牛骨酶解制备血管紧张素转换酶抑制肽的研究
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摘要
本文利用牛骨蛋白酶解制备血管紧张素转换酶抑制肽,在以下几个方面开展了研究：
研究了牛骨软化过程中骨蛋白的变化及实验室制备牛骨粉的营养价值；通过对牛骨的水解发现碱性蛋白酶、中性蛋白酶、胰蛋白酶对牛骨的水解效果较好；研究了酶解条件对牛骨蛋白酶解动力学参数的影响,根据动力学试验得到：碱性蛋白酶作用于骨蛋白的最适温度为55℃、pH为12,此时Vmax为0.10824 g/L·min,Km为11.71892g/L；中性蛋白酶作用于骨蛋白的最适条件温度为45℃、pH为7,此时Vmax为0.04724 g/L·min,Km为14.35711g/L；胰蛋白酶作用于骨蛋白的最适温度为55℃、pH为9,此时Vmax为0.05073g/L·min,Km为4.31516g/L。
从新鲜猪肺中提取了血管紧张素转换酶(ACE)的粗酶液,纯化后得到了比活力为5.21u的ACE酶液；通过水解度与ACE抑制率的关系研究发现不同蛋白酶水解的水解度与ACE抑制率的关系不同；在此基础上选用碱性蛋白酶作为酶解骨粉制备ACE抑制肽的工具酶,通过条件的优化得到的最适酶解参数为50℃、pH 10、底物浓度25%、E/S为0.4、酶解时间为3h。
利用葡聚糖凝胶色谱测定A3h的分子量分布,发现该混合肽主要分为3个组分,其中组分111分子量小于1000Da,且半抑制浓度最小为0.561mg/ml,抑制活性较高；利用5000Da的中空纤维膜进行超滤,得到的小分子混合肽进行氨基酸分析等定性分析。通过ACE抑制活性消化稳定性实验和ACE抑制动力学研究发现该混合肽为真抑制型的竞争性抑制剂。
收集已报道数据进行分析发现肽中疏水性氨基酸和芳香族氨基酸对其ACE抑制活性有较大影响；利用大孔树脂富集疏水性氨基酸,确定了操作条件以50mg/ml骨肽上样浓度为佳,100%乙醇洗脱得到的组分ACE抑制活性最高,其半抑制浓度(IC50)为0.45mg/ml,组分得率为24.59%；利用颗粒活性炭富集芳香族氨基酸,确定了30mg/ml上样浓度为佳,利用100%乙醇+5%乙酸洗脱得到的组分IC50为0.59mg/ml。
In this paper, angiotensin-converting enzyme inhibitory peptides were prepared by hydrolysis cattle bone. Therefore, the work was carried out from the following aspects:
The changes of bone protein by softening process and nutritional value of cattle bone meal by preparating in the laboratory were studied. The hydrolysis effect of three kinds of enzymes which were alkaline protease, neutral protease and trypsin were better than others. The bone protease solution conditions impacted on the kinetic parameters. According to the dynamic test, the optimum conditions of alkaline protease acted on the bone protein at 55℃and pH 12 and the kinetic parameters was 0.10824 g/L·min for Vmax and 11.71892g/L for Km. The optimum conditions of Neutral protease was at 45℃and pH 7 and the Vmax was 0.04724g/L·min with Km for 14.35711g/L at the same time. The optimum conditions of Trypsin was at 55℃,pH 9 and this time Vmax for 0.05073g/L·min and Km for 4.31516g/L,
The crude enzyme solution of angiotensin-converting enzyme (ACE) was extracted from fresh lung. After purification, the specific activity of ACE enzyme solution was improved to 5.21u. The relationship between the degree of hydrolysis and the rate of ACE inhibition was researched. The results were that the regulation was inconsistent in different enzymes. On this basis, the alkaline protease was selected as a tool to preparate ACE inhibitory peptide from cattle bone meal. Optimum hydrolysis conditions were obtained with an enzyme to substrate ratio (E:S) of 0.04 for 3 h at 50℃and pH 10.
The molecular weight distribution of hydrolysates was determined by Sephadex G-25 chromatography. There are three peaks in which molecular weight of the peakⅢis less than 1000 Da, the half maximal inhibitory concentration (IC50) was determined as 0.561mg/ml. Ultrafiltrate (5000Da) and hydrolysates were qualitative analyzed by amino acid analyzers. Digestive stability test shows that ACE inhibitory activity is stability because the mixture can not be hydrolysis easily by digestive enzyme.ACE inhibition kinetics study found that the ultra-filtrate was a competitive inhibitor.
Scatter and non-linear regression model demonstrates that the proportion of hydrophobic amino acids and aromatic amino acids in protein have great impacted on ACE inhibitory activity of hydrolysis.Based on the above conclusions, macroporous resin and activated carbon were used for enrichment of hydrophobic amino acids and aromatic amino acid to improve ACE inhibitory activity of bone peptide. The optimum sample concentration of macroporous resin purification was 50mg/ml.100% ethanol eluted fraction obtained the highest ACE inhibitory activity and its half-inhibitory concentration (IC50) as 0.45mg/ml, the yield of component as 24.59%.The optimum sample concentration of activated carbon purification was 30mg/ml.100% ethanol and 5% acetic acid elution fraction obtained IC50 as 0.59mg/ml.
研究了牛骨软化过程中骨蛋白的变化及实验室制备牛骨粉的营养价值；通过对牛骨的水解发现碱性蛋白酶、中性蛋白酶、胰蛋白酶对牛骨的水解效果较好；研究了酶解条件对牛骨蛋白酶解动力学参数的影响,根据动力学试验得到：碱性蛋白酶作用于骨蛋白的最适温度为55℃、pH为12,此时Vmax为0.10824 g/L·min,Km为11.71892g/L；中性蛋白酶作用于骨蛋白的最适条件温度为45℃、pH为7,此时Vmax为0.04724 g/L·min,Km为14.35711g/L；胰蛋白酶作用于骨蛋白的最适温度为55℃、pH为9,此时Vmax为0.05073g/L·min,Km为4.31516g/L。
从新鲜猪肺中提取了血管紧张素转换酶(ACE)的粗酶液,纯化后得到了比活力为5.21u的ACE酶液；通过水解度与ACE抑制率的关系研究发现不同蛋白酶水解的水解度与ACE抑制率的关系不同；在此基础上选用碱性蛋白酶作为酶解骨粉制备ACE抑制肽的工具酶,通过条件的优化得到的最适酶解参数为50℃、pH 10、底物浓度25%、E/S为0.4、酶解时间为3h。
利用葡聚糖凝胶色谱测定A3h的分子量分布,发现该混合肽主要分为3个组分,其中组分111分子量小于1000Da,且半抑制浓度最小为0.561mg/ml,抑制活性较高；利用5000Da的中空纤维膜进行超滤,得到的小分子混合肽进行氨基酸分析等定性分析。通过ACE抑制活性消化稳定性实验和ACE抑制动力学研究发现该混合肽为真抑制型的竞争性抑制剂。
收集已报道数据进行分析发现肽中疏水性氨基酸和芳香族氨基酸对其ACE抑制活性有较大影响；利用大孔树脂富集疏水性氨基酸,确定了操作条件以50mg/ml骨肽上样浓度为佳,100%乙醇洗脱得到的组分ACE抑制活性最高,其半抑制浓度(IC50)为0.45mg/ml,组分得率为24.59%；利用颗粒活性炭富集芳香族氨基酸,确定了30mg/ml上样浓度为佳,利用100%乙醇+5%乙酸洗脱得到的组分IC50为0.59mg/ml。
In this paper, angiotensin-converting enzyme inhibitory peptides were prepared by hydrolysis cattle bone. Therefore, the work was carried out from the following aspects:
The changes of bone protein by softening process and nutritional value of cattle bone meal by preparating in the laboratory were studied. The hydrolysis effect of three kinds of enzymes which were alkaline protease, neutral protease and trypsin were better than others. The bone protease solution conditions impacted on the kinetic parameters. According to the dynamic test, the optimum conditions of alkaline protease acted on the bone protein at 55℃and pH 12 and the kinetic parameters was 0.10824 g/L·min for Vmax and 11.71892g/L for Km. The optimum conditions of Neutral protease was at 45℃and pH 7 and the Vmax was 0.04724g/L·min with Km for 14.35711g/L at the same time. The optimum conditions of Trypsin was at 55℃,pH 9 and this time Vmax for 0.05073g/L·min and Km for 4.31516g/L,
The crude enzyme solution of angiotensin-converting enzyme (ACE) was extracted from fresh lung. After purification, the specific activity of ACE enzyme solution was improved to 5.21u. The relationship between the degree of hydrolysis and the rate of ACE inhibition was researched. The results were that the regulation was inconsistent in different enzymes. On this basis, the alkaline protease was selected as a tool to preparate ACE inhibitory peptide from cattle bone meal. Optimum hydrolysis conditions were obtained with an enzyme to substrate ratio (E:S) of 0.04 for 3 h at 50℃and pH 10.
The molecular weight distribution of hydrolysates was determined by Sephadex G-25 chromatography. There are three peaks in which molecular weight of the peakⅢis less than 1000 Da, the half maximal inhibitory concentration (IC50) was determined as 0.561mg/ml. Ultrafiltrate (5000Da) and hydrolysates were qualitative analyzed by amino acid analyzers. Digestive stability test shows that ACE inhibitory activity is stability because the mixture can not be hydrolysis easily by digestive enzyme.ACE inhibition kinetics study found that the ultra-filtrate was a competitive inhibitor.
Scatter and non-linear regression model demonstrates that the proportion of hydrophobic amino acids and aromatic amino acids in protein have great impacted on ACE inhibitory activity of hydrolysis.Based on the above conclusions, macroporous resin and activated carbon were used for enrichment of hydrophobic amino acids and aromatic amino acid to improve ACE inhibitory activity of bone peptide. The optimum sample concentration of macroporous resin purification was 50mg/ml.100% ethanol eluted fraction obtained the highest ACE inhibitory activity and its half-inhibitory concentration (IC50) as 0.45mg/ml, the yield of component as 24.59%.The optimum sample concentration of activated carbon purification was 30mg/ml.100% ethanol and 5% acetic acid elution fraction obtained IC50 as 0.59mg/ml.
引文
1.曹文红,章超桦.食品蛋白降血压肽及其酶法制备.食品科技,2002,5：9-10.
2.曹文红.酶解中国毛虾蛋白制备血管紧张素转换酶抑制肽的研究.[硕士学位论文].湛江海洋大学,2002：54-60.
3.陈兰英,田敏,谢贵福等.猪肺血管紧张素转化酶的提纯.生物化学杂志,1988,4(4)：345-350.
4.陈新,陈庆森,庞广昌.Alcalase AF 2.4L酶解玉米蛋白动力学研究.食品科学,2004,25(10)：29-32.
5.陈斌,裴国献,刘晓霞.骨组织结构显微形态.第一军医大学学报,2005,25(4)：403-406.
6.陈瑞锋,陈璐,陈廷登.用活性炭分离纯化苯丙氨酸与酪氨酸的研究.浙江工业大学学报,2006,34(4)：373-376.
7.程云辉,王璋,许时婴.大孔吸附树脂对麦胚肽的吸附特性研究.食品与机械,2005,21：7-12.
8.Cheison S C,王璋,许时婴.大孔吸附树脂提取苦味乳清蛋白水解物及其功能性质和生物活性的研究.食品科学,2006,27：103-107.
9.丁小燕,张雯,陈延锋等.复合风味蛋白酶水解鸡骨泥工艺条件的研究.中国食品学报.2006(3)：88-92.
10.董瑞新,闰循领.胶原蛋白温度效应的Ranlall光谱研究.光谱学与光谱分析,2004,24(11)：1367-1369.
11.郭明勋,史玉敏.骨的成分与胶原蛋白.明胶科学与技术,2001,21(4)：204-209.
12.韩继福,任建波,王海波等.活性炭吸附芳香族氨基酸制备高F值寡肽混合物的研究.安徽农业大学学报,2005,32(1)：77-80.
13.郝记明,郭顺堂,章超桦.酶法水解马氏珠母贝肉制备血管紧张素转换酶抑制肽的研究.[硕士学位论文].中国农业大学,2005.
14.黄素珍.超微鲜骨泥食品得研究与开发.中国食品与畜产科学,2003,10(4)：149-151.
15.黄朝明,孔繁东,祖国仁等.降血压肽分离的研究现状与展望.食品研究与开 发,2006,27(4)：161-164.
16.黄晓任,刘邻渭.食品化学综合实验.北京：中国农业出版,2002.
17.蒋挺大.胶原与胶原蛋白.[博士学位论文].化学工业出版社,2006.
18.姬莹莹,范远景.珠蛋白提取、ACE抑制肽的酶法制备及酶动力学的研究.[硕士学位论文].合肥工业大学,2007.
19.李帆,贾冬英,姚开等.牦牛骨蛋白的酶解条件研究.氨基酸和生物资源,2006,28(4)：7-10.
20.李培骏,袁永俊,胡婷等.胰蛋白酶水解酪蛋白进程研究.食品与机械,2005,11.
21.廖丹葵.鸡蛋蛋黄蛋白质制备降血压肽的研究.[硕士学位论文].广西大学,2006.
22.刘忠义.草鱼肠道胰蛋白酶同工酶GT-A的动力学和热力学性质.西北农林科技大学学报,2008,36(1)：211-217.
23.吕炜锋,储瑞霭,金建勤等.用活性炭分离纯化苯丙氨酸的研究.中国生化药物杂志,1997,18(2)：82-83.
24.任建东,乐国伟.酶解鹿茸血疏水性肽的分离及活性研究.[硕士学位论文].江南大学,2008.
25.孙淑斌,王传怀.活性炭色谱法分离纯化苯丙氨酸的研究.氨基酸和生物资源,1995,17(1)：9-12.
26.汤鸣强,郑挺,曾小芳等.酿造酱油米曲霉产中性蛋白酶提取与酶学性质研究.中国调味品,2008,2：38-50.
27.王英超,李天俊,王敏华等.羊骨酶解物中降血压肽的分离、纯化工艺研究.食品研究与开发,2007,28(9)：7-10.
28.吴建平,丁霄霖.食品蛋白质降血压肽的研究进展.中国粮油学报,1998,13(5)：10-14.
29.夏杨毅.超微粉碎骨泥的流变学加工特性研究.[博士学位论文].西南农业大学,2005.
30.杨桂苹.骨粉营养成分的分析.食品科技,1997,6：41-43.
31.杨严俊,吉川正明.高效液相色谱分离纯化血管紧张素转换酶活性抑制肽.色谱,2003,21(3)：202-205.
32.余奕珂,胡建恩,白雪芳等.胃蛋白酶水解珠蛋白的研究.河南工业大学学报(自然科学版),2006,6：45-56.
33.袁勤生,赵健.酶与酶工程.[博士学位论文].上海：华东理工大学出版社,2005.
34.袁玉燕,韩爱军.超细鲜骨粉的粒度及营养特性.食品工业,2001,3：10-12.
35.王朝旭.酶法水解骨蛋白最佳条件的研究.食品科学,2001(2)：48-49.
36.王梅,谷文英,沈蓓英.活性炭色谱法分离制备高F值寡肽混合物.无锡轻工大学学报,1998,17(4)：41-4576.
37.王喜波,迟玉杰.活性炭色谱法分离芳香族氨基酸制备高F值蛋清低聚肽混合物的研究.食品工业科技,2005,26(6)：55-57.
38.王英超,李天俊,王敏华等.羊骨酶解物中降血压肽的分离、纯化工艺研究.食品研究与开发,2007,28(9)：7-10.
39.伍先绍,贺稚非,刘琳等.碱性蛋白酶产生菌株的筛选及其酶学性质研究进展.中国食品添加剂,58-61.
40.詹莉,刘书亮,吴琦.枯草芽孢杆菌弹性蛋白酶的纯化及酶学性质的研究.[硕士学位论文].四川农业大学,2008.
41.张昊宇,王强.花生短肽制备及其功能活性研究.[博士学位论文].中国农业科学院,2007.
42.张永秀,王世平.牛骨蛋白的酶解研究.[硕士学位论文].中国农业大学,2006.
43.张青,许学书,谢静莉等.米曲霉蛋白酶动力学特性的研究.食品科学,2007,28(6)：213-217.
44.张焱.大豆蛋白ACE抑制肤的酶法制备及分离纯化.[硕士学位论文].合肥工业大学,2007.
45.张卓睿.高电压脉冲电场作用下牛骨钙快速离子化的研究.[博士学位论文].吉林大学,2006.
46.赵谋明,任娇艳,崔春等.活性炭静态吸附草鱼蛋白水解物种氨基酸特性的研究.食品与机械,2005,21(6)：13-16.
47.赵谋明,崔春,刘珊.低值鱼蛋白酶解产物制备不同肉香型热反应物.食品与生物技术学报.2006(5)：1-6.
48.赵霞,马俪珍.羊骨的酶解和多肽饮料的开发.[硕士学位论文].山西农业大学, 2004.
49.赵一明,王璋.酪蛋白非磷肽的制备及其ACE抑制活性的研究.[硕士学位论文].江南大学,2008.
50.钟芳,张晓梅,麻建国.大豆肽的大孔吸附树脂以及凝胶过滤色谱分离.食品与机械,2006,22：25-28.
51.朱丽娟,熊幼翎.玉米蛋白水解物体外消化产物的抗氧化及ACE抑制活性研究.[硕士学位论文].江南大学,2008.
52.Atkinson P J, Hallsworth A S.The spatial structure of bone. In:Harrision RJ, Navartman V, eds.Progress in Anatomy [PhD dissertation].Cambridge:Cambridge University Press,1982:179-99.
53.Atkins P W. The Elements of Physical Chemistry, Oxford University Press, 1996:354-362.
54. Astawan M, Wathuni M, Yasuhara T, et al. Effect of Angiotensin I-Converting Enzyme inhibitory substance derived Indonesian dried-salted fish on blood pressure of rats[J].Biosci Biotech Bioehem,1995,59(3):425-428.
55.BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of proteindye binding. Anal Biochem,1976, 72:248-254.
56. Chen J R, Yang S C, Suetsana K,& Chao J C J. Soybean protein-derived hydrolysate affects blood pressure in spontaneously hypertensive rats. Journal of Food Biochemistry,2004,28:61-73.
57. Cushman D W,& Cheung H S.Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology,1971, 20:1637-1648.
58.Erdos E G & Skidgel R A.The angiotensin Ⅰ-converting enzyme.Laboratory Investigation,1987,56:345-353.
59.Fujita H, Yoshikawa M. A prodrug type ACE inhibitory peptide derivedf rom fish protein. ImmunoPharmacology.1999,44:123-127.
60.Fujita H, Yokoyama K, Yoshikawa M. Classification and antihypertensive of angiotensin I-coverting enzyme inhibitory peptides derived from food proteins. Journal of Food Seienee.2000,65:564-5691.
61.Fujita H, Sasaki R, Yoshikawa M.Potentiation of the antihypertensive activity of orally, a vasorelaxing peptide derived from ovalbumin, by emulsifieation in egg hosphatidyleholine.Biosci Biotech Biochem.1995,59:2344-2345.
62.Fu-Yuan CHENG,Tien-Chun WAN, Yu-Tse LIU, Kung-Ming LAI, Liang-Chuan LIN,& Ryoichi SAKAT. A study of in vivo antihypertensive properties of enzymatic hydrolysate from chicken leg bone protein. Animal Science Journal, 2008,79:614-619.
63. Fu-Yuan CHENG, Tien-Chun WAN,Yu-Tse LIU,Chi-Ming CHEN,Liang-Chuan LIN,& Ryoichi SAKATA.Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase. Animal Science Journal,2009,80:91-97.
64. Ge shijun. Continuous Production of high degree case in hydrolysates by immobilized proteases in column reaetor.Journal of Biotechnology.1996,50: 161-170.
65.Gouda K G M, Gowda L R, Rao A G A,& Prakash V. Angiotensin I-converting enzyme inhibitory peptise derived from glycinin, the 11s globulin of soybean (Glycine max).Journal of Agriculture and Food Chemistry,2006,54:4568-4573.
66.Haque E& Chand R. Antihypertensive and antimicrobial bioactive peptides from milk proteins.European Food Research and Technology,2008,227:7-15.
67.Je J Y, Park P J, ByunH G,et al. Angiotensinl converting enzyme(ACE) inhibitory PePtide derived from the sauce of fermented blue mussel (Mytilus edulis). Bioresource Technology.2005,96:1624-1629.
68.Jeanette otte, Shalaby, Mila Zakora, Are H.Pripp and Sayed A.El-Shabrawy. Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis.International Dairy Journal,2007, 17(5):488-503.
69. Jung W K, Mendis E, Je J Y, Park P J, Son B W, Kim H C,Choi Y K,& Kim S K. 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.
70.Katayama K, Tomatsu M, Fuchu H, Sugiyama M, Kawahara S, Yamauchi K, Kawamura Y,& Muguruma M. Purification and characterization of an angiotensin I-converting enzyme inhibitory peptide derived from porcine troponin C.Animal Science Journal,2003,74:53-58.
71.Katayama K, Tomatsu M, Kawahara S,Yamauchi K, Fuchu H, Kodama Y, Kawamura Y,& Muguruma M.Inhibitory profile of nonapeptide derived from porcine troponin C against angiotensin I-converting enzyme. Journal of Agricultural and Food Chemistry,2004,52:771-775.
72. Kohama Y, Matsumoto S,Oka H T. Isolation of angiotensin-converting enzyme inhibitor from tuna musele. Biochem Biophys Res Commun.1988,155:332-337.
73.Koji NAKADE, Ryosuke KAMISHIMA, Yusuke INOUE, Abdulatef AHHMED, Satoshi KAWAHARA,Tatsuo NAKAYAMA, Masugi MARUYAMA, Masahiro NUMATA, Kazuyoshi OHTA, Takayoshi AOKI,& Michio MUGURUMA. Identification of an antihypertensive peptide derived from chicken bone extract. Animal Science Journal,2008,79:710-715.
74.Li G, Le G, Shi Y,& Shrestha S.Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects.Nutrition Research,2004,24:469-486.
75.Matsufuji H, Matsui T, Seki E, Osajima K, Nakashima M,& Osajima Y. Angiotensin Ⅰ converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle.Bioscience Biotechnology and Biochemistry,1994,58:2244-2245.
76.Matsui T & Kawasaki T. Antihypertensive effects of bioactive peptides derived from protein.Journal of Japanese Society of Nutrition and Food Science,2000, 53:77-85.
77.Miguel M, Lopez-Fandino R, Ramos M,& Aleixandre A.Short-term effect of egg-white hydrolysate products on Journal of Nutrition,2005,94:731-937.
78.Muguruma M, Katayama K, Fuchu H,& Sugiyama M.Peptic hydrolysate of porcine crude myosin has many active fractions inhibiting angiotensin I-converting enzyme. Asian-Australasian Journal of Animal Science,2003,16: 1384-1389.
79. Muguerza B,Ramos M, Sa'nchezE, et al.Antihypertensive aetivity of milks fermented by Enterococcus faecalis strains isolated from raw milk. Iniernational Dairy Journal.2006,16:61-69.
80.Masuda O,Nakamura Y, TakanoT.Antihypwrtensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive Rats.Nutrient Metabolism.1996,26:3063-3068.
81.Nakamura Y, Masuda O, Takano T. Decrease of tissue Angiotensin 1-converting enzyme activity upon feeding sour milk in sponianeously hypertensive rats. Biosci Biotech Biochem.1996,60:488-489.
82.Otte J, Shalaby Samah M A, Zakora M & Nielsen M S.Fractionation and identication of ACE-inhibitory.peptides from a-lactalbumin and b-casein produced by thermolysin-catalysed hydrolysis.International Dairy Journal,2007, 17(12):1460-1472.
83. Saiga A, Okumura T, Makihara T et al.Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract. Journal of Agricultural and Food Chemistry,2003,51:1741-1745.
84.Saito Y, Wanezaki K,& Kawato A.Structure and angiotensin Ⅰ-converting enzyme inhibitory peptides from sake and sake lees.Biosce Biotech Bioehem, 1994,58:1767-1771.
85.Suetsuna K & Nakano T. Identication of anantihypertensive peptide fromPeptic digest of wakame (Undaria pinnatida).The Journal of Nutritional Biochemistry, 2000,11(9):450-454.
86. Suetsuna K, Yamagami M, Kuwata K, et al.Inhibitory activity against Angiotensin Ⅰ-Conerting Enzyme of peptides originating from fish and shellfish musele[J].Nippon Suisan Gakkaishi,1988,54(10):1852-1856.
87.Suetsuna K. Isolation and eharaeterization of angiotensin Ⅰ-converting enzyme inhibitor dipeptides derived from allium sativum. Nutr Bioehem.1998, 9:415-419.
88.Taguchi T & Yokoyama Y Wu.Quality Engineering Series,1993, Vol.4. Taguchi Methods:Design of Experiments (ASI Press,Tokyo).
89.Won-Kyo Jung, EreshaMendis, Jae-Young Je, et al.Angiotensin Ⅰ-eonverting enzyme inhibitory peptide from yellowfin sole(Limanda aspera)frame protein and its antihypertensive effeet in spontaneously hypertensive rats.Food Chemistry, 2006,94:26-29.
90.Ukeda H,Matsuda H, Osajima K. Peptides from peptic hydrolyzate of heated sardine meat that inhibit angiotensin I-converting enzyme.Nippon Nogeikagaku Kaishii,1992,66:25-29.
91.Wu J,& Ding X.Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats.Journal of Agricultural and Food Chemistry,2001,49:501-506.
92.Zhang J H, Tatsumi E,& Ding C H. Angiotensinl-eonverting enzyme inhibitory peptides in douche,a Chinese traditional fermented soybean produet. Food Chem, 2006,98:551-557.
2.曹文红.酶解中国毛虾蛋白制备血管紧张素转换酶抑制肽的研究.[硕士学位论文].湛江海洋大学,2002：54-60.
3.陈兰英,田敏,谢贵福等.猪肺血管紧张素转化酶的提纯.生物化学杂志,1988,4(4)：345-350.
4.陈新,陈庆森,庞广昌.Alcalase AF 2.4L酶解玉米蛋白动力学研究.食品科学,2004,25(10)：29-32.
5.陈斌,裴国献,刘晓霞.骨组织结构显微形态.第一军医大学学报,2005,25(4)：403-406.
6.陈瑞锋,陈璐,陈廷登.用活性炭分离纯化苯丙氨酸与酪氨酸的研究.浙江工业大学学报,2006,34(4)：373-376.
7.程云辉,王璋,许时婴.大孔吸附树脂对麦胚肽的吸附特性研究.食品与机械,2005,21：7-12.
8.Cheison S C,王璋,许时婴.大孔吸附树脂提取苦味乳清蛋白水解物及其功能性质和生物活性的研究.食品科学,2006,27：103-107.
9.丁小燕,张雯,陈延锋等.复合风味蛋白酶水解鸡骨泥工艺条件的研究.中国食品学报.2006(3)：88-92.
10.董瑞新,闰循领.胶原蛋白温度效应的Ranlall光谱研究.光谱学与光谱分析,2004,24(11)：1367-1369.
11.郭明勋,史玉敏.骨的成分与胶原蛋白.明胶科学与技术,2001,21(4)：204-209.
12.韩继福,任建波,王海波等.活性炭吸附芳香族氨基酸制备高F值寡肽混合物的研究.安徽农业大学学报,2005,32(1)：77-80.
13.郝记明,郭顺堂,章超桦.酶法水解马氏珠母贝肉制备血管紧张素转换酶抑制肽的研究.[硕士学位论文].中国农业大学,2005.
14.黄素珍.超微鲜骨泥食品得研究与开发.中国食品与畜产科学,2003,10(4)：149-151.
15.黄朝明,孔繁东,祖国仁等.降血压肽分离的研究现状与展望.食品研究与开 发,2006,27(4)：161-164.
16.黄晓任,刘邻渭.食品化学综合实验.北京：中国农业出版,2002.
17.蒋挺大.胶原与胶原蛋白.[博士学位论文].化学工业出版社,2006.
18.姬莹莹,范远景.珠蛋白提取、ACE抑制肽的酶法制备及酶动力学的研究.[硕士学位论文].合肥工业大学,2007.
19.李帆,贾冬英,姚开等.牦牛骨蛋白的酶解条件研究.氨基酸和生物资源,2006,28(4)：7-10.
20.李培骏,袁永俊,胡婷等.胰蛋白酶水解酪蛋白进程研究.食品与机械,2005,11.
21.廖丹葵.鸡蛋蛋黄蛋白质制备降血压肽的研究.[硕士学位论文].广西大学,2006.
22.刘忠义.草鱼肠道胰蛋白酶同工酶GT-A的动力学和热力学性质.西北农林科技大学学报,2008,36(1)：211-217.
23.吕炜锋,储瑞霭,金建勤等.用活性炭分离纯化苯丙氨酸的研究.中国生化药物杂志,1997,18(2)：82-83.
24.任建东,乐国伟.酶解鹿茸血疏水性肽的分离及活性研究.[硕士学位论文].江南大学,2008.
25.孙淑斌,王传怀.活性炭色谱法分离纯化苯丙氨酸的研究.氨基酸和生物资源,1995,17(1)：9-12.
26.汤鸣强,郑挺,曾小芳等.酿造酱油米曲霉产中性蛋白酶提取与酶学性质研究.中国调味品,2008,2：38-50.
27.王英超,李天俊,王敏华等.羊骨酶解物中降血压肽的分离、纯化工艺研究.食品研究与开发,2007,28(9)：7-10.
28.吴建平,丁霄霖.食品蛋白质降血压肽的研究进展.中国粮油学报,1998,13(5)：10-14.
29.夏杨毅.超微粉碎骨泥的流变学加工特性研究.[博士学位论文].西南农业大学,2005.
30.杨桂苹.骨粉营养成分的分析.食品科技,1997,6：41-43.
31.杨严俊,吉川正明.高效液相色谱分离纯化血管紧张素转换酶活性抑制肽.色谱,2003,21(3)：202-205.
32.余奕珂,胡建恩,白雪芳等.胃蛋白酶水解珠蛋白的研究.河南工业大学学报(自然科学版),2006,6：45-56.
33.袁勤生,赵健.酶与酶工程.[博士学位论文].上海：华东理工大学出版社,2005.
34.袁玉燕,韩爱军.超细鲜骨粉的粒度及营养特性.食品工业,2001,3：10-12.
35.王朝旭.酶法水解骨蛋白最佳条件的研究.食品科学,2001(2)：48-49.
36.王梅,谷文英,沈蓓英.活性炭色谱法分离制备高F值寡肽混合物.无锡轻工大学学报,1998,17(4)：41-4576.
37.王喜波,迟玉杰.活性炭色谱法分离芳香族氨基酸制备高F值蛋清低聚肽混合物的研究.食品工业科技,2005,26(6)：55-57.
38.王英超,李天俊,王敏华等.羊骨酶解物中降血压肽的分离、纯化工艺研究.食品研究与开发,2007,28(9)：7-10.
39.伍先绍,贺稚非,刘琳等.碱性蛋白酶产生菌株的筛选及其酶学性质研究进展.中国食品添加剂,58-61.
40.詹莉,刘书亮,吴琦.枯草芽孢杆菌弹性蛋白酶的纯化及酶学性质的研究.[硕士学位论文].四川农业大学,2008.
41.张昊宇,王强.花生短肽制备及其功能活性研究.[博士学位论文].中国农业科学院,2007.
42.张永秀,王世平.牛骨蛋白的酶解研究.[硕士学位论文].中国农业大学,2006.
43.张青,许学书,谢静莉等.米曲霉蛋白酶动力学特性的研究.食品科学,2007,28(6)：213-217.
44.张焱.大豆蛋白ACE抑制肤的酶法制备及分离纯化.[硕士学位论文].合肥工业大学,2007.
45.张卓睿.高电压脉冲电场作用下牛骨钙快速离子化的研究.[博士学位论文].吉林大学,2006.
46.赵谋明,任娇艳,崔春等.活性炭静态吸附草鱼蛋白水解物种氨基酸特性的研究.食品与机械,2005,21(6)：13-16.
47.赵谋明,崔春,刘珊.低值鱼蛋白酶解产物制备不同肉香型热反应物.食品与生物技术学报.2006(5)：1-6.
48.赵霞,马俪珍.羊骨的酶解和多肽饮料的开发.[硕士学位论文].山西农业大学, 2004.
49.赵一明,王璋.酪蛋白非磷肽的制备及其ACE抑制活性的研究.[硕士学位论文].江南大学,2008.
50.钟芳,张晓梅,麻建国.大豆肽的大孔吸附树脂以及凝胶过滤色谱分离.食品与机械,2006,22：25-28.
51.朱丽娟,熊幼翎.玉米蛋白水解物体外消化产物的抗氧化及ACE抑制活性研究.[硕士学位论文].江南大学,2008.
52.Atkinson P J, Hallsworth A S.The spatial structure of bone. In:Harrision RJ, Navartman V, eds.Progress in Anatomy [PhD dissertation].Cambridge:Cambridge University Press,1982:179-99.
53.Atkins P W. The Elements of Physical Chemistry, Oxford University Press, 1996:354-362.
54. Astawan M, Wathuni M, Yasuhara T, et al. Effect of Angiotensin I-Converting Enzyme inhibitory substance derived Indonesian dried-salted fish on blood pressure of rats[J].Biosci Biotech Bioehem,1995,59(3):425-428.
55.BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of proteindye binding. Anal Biochem,1976, 72:248-254.
56. Chen J R, Yang S C, Suetsana K,& Chao J C J. Soybean protein-derived hydrolysate affects blood pressure in spontaneously hypertensive rats. Journal of Food Biochemistry,2004,28:61-73.
57. Cushman D W,& Cheung H S.Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology,1971, 20:1637-1648.
58.Erdos E G & Skidgel R A.The angiotensin Ⅰ-converting enzyme.Laboratory Investigation,1987,56:345-353.
59.Fujita H, Yoshikawa M. A prodrug type ACE inhibitory peptide derivedf rom fish protein. ImmunoPharmacology.1999,44:123-127.
60.Fujita H, Yokoyama K, Yoshikawa M. Classification and antihypertensive of angiotensin I-coverting enzyme inhibitory peptides derived from food proteins. Journal of Food Seienee.2000,65:564-5691.
61.Fujita H, Sasaki R, Yoshikawa M.Potentiation of the antihypertensive activity of orally, a vasorelaxing peptide derived from ovalbumin, by emulsifieation in egg hosphatidyleholine.Biosci Biotech Biochem.1995,59:2344-2345.
62.Fu-Yuan CHENG,Tien-Chun WAN, Yu-Tse LIU, Kung-Ming LAI, Liang-Chuan LIN,& Ryoichi SAKAT. A study of in vivo antihypertensive properties of enzymatic hydrolysate from chicken leg bone protein. Animal Science Journal, 2008,79:614-619.
63. Fu-Yuan CHENG, Tien-Chun WAN,Yu-Tse LIU,Chi-Ming CHEN,Liang-Chuan LIN,& Ryoichi SAKATA.Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase. Animal Science Journal,2009,80:91-97.
64. Ge shijun. Continuous Production of high degree case in hydrolysates by immobilized proteases in column reaetor.Journal of Biotechnology.1996,50: 161-170.
65.Gouda K G M, Gowda L R, Rao A G A,& Prakash V. Angiotensin I-converting enzyme inhibitory peptise derived from glycinin, the 11s globulin of soybean (Glycine max).Journal of Agriculture and Food Chemistry,2006,54:4568-4573.
66.Haque E& Chand R. Antihypertensive and antimicrobial bioactive peptides from milk proteins.European Food Research and Technology,2008,227:7-15.
67.Je J Y, Park P J, ByunH G,et al. Angiotensinl converting enzyme(ACE) inhibitory PePtide derived from the sauce of fermented blue mussel (Mytilus edulis). Bioresource Technology.2005,96:1624-1629.
68.Jeanette otte, Shalaby, Mila Zakora, Are H.Pripp and Sayed A.El-Shabrawy. Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis.International Dairy Journal,2007, 17(5):488-503.
69. Jung W K, Mendis E, Je J Y, Park P J, Son B W, Kim H C,Choi Y K,& Kim S K. 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.
70.Katayama K, Tomatsu M, Fuchu H, Sugiyama M, Kawahara S, Yamauchi K, Kawamura Y,& Muguruma M. Purification and characterization of an angiotensin I-converting enzyme inhibitory peptide derived from porcine troponin C.Animal Science Journal,2003,74:53-58.
71.Katayama K, Tomatsu M, Kawahara S,Yamauchi K, Fuchu H, Kodama Y, Kawamura Y,& Muguruma M.Inhibitory profile of nonapeptide derived from porcine troponin C against angiotensin I-converting enzyme. Journal of Agricultural and Food Chemistry,2004,52:771-775.
72. Kohama Y, Matsumoto S,Oka H T. Isolation of angiotensin-converting enzyme inhibitor from tuna musele. Biochem Biophys Res Commun.1988,155:332-337.
73.Koji NAKADE, Ryosuke KAMISHIMA, Yusuke INOUE, Abdulatef AHHMED, Satoshi KAWAHARA,Tatsuo NAKAYAMA, Masugi MARUYAMA, Masahiro NUMATA, Kazuyoshi OHTA, Takayoshi AOKI,& Michio MUGURUMA. Identification of an antihypertensive peptide derived from chicken bone extract. Animal Science Journal,2008,79:710-715.
74.Li G, Le G, Shi Y,& Shrestha S.Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects.Nutrition Research,2004,24:469-486.
75.Matsufuji H, Matsui T, Seki E, Osajima K, Nakashima M,& Osajima Y. Angiotensin Ⅰ converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle.Bioscience Biotechnology and Biochemistry,1994,58:2244-2245.
76.Matsui T & Kawasaki T. Antihypertensive effects of bioactive peptides derived from protein.Journal of Japanese Society of Nutrition and Food Science,2000, 53:77-85.
77.Miguel M, Lopez-Fandino R, Ramos M,& Aleixandre A.Short-term effect of egg-white hydrolysate products on Journal of Nutrition,2005,94:731-937.
78.Muguruma M, Katayama K, Fuchu H,& Sugiyama M.Peptic hydrolysate of porcine crude myosin has many active fractions inhibiting angiotensin I-converting enzyme. Asian-Australasian Journal of Animal Science,2003,16: 1384-1389.
79. Muguerza B,Ramos M, Sa'nchezE, et al.Antihypertensive aetivity of milks fermented by Enterococcus faecalis strains isolated from raw milk. Iniernational Dairy Journal.2006,16:61-69.
80.Masuda O,Nakamura Y, TakanoT.Antihypwrtensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive Rats.Nutrient Metabolism.1996,26:3063-3068.
81.Nakamura Y, Masuda O, Takano T. Decrease of tissue Angiotensin 1-converting enzyme activity upon feeding sour milk in sponianeously hypertensive rats. Biosci Biotech Biochem.1996,60:488-489.
82.Otte J, Shalaby Samah M A, Zakora M & Nielsen M S.Fractionation and identication of ACE-inhibitory.peptides from a-lactalbumin and b-casein produced by thermolysin-catalysed hydrolysis.International Dairy Journal,2007, 17(12):1460-1472.
83. Saiga A, Okumura T, Makihara T et al.Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract. Journal of Agricultural and Food Chemistry,2003,51:1741-1745.
84.Saito Y, Wanezaki K,& Kawato A.Structure and angiotensin Ⅰ-converting enzyme inhibitory peptides from sake and sake lees.Biosce Biotech Bioehem, 1994,58:1767-1771.
85.Suetsuna K & Nakano T. Identication of anantihypertensive peptide fromPeptic digest of wakame (Undaria pinnatida).The Journal of Nutritional Biochemistry, 2000,11(9):450-454.
86. Suetsuna K, Yamagami M, Kuwata K, et al.Inhibitory activity against Angiotensin Ⅰ-Conerting Enzyme of peptides originating from fish and shellfish musele[J].Nippon Suisan Gakkaishi,1988,54(10):1852-1856.
87.Suetsuna K. Isolation and eharaeterization of angiotensin Ⅰ-converting enzyme inhibitor dipeptides derived from allium sativum. Nutr Bioehem.1998, 9:415-419.
88.Taguchi T & Yokoyama Y Wu.Quality Engineering Series,1993, Vol.4. Taguchi Methods:Design of Experiments (ASI Press,Tokyo).
89.Won-Kyo Jung, EreshaMendis, Jae-Young Je, et al.Angiotensin Ⅰ-eonverting enzyme inhibitory peptide from yellowfin sole(Limanda aspera)frame protein and its antihypertensive effeet in spontaneously hypertensive rats.Food Chemistry, 2006,94:26-29.
90.Ukeda H,Matsuda H, Osajima K. Peptides from peptic hydrolyzate of heated sardine meat that inhibit angiotensin I-converting enzyme.Nippon Nogeikagaku Kaishii,1992,66:25-29.
91.Wu J,& Ding X.Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats.Journal of Agricultural and Food Chemistry,2001,49:501-506.
92.Zhang J H, Tatsumi E,& Ding C H. Angiotensinl-eonverting enzyme inhibitory peptides in douche,a Chinese traditional fermented soybean produet. Food Chem, 2006,98:551-557.