花生蛋白ACE抑制肽的制备和降血压效果的研究
|
摘要
本文以花生粕为原料，运用二次回归正交旋转组合设计对花生蛋白的提取工艺进行了优化；以血管紧张素转化酶(Angiotensin converting enzyme，ACE)抑制率为指标，从六种商业蛋白酶中筛选出碱性蛋白酶进行酶解，制备高活性的ACE抑制肽；选用凝胶层析法进行纯化分离；用凝胶电泳研究了分子量的分布；使用高效液相和质谱联用对产物结构进行了初步分析；选用自发性高血压大鼠(Spontaneously hypertensive rat，SHR)进行了降血压的体内动物实验。研究结果如下：
1．二次浸提对蛋白的提取率有显著的影响。采用碱提酸沉淀法，结合DPS软件分析，研究了温度、pH和料液比对蛋白质提取率的影响。结果表明：花生蛋白质提取条件较优的范围为：温度为52-55℃，pH为9.3-9.6，料液比为1：9.7-1：10.3，在相同的条件下，进行二次浸提，每次浸提2h。
2．采用响应面法筛选出碱性蛋白酶(Alcalase)酶解花生蛋白制备ACE抑制肽，以ACE抑制率为响应值，研究了温度、底物浓度和酶与底物浓度比对ACE活性的影响。结果表明：生产ACE抑制肽的最优组合为温度53.7℃，底物浓度7.72％，酶与底物浓度比4.18％，pH8.0，水解时间为120min。
3．经凝胶层析的方法对ACE抑制肽进行了分离和纯化，通过Tricine-SDS-PAGE电泳法分析了各组分的分子量的分布，结果表明：分子量小于1500D的小肽具有较高的ACE抑制率。
4．对于高活性的ACE抑制肽通过高效液相和质谱分析表明：经凝胶柱分离的单一组分仍然是由分子量不同的许多肽组成的混合物，其分子量主要分布在300-700D。
5．通过自发性高血压大鼠的急性降压实验表明：在给药后120min血压开始明显下降，在给药后120min、150min、180min、210min时间段的血压与给药前比较均有明显的降压效果，平均动脉血压分别下降了9.9％、12.2％、14.0％和13.1％；慢性降压实验表明：连续灌服ACE抑制肽后，高血压大鼠血压呈现下降趋势，其中给药后2h、第2d、5d、7d血压明显低于模型组。
Extraction process of peanut protein from peanut dregs was optimized by means oforthogonally rotational combination design in this paper. Monitored by Angiotensin convertingenzyme (ACE) inhibitory rate, the alkali proteinase was selected from six commercialproteinases and used for enzymolysis to produce highly active ACE inhibitory peptides;the gel chromatography method was used for the separation and purification, the gelelectrophoresis was used to investigate the distribution of molecular weights, HPLC-MSwas used to preliminarily analyze the product structure, and SHR was selected to testhypotensive activity in animal in vivo. The experimental results were as follows:
1. Twice digestion and extraction had a significant effect on protein extraction rate.The alkali treatment and acid precipitation method was adopted and the effects of itstemperature, pH and liquid and material ratio on the extraction rate of protein wereinvestigated in combination with the DPS software analysis. The results indicated that theoptimized range of peanut protein extraction conditions were temperature 32-35℃, pH9.3-9.6, material and liquid ratio 1: 9.7-1: 10.3, twice digestion was performed under thesame conditions with extraction time of 2h each time.
2. Alcalase was selected for enzymolysis of peanut protein by the method of responsesurface to produce ACE inhibitory peptides. The effects of temperature, substrateconcentration and the ratio of enzyme and substrate concentration on ACE activity wereinvestigated by using the ACE inhibitory rates as the response value. The results indicatedthat the proper conditions were temperature 53.7℃, substrate concentration 7.72%, theratio of enzyme and substrate concentration 4.18%, pH8.0, and hydrolysis time 120min.
3. ACE inhibitory peptides were separated and purified by the method of gelchromatography, and the distributions of molecular weights of different components wereanalyzed by Tricine-SDS-PAGE electrophoresis. The results indicated that the short chainpeptides with the molecular weights of less than 1500D had higher inhibitory rate onACE.
4. The highly active ACE inhibitory peptides were analyzed by HPLC-MS and theresults indicated that the single component separated from gel column was the mixture ofpeptides of different molecular weights, and its molecular weights were mainlydistributed in the range of 300-700D.
5. The result of acute antihypertensive experiments with spontaneously hypertensiverats (SHR) indicated that blood pressures began to decrease significantly 120min afteroral administration. Compared with the blood pressure before oral administration, theblood pressures 120min, 150min, 180min, and 210min after oral administration were decreased significantly, with the mean arterial pressure (MAP) reduction of 9.9%, 12.2%,14.0% and 13.1%, respectively; and chronic antihypertensive experiments with SHRindicated that the blood pressure of SHR tended to decrease after consecutive oraladministrations, and the blood pressures 2h after oral administration, on day 2, day 5, andday 7 were significantly lower than that of the model group.
1．二次浸提对蛋白的提取率有显著的影响。采用碱提酸沉淀法，结合DPS软件分析，研究了温度、pH和料液比对蛋白质提取率的影响。结果表明：花生蛋白质提取条件较优的范围为：温度为52-55℃，pH为9.3-9.6，料液比为1：9.7-1：10.3，在相同的条件下，进行二次浸提，每次浸提2h。
2．采用响应面法筛选出碱性蛋白酶(Alcalase)酶解花生蛋白制备ACE抑制肽，以ACE抑制率为响应值，研究了温度、底物浓度和酶与底物浓度比对ACE活性的影响。结果表明：生产ACE抑制肽的最优组合为温度53.7℃，底物浓度7.72％，酶与底物浓度比4.18％，pH8.0，水解时间为120min。
3．经凝胶层析的方法对ACE抑制肽进行了分离和纯化，通过Tricine-SDS-PAGE电泳法分析了各组分的分子量的分布，结果表明：分子量小于1500D的小肽具有较高的ACE抑制率。
4．对于高活性的ACE抑制肽通过高效液相和质谱分析表明：经凝胶柱分离的单一组分仍然是由分子量不同的许多肽组成的混合物，其分子量主要分布在300-700D。
5．通过自发性高血压大鼠的急性降压实验表明：在给药后120min血压开始明显下降，在给药后120min、150min、180min、210min时间段的血压与给药前比较均有明显的降压效果，平均动脉血压分别下降了9.9％、12.2％、14.0％和13.1％；慢性降压实验表明：连续灌服ACE抑制肽后，高血压大鼠血压呈现下降趋势，其中给药后2h、第2d、5d、7d血压明显低于模型组。
Extraction process of peanut protein from peanut dregs was optimized by means oforthogonally rotational combination design in this paper. Monitored by Angiotensin convertingenzyme (ACE) inhibitory rate, the alkali proteinase was selected from six commercialproteinases and used for enzymolysis to produce highly active ACE inhibitory peptides;the gel chromatography method was used for the separation and purification, the gelelectrophoresis was used to investigate the distribution of molecular weights, HPLC-MSwas used to preliminarily analyze the product structure, and SHR was selected to testhypotensive activity in animal in vivo. The experimental results were as follows:
1. Twice digestion and extraction had a significant effect on protein extraction rate.The alkali treatment and acid precipitation method was adopted and the effects of itstemperature, pH and liquid and material ratio on the extraction rate of protein wereinvestigated in combination with the DPS software analysis. The results indicated that theoptimized range of peanut protein extraction conditions were temperature 32-35℃, pH9.3-9.6, material and liquid ratio 1: 9.7-1: 10.3, twice digestion was performed under thesame conditions with extraction time of 2h each time.
2. Alcalase was selected for enzymolysis of peanut protein by the method of responsesurface to produce ACE inhibitory peptides. The effects of temperature, substrateconcentration and the ratio of enzyme and substrate concentration on ACE activity wereinvestigated by using the ACE inhibitory rates as the response value. The results indicatedthat the proper conditions were temperature 53.7℃, substrate concentration 7.72%, theratio of enzyme and substrate concentration 4.18%, pH8.0, and hydrolysis time 120min.
3. ACE inhibitory peptides were separated and purified by the method of gelchromatography, and the distributions of molecular weights of different components wereanalyzed by Tricine-SDS-PAGE electrophoresis. The results indicated that the short chainpeptides with the molecular weights of less than 1500D had higher inhibitory rate onACE.
4. The highly active ACE inhibitory peptides were analyzed by HPLC-MS and theresults indicated that the single component separated from gel column was the mixture ofpeptides of different molecular weights, and its molecular weights were mainlydistributed in the range of 300-700D.
5. The result of acute antihypertensive experiments with spontaneously hypertensiverats (SHR) indicated that blood pressures began to decrease significantly 120min afteroral administration. Compared with the blood pressure before oral administration, theblood pressures 120min, 150min, 180min, and 210min after oral administration were decreased significantly, with the mean arterial pressure (MAP) reduction of 9.9%, 12.2%,14.0% and 13.1%, respectively; and chronic antihypertensive experiments with SHRindicated that the blood pressure of SHR tended to decrease after consecutive oraladministrations, and the blood pressures 2h after oral administration, on day 2, day 5, andday 7 were significantly lower than that of the model group.
引文
1．王瑛瑶，王璋，许时婴。水酶法从花生中提取油和水解蛋白．中国粮油学报，2004，19(5)：59-61
2．张建成，宁维光．花生的药用及保健功能．中国食物与营养，2005，9：68-69
3．关荣发，叶兴乾，朱河水．粮油作物中降血压肽的研究．粮油加工与食品机械，2005，6：60-64
4．徐榕榕，陶冠军，杨严俊．高效液相色谱测定血管紧张素转换酶抑制肽的活性．河南工业大学学报(自然科学版)，2005，26(4)：18-21
5．何东平，张世宏．冷榨花生饼制备花生多肽的研究．中国油脂，2004，29(11)：18-20
6．蒲首丞，王金水．酶解蛋白制备生物活性肽进展．粮食与油脂，2005，3：16-17
7．郑冬梅，孔保华，李升福．玉米蛋白及其水解肽的研究动态．食品与发酵工业，2000，28(11)：55-59
8．励建荣，封平．功能肽的研究进展．食品科学，2004，25(11)：415-419
9．毕吴，刘志国．米糠蛋白酶解产物中血管紧张素转化酶抑制剂的研究．华中科技大学学报(医学版)，2005，34(6)：699-706
10．徐满清，郑为完，祝团结．花生蛋白部分水解制取微胶囊速溶花生粉壁材的研究．南昌大学学报(工科版)，2004，26(1)：81-84
11．杨晓泉，陈中，赵谋民．花生蛋白的分离及部分性质研究．中国粮油学报，2001，16(5)：25-28
12．刘大川，张维农，胡小泓．花生蛋白制备工艺和功能特性的研究．武汉工业学院学报，2001，4：1-10
13．刘志强，邓光炳．花生水酶法蛋白质提取及制油研究．中国油脂，1999，24(2)：3-6
14．李明妹，姚开，贾冬英．碱提酸沉法制取花生分离蛋白的优化条件．中国油脂，2004，29(1)：21-23
15．李艳丽，陈光，丛建民．碱性蛋白酶水解法制备玉米肽的研究．吉林农业大学学报，2004，26(2)：142-144
16．刘志强，何昭青．水酶法花生蛋白质提取及制油研究．中国粮油学报，1999，14(1)：36-39
17．刘志强，曾云龙．水酶法提油工艺对花生蛋白功能特性的影响．食品工业科技，2004，25(1)：61-65
18．许萍，薛长湖，张香治．3942中性蛋白酶酶解中国毛虾蛋白制备血管紧张素转移酶抑制肽．中国水产科学，2005，12(4)：501-505
19．辛志宏，马海乐，吴守一．从小麦胚芽蛋白中分离和鉴定血管紧张素转化酶抑制肽的研究．食品科学，2003，24(7)：130-133
20．吴建平，丁霄霖．大豆降压肽的研制(Ⅱ)酶E作用条件的优化．中国油脂，1998，23(3)：6-8
21．吴建平，丁霄霖．大豆降压肽的研制(Ⅲ)酶E水解进程参数的研究．中国油脂，1998，23(4)：12-16
22．宫霞，乐国伟，施用晖．电泳制备家蝇幼虫抗菌肽及其性质．无锡轻工大学学报，2003，22(6)：26-38
23. Xue Zhaohui, Wu Moucheng, Yin Jingzhang. Technological optimizatation for hydrolysisof rapeseed albuminwithalcalase. 农业工程学报, 2003, 19 (5): 176-181
24．宫霞，郭本恒，李云飞．酶解酪蛋白生产生物活性肽的研究现状及开发前景．中国乳品工业，2005，33(4)：44-47
25．欧成坤，杨瑞金．牡蛎酶解产物的ACE抑制活性和清除自由基活性研究．食品工业科技，2005，26(3)：59-62
26．李朝慧，罗永康，王全宇．乳清蛋白酶解制备ACE抑制肽的研究．中国乳品工业，2005，33(2)：8-11
27．倪莉，陶冠军，戴军．血管紧张素转化酶活性抑制剂丝素肽的分离、纯化和结构鉴定．色谱，2001，19(3)：222-225
28．黄文奕．血管紧张素转换酶抑制剂的药理机制及其临床应用．抗感染药学，2005，2(3)：115-117
29．韩继福，任建波．Alcalase酶水解玉米蛋白粉制备可溶性肽最佳条件的研究．吉林农业科学，2003，28(3)：46-49
30．顿新鹏，陈正望．酶法水解米渣蛋白制备大米小分子肽．食品科学，2004，25(6)：113-116
31．陈平，梁宋平．一种优化的MALDI-TOF质谱分析多肽C端序列方法．中国生物化学与分子生物学报，2001，17(5)：656-660
32．齐威，何明霞，何志敏．胰蛋白酶水解全酪蛋白反应过程中的色谱分析．色谱，2002，20(1)：1-5
33．邹远东．生物活性肽：21世纪人类健康的宠儿．科学中国人，2004，8：78-79
34．罗国安，王义明，朱瑛．质谱在肽和蛋白分析中的应用．药学学报，2000，35(4)：316-320
35．仪凯，周瑞宝．中性蛋白酶水解花生粕的研究．中国油脂，2005，30(7)：71-73
36．万书波．中国花生生产与科研动态．花生学报2001，30(4)：11-13
37．周雪松，赵谋明．我国花生食品产业现状与发展趋势．食品与发酵工业，2004，30(6)：84-89
38．刘海梅，熊善柏，赵山．脱脂油菜籽饼粕蛋白质分步酶水解研究碱性蛋白酶与木瓜蛋白酶分步水解．粮食与油脂，2002，3：2-4
39．王瑛瑶，王璋．水酶法从花生中提取蛋白质与油—酶解工艺参数．无锡轻工大学学报，2003，22(4)：60-64
40．禹山林，陶寿祥，宋连生．阿根廷花生科技考察报告．花生学报，2003，32(1)：26-28
41．陈正望，蔡小丽，顿新鹏．狗小肠新生物活性肽的分离纯化及结构测定．华中科技大学学报(自然科学版)，33(8)：87-89
42．陈永森，邱芋，姚忠．固相合成胸腺五肽的分离与纯化．南京工业大学学报，2005，27(2)：13-17
43．赵成萍，张映．鸡胰多肽、猪胰多肽的提取、纯化和鉴定研究．山西农业大学学报，2006，26(2)：141-158
44．黄朝明，孔繁东，祖国仁．降血压肽分离的研究现状与展望．食品研究与开发，2006，127(4)：161-164
45．刘友明，赵思明，熊善柏．米糠的蛋白酶水解提取物抗氧化活性及分子量分布研究．中国粮油学报，2006，21(2)：1-4
46．冯晓梅，韩玉谦，赵志强．牡蛎活性肽的制备及其理化性质的初步研究．中国海洋药物杂志，2006，25(2)：22-26
47．邹存媛，郑永杰，卢鹤．凝胶过滤法分离纯化大豆蛋白肽．齐齐哈尔大学学报，2006，22(1)：27-30
48．滕宇，邱芳萍，严铭铭．人参多肽的分离纯化．长春工业大学学报(自然科学版)，2005，26(3)：181-183
49．潘道东．酸乳中抗高血压肽的分离及其特性研究．食品科学，2005，26(1)：205-210
50．刘志国，陈江源，王亚林．玉米醇溶蛋白酶解制备ACE抑制肽的研究．食品研究与开发，2006，27(2)：138-141
51．曹佐武．小分子肽的Tricine-SDS-PAGE方法．生物学通报，2003，38(3)：55-56
52．曹佐武．有效分离1kDa小肽Tricine-SDS-PAGE方法．中国生物工程杂志，2004，24(1)：74-76
53．石继红，赵水同，王俊楼．SDS-聚丙烯酰胺凝胶电泳分析低分子多肽．第四军医大学学报，2000，21(6)：761-763
54．巩发永，齐桂年，李静．四川边茶中茶多糖提取条件的优化研究．茶叶科学，2005，25(3)：229-236
55. Seung-Cheol Lee, Seok-Moon Jeong, So-Young Kim. Effect of far-infrared radiation and heat treatment on the antioxidant activity of water extracts from peanut hulls. Food Chemistry, 2006, 94: 489-493
56. Renmin Gong, Yingzhi Sun, Jian Chen. Effect of chemical modification on dye adsorption capacity of peanut hull. Dyes and Pigments, 2005, 67: 175-181
57. Jianmei Yu, Mohamed Ahmedna, Ipek Goktepe. Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. Food Chemistry, 2005, 90: 199-206
58. Jean-Yu Hwang, Yeong-Shin Shue, Hung-Min Chang. Antioxidative activity of roasted and defatted peanut kernels. Food Research International, 2001, 34:639-647
59. Min-Suk Ma, In Young Bae, Hyeon Gyu Lee. Purification and identification of angiotensin I-converting enzyme inhibitory peptide from buckwheat. Food Chemistry, 2006, 94:36-42
60. Fahmi A., Morimura S., Guo H.C.. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochemistry, 2004,39:1195-1200
61. Justine Mouecoucou, Christian Villaume , Christian Sanchez. Effects of gum arabic, low methoxy pectin and xylan on in vitro digestibility of peanut protein. Food Research International. 2004, 37:777-783
62. van de Wai R.M.A., Plokker H.W.M., Lokb D.J.A.. Determinants of increased angiotensin II levels in severe chronic heart failure patients despite ACE inhibition. International Journal of Cardiology, 2006,106:367-372
63. Won-Kyo Jung, Eresha Mendis, Jae-Young Je. Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry, 2006, 94:26-32
64. Angeles Pozo-Bayon M., Alcaide Juan M., Carmen Polo M.. Angiotensin I-converting enzyme inhibitory compounds in white and red wines. Food Chemistry, 2005, 86:25-30
65. Ju-Eun Lee, In Young Bae, Hyeon Gyu Lee. Tyr-Pro-Lys, an angiotensin I-converting enzyme inhibitory peptide derived from broccoli (Brassica oleracea Italica). Food Chemistry, 2006, 69:106-114
66. Jae-Young Je, Pyo-Jam Park, Hee-Guk Byun. Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel (Mytilus edulis). Bioresource Technology, 2005,96:1624-1629
67. Wen-Dee Chiang, May-June Tsou, Zong-Yao Tsai. Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry, 2005,58:85-91
68. Jian-Hua Zhang, Eizo Tatsumi, Chang-He Ding. Angiotensin I-converting enzyme inhibitory peptides in douchi, a Chinese traditional fermented soybean product. Food Chemistry, 2005, 67:112-118
69. Fida Hasan, Megumi Kitagawa, Yoichi Kumada. Production kinetics of angiotensin-I converting enzyme inhibitory peptides from bonito meat in artificial gastric juice. Process Biochemistry, 2006, 41:505-511
70. Kuba M., Tana C, Tawata S.. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase. Process Biochemistry, 2005, 40:2191-2196
71. Jang A., Lee M.. Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Science, 2005, 69:653-661
72. Fahmi A., Morimura S., Guo H.C.. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochemistry, 2004,39:1195-1200
73. Chang-Kee Hyun, Heuyn-Kil Shin. Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides. Process Biochemistry, 2000, 36:65-71
74. Jianping Wu, Xiaolin Ding. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International, 2002, 35:367-375
2．张建成，宁维光．花生的药用及保健功能．中国食物与营养，2005，9：68-69
3．关荣发，叶兴乾，朱河水．粮油作物中降血压肽的研究．粮油加工与食品机械，2005，6：60-64
4．徐榕榕，陶冠军，杨严俊．高效液相色谱测定血管紧张素转换酶抑制肽的活性．河南工业大学学报(自然科学版)，2005，26(4)：18-21
5．何东平，张世宏．冷榨花生饼制备花生多肽的研究．中国油脂，2004，29(11)：18-20
6．蒲首丞，王金水．酶解蛋白制备生物活性肽进展．粮食与油脂，2005，3：16-17
7．郑冬梅，孔保华，李升福．玉米蛋白及其水解肽的研究动态．食品与发酵工业，2000，28(11)：55-59
8．励建荣，封平．功能肽的研究进展．食品科学，2004，25(11)：415-419
9．毕吴，刘志国．米糠蛋白酶解产物中血管紧张素转化酶抑制剂的研究．华中科技大学学报(医学版)，2005，34(6)：699-706
10．徐满清，郑为完，祝团结．花生蛋白部分水解制取微胶囊速溶花生粉壁材的研究．南昌大学学报(工科版)，2004，26(1)：81-84
11．杨晓泉，陈中，赵谋民．花生蛋白的分离及部分性质研究．中国粮油学报，2001，16(5)：25-28
12．刘大川，张维农，胡小泓．花生蛋白制备工艺和功能特性的研究．武汉工业学院学报，2001，4：1-10
13．刘志强，邓光炳．花生水酶法蛋白质提取及制油研究．中国油脂，1999，24(2)：3-6
14．李明妹，姚开，贾冬英．碱提酸沉法制取花生分离蛋白的优化条件．中国油脂，2004，29(1)：21-23
15．李艳丽，陈光，丛建民．碱性蛋白酶水解法制备玉米肽的研究．吉林农业大学学报，2004，26(2)：142-144
16．刘志强，何昭青．水酶法花生蛋白质提取及制油研究．中国粮油学报，1999，14(1)：36-39
17．刘志强，曾云龙．水酶法提油工艺对花生蛋白功能特性的影响．食品工业科技，2004，25(1)：61-65
18．许萍，薛长湖，张香治．3942中性蛋白酶酶解中国毛虾蛋白制备血管紧张素转移酶抑制肽．中国水产科学，2005，12(4)：501-505
19．辛志宏，马海乐，吴守一．从小麦胚芽蛋白中分离和鉴定血管紧张素转化酶抑制肽的研究．食品科学，2003，24(7)：130-133
20．吴建平，丁霄霖．大豆降压肽的研制(Ⅱ)酶E作用条件的优化．中国油脂，1998，23(3)：6-8
21．吴建平，丁霄霖．大豆降压肽的研制(Ⅲ)酶E水解进程参数的研究．中国油脂，1998，23(4)：12-16
22．宫霞，乐国伟，施用晖．电泳制备家蝇幼虫抗菌肽及其性质．无锡轻工大学学报，2003，22(6)：26-38
23. Xue Zhaohui, Wu Moucheng, Yin Jingzhang. Technological optimizatation for hydrolysisof rapeseed albuminwithalcalase. 农业工程学报, 2003, 19 (5): 176-181
24．宫霞，郭本恒，李云飞．酶解酪蛋白生产生物活性肽的研究现状及开发前景．中国乳品工业，2005，33(4)：44-47
25．欧成坤，杨瑞金．牡蛎酶解产物的ACE抑制活性和清除自由基活性研究．食品工业科技，2005，26(3)：59-62
26．李朝慧，罗永康，王全宇．乳清蛋白酶解制备ACE抑制肽的研究．中国乳品工业，2005，33(2)：8-11
27．倪莉，陶冠军，戴军．血管紧张素转化酶活性抑制剂丝素肽的分离、纯化和结构鉴定．色谱，2001，19(3)：222-225
28．黄文奕．血管紧张素转换酶抑制剂的药理机制及其临床应用．抗感染药学，2005，2(3)：115-117
29．韩继福，任建波．Alcalase酶水解玉米蛋白粉制备可溶性肽最佳条件的研究．吉林农业科学，2003，28(3)：46-49
30．顿新鹏，陈正望．酶法水解米渣蛋白制备大米小分子肽．食品科学，2004，25(6)：113-116
31．陈平，梁宋平．一种优化的MALDI-TOF质谱分析多肽C端序列方法．中国生物化学与分子生物学报，2001，17(5)：656-660
32．齐威，何明霞，何志敏．胰蛋白酶水解全酪蛋白反应过程中的色谱分析．色谱，2002，20(1)：1-5
33．邹远东．生物活性肽：21世纪人类健康的宠儿．科学中国人，2004，8：78-79
34．罗国安，王义明，朱瑛．质谱在肽和蛋白分析中的应用．药学学报，2000，35(4)：316-320
35．仪凯，周瑞宝．中性蛋白酶水解花生粕的研究．中国油脂，2005，30(7)：71-73
36．万书波．中国花生生产与科研动态．花生学报2001，30(4)：11-13
37．周雪松，赵谋明．我国花生食品产业现状与发展趋势．食品与发酵工业，2004，30(6)：84-89
38．刘海梅，熊善柏，赵山．脱脂油菜籽饼粕蛋白质分步酶水解研究碱性蛋白酶与木瓜蛋白酶分步水解．粮食与油脂，2002，3：2-4
39．王瑛瑶，王璋．水酶法从花生中提取蛋白质与油—酶解工艺参数．无锡轻工大学学报，2003，22(4)：60-64
40．禹山林，陶寿祥，宋连生．阿根廷花生科技考察报告．花生学报，2003，32(1)：26-28
41．陈正望，蔡小丽，顿新鹏．狗小肠新生物活性肽的分离纯化及结构测定．华中科技大学学报(自然科学版)，33(8)：87-89
42．陈永森，邱芋，姚忠．固相合成胸腺五肽的分离与纯化．南京工业大学学报，2005，27(2)：13-17
43．赵成萍，张映．鸡胰多肽、猪胰多肽的提取、纯化和鉴定研究．山西农业大学学报，2006，26(2)：141-158
44．黄朝明，孔繁东，祖国仁．降血压肽分离的研究现状与展望．食品研究与开发，2006，127(4)：161-164
45．刘友明，赵思明，熊善柏．米糠的蛋白酶水解提取物抗氧化活性及分子量分布研究．中国粮油学报，2006，21(2)：1-4
46．冯晓梅，韩玉谦，赵志强．牡蛎活性肽的制备及其理化性质的初步研究．中国海洋药物杂志，2006，25(2)：22-26
47．邹存媛，郑永杰，卢鹤．凝胶过滤法分离纯化大豆蛋白肽．齐齐哈尔大学学报，2006，22(1)：27-30
48．滕宇，邱芳萍，严铭铭．人参多肽的分离纯化．长春工业大学学报(自然科学版)，2005，26(3)：181-183
49．潘道东．酸乳中抗高血压肽的分离及其特性研究．食品科学，2005，26(1)：205-210
50．刘志国，陈江源，王亚林．玉米醇溶蛋白酶解制备ACE抑制肽的研究．食品研究与开发，2006，27(2)：138-141
51．曹佐武．小分子肽的Tricine-SDS-PAGE方法．生物学通报，2003，38(3)：55-56
52．曹佐武．有效分离1kDa小肽Tricine-SDS-PAGE方法．中国生物工程杂志，2004，24(1)：74-76
53．石继红，赵水同，王俊楼．SDS-聚丙烯酰胺凝胶电泳分析低分子多肽．第四军医大学学报，2000，21(6)：761-763
54．巩发永，齐桂年，李静．四川边茶中茶多糖提取条件的优化研究．茶叶科学，2005，25(3)：229-236
55. Seung-Cheol Lee, Seok-Moon Jeong, So-Young Kim. Effect of far-infrared radiation and heat treatment on the antioxidant activity of water extracts from peanut hulls. Food Chemistry, 2006, 94: 489-493
56. Renmin Gong, Yingzhi Sun, Jian Chen. Effect of chemical modification on dye adsorption capacity of peanut hull. Dyes and Pigments, 2005, 67: 175-181
57. Jianmei Yu, Mohamed Ahmedna, Ipek Goktepe. Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. Food Chemistry, 2005, 90: 199-206
58. Jean-Yu Hwang, Yeong-Shin Shue, Hung-Min Chang. Antioxidative activity of roasted and defatted peanut kernels. Food Research International, 2001, 34:639-647
59. Min-Suk Ma, In Young Bae, Hyeon Gyu Lee. Purification and identification of angiotensin I-converting enzyme inhibitory peptide from buckwheat. Food Chemistry, 2006, 94:36-42
60. Fahmi A., Morimura S., Guo H.C.. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochemistry, 2004,39:1195-1200
61. Justine Mouecoucou, Christian Villaume , Christian Sanchez. Effects of gum arabic, low methoxy pectin and xylan on in vitro digestibility of peanut protein. Food Research International. 2004, 37:777-783
62. van de Wai R.M.A., Plokker H.W.M., Lokb D.J.A.. Determinants of increased angiotensin II levels in severe chronic heart failure patients despite ACE inhibition. International Journal of Cardiology, 2006,106:367-372
63. Won-Kyo Jung, Eresha Mendis, Jae-Young Je. Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry, 2006, 94:26-32
64. Angeles Pozo-Bayon M., Alcaide Juan M., Carmen Polo M.. Angiotensin I-converting enzyme inhibitory compounds in white and red wines. Food Chemistry, 2005, 86:25-30
65. Ju-Eun Lee, In Young Bae, Hyeon Gyu Lee. Tyr-Pro-Lys, an angiotensin I-converting enzyme inhibitory peptide derived from broccoli (Brassica oleracea Italica). Food Chemistry, 2006, 69:106-114
66. Jae-Young Je, Pyo-Jam Park, Hee-Guk Byun. Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel (Mytilus edulis). Bioresource Technology, 2005,96:1624-1629
67. Wen-Dee Chiang, May-June Tsou, Zong-Yao Tsai. Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry, 2005,58:85-91
68. Jian-Hua Zhang, Eizo Tatsumi, Chang-He Ding. Angiotensin I-converting enzyme inhibitory peptides in douchi, a Chinese traditional fermented soybean product. Food Chemistry, 2005, 67:112-118
69. Fida Hasan, Megumi Kitagawa, Yoichi Kumada. Production kinetics of angiotensin-I converting enzyme inhibitory peptides from bonito meat in artificial gastric juice. Process Biochemistry, 2006, 41:505-511
70. Kuba M., Tana C, Tawata S.. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase. Process Biochemistry, 2005, 40:2191-2196
71. Jang A., Lee M.. Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Science, 2005, 69:653-661
72. Fahmi A., Morimura S., Guo H.C.. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process Biochemistry, 2004,39:1195-1200
73. Chang-Kee Hyun, Heuyn-Kil Shin. Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides. Process Biochemistry, 2000, 36:65-71
74. Jianping Wu, Xiaolin Ding. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International, 2002, 35:367-375