大豆低聚肽制备及其ACE抑制活性和螯合特性的研究
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摘要
大豆分离蛋白(SPI)作为一种植物源蛋白质,具有很高的营养价值,通过蛋白酶酶解SPI后得到的大豆低聚肽作为一种去抗营养因子、并具有一定生理活性的功能性食品基料,具有广泛的应用空间。利用低聚肽的特殊配体特征,开发新的微量元素添加剂,是增加大豆低聚肽应用范围的有益探索。本文在考虑大豆低聚肽利用方式的前提下,对利用木瓜蛋白酶和菠萝蛋白酶酶解大豆分离蛋白制备的大豆低聚肽的成分、酶解工艺优化条件、酶解产物体外ACE(血管紧张素转化酶)抑制活性组分筛选及其微量元素螯合物的制备工艺进行了研究,得到的具体结论如下:
酶解产物SDS-PAGE、HPLC和质谱分析结果表明两种酶的酶解产物分子量为1000Da左右,多数为低聚肽。酶解反应初始阶段酶浓度、底物浓度、酶解反应温度对SPI水解率影响的研究表明,在反应初始阶段存在着产物和底物对反应速率的抑制现象,此外反应温度对反应初始阶段速率有一定的提高效果。木瓜蛋白酶酶解SPI的Km值为0.1404g/L,Vmax为7.51 g·L-1·min-1,菠萝蛋白酶酶解SPI的Km值为0.1173g/L,Vmax为4.95 g·L-1·min-1。
通过对影响SPI酶解产物DH的主要因素:酶解时间、酶解温度、加酶量、pH、底物浓度的单因素和双因素实验,得出木瓜蛋白酶酶解SPI的最佳工艺为:酶解时间3h、酶解温度60℃、加酶量0.35g、pH7.5、底物浓度5g/100mL;菠萝蛋白酶酶解SPI的最佳工艺为:酶解时间10h、酶解温度40℃、加酶量0.35g、pH7.5、底物浓度5g/100mL。
对影响大豆低聚肽ACE活性相关因素的研究,通过研究得出木瓜蛋白酶酶解SPI的时间基本不影响低聚肽ACE抑制活性,但酶解72h以后的产物抑制活性最强;菠萝蛋白酶酶解SPI制备的大豆低聚肽抑制ACE活性随时间增加而减小。而底物中的小分子糖类会降低ACE抑制活性,此外通过研究得出木瓜蛋白酶和菠萝蛋白酶酶解制备的大豆低聚肽的IC50约在70μg/mL左右。通过HPLC和SphadexG-25对大豆低聚肽的分离和抑制活性的研究,得到高ACE抑制活性抑制组分,并对其进行了初步的分离纯化,得到出峰时间为29.527min的高活性组分。
通过研究影响大豆低聚肽金属离子螯合物的制备因素,得出大豆低聚肽铜螯合物的最佳工艺条件为:铜离子浓度0.67.0×10-4mol/L、时间10min、反应温度50℃、pH4.0、加入沉淀剂比例为1:2,最终螯合物收率为76.06%。大豆低聚肽锌螯合物的最佳工艺条件为:锌离子浓度0.67×10-4mo/Ll、时间10min、反应温度50℃、pH6.0、加入沉淀剂比例为1:1.75,最终收率为74.83%。铜低聚肽螯合物具有一定酸稳定性,当pH为3~4时,能够较好的以螯合物的形式存在。
The soy protein isolates (SPI) have high nutritional values. Soy oligo-peptides, which are the products of SPI hydrolysis by enzyme, are kinds of functional food-bases. The metal-chelate complex of soy oligo-peptides can increase the soy oligo-peptide application scopes on food and feed. In this paper, the main study is to found a method to preparie soy oligo-peptide from SPI with papain and bromelain.The enzyme hydrolysis process conditions, including the in vitro ACE (Angiotensin-converting enzyme) inhibitory activity of hydrolysate are the research contents . Seperation of the high activie components and preparation of soy oligo-peptides’metal-chelate complex are included in this paper. The results of the experiments were as follows:
a)Through the preliminary enzyme hydrolysis experiments, the enzyme hydrolysis products of SPI were analyzed by SDS-PAGE, HPLC and mass spectrometry, the results showed that the majority molecular weight of oligo-peptides of the two enzymes hydrolysis are about 1000Da. Among the relationship of the enzyme concentration, the substrate concentration, the reaction temperature and the hydrolysis degree of SPI,the hydrolysis products and the reaction substrates could inhibit the reaction rates during the initial reaction times. However the reaction temperature could increase the reaction rate at the same time. The Km of hydrolyzing SPI by Papain is 0.1404g/L and the Vmax is 7.51 g·L-1·min-1.The Km of hydrolyzing SPI by Bromelain is 0.1173g/L and the Vmax is 4.95 g·L-1·min-1.
b) The impact of the main factors on the enzyme reaction of SPI: enzymolysis time, hydrolysis temperature, enzyme concentration, pH, substrate concentration was studied according to the single-factor and two-factor experiment. The best process for papain were that recation time was 3h, hydrolysis temperature was 60℃, the amount of enzyme was 0.35g, pH was 7.5, substrate concentration was 5g/100mL. The best enzyme technology for bromelain were that enzymolysis time was 10h, digestion temperature was 40℃, the amount of enzyme was 0.35g, pH was 7.5, substrate concentration was 5g/100mL.
c)The study on related factors of impacting ACE inhibitory activity had shown that the proteinase hydrolysis time did not affect the ACE inhibitory activity of soy oligo-peptides deeply by papain and reached the peak at 72 hours, but the soy oligo-peptides by bromelain reducing the ACE inhibitory activity with increasing time. the small molecule carbohydrates would decreased ACE inhibitory activity. In addition, through the study of soy oligo-peptides prapared by papain and bromelain, the IC50 of hydrolysate were around 70μg/mL. Through separated soy oligo-peptide by HPLC and SphadexG-25 and study of inhibitory activity, A kind of high ACE inhibitory activity components was separated from the soy oligo-peptides by HPLC and SphadexG-25. The high active component was at 29.527min of HPLC.
d)The preparation of copper chelate and zinc chelate of soy oligo-peptides were studied. For copper chelate of soy oligo-peptides, the optimum reaction conditions were that the copper ion concentration was 0.67×10-4mol/L, the reactin time was 10min, the reaction temperature was 50℃, the pH was 4.0, the precipitation agent (ethanol) ratio was 1:2. The recovery ratio is 76.06%. For chelate zinc of soy oligo-peptides, the optimum reaction conditions were that the zinc ion concentration was 0.67×10-4mol/L, the reaction time was 10min, the reaction temperature was 50℃, the pH was 6.0, the precipitation agent (ethanol) ratio was 1:1.75, the recovery ratio was 74.83%.The copper chelate of Oligo-peptides shew that it had a certain degree of resistance to acid. The compound could exist in the form of chelate when the solvent pH was between 3 ~ 4.
酶解产物SDS-PAGE、HPLC和质谱分析结果表明两种酶的酶解产物分子量为1000Da左右,多数为低聚肽。酶解反应初始阶段酶浓度、底物浓度、酶解反应温度对SPI水解率影响的研究表明,在反应初始阶段存在着产物和底物对反应速率的抑制现象,此外反应温度对反应初始阶段速率有一定的提高效果。木瓜蛋白酶酶解SPI的Km值为0.1404g/L,Vmax为7.51 g·L-1·min-1,菠萝蛋白酶酶解SPI的Km值为0.1173g/L,Vmax为4.95 g·L-1·min-1。
通过对影响SPI酶解产物DH的主要因素:酶解时间、酶解温度、加酶量、pH、底物浓度的单因素和双因素实验,得出木瓜蛋白酶酶解SPI的最佳工艺为:酶解时间3h、酶解温度60℃、加酶量0.35g、pH7.5、底物浓度5g/100mL;菠萝蛋白酶酶解SPI的最佳工艺为:酶解时间10h、酶解温度40℃、加酶量0.35g、pH7.5、底物浓度5g/100mL。
对影响大豆低聚肽ACE活性相关因素的研究,通过研究得出木瓜蛋白酶酶解SPI的时间基本不影响低聚肽ACE抑制活性,但酶解72h以后的产物抑制活性最强;菠萝蛋白酶酶解SPI制备的大豆低聚肽抑制ACE活性随时间增加而减小。而底物中的小分子糖类会降低ACE抑制活性,此外通过研究得出木瓜蛋白酶和菠萝蛋白酶酶解制备的大豆低聚肽的IC50约在70μg/mL左右。通过HPLC和SphadexG-25对大豆低聚肽的分离和抑制活性的研究,得到高ACE抑制活性抑制组分,并对其进行了初步的分离纯化,得到出峰时间为29.527min的高活性组分。
通过研究影响大豆低聚肽金属离子螯合物的制备因素,得出大豆低聚肽铜螯合物的最佳工艺条件为:铜离子浓度0.67.0×10-4mol/L、时间10min、反应温度50℃、pH4.0、加入沉淀剂比例为1:2,最终螯合物收率为76.06%。大豆低聚肽锌螯合物的最佳工艺条件为:锌离子浓度0.67×10-4mo/Ll、时间10min、反应温度50℃、pH6.0、加入沉淀剂比例为1:1.75,最终收率为74.83%。铜低聚肽螯合物具有一定酸稳定性,当pH为3~4时,能够较好的以螯合物的形式存在。
The soy protein isolates (SPI) have high nutritional values. Soy oligo-peptides, which are the products of SPI hydrolysis by enzyme, are kinds of functional food-bases. The metal-chelate complex of soy oligo-peptides can increase the soy oligo-peptide application scopes on food and feed. In this paper, the main study is to found a method to preparie soy oligo-peptide from SPI with papain and bromelain.The enzyme hydrolysis process conditions, including the in vitro ACE (Angiotensin-converting enzyme) inhibitory activity of hydrolysate are the research contents . Seperation of the high activie components and preparation of soy oligo-peptides’metal-chelate complex are included in this paper. The results of the experiments were as follows:
a)Through the preliminary enzyme hydrolysis experiments, the enzyme hydrolysis products of SPI were analyzed by SDS-PAGE, HPLC and mass spectrometry, the results showed that the majority molecular weight of oligo-peptides of the two enzymes hydrolysis are about 1000Da. Among the relationship of the enzyme concentration, the substrate concentration, the reaction temperature and the hydrolysis degree of SPI,the hydrolysis products and the reaction substrates could inhibit the reaction rates during the initial reaction times. However the reaction temperature could increase the reaction rate at the same time. The Km of hydrolyzing SPI by Papain is 0.1404g/L and the Vmax is 7.51 g·L-1·min-1.The Km of hydrolyzing SPI by Bromelain is 0.1173g/L and the Vmax is 4.95 g·L-1·min-1.
b) The impact of the main factors on the enzyme reaction of SPI: enzymolysis time, hydrolysis temperature, enzyme concentration, pH, substrate concentration was studied according to the single-factor and two-factor experiment. The best process for papain were that recation time was 3h, hydrolysis temperature was 60℃, the amount of enzyme was 0.35g, pH was 7.5, substrate concentration was 5g/100mL. The best enzyme technology for bromelain were that enzymolysis time was 10h, digestion temperature was 40℃, the amount of enzyme was 0.35g, pH was 7.5, substrate concentration was 5g/100mL.
c)The study on related factors of impacting ACE inhibitory activity had shown that the proteinase hydrolysis time did not affect the ACE inhibitory activity of soy oligo-peptides deeply by papain and reached the peak at 72 hours, but the soy oligo-peptides by bromelain reducing the ACE inhibitory activity with increasing time. the small molecule carbohydrates would decreased ACE inhibitory activity. In addition, through the study of soy oligo-peptides prapared by papain and bromelain, the IC50 of hydrolysate were around 70μg/mL. Through separated soy oligo-peptide by HPLC and SphadexG-25 and study of inhibitory activity, A kind of high ACE inhibitory activity components was separated from the soy oligo-peptides by HPLC and SphadexG-25. The high active component was at 29.527min of HPLC.
d)The preparation of copper chelate and zinc chelate of soy oligo-peptides were studied. For copper chelate of soy oligo-peptides, the optimum reaction conditions were that the copper ion concentration was 0.67×10-4mol/L, the reactin time was 10min, the reaction temperature was 50℃, the pH was 4.0, the precipitation agent (ethanol) ratio was 1:2. The recovery ratio is 76.06%. For chelate zinc of soy oligo-peptides, the optimum reaction conditions were that the zinc ion concentration was 0.67×10-4mol/L, the reaction time was 10min, the reaction temperature was 50℃, the pH was 6.0, the precipitation agent (ethanol) ratio was 1:1.75, the recovery ratio was 74.83%.The copper chelate of Oligo-peptides shew that it had a certain degree of resistance to acid. The compound could exist in the form of chelate when the solvent pH was between 3 ~ 4.
引文
1.曹玉华,杨慧萍.固定化胰蛋白酶制备大豆肽正交实验的研究.食品科技,2003,10:33~35.
2.陈成,刘文玉,杨青.大豆蛋白活性肽的生物学功能及其应用.黑龙江农业科学, 2004, (3): 40~42.
3.陈历俊,卢阳,骆承庠,刘爱萍.连续搅拌罐膜反应器(CSTMR)法生产大豆肽动力学研究食品工业与科技,2003,24(6):26~28.
4.陈美珍,余杰,郭慧敏.大豆分离蛋白酶解物清除羟自由基作用的研究.食品科学, 2002, 23(1): 43~46.
5.范远景,姬莹莹,张焱大豆蛋白酶解肽的分子量分布及抑制ACE活性关系研究.食品科学,2007,28(10):57~61.
6.高荣丽,陶冠军,杨严俊.葵花籽粕的综合利用.食品工业科技,2006,27(7):138~140.
7.高素蕴,潘思轶,郭康权.大豆分离蛋白水解物螯合锌(II)的合成与制备.食品科学,2003,24(10):117~120.
8.龚树立,文剑,吴逸民,杨玉铮,杨则宜,周丽丽,高红,徐葆华,李肃反.大豆多肽运动饮料的研制,食品与发酵工业,2003,29(4):86~87.
9.龚树立.大豆多肽研究概况及其在运动饮料中的应用.食品与发酵工业,2004,30(6):112~116.
10.郭敏亮,陈军,姜勇明,郑杰军.用豆粕生产大豆蛋白肽饮料.食品科学,1992,10:1~3.
11.郭兴风.蛋白质水解度的测定.中国油脂,2000,25(6):176~177.
12.国家食品药品监督管理局.保健食品注册管理办法(试行).2005.
13.国明明,华欲飞.大豆肽免疫调节作用的研究.食品科技,2007,7:242~244.
14.韩飞,于婷婷,周孟良,酶法生产大豆蛋白ACE抑制肽的研究.食品科学,2008,29(11):369~374.
15.厚生省.食品与科学(日),1989,31:41.
16.金英姿,庞彩霞.玉米蛋白的深加工及应用.新疆农业科技, 2004(3): 10.
17.瞿水忠,周瑞宝.饲用菜籽蛋白营养特性的研究.郑州工程学院学报.1992,1:51~60.
18.李书国,陈辉,庄玉亭,李雪梅.复合酶法制备活性大豆寡肽研究.粮食与油脂,2001,3:5~7.
19.李艳玲,李松彪.棉籽蛋白的开发利用.饲料广角.2001,15:6~8.
20.梁金钟,范洪臣,程丽,王淑杰.微生物液态发酵法生产大豆蛋白活性肽的研究.食品与发酵工业,2008,34(1):88~92.
21.刘炳智.大豆肽及其开发应用前景.日用化学工业.1997,1:61~62.
22.刘佳,王兴国,杨严俊.酶法制备大豆蛋白降血压肽的研究.中国油脂,2008,33(9):6~9.
23.刘明,倪辉,吴永沛.大豆抗氧化活性肽研究进展.食品科学,2006, 27(12):897~900.
24.刘忆梅,陈朝晖.大豆蛋白肽降血脂功能性的研究.大豆通报,2004,3:22.
25.刘志伟,刘秀梅,郑梦云.大豆蛋白体外酶解物中血管紧张素转化酶抑制剂活性肽研究.营养学报,2007 ,29 (1):69~73.
26.陆旋,朱正华.家蚕丝素蛋白铜元素螯合物的制备及其对动物生理影响研究.蚕桑通报,2005,36(3):16~20.
27.梅娜,周文明,胡晓玉,李玲玲.花生粕营养成分分析.西北农业学报,2007,16(3):96~99.
28.宁正祥.食品成分分析手册.北京,中国轻工业出版社,1998:119~124.
29.邱冬玲,胡长利,崔建云.丝素肽与锌螯合工艺条件的研究.食品研究与开发,2008,29(7):38~42.
30.沈仓良,姚文,陈伟华,朱伟云,邹思湘.伴大豆球蛋白水解肽对灌喂大肠杆菌( E.coli)的小鼠肠道微生物区系和健康的影响.中国现代医学杂志,2006,16(3):354~358.
31.孙占田,计成,王碧莲.小肽的吸收机制及在动物生产中的应用研究.饲料研究,2007,1:28~31
32.檀志芬,生庆海,邱泉若,刘建光.蛋白质水解度的测定方法.食品工业科技,2005,26(7):174~175.
33.陶红,梁岐.双酶水解降低大豆寡肽苦味研究.食品工业科技,2003,(增刊):35~37
34.田仁林.蓖麻子的成分和化学.中国油脂,1981(S1):118~123.
35.王芳.微生物发酵法水解大豆蛋白制备大豆蛋白活性肽的研究.黑龙江粮食,2008(4):41~43.
36.王镜岩,朱圣庚,徐长法.生物化学(上册).北京:高等教育出版社,2002:320 ~432.
37.王丽彬,张弢,王旻.高纯度木瓜蛋白酶的分离纯化和性质研究.中国生化药物杂志,2006,27(3):159~162.
38.王世英.阿片肽研究的回顾和展望[J].生物学通报, 2001,36(1): 5~7.
39.卫琳,宋俊梅,宁维颖.米曲霉固态发酵豆粕制备大豆肽的研究.粮食加工,2009,34(1):34~36
40.吴建平.大豆降血压肽的研制.中国油脂,1998,23(5):22~25.
41.吴炜亮,吴国杰,梁道双杨帆. ACE抑制肽的生理功能和研究进展.现代食品科技,2006,22(3):251~254.
42.徐广飞,黄金华,吴杰,仇梁林.大豆低聚肽对自发性高血压大鼠降血压作用的研究中华老年多器官疾病杂志,2004,3(4):288~291
43.徐力,赵忠岩,李鸿梅,王智,黄宜兵,张学忠.大豆蛋白的小分子酶解产物抗氧化活性研究.吉林农业大学学报,2007,29(1):48~52.
44.曾琦斐.微量元素与人体健康.中国科技信息.2008,3:158~159
45.张国胜,孔繁东,祖国仁等.大豆蛋白抗高血压活性肽的研究.中国乳品工业, 2004, 32(8)12~14.
46.张全才,熊华,曹银娣,李庭,齐金峰.米蛋白肽锌的制备工艺研究.食品科技2008,33(2):110~133
47.张延坤,刘炳智.大豆肽在食品工业中的应用.食品工业,1997,(3):5~6.
48.赵东海;张洪;黄建韶.蚕豆蛋白提取工艺的研究.食品与机械.2005,2:32~33.
49.赵秀娟,王小雪,吴博学,宋会娟.大豆活性肽粉对喂饲高血脂饲料大鼠的影响.中国卫生检疫杂志,2002,12(8):421~422.
50.郑建仙,高孔荣.功能性食品及其在我国的开发前景.食品机械,1994, 41(3):6~10.
51.郑炯.血红蛋白多肽螯合铁的制备及其抗贫血功能研究[硕士学位论文].西南农业大学.2008
52.朱桂红.快速配制铬黑T指示剂方法.湖南造纸,2005,2:37.
53.朱建华.豌豆蛋白质的特性.粮油食品科技.1996,4:32~33.
54.左伟勇,陈伟华,邹思湘.伴大豆球蛋白胃蛋白酶水解肽对小鼠免疫功能及肠道内环境的影响南京农业大学学报,2005, 28 (3) : 71～74.
55.左伟勇,陈伟华,邹思湘.伴大豆球蛋白胃蛋白酶水解肽对双歧杆菌增值的影响.食品与发酵工业,2004,30(5):10~13.
56. A..Constantinides, B. Adu-Amankwa Enzymatic modification of vegetable protein: Mechanism, kinetics, and production of soluble and partially soluble protein in a batch reactor. Biotechnology and Bioengineering , 1978, 22 (8):1543~1565.
57. A.G.Marangoni. Enzyme Kinetics-A Modern Approach. New York, John Wiley & Sons,2001.
58. Additions and Corrections. The Complete Amino Acid Sequence of Papain. The Journal of Biological Chemistry1970, 245(14):3485~3492.
59. Adler-Nissen J. Determination of the degree of hydrolysis of food protein hydrolysate by trinitrobenzensulfonic acid. Journal of Agriculture food chemistry,1979,27(6):1256~1262.
60. Adler-Nissen J. Enzymatic hydrolysis of food protein. NEW York,Elsevier Applied Science Publishers,1986.
61. Adler-Nissen J. Enzymatic hydrolysis of soy protein for nutritional fortification of low pH food. Ann Nutrition Aliment,1978,32(2~3):205~216.
62. Ajjipura B Hemavathi, Hangalore Umesh Hebbar and Karumanchi SMS Raghavarao. Reverse micellar extraction of bromelain from Ananas comosus L.Merryl. Journal of Chemical Technology and Biotechnology, 2007 (82): 985~992.
63. Andrés Moure, Herminia Domínguez, Juan Carlos Parajó. Antioxidant properties of ultrafiltration-recovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochemistry , 2006,41(2): 447~456.
64. B.Adu-Amankwa, A. Constantinides, W. R. Vieth Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor. Biotechnology and Bioengineering, 1981, 23(11):2609~2627.
65. Bernard P.O’Hara, Andrew, M. Hemmings, David J. Buttle, and Laurence H. Pearl.Crystal structure of-glycyl endopeptidase from Carica papaya:a cysteine of unusual substrate specificity. Biochemistry,1995,34:13190~13195.
66. Bradley W. Bolling, Jeffrey B. Blumberg, C.-Y. Oliver Chen. Extraction methods determine the antioxidant capacity and induction of quinone reductase by soy products in vitro. Food Chemistry, 2009, 116(1):351~355.
67. CHEN Cui-lin, SHI Yan-guo, WANG Zhen-yu, ZHAO Hai-tian. Effect of protein subunits and 7S/11S on functional properties of SPI. Food science, 2006, 27(3):70~74.
68. Chen H M.Muramoto K.Y amauchi F, et al. Antioxidative properties of histidine containing peptides desinned from peptide fragments found in the digests of soybean protein. Journal of Agriculture Food Chemistry,1998, 46: 49~53.
69. Chen J R,Kunio S,Fumio Y.Isolation and characterization of immunostimulative peptides fromsoybean. Journal of Nutrition Biochemistry,1995,6(6):310~313.
70. D.Spellman, E.McEvory, G.O’Cuinn, R.J.FitzGerald. Proteinase and exopeptidase hydrolysis of whey protein: comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. International Dairy Journal, 2003, 13:447~453.
71. Danji Fukushima. Recent progress of soybean protein foods: chemistry, technology and nutrition. Food Review International, 1991,7(3): 323~351.
72. Fang Zhong, Jianmin Liu, Jianguo Ma, Charles F. Shoemakerb. Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates.Food Research International, 2007, 40( 6):756~762.
73. Fang Zhong, Jianmin Liu, Jianguo Ma, Charles F. Shoemakerb. Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice.Food Research International, 2007, 40(6):661~667.
74. Fouad M.T. Chelation and chelated minerals. Journal of Applied Nutrition, 1974,26:6.
75. Fujimaki M, Kato H, Arai S, Yamashita M. Symposium on microbial changes in foods, Application of microbial proteases to soybean and other materials to improve acceptability, especially through the formation of plastein. Journal of Apply Bacteriol,1971,34(4):119~131.
76. Hsin-Yi Yang, Suh-Ching Yang, Shu-Tzu Chen, Jiun-Rong Chen .Soy protein hydrolysate ameliorates cardiovascular remodeling in rats with l-NAME-induced hypertension. The Journal of Nutritional Biochemistry, 2008,19(12): 833~839.
77. J. Dziuba, P. Minkiewicz, D. and Na?ecz, A. Iwaniak. Database of biologically active peptide sequences. Nahrung,1999,43(3):190~l95.
78. 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.
79. Laura P. Hale, Paula K. Greer, Chau T. Trinh, Cindy L James. Proteinase activity and stability of natural bromelain preparations. International Immuopharmacology, 2005, 5:783~793.
80. Lei Wei, Eric Clauser, Frangoi As lhenc-Gelas, and PierreC orvolThe Two Homologous Domains of Human Angiotensin I-convertingEnzyme Interact Differently with Competitive Inhibitors. Journal of Biological Chemistry,1992, 267(19): 13398~13405.
81. Lovati M R, Manzoni C, Gianazza E,etal. Soy protein peptides regulate cholesterol homeostasis in Hep G2 cells. Journal of Nutrition, 2000, 130: 2543~2549.
82. Lynch GL, van der Aar PJ, Berger LL, Fahey GC Jr, Merchen NR. Proteolysis of alcohol-treated soybean meal proteins by Bacteroides ruminicola, Bacteroides amylophilus, pepsin, trypsin, and in the rumen of steers. Journal of Dairy Science. 1988, 71(9):2416~2427.
83. M. Kuba, C. Tana, S. Tawata and M. Yasuda. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase .Process Biochemistry, 2005,40(6): 2191~2196.
84. Manzoni C, Lovati M R, Gianazza E,et al. Soybean protein products as regulators of liverlow-density lipoprotein receptors. II. 1α-α' rich commercial soy concentrate andα' deficient mutant differently affect tlow-density lipoprotein receptoractivation. Journal of Agriculture Food Chemistry, 1998,46:2481~2484.
85. María D. Mesa, Jose M. Silván, Josune Olza,ángel Gila , María D. del Castillob, Antioxidant properties of soy protein–fructooligosaccharide glycation systems and its hydrolyzates Food Research International, 2008,41(6):606~615.
86. Megumi Kuba, Kumi Tanaka, Shinkichi Tawata, Yasuhito Takeda. AngiotensinⅠ-Converting Enzyme inhibitory peptides isolated from Tofuyo fermented soybean food. Bioscience, Biotechnology and Biochemistry.2003,67(6):1278~1283.
87. Min-Liang Guo, Yong-Ming Jiang, Zhong-Liang Ma and Yan-Li Li. Hydrolytic characteristics of chitosan-immobilized As 1.398 neutral proteinase (from B. subtilis) to soybean protein. Food Chemistry, 1996,55(4): 373~377.
88. Munir Cheryan, David Deeslie. Soy protein hydrolysis in membrane reactors. Journal of the American Oil Chemists' Society,1983,60(6):1112~1115.
89. Nagaoka S,Awano R,Nagata N,et a1.Serum cholesterol re—duction and cholesterol absorption inhibition in Caco-2cells by a soy protein peptide hydrolyzate.Bioscience, Biotechnology and Biochemistry, 1997,61:354~356
90. P.M. Nielsen, D. Petersen, C. Dambmann. Improved Method for Determining Food Protein Degree of Hydrolysis. Journal of Food Science, 2006, 66 ( 5):642~646.
91. Qinyun Chen. Effect of Amide contents in wheat gluten hydrolysates on the thermal flavor generation ,ph. D. thesis ,Rutgers university , 1995 :54 ~56.
92. R. Bhattacharya and D. Bhattacharyya. Preservationof natural stability of fruit bromelain from Ananas comosus L (pineapple). Journal of Food Biochemistry,2009, (33) :1~19.
93. Satoshi Nagaoka1, Keiji Miwa, Michiko Eto, Yasuo Kuzuya, Goro Hori ,Kazuhiro Yamamoto. Soy Protein Peptic Hydrolysate with Bound Phospholipids Decreases Micellar Solubility and Cholesterol Absorption in Rats and Caco-2 Cells. Journal of Nutrition.1999, 129:1725~1730.
94. Shin Joung Rho, Ji-Soo Lee, Yong Il Chung, Young-Wan Kim, Hyeon Gyu Lee.Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract. Process Biochemistry, 2009, 44(4):490~493.
95. Shoshi Ota, Eiko Muta, Yasuko Katahra and Yoshiko Okamoto. Reinvestigation of Fractionation and Some Properties of the Proteolytically Active Components of Stem and Fruit Bromelains. Japanese Biochemical Society, 1985, 98(1): 219~228.
96. T. Kodera, N. Nio, Identification of an angiotensin I-converting enzyme inhibitory peptides from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in spontaneously hypertensive model rats Journal Food Science 2006, 71:164~173.
97. Thanh V H , Shbasaki K. Major proteins of soybean seeds , A straightforward fraction and their characterization. Journal of Agriculture Food Chemistry,24 :1117 ~1121.
98. Tsuruki T, Kishi K, Takahashi M, et al. Soymetide, an immunostimulating peptide derived fromsoybeanβ-conglycinin,is an fMLP agonist. FEBS Letters, 2003,540:206~210.
99. Wen-Dee Chiang, Chieh-Jen Shih and Yan-Hwa Chu. Functional properties of soy protein hydrolysate produced from a continuous membrane reactor system. Food Chemistry, 1999, 65(2): 189~194
100. Wen-Dee Chiang, May-June Tsou, Zong-Yao Tsai,Tsun-Chung Tsai. Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry, 2006, 98(4):725~732
101. XiangZhen Kong, MingMing Guo, YuFei Hua, Dong Cao, CaiMeng Zhang. Enzymatic preparation of immunomodulating hydrolysates from soy proteins. Bioresource Technology, 2008, 99(18):8873~8879.
102. Yoshikawa M, Kishi K, Takahashi M, Watanabe A, Miyamura T, Yamazaki M, Chiba H. Immunostimulating peptide derived from soybean protein. Annals of the New York Academy of Sciences. 1993, 23(685):375~376.
2.陈成,刘文玉,杨青.大豆蛋白活性肽的生物学功能及其应用.黑龙江农业科学, 2004, (3): 40~42.
3.陈历俊,卢阳,骆承庠,刘爱萍.连续搅拌罐膜反应器(CSTMR)法生产大豆肽动力学研究食品工业与科技,2003,24(6):26~28.
4.陈美珍,余杰,郭慧敏.大豆分离蛋白酶解物清除羟自由基作用的研究.食品科学, 2002, 23(1): 43~46.
5.范远景,姬莹莹,张焱大豆蛋白酶解肽的分子量分布及抑制ACE活性关系研究.食品科学,2007,28(10):57~61.
6.高荣丽,陶冠军,杨严俊.葵花籽粕的综合利用.食品工业科技,2006,27(7):138~140.
7.高素蕴,潘思轶,郭康权.大豆分离蛋白水解物螯合锌(II)的合成与制备.食品科学,2003,24(10):117~120.
8.龚树立,文剑,吴逸民,杨玉铮,杨则宜,周丽丽,高红,徐葆华,李肃反.大豆多肽运动饮料的研制,食品与发酵工业,2003,29(4):86~87.
9.龚树立.大豆多肽研究概况及其在运动饮料中的应用.食品与发酵工业,2004,30(6):112~116.
10.郭敏亮,陈军,姜勇明,郑杰军.用豆粕生产大豆蛋白肽饮料.食品科学,1992,10:1~3.
11.郭兴风.蛋白质水解度的测定.中国油脂,2000,25(6):176~177.
12.国家食品药品监督管理局.保健食品注册管理办法(试行).2005.
13.国明明,华欲飞.大豆肽免疫调节作用的研究.食品科技,2007,7:242~244.
14.韩飞,于婷婷,周孟良,酶法生产大豆蛋白ACE抑制肽的研究.食品科学,2008,29(11):369~374.
15.厚生省.食品与科学(日),1989,31:41.
16.金英姿,庞彩霞.玉米蛋白的深加工及应用.新疆农业科技, 2004(3): 10.
17.瞿水忠,周瑞宝.饲用菜籽蛋白营养特性的研究.郑州工程学院学报.1992,1:51~60.
18.李书国,陈辉,庄玉亭,李雪梅.复合酶法制备活性大豆寡肽研究.粮食与油脂,2001,3:5~7.
19.李艳玲,李松彪.棉籽蛋白的开发利用.饲料广角.2001,15:6~8.
20.梁金钟,范洪臣,程丽,王淑杰.微生物液态发酵法生产大豆蛋白活性肽的研究.食品与发酵工业,2008,34(1):88~92.
21.刘炳智.大豆肽及其开发应用前景.日用化学工业.1997,1:61~62.
22.刘佳,王兴国,杨严俊.酶法制备大豆蛋白降血压肽的研究.中国油脂,2008,33(9):6~9.
23.刘明,倪辉,吴永沛.大豆抗氧化活性肽研究进展.食品科学,2006, 27(12):897~900.
24.刘忆梅,陈朝晖.大豆蛋白肽降血脂功能性的研究.大豆通报,2004,3:22.
25.刘志伟,刘秀梅,郑梦云.大豆蛋白体外酶解物中血管紧张素转化酶抑制剂活性肽研究.营养学报,2007 ,29 (1):69~73.
26.陆旋,朱正华.家蚕丝素蛋白铜元素螯合物的制备及其对动物生理影响研究.蚕桑通报,2005,36(3):16~20.
27.梅娜,周文明,胡晓玉,李玲玲.花生粕营养成分分析.西北农业学报,2007,16(3):96~99.
28.宁正祥.食品成分分析手册.北京,中国轻工业出版社,1998:119~124.
29.邱冬玲,胡长利,崔建云.丝素肽与锌螯合工艺条件的研究.食品研究与开发,2008,29(7):38~42.
30.沈仓良,姚文,陈伟华,朱伟云,邹思湘.伴大豆球蛋白水解肽对灌喂大肠杆菌( E.coli)的小鼠肠道微生物区系和健康的影响.中国现代医学杂志,2006,16(3):354~358.
31.孙占田,计成,王碧莲.小肽的吸收机制及在动物生产中的应用研究.饲料研究,2007,1:28~31
32.檀志芬,生庆海,邱泉若,刘建光.蛋白质水解度的测定方法.食品工业科技,2005,26(7):174~175.
33.陶红,梁岐.双酶水解降低大豆寡肽苦味研究.食品工业科技,2003,(增刊):35~37
34.田仁林.蓖麻子的成分和化学.中国油脂,1981(S1):118~123.
35.王芳.微生物发酵法水解大豆蛋白制备大豆蛋白活性肽的研究.黑龙江粮食,2008(4):41~43.
36.王镜岩,朱圣庚,徐长法.生物化学(上册).北京:高等教育出版社,2002:320 ~432.
37.王丽彬,张弢,王旻.高纯度木瓜蛋白酶的分离纯化和性质研究.中国生化药物杂志,2006,27(3):159~162.
38.王世英.阿片肽研究的回顾和展望[J].生物学通报, 2001,36(1): 5~7.
39.卫琳,宋俊梅,宁维颖.米曲霉固态发酵豆粕制备大豆肽的研究.粮食加工,2009,34(1):34~36
40.吴建平.大豆降血压肽的研制.中国油脂,1998,23(5):22~25.
41.吴炜亮,吴国杰,梁道双杨帆. ACE抑制肽的生理功能和研究进展.现代食品科技,2006,22(3):251~254.
42.徐广飞,黄金华,吴杰,仇梁林.大豆低聚肽对自发性高血压大鼠降血压作用的研究中华老年多器官疾病杂志,2004,3(4):288~291
43.徐力,赵忠岩,李鸿梅,王智,黄宜兵,张学忠.大豆蛋白的小分子酶解产物抗氧化活性研究.吉林农业大学学报,2007,29(1):48~52.
44.曾琦斐.微量元素与人体健康.中国科技信息.2008,3:158~159
45.张国胜,孔繁东,祖国仁等.大豆蛋白抗高血压活性肽的研究.中国乳品工业, 2004, 32(8)12~14.
46.张全才,熊华,曹银娣,李庭,齐金峰.米蛋白肽锌的制备工艺研究.食品科技2008,33(2):110~133
47.张延坤,刘炳智.大豆肽在食品工业中的应用.食品工业,1997,(3):5~6.
48.赵东海;张洪;黄建韶.蚕豆蛋白提取工艺的研究.食品与机械.2005,2:32~33.
49.赵秀娟,王小雪,吴博学,宋会娟.大豆活性肽粉对喂饲高血脂饲料大鼠的影响.中国卫生检疫杂志,2002,12(8):421~422.
50.郑建仙,高孔荣.功能性食品及其在我国的开发前景.食品机械,1994, 41(3):6~10.
51.郑炯.血红蛋白多肽螯合铁的制备及其抗贫血功能研究[硕士学位论文].西南农业大学.2008
52.朱桂红.快速配制铬黑T指示剂方法.湖南造纸,2005,2:37.
53.朱建华.豌豆蛋白质的特性.粮油食品科技.1996,4:32~33.
54.左伟勇,陈伟华,邹思湘.伴大豆球蛋白胃蛋白酶水解肽对小鼠免疫功能及肠道内环境的影响南京农业大学学报,2005, 28 (3) : 71～74.
55.左伟勇,陈伟华,邹思湘.伴大豆球蛋白胃蛋白酶水解肽对双歧杆菌增值的影响.食品与发酵工业,2004,30(5):10~13.
56. A..Constantinides, B. Adu-Amankwa Enzymatic modification of vegetable protein: Mechanism, kinetics, and production of soluble and partially soluble protein in a batch reactor. Biotechnology and Bioengineering , 1978, 22 (8):1543~1565.
57. A.G.Marangoni. Enzyme Kinetics-A Modern Approach. New York, John Wiley & Sons,2001.
58. Additions and Corrections. The Complete Amino Acid Sequence of Papain. The Journal of Biological Chemistry1970, 245(14):3485~3492.
59. Adler-Nissen J. Determination of the degree of hydrolysis of food protein hydrolysate by trinitrobenzensulfonic acid. Journal of Agriculture food chemistry,1979,27(6):1256~1262.
60. Adler-Nissen J. Enzymatic hydrolysis of food protein. NEW York,Elsevier Applied Science Publishers,1986.
61. Adler-Nissen J. Enzymatic hydrolysis of soy protein for nutritional fortification of low pH food. Ann Nutrition Aliment,1978,32(2~3):205~216.
62. Ajjipura B Hemavathi, Hangalore Umesh Hebbar and Karumanchi SMS Raghavarao. Reverse micellar extraction of bromelain from Ananas comosus L.Merryl. Journal of Chemical Technology and Biotechnology, 2007 (82): 985~992.
63. Andrés Moure, Herminia Domínguez, Juan Carlos Parajó. Antioxidant properties of ultrafiltration-recovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochemistry , 2006,41(2): 447~456.
64. B.Adu-Amankwa, A. Constantinides, W. R. Vieth Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor. Biotechnology and Bioengineering, 1981, 23(11):2609~2627.
65. Bernard P.O’Hara, Andrew, M. Hemmings, David J. Buttle, and Laurence H. Pearl.Crystal structure of-glycyl endopeptidase from Carica papaya:a cysteine of unusual substrate specificity. Biochemistry,1995,34:13190~13195.
66. Bradley W. Bolling, Jeffrey B. Blumberg, C.-Y. Oliver Chen. Extraction methods determine the antioxidant capacity and induction of quinone reductase by soy products in vitro. Food Chemistry, 2009, 116(1):351~355.
67. CHEN Cui-lin, SHI Yan-guo, WANG Zhen-yu, ZHAO Hai-tian. Effect of protein subunits and 7S/11S on functional properties of SPI. Food science, 2006, 27(3):70~74.
68. Chen H M.Muramoto K.Y amauchi F, et al. Antioxidative properties of histidine containing peptides desinned from peptide fragments found in the digests of soybean protein. Journal of Agriculture Food Chemistry,1998, 46: 49~53.
69. Chen J R,Kunio S,Fumio Y.Isolation and characterization of immunostimulative peptides fromsoybean. Journal of Nutrition Biochemistry,1995,6(6):310~313.
70. D.Spellman, E.McEvory, G.O’Cuinn, R.J.FitzGerald. Proteinase and exopeptidase hydrolysis of whey protein: comparison of the TNBS, OPA and pH stat methods for quantification of degree of hydrolysis. International Dairy Journal, 2003, 13:447~453.
71. Danji Fukushima. Recent progress of soybean protein foods: chemistry, technology and nutrition. Food Review International, 1991,7(3): 323~351.
72. Fang Zhong, Jianmin Liu, Jianguo Ma, Charles F. Shoemakerb. Fractionation and identification of a novel hypocholesterolemic peptide derived from soy protein Alcalase hydrolysates.Food Research International, 2007, 40( 6):756~762.
73. Fang Zhong, Jianmin Liu, Jianguo Ma, Charles F. Shoemakerb. Preparation of hypocholesterol peptides from soy protein and their hypocholesterolemic effect in mice.Food Research International, 2007, 40(6):661~667.
74. Fouad M.T. Chelation and chelated minerals. Journal of Applied Nutrition, 1974,26:6.
75. Fujimaki M, Kato H, Arai S, Yamashita M. Symposium on microbial changes in foods, Application of microbial proteases to soybean and other materials to improve acceptability, especially through the formation of plastein. Journal of Apply Bacteriol,1971,34(4):119~131.
76. Hsin-Yi Yang, Suh-Ching Yang, Shu-Tzu Chen, Jiun-Rong Chen .Soy protein hydrolysate ameliorates cardiovascular remodeling in rats with l-NAME-induced hypertension. The Journal of Nutritional Biochemistry, 2008,19(12): 833~839.
77. J. Dziuba, P. Minkiewicz, D. and Na?ecz, A. Iwaniak. Database of biologically active peptide sequences. Nahrung,1999,43(3):190~l95.
78. 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.
79. Laura P. Hale, Paula K. Greer, Chau T. Trinh, Cindy L James. Proteinase activity and stability of natural bromelain preparations. International Immuopharmacology, 2005, 5:783~793.
80. Lei Wei, Eric Clauser, Frangoi As lhenc-Gelas, and PierreC orvolThe Two Homologous Domains of Human Angiotensin I-convertingEnzyme Interact Differently with Competitive Inhibitors. Journal of Biological Chemistry,1992, 267(19): 13398~13405.
81. Lovati M R, Manzoni C, Gianazza E,etal. Soy protein peptides regulate cholesterol homeostasis in Hep G2 cells. Journal of Nutrition, 2000, 130: 2543~2549.
82. Lynch GL, van der Aar PJ, Berger LL, Fahey GC Jr, Merchen NR. Proteolysis of alcohol-treated soybean meal proteins by Bacteroides ruminicola, Bacteroides amylophilus, pepsin, trypsin, and in the rumen of steers. Journal of Dairy Science. 1988, 71(9):2416~2427.
83. M. Kuba, C. Tana, S. Tawata and M. Yasuda. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase .Process Biochemistry, 2005,40(6): 2191~2196.
84. Manzoni C, Lovati M R, Gianazza E,et al. Soybean protein products as regulators of liverlow-density lipoprotein receptors. II. 1α-α' rich commercial soy concentrate andα' deficient mutant differently affect tlow-density lipoprotein receptoractivation. Journal of Agriculture Food Chemistry, 1998,46:2481~2484.
85. María D. Mesa, Jose M. Silván, Josune Olza,ángel Gila , María D. del Castillob, Antioxidant properties of soy protein–fructooligosaccharide glycation systems and its hydrolyzates Food Research International, 2008,41(6):606~615.
86. Megumi Kuba, Kumi Tanaka, Shinkichi Tawata, Yasuhito Takeda. AngiotensinⅠ-Converting Enzyme inhibitory peptides isolated from Tofuyo fermented soybean food. Bioscience, Biotechnology and Biochemistry.2003,67(6):1278~1283.
87. Min-Liang Guo, Yong-Ming Jiang, Zhong-Liang Ma and Yan-Li Li. Hydrolytic characteristics of chitosan-immobilized As 1.398 neutral proteinase (from B. subtilis) to soybean protein. Food Chemistry, 1996,55(4): 373~377.
88. Munir Cheryan, David Deeslie. Soy protein hydrolysis in membrane reactors. Journal of the American Oil Chemists' Society,1983,60(6):1112~1115.
89. Nagaoka S,Awano R,Nagata N,et a1.Serum cholesterol re—duction and cholesterol absorption inhibition in Caco-2cells by a soy protein peptide hydrolyzate.Bioscience, Biotechnology and Biochemistry, 1997,61:354~356
90. P.M. Nielsen, D. Petersen, C. Dambmann. Improved Method for Determining Food Protein Degree of Hydrolysis. Journal of Food Science, 2006, 66 ( 5):642~646.
91. Qinyun Chen. Effect of Amide contents in wheat gluten hydrolysates on the thermal flavor generation ,ph. D. thesis ,Rutgers university , 1995 :54 ~56.
92. R. Bhattacharya and D. Bhattacharyya. Preservationof natural stability of fruit bromelain from Ananas comosus L (pineapple). Journal of Food Biochemistry,2009, (33) :1~19.
93. Satoshi Nagaoka1, Keiji Miwa, Michiko Eto, Yasuo Kuzuya, Goro Hori ,Kazuhiro Yamamoto. Soy Protein Peptic Hydrolysate with Bound Phospholipids Decreases Micellar Solubility and Cholesterol Absorption in Rats and Caco-2 Cells. Journal of Nutrition.1999, 129:1725~1730.
94. Shin Joung Rho, Ji-Soo Lee, Yong Il Chung, Young-Wan Kim, Hyeon Gyu Lee.Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract. Process Biochemistry, 2009, 44(4):490~493.
95. Shoshi Ota, Eiko Muta, Yasuko Katahra and Yoshiko Okamoto. Reinvestigation of Fractionation and Some Properties of the Proteolytically Active Components of Stem and Fruit Bromelains. Japanese Biochemical Society, 1985, 98(1): 219~228.
96. T. Kodera, N. Nio, Identification of an angiotensin I-converting enzyme inhibitory peptides from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in spontaneously hypertensive model rats Journal Food Science 2006, 71:164~173.
97. Thanh V H , Shbasaki K. Major proteins of soybean seeds , A straightforward fraction and their characterization. Journal of Agriculture Food Chemistry,24 :1117 ~1121.
98. Tsuruki T, Kishi K, Takahashi M, et al. Soymetide, an immunostimulating peptide derived fromsoybeanβ-conglycinin,is an fMLP agonist. FEBS Letters, 2003,540:206~210.
99. Wen-Dee Chiang, Chieh-Jen Shih and Yan-Hwa Chu. Functional properties of soy protein hydrolysate produced from a continuous membrane reactor system. Food Chemistry, 1999, 65(2): 189~194
100. Wen-Dee Chiang, May-June Tsou, Zong-Yao Tsai,Tsun-Chung Tsai. Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry, 2006, 98(4):725~732
101. XiangZhen Kong, MingMing Guo, YuFei Hua, Dong Cao, CaiMeng Zhang. Enzymatic preparation of immunomodulating hydrolysates from soy proteins. Bioresource Technology, 2008, 99(18):8873~8879.
102. Yoshikawa M, Kishi K, Takahashi M, Watanabe A, Miyamura T, Yamazaki M, Chiba H. Immunostimulating peptide derived from soybean protein. Annals of the New York Academy of Sciences. 1993, 23(685):375~376.