【作者】 王伟；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品安全， 2008， 博士

【摘要】 高血压是最常见的心血管疾病之一,它能造成大脑、心血管、肾脏的损害,是导致脑卒中、心力衰竭和冠心病等疾病的重要因素。天然食品中的降血压功能因子由于无副作用,已成为非药物控制高血压进程的理想选择。利用生物酶水解食物蛋白所生成的肽类能够表现出不同的生物活性,这些生物活性肽在消化降解和食物加工制作过程中可从其前体蛋白中释放出来。蚕蛹蛋白是一种高营养蛋白,我国每年的蚕蛹产量非常高,但是至今蚕蛹蛋白的综合利用途径还不完善,形成的产品附加值也不高。本研究通过可控酶解的办法,对利用蚕蛹蛋白制备具有降血压效果的血管紧张素转换酶抑制肽进行了系统研究。本文首先建立起了一种快速方便检测血管紧张素转换酶抑制肽活性的RP-HPLC方法,并优化了底物HHL与ACE酶作用转化为马尿酸（HA）的最佳反应当量,得出了HHL和ACE之间最优的定量关系是:HHL为5mM浓度时体积为6μL,ACE为0.1U/ml浓度下10μL。通过对蚕蛹蛋白酶解产物活性的测定,证明该方法具有快速、方便、准确的特点。通过利用不同蛋白酶对蚕蛹蛋白进行水解,得出酸性蛋白酶（Aspergillus usamii NO.537）水解的产物具有ACE抑制活性高、苦味低、色泽好、溶液稳定性好的特点,确定利用酸性蛋白酶作为水解蚕蛹蛋白的首选酶。通过单因子实验、部分因子优化和中心点组合设计实验,得出了酸性蛋白酶水解蚕蛹蛋白的最优的水解条件为水解温度35℃,水解时间4.92 h,酶比底物（w/w）为1%（相当于500U/g底物）,底物浓度（水/蚕蛹蛋白）为14.94（w/v）,pH为2.18,使水解产物的ACE抑制活性IC₅₀从单因子实验结果的2.5mg/mL增强到1.38mg/mL。利用传统的Osborne蛋白提取方法,根据每种蛋白组分的溶解特性的区别,提取到了四种蛋白组分,在蚕蛹蛋白中,蛋白组分的含量特点为清蛋白＞谷蛋白＞醇溶蛋白＞球蛋白。清蛋白组分为蚕蛹粉中的优势蛋白组分。对四种蛋白组分的氨基酸组成分析表明,清蛋白的必需氨基酸占总氨基酸的44.58%,必需氨基酸与非必需氨基酸的比值为80.43%,比WHO/FAO提出的必需氨基酸占总氨基酸的40%,和必需氨基酸与非必需氨基酸之比为60%的参考蛋白模式要高。通过中心点实验优化方法得出了清蛋白的最佳提取条件为:水/蛋白原料在6.8,提取时间5h,使清蛋白的纯蛋白得率从17.39g/100g蛋白原料上升到26.41g/100g蛋白原料,提高了34%。总蛋白得率达到61.83%。了解潜在的具有抗高血压活性蛋白原料的具体组成成分,以及明确哪种蛋白组分对ACE抑制活性起主要贡献作用,对更好的开发该蛋白原料具有重要意义。通过对蚕蛹蛋白组分的分子量分布特点和其水解产物的分子量分布情况与其ACE抑制活性之间的关系进行研究,得出蚕蛹蛋白组分中,清蛋白组分的ACE抑制率最高,水解度也最高,表明清蛋白易于被酸性蛋白酶水解,所得小肽分子生物活性强。对清蛋白组分经酸性蛋白酶酶解后的产物,经过超滤、凝胶过滤、多维液相系统和离子阱质谱的逐步分离和检测,得出一个新的ACE抑制肽,其结构为“SEPTVF”,通过化学固相合成法合成六肽SEPTVF,检测ACE抑制活性,得出此活性肽ACE抑制活性的IC₅₀值为324μM。以20个已经公布了结构和活性数据的ACE抑制肽作为训练集,生成了ACE抑制肽的药效团模型（Hypo 1）。将从蚕蛹蛋白中得到的血管紧张素转化酶抑制肽（ser-glu-pro-thr-val-phe）与该选择的药效团模型Hypo 1进行叠合比较,识别其对ACE抑制活性起关键作用的结构部分,证明此活性肽的thr-val-phe部分对其ACE的抑制活性起主要作用。通过研究自发性高血压大鼠血压（spontaneously hypertensive rat,SHR）摄入清蛋白酶解产物前后动脉收缩压的变化,得出清蛋白酶解产物对自发性高血压大鼠具有短期和长期降低动脉收缩压的效果。本论文的研究结果为综合利用蚕蛹蛋白,利用蚕蛹蛋白制备具有高经济附加值的降血压活性肽制品提供了基础研究数据。更多还原

【Abstract】 The hypertension is one of the most common cardiovascular diseases, it can cause the damage of cerebra, heart blood vessel and kidney, and it is also the most important reason of causing apoplexy, heart failure and coronary. Treatment and prevention hypertension was helpful to improve humanity’s health and lengthen life. The functional factors of anti-hypertension in natural food materials were ideal choices for non-druggery treat hypertension, for the functional factors with no side effects. Peptides hydrolyzed from food protein by proteases were with different bioactivities, these peptides could be released from its former proteins during digesting and food processing.Silkworm pupae protein was with high nutritional value, and the output was abundant each year in China, but the thoroughly utilizing approach of silkworm protein was still faultiness, and its add-ons value were also not ideal. In this study, by the method of controllable enzymatic hydrolyzation, we pay our attention on the preparation of Angiotensin I-Converting enzyme inhibitory peptides from silkworm pupae protein and then study on the peptides Quantitative Structure-Activity Relationship （QSAR）.Firstly, we set up a novel fast and convenient RP-HPLC method for Angiotensin I-Converting enzyme inhibitory peptides assay. And, we also optimized the super quantity of substrate HHL with the enzyme of ACE. The results was, 5mM HHL 6μL with 0.1U/mL ACE 10μL. Following assayed the hydrolysates inhibitory activity on Angiotensin I-Converting enzyme from silkworm protein, the results showed this RP-HPLC method was fast, convinent and precise.We utilized several kinds of protease to hydrolyze silkworm pupae protein, and got the hydrolysates by acidic protease （Aspergillus usamii NO. 537） with the highest inhibitory rate on ACE, and the hydrolysate was also with lower bitterness and super colour and stability. So, we took acidic protease as the preferred enzyme for hydrolyzing silkworm pupae protein.And after one factor experiment, fractional factorial design （FFD）, and center composite （CCD） design methods, the optimal hydrolyzing conditions were as the follows: temperature was 35°C, the hydrolysis time was 4.92 hour, the E/S （w/w） was 1% （equal to 500U/g substrate）, the substrate concentration （water: silkworm pupae protein） was 14.94 （w/v）, and the suitable pH was 2.18. The ACE inhibition was improved from IC₅₀ 2.5 mg/mL of one-factor design to 1.38mg/mL.According to the different dissolve ability of protein components, we distilled four kinds of protein components by the method of Osborne, the results showed that the content characteristics of protein components was albumin > glutelin > prolamine > globulin. Albumin was the predominant component in silkworm pupae protein. And the composing analysis of amino acids showed, the necessary amino acids were 44.48% to total amino acids, and the necessary to un-necessary amino acids was 80.43%, these results was higher than the standard of WHO/FAO that necessary amino acid was account for 40% to total amino acids and the necessary to unnecessary amino acids was 60%.By the center composite design （CCD） methods, we got the optimal distilling conditions of albumin was, water/silkworm material was 6.8, distilling time was 5hour. The distilling rate of pure protein was increased from 17.39g/100g silkworm material to 26.41g/100g, the increase rate was 34%, and total protein distilling rate reached to 61.83%.Make clear the detail components of the protein which with potential anti-hypertension activity, and make sure which kind of component contribute more on the ACE inhibitory activity, were with great value for the protein further application. By studying on the molecular weight distribute characteristics of components in silkworm pupae protein and the relationships between the hydrolyzing characteristics of different protein components in silkworm pupae protein and Angiotensin I-Converting enzyme inhibitory activity （ACEIP）, we got the hydrolysates of albumin with the highest ACE inhibitory rate and degree of hydrolysis （DH）, it showed albumin was proned to be hydrolyzed by acidic protease （ Aspergillus usamii NO. 537）, and the inhibitory activity of the hydrolysates peptides were very high.We separated the hydrolysates of albumin with ultrafiltration, Sephadex G-15 filtration, and the Ettan MDLC nanoflow/capillary LC system separation, and then by nanoscale reversed-phase LC-MS/MS analysis, we got a ACE inhibitory peptide, the sequence was "SEPTVF", and we synthesized this peptide by chemical method, assayed its inhibitory activity on ACE, the IC₅₀ was 324μM.Based on the published literature, we collected 20 compounds as training set to generate pharmacophore models for ACE inhibitory peptides （Hypo 1）. The peptide which we got （ser-glu-pro-thr-val-phe） from silkworm pupae protein was docking onto the best hypothesis Hypo 1 to determine which amino acid compositions best adhere to the requirements of the pharmacophore model, the result showed the best pharmacophore model maps to later three amino acids " thr-val-phe".By study on the changing of artery shrink pression of spontaneously hypertensive rat （SHR） before and after they ate the hydrolysate of albumin, we got the hydrolysates with the effect of decreasing the artery shrink pression of SHR -22.7mmHg at the concentration of 80mg/kg for 4 weeks, and with no side effects.The results of this paper provided basic research data for thoroughly application silkworm pupae protein and utilizing it to produce high economical add-on antihypertensive peptides products.更多还原