析，结果表明：Asp1.398枯草蛋白酶酶解豆粕效果最好，其次是胰蛋白酶，木瓜蛋白酶最差，豆粕水解度越大的，其低分子量的蛋白质与肽类等物质越多，水解度为5.68％，30KD以上占73.09％，10KD-30KD占2.75％，3KD-10KD占13.42％，3KD以下10.75％；水解度为8.73％，30KD以上占44.87％，10KD-30KD占8.83％，3KD-10KD占14.63％，3KD以下31.67％；而原料蛋白未水解，30KD以上占94.12％，10KD-30KD占3.06％，3KD-10KD占1.86％，3KD以下只有0.97％。在相同条件下，Asp1.398枯草蛋白酶对豆粕蛋白酶解程度最高，10KD以下的小分子蛋白质和氨基酸总比例为46.3％，其次为棉粕25.13％，花生粕和菜粕较小，分别为13.38％和12.58％。这在SDS-PAGE电泳图谱中也能反映出来，其水解度越大，蛋白带越少。3、植物蛋白酶解产物对小鼠胚胎成纤维细胞生长的影响用木瓜蛋白酶、胰蛋白酶、As1.398枯草蛋白酶分别对豆粕、棉粕、菜粕和花生粕等四种植物蛋白进行酶解，用酶解产物对小鼠胚胎成纤维细胞进行培养试验研究，探讨不同酶解产物的蛋白质组分对细胞生长的影响。结果表明，各种酶解产物对小鼠胚胎成纤维细胞的生长都有促进作用，而棉粕的As1.398枯草蛋白酶酶解产物对小鼠胚胎成纤维细胞促生长效果最好。其对小鼠胚胎成纤维细胞促生长速度比豆粕、菜粕和花生粕酶解产物都快，且差异显著（P＜0.05）。4、饼粕蛋白及其酶解物对鱼类肠道微生物体外培养的影响用平板法从健康鱼类肠道中分离得到三种不同的微生物：大肠杆菌、芽孢杆菌、乳酸杆菌，再用液体培养法研究了四种植物性蛋白及其酶解物在体外对这三种微生物生长的影响。结果表明，除菜粕原料蛋白外，棉粕、豆粕、花生粕等原料蛋白及棉粕、豆粕、菜粕、花生粕等蛋白酶解物对芽孢杆菌、乳酸杆菌促生长作用明显，差异均显著（P＜0.05）；除棉粕酶解物对大肠杆菌有促生长作用外，四种原料蛋白及其酶解物对大肠杆菌的促生长作用均不明显，差异不显著（P＞0.05）。5、饼粕蛋白酶解产物对异育银鲫生长和免疫指标的影响以基础日粮为对照，将棉粕、菜粕、豆粕、花生粕的酶解物和鱼粉添加于异育银鲫饲料中，喂养61天，结果表明：试验各组异育银鲫增重率与对照组相比均有提高，饵料系数也相应下降，添加棉粕酶解物组增重率最高，与对照组相比提高32.13％，且差异显著（P＜0.05），其它各组增重率比对照组分别提高了3.67％、5.83％、12.17％、11.66％，但差异不显著（P＞0.05）。棉粕酶解物组的酸性磷酸酶（ACP）、溶菌酶活力与对照组相比有显著提高（P＜0.05），其它各试验组的超氧化物歧化酶（SOD），酸性磷酸酶（ACP），碱性磷酸酶（AKP）和溶菌酶（UL）的活力与对照组相比的活力均无显著差异。在以上几个试验中，通过对饼粕蛋白酶解产物的化学分析和生物效价分析得出初步结论，棉粕的枯草蛋白酶酶解产物的小肽数量和生物效价均较高，所以在以下两个试验中，我们进一步对棉粕酶解产物进行高效液相分析及其对异育银鲫生长发育、消化机能和胰蛋白酶基因表达量的研究，探讨蛋白酶解产物对鱼类的影响效果和作用机制。6、棉粕蛋白酶解产物组份的凝胶层析和HPLC分析通过常规测定、凝胶层析、高效液相色谱等三种方法，测定和分析了棉粕及其As1.398枯草蛋白酶酶解产物的蛋白质组份，并进行比较研究。结果表明：棉粕经过枯草杆菌蛋白酶酶解后，可溶性蛋白比例比原来增加了1倍，游离氨基酸的含量是原来的3.9倍；棉粕原料和酶解后的蛋白质产物经过SephadexG-50、SephadexG-25凝胶层析分离后均得到两个主要蛋白峰，期分子量均超过6000KD，但酶解物的两个峰所含蛋白量均比原料中多；用高效液相色谱法分析可知棉粕原料含有较多大分子蛋白，而酶解物中大分子蛋白含量下降了7.71％，小肽（2-10肽）的含量由27.04％增加到66.78％，游离氨基酸的含量也增加较多。由此可见，棉粕蛋白酶解后能产生丰富的肽类物质，小肽及游离氨基酸的增加有利于动物机体的直接吸收利用，能更快地促进动物的生长发育。7、棉粕蛋白酶解物对异育银鲫消化、生长和胰蛋白酶mRNA表达量的影响为了探讨棉粕蛋白酶产物及添加量对异育银鲫生长，消化蛋白酶活性及胰蛋白酶mRNA表达水平的影响，并研究鱼类生产性能及各项生化指标与蛋白酶活性之间的相互关系。本研究以棉粕蛋白酶解底物，用枯草杆菌蛋白酶对其进行酶解，以酶解产物的混合物1.5％和3.0％两个梯度等量替代鱼饲料中棉粕在室内流水养殖系统中喂养异育银鲫鱼种（体重30±2g）65天。测定鱼类的生长，营养物质表观消化率、消化蛋白酶活性及肝胰脏中蛋白酶mRNA表达水平等指标。结果表明：添加1.5％和3.0％棉粕酶解产物两组鱼类在饲养35天后的增重率分别比对照组高8.96％和44.24％，且差异显著（p＜0.05），在饲养65天后，两组增重率分别比对照组高33.19％和69.62％，且差异显著（p＜0.01），肝胰脏中蛋白酶mRNA表达水平也相应提高，并且以3.0％添加梯度的效果最好（p＜0.05），表明鱼类的生长与消化蛋白酶mRNA表达水平相关，同时棉粕蛋白酶解物对肠道蛋白酶的活性和营养物质表观消化率都促进作用，这可能是棉粕蛋白酶解物的对鱼类生长调控机制之一。更多还原

【Abstract】 The plant protein hydrolysates include some active peptides, this active peptide could not only have some nutritional function ,but also could promote animal growth, enhance immunity ability, advance the somatic ability of antivirus and so on. The research use plant protein hydrolysates enzymed by endoproteinase to do biological valuable analysis, and use the hydrolysates to do in vivo experiment to explore the hydrolysates’ function on fish development and its mechanism, to offer the theory evidence for the The plant protein hydrolysates’ exploration and application.1 Selection of the Optimum Conditions of Enzymatic Hydrolysis of Soybean meal with different EnzymesThe optimum enzymatic conditions of papain , trypsin and As1.398 subtilisin which hydrolyze Soybean meal（SBM） were studied with the degree of hydrolysis（DH） through mono-factor analysis and orthogonal test（ L₉ （3⁴） ）.The result showed that the optimum enzymatic conditions of papain were PH 7.5, temperature 50℃, time 7 hours ,enzyme concentration 5 percent of Soybean meal（SBM） protein .In such conditions, the degree of hydrolysis of soybean meal（SBM） can go up to 3.67%.The optimum conditions of trypsin were PH 7.0, temperature 50℃.time 7 hours ,enzyme concentration 5 percent of Soybean meal（SBM） protein. In such conditions, the degree of hydrolysis of soybean meal can go up to 5.53%; The optimum conditions of Asl.398 subtilisin were PH 6.5, temperature 50℃,time 7 hours ,enzyme concentration 5 percent of Soybean meal（SBM） protein. In such conditions, the degree of hydrolysis of soybean meal can go up to 8.73%.The hydrolysis activity of As1.398 subtilisin is the highest of three enzyme.2 Ultrafiltratione and SDS-PAGE analysis of common feeds plant protein hydrolysatesSoybean meal, cottonseed meal, rapeseed meal and peanut meal were hydrolyzed with the papain, trypsin and As1.398 subtilisin. Enzymolysis products of them were analysed with the method of ultrafiltratione and SDS-PAGE electrophoresis. The result showed that: As1.398 subtilisin was the best proteinase among three enzyme which hydrolyze soybean meal. The higher hydrolysis degree（DH） of soybean meal is, the more small molecular weights of enzymatic products of the soybean meal is, when soybean meal DH is 5.68％, the molecular weights which are bigger than 30KD is 73.09％, from 10KD to 30KD is 2.75％, from 3KD to 10KD is 13.42％, the smaller than 3KD is 10.75％,when soybean meal DH is 8.73％, the bigger than 30KD is 44.87％, from 10Kd to 30Kd is 8.83％, from 3Kd to 10Kd is 14.63％, the smaller than 3KD is 31.67％,when soybean meal was not hydrolyzed, the bigger than 30KD is 94.12％, from 10KD to 30KD is 3.06％, from 3Kd to 10Kd is 1.86％, the smaller than 3KD is 0.97％. As the optional conditions of enzymatic protein hydrolysis were the same, the soybean meal hydrolyzed by As1.398 subtilisin was the highest degree of hydrolysis, the second is cottonseed meal, the third is peanut meal, the lowest is rapeseed meal, the molecular weights below 10KD of enzymatic products were 46.3％, 25.13％, 13.38％and 12.58％. The molecular weights of enzymatic products were measured by sodium dodecy sulfafce-polyactivity gel electrophoresis（SDS-PAGE） in this experiment.3 Effect of plant protein hydrolysates on the growth of the mouse embryo fibroblasts cellPapain, trypsin and As1.398subtilisin were used to degrade soybean meal, cottonseed meal, rapeseed meal and peanut meal separately, then used hydrolysates to study the effect on the culture of the mouse embryo fibroblasts cell. The result indicated that all plant protein hydrolysates could promote the growth of the mouse embryo fibroblasts cell, and the most effective hydrolysates was the cottonseed meal degraded by As1.398subtilisin, the speed of cell growth of that was improved than that of hydrolysates of the peanut meal, rapeseed meal and soybean meal.4 Effects of plant proteins hydrolysates on proliferation of intestinal microorganisms of fishEscherichia coli, Bacillus and Lactobacillus were isolated from intestines of healthy carps by plate cultivation. Effects of plant proteins and their hydrolysates on the growth of three microorganisms were studied by liquid cultivation in vitro. The result was that: cottonseed meal, soybean meal, peanut meal and the hydrolysates of cottonseed, soybean, rapeseed meal and peanut meal observably promoted the proliferation of Bacillus and Lactobacillus. and than the effect was significantly different（p＜0.05）: all of four meals and their hydrolysates could promote the proliferation of Escherichia coli except hydrotysates of cottonseed meal., but had no significant effects（p＞0.05）.5 Effect of hydrolysates on the growth and immune indices of Alloggnogeuetic Crucian CarpThe basal diet as the control group, four hydrolysates and fish meal were added in test groups teed for Alloggnogeuetic crucian, fish raised for 61 days, the results demonstrated that: the gained weight rates of all experiment groups were higher than the control and the feed coefficient was reduced, the highest weight gain which was 32.13％higher than control group was in Cottonseed meal hydrolysates group（P＜0.05）, gained weight rate of others were 3.67％,5.83％,12.17％and 11.66％higher than control （P＞0.05）.The activity of Acidity Phosphatase（ACP） and lysozyme in Cottonseed meal hydrolysates was significant higher than control（P＜0.05）, the activity of Superoxide Dismutase（SOD）, Acidity Phosphatase（ACP）, Alkaline Phosphatase（ALP） and lysozyme was no-significance with the control.In finished experiments, based on analysis of chemistry and bio-active valve of plant protein hydrolysates, we have inclusion that cottonseed meal protein hydrolysates contain a lot of small molecular peptides and high bio-active valve, so we do next two experiments in which we used HPLC to analyses peptides of cottonseed meal protein hydrolysates and raising fish indoor, and then study how cottonseed meal protein hydrolystaes affect the group development, digestive performance and trypsin Mrna levels of Alloggnogeuetic crucian in order to evidence affecting mechanism.6 Analysis of protein and peptides components of cottonseed hydrolysates by HPLCIn this experiment, protein components in cottonseed and enzymatic hydrolysates of cottonseed were analyzed by general methods, gel filtration chromatography and high performance liquid chromatography （HPLC）. It showed that: soluble proteins were increased by 1 time and free amino acids were increased by 2.9 times after been hydrolyzed by subtilisin; two main peaks were found by chromaography column of sephadexG-50, proteins in the cottonseed’s peak were more than those in hydrolysate’s. The molecular weight of the cottonseed and hydrolysate’s two peaks were higher than 7000Da with a standard curve, but protein concentition of hydrolysates was less than that of cottonseed meal:the HPLC method was used to determine that the hydrolysates contained more proteins than cottonseed, the contents were decreased 7.71％, but small peptides（2-10 amino acids） were increased 24.73％, and tree amino acids were also increased. Animals can absorb small peptides and free amino acids firsthand ,so cottonseed hydrolysates were better for animals contrast with cottonseed。7 Effect of cottonseed meal hydrolysates on group, trypsin’s activity and expressing level of trypsinogen’s mRNA of Allogygenetic Crucian CarpTo explore the effect of Cottonseed meal hydrolysates and additive quantity in diets on Allogynogenetic Crucian’s growth, trypsin’s activity and expressing level of trypsinogen’s mRNA, and to study the relationship of proteinase’s activity and Allogynogenetic Crucian’s productive performance and biochemistry index. Firstly, we use the Asl.398 Neutrase to hydrolyze the Cottonseed meal hydrolysates, secondly, use the hydrolysates to feed Allogynogenetic Crucian （weight30±2g） raised in the inner flowing cultivating system by 1.5 and 3.0 percentage for 65 days, finally, measure the fish’s growth index, ADC of nutriment, the proteinase’s activity and the expressing level of trypsinogen’s mRNA. The result stated that, the body weight of 1.5％and 3.0％groups fish were higher then the control by 8.96％and 44.24％respectively raising for 35 days, and significantly different （p＜0.05）, furthermore after 65 days the higher was 33.19％and 69.62％（p＜0.01）, and the expressing level of trypsinogen’s mRNA were also enhanced, and all the data suggest the 3.0％was the best level, and the correlation of the Allogynogenetic Crucian’s growth and expressing level of trypsinogen’s mRNA ,simultaneously the hydrolysates could accelerate the trypsin’s activity and ADC of nutriment.更多还原