核桃蛋白酶解产物的活性初步探究
|
摘要
我国的核桃资源非常丰富,其中所含的核桃蛋白是一种极优质的植物蛋白,除用于初级食品的制作外,还可作为食品基料或食品添加剂使用,应用前景广阔。目前,具有高附加值的核桃多肽已成为核桃蛋白的主要加工方向之一。生物活性肽是现今研究的热点活性物质之一,具有多种生理活性,而且与其它蛋白质类药物相比,它被机体吸收的效率更高,因此受到越来越多的关注。目前的研究主要是利用来源丰富的蛋白质通过蛋白酶水解来制备出大量具有活性的多肽。
本论文选取核桃蛋白作为蛋白质来源,选用胰蛋白酶、胃蛋白酶和木瓜蛋白酶三种常用水解酶对核桃蛋白进行酶解,得到一系列酶解产物,并对酶解产物的活性进行了筛选。
首先参考各种文献资料,确定三种蛋白酶的酶解条件,其中胰蛋白酶酶解条件为：加酶量E/S为1×104U/g,pH8.0,温度55℃,酶解时间8h；胃蛋白酶酶解条件为：pH 2,温度设定为37℃,加酶量E/S为1×104U/g,酶解时间8h；木瓜蛋白酶酶解条件为：pH8.0,温度55℃,加酶量E/S为1×104U/g,酶解时间8h。根据选定的酶解条件对核桃蛋白进行酶解,得到三种酶解产物：胰蛋白酶酶解产物(Y)、胃蛋白酶酶解产物(W)和木瓜蛋白酶酶解产物(M),并通过SDS-PAGE电泳对酶解产物的分子量进行了初步研究,发现三种酶解产物均为分子量在15KD以下的小肽,为后续的活性研究准备好了样品。
接着对三种酶解产物(Y、W和M)进行了酶活性、血液活性、抑菌活性和抗肿瘤活性的筛选。结果表明：Y、W、M均具有一定的丝氨酸蛋白酶活性,无丝氨酸蛋白酶抑制活性；三种酶解产物中只能确定W具有一定的纤维蛋白原水解活性,并很有研究价值：W能水解纤维蛋白原的Aα、Bβ链；三种酶解产物均无磷脂酶A2活性及磷脂酶A2抑制活性；Y、W、M均具有一定的溶血活性,其中Y的溶血活性较高,溶血率高达199.33%,W和M的溶血率分别为47.49%和27.09%；三者均无红细胞凝集活性及血小板聚集活性；Y和M不具有血小板聚集抑制活性,而W的该活性较强,最大抑制率为70%左右；Y、W、M均对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、蜡样芽孢杆菌、巨大芽孢杆菌和白色念球菌六种细菌无抑制作用；Y、M对Hela细胞的抑制效果不明显,抑制率低于20%,W对Hela细胞的抑制率可达到75%,抑制效果明显；三者对PC-12细胞的抑制率都不超过15%；W对MCF-7细胞的抑制率为40%,其它两者不明显；Y、W、M均对MDA-435细胞具有一定的抑制作用,抑制率分别为56.18%、71.45%和26.56%；三者对Hep G2细胞几乎没有抑制作用。
最后对酶解产物的丝氨酸蛋白酶活性进行了初步研究,排除了丝氨酸蛋白酶水解活性中的多种干扰因素,包括反应体系、酶解底物等,基本确证了丝氨酸蛋白酶活性的存在；初步确定了三种蛋白酶酶解核桃蛋白的最佳酶解时间；而且对M和W进行了初步的分离纯化,M分别选用Sephadex G-75、Sephadex G-25凝胶层析,W选用了Sephadex G-25凝胶层析和Sephadex C-25离子交换层析两种方法,结果得到了12个组分：MG-75Ⅰ、MG-75Ⅱ、MG-25Ⅰ、MG-25Ⅱ、MG-25Ⅲ、MG-25Ⅳ、WG-25Ⅰ、WG-25Ⅱ、WC-25Ⅰ、WC-25Ⅱ、WC-25Ⅲ、WC-25Ⅳ,但在对这12个组分进行丝氨酸蛋白酶活性的筛选过程中发现,各组分均不具有该活性。
Walnut resources are very rich in China. Walnut protein is one of them, which is an extremely high-quality plant protein resource. In addtion to the primary food production, walnut protein can be used as food base material or food additives. Nowadays, high value-added walnut protein peptide is a main direction of processing. Bioactive peptide is one of the most popular active substances in current study, which has a variety of biological activities. Moreover, bioactive peptides, compared with other proteinic drugs, can be absorbed by the human body more efficiently and therefore gain more and more attention. The current main method of active peptides preparing is performed by hydrolysing a rich source of protein with protease.
In this paper, walnut protein was selected as a protein source, three kinds of common enzymes:trypsin, pepsin and papain were selected for hydrolyzation of walnut protein, then a series of hydrolysates were obtained and activities of hydrolysates of enzymes digestion were screened.
Firstly, three kinds of protease digestion conditions were identified refered to a variety of literature, trypsin digestion conditions were:the amount of enzyme E/S is 1×104U/g, pH8.0, temperature 55℃, hydrolysis time is 8h; pepsin hydrolysis conditions:pH2.0, the temperature set to 37℃, the amount of enzyme E/S is1×104U/g, hydrolysis time is 8h; papain hydrolysis conditions:pH8.0, temperature 55℃, the amount of enzyme E/S is 1×104U/g, hydrolysis time is 8h. According to the selected enzyme hydrolysis conditions, walnut proteins were hydrolysed and three kinds of hydrolysates were obtained:trypsin hydrolysates (Y), pepsin hydrolysates (W), and papain hydrolysates (M), and through SDS-PAGE, a preliminary study of the molecular weight of hydrolysates was conducted, which suggested that three kinds of hydrolysates were small peptides with the molecular weight of below 15KD, by doing this, the samples of the follow-up activity study were ready.
Enzymatic activity, blood activity, antibacterial activity and anti-tumor activity of the three kinds of hydrolysates (Y, W and M) were screened. The results showed that:Y, W, M all have a certain degree of serine protease activity, no serine protease inhibitory activity; In these three kinds of hydrolysates, W has fibrinogen hydrolysis activity, while the other two are uncertain. The fibrinogen hydrolysis activity of W is of great research value:W can hydrolyze fibrinogen Aα, Bβchain; All three kinds of hydrolysates have no phospholipase A2 activity and also no phospholipase A2 inhibitory activity; Y, W, M all have certain hemolytic activity, in which the hemolytic activity is higher in Y, hemolysis rate is as high as 199.33%, while for W and M, hemolysis rates are respectively 47.49% and 27.09%; none of them has hemagglutination activity and platelet aggregation activity; Y, and M do not have platelet aggregation inhibitory activity, while the W, has strong activity with maximum inhibition rate of about 70%; Y, W, M all have no inhibitory effect on E. coli, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Bacillus megaterium and Monilia albican bacteria; the inhibitory effect of Y, M on Hela cells is not obvious, whice has a inhibition rate of less than 20%, while the inhibitory rate of W on Hela cells could reach 75% and its effect is obvious; the inhibitory rates of all three on PC-12 cells are no more than 15%; the inhibition rate of W on MCF-7 cells is 40% while the other two is not obvious; Y, W, M all have the certain inhibition effect on MDA-435 cells with a inhibitory rates of 56.18%,71.45% and 26.56% respectively; all three almost have no inhibitory effect on Hep G2 cells.
Finally, the serine protease hydrolysis activities of the hydrolysates were elementrily studied, numbers of interfering factors in the hydrolysates, including reactive systems, walnut protein and the enzymes, were excluded. At last, the existence of serine protease activity were confirmed; and a preliminary separation and purification for the M and W were carried out, Sephadex G-75, Sephadex G-25 gel chromatography were selected for the M, Sephadex G-25 gel chromatography and Sephadex C-25 ion-exchange chromatography were selected for the W, the 12 components obtained are MG-75Ⅰ, MG-75Ⅱ, MG-25Ⅰ, MG-25Ⅱ, MG-25Ⅲ, MG-25Ⅳ, WG-25Ⅰ, WG-25Ⅱ, WC-25Ⅰ, WC-25Ⅱ, WC-25Ⅲ, WC-25Ⅳ, however none of these 12 compositions has serine protease activity.
本论文选取核桃蛋白作为蛋白质来源,选用胰蛋白酶、胃蛋白酶和木瓜蛋白酶三种常用水解酶对核桃蛋白进行酶解,得到一系列酶解产物,并对酶解产物的活性进行了筛选。
首先参考各种文献资料,确定三种蛋白酶的酶解条件,其中胰蛋白酶酶解条件为：加酶量E/S为1×104U/g,pH8.0,温度55℃,酶解时间8h；胃蛋白酶酶解条件为：pH 2,温度设定为37℃,加酶量E/S为1×104U/g,酶解时间8h；木瓜蛋白酶酶解条件为：pH8.0,温度55℃,加酶量E/S为1×104U/g,酶解时间8h。根据选定的酶解条件对核桃蛋白进行酶解,得到三种酶解产物：胰蛋白酶酶解产物(Y)、胃蛋白酶酶解产物(W)和木瓜蛋白酶酶解产物(M),并通过SDS-PAGE电泳对酶解产物的分子量进行了初步研究,发现三种酶解产物均为分子量在15KD以下的小肽,为后续的活性研究准备好了样品。
接着对三种酶解产物(Y、W和M)进行了酶活性、血液活性、抑菌活性和抗肿瘤活性的筛选。结果表明：Y、W、M均具有一定的丝氨酸蛋白酶活性,无丝氨酸蛋白酶抑制活性；三种酶解产物中只能确定W具有一定的纤维蛋白原水解活性,并很有研究价值：W能水解纤维蛋白原的Aα、Bβ链；三种酶解产物均无磷脂酶A2活性及磷脂酶A2抑制活性；Y、W、M均具有一定的溶血活性,其中Y的溶血活性较高,溶血率高达199.33%,W和M的溶血率分别为47.49%和27.09%；三者均无红细胞凝集活性及血小板聚集活性；Y和M不具有血小板聚集抑制活性,而W的该活性较强,最大抑制率为70%左右；Y、W、M均对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、蜡样芽孢杆菌、巨大芽孢杆菌和白色念球菌六种细菌无抑制作用；Y、M对Hela细胞的抑制效果不明显,抑制率低于20%,W对Hela细胞的抑制率可达到75%,抑制效果明显；三者对PC-12细胞的抑制率都不超过15%；W对MCF-7细胞的抑制率为40%,其它两者不明显；Y、W、M均对MDA-435细胞具有一定的抑制作用,抑制率分别为56.18%、71.45%和26.56%；三者对Hep G2细胞几乎没有抑制作用。
最后对酶解产物的丝氨酸蛋白酶活性进行了初步研究,排除了丝氨酸蛋白酶水解活性中的多种干扰因素,包括反应体系、酶解底物等,基本确证了丝氨酸蛋白酶活性的存在；初步确定了三种蛋白酶酶解核桃蛋白的最佳酶解时间；而且对M和W进行了初步的分离纯化,M分别选用Sephadex G-75、Sephadex G-25凝胶层析,W选用了Sephadex G-25凝胶层析和Sephadex C-25离子交换层析两种方法,结果得到了12个组分：MG-75Ⅰ、MG-75Ⅱ、MG-25Ⅰ、MG-25Ⅱ、MG-25Ⅲ、MG-25Ⅳ、WG-25Ⅰ、WG-25Ⅱ、WC-25Ⅰ、WC-25Ⅱ、WC-25Ⅲ、WC-25Ⅳ,但在对这12个组分进行丝氨酸蛋白酶活性的筛选过程中发现,各组分均不具有该活性。
Walnut resources are very rich in China. Walnut protein is one of them, which is an extremely high-quality plant protein resource. In addtion to the primary food production, walnut protein can be used as food base material or food additives. Nowadays, high value-added walnut protein peptide is a main direction of processing. Bioactive peptide is one of the most popular active substances in current study, which has a variety of biological activities. Moreover, bioactive peptides, compared with other proteinic drugs, can be absorbed by the human body more efficiently and therefore gain more and more attention. The current main method of active peptides preparing is performed by hydrolysing a rich source of protein with protease.
In this paper, walnut protein was selected as a protein source, three kinds of common enzymes:trypsin, pepsin and papain were selected for hydrolyzation of walnut protein, then a series of hydrolysates were obtained and activities of hydrolysates of enzymes digestion were screened.
Firstly, three kinds of protease digestion conditions were identified refered to a variety of literature, trypsin digestion conditions were:the amount of enzyme E/S is 1×104U/g, pH8.0, temperature 55℃, hydrolysis time is 8h; pepsin hydrolysis conditions:pH2.0, the temperature set to 37℃, the amount of enzyme E/S is1×104U/g, hydrolysis time is 8h; papain hydrolysis conditions:pH8.0, temperature 55℃, the amount of enzyme E/S is 1×104U/g, hydrolysis time is 8h. According to the selected enzyme hydrolysis conditions, walnut proteins were hydrolysed and three kinds of hydrolysates were obtained:trypsin hydrolysates (Y), pepsin hydrolysates (W), and papain hydrolysates (M), and through SDS-PAGE, a preliminary study of the molecular weight of hydrolysates was conducted, which suggested that three kinds of hydrolysates were small peptides with the molecular weight of below 15KD, by doing this, the samples of the follow-up activity study were ready.
Enzymatic activity, blood activity, antibacterial activity and anti-tumor activity of the three kinds of hydrolysates (Y, W and M) were screened. The results showed that:Y, W, M all have a certain degree of serine protease activity, no serine protease inhibitory activity; In these three kinds of hydrolysates, W has fibrinogen hydrolysis activity, while the other two are uncertain. The fibrinogen hydrolysis activity of W is of great research value:W can hydrolyze fibrinogen Aα, Bβchain; All three kinds of hydrolysates have no phospholipase A2 activity and also no phospholipase A2 inhibitory activity; Y, W, M all have certain hemolytic activity, in which the hemolytic activity is higher in Y, hemolysis rate is as high as 199.33%, while for W and M, hemolysis rates are respectively 47.49% and 27.09%; none of them has hemagglutination activity and platelet aggregation activity; Y, and M do not have platelet aggregation inhibitory activity, while the W, has strong activity with maximum inhibition rate of about 70%; Y, W, M all have no inhibitory effect on E. coli, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Bacillus megaterium and Monilia albican bacteria; the inhibitory effect of Y, M on Hela cells is not obvious, whice has a inhibition rate of less than 20%, while the inhibitory rate of W on Hela cells could reach 75% and its effect is obvious; the inhibitory rates of all three on PC-12 cells are no more than 15%; the inhibition rate of W on MCF-7 cells is 40% while the other two is not obvious; Y, W, M all have the certain inhibition effect on MDA-435 cells with a inhibitory rates of 56.18%,71.45% and 26.56% respectively; all three almost have no inhibitory effect on Hep G2 cells.
Finally, the serine protease hydrolysis activities of the hydrolysates were elementrily studied, numbers of interfering factors in the hydrolysates, including reactive systems, walnut protein and the enzymes, were excluded. At last, the existence of serine protease activity were confirmed; and a preliminary separation and purification for the M and W were carried out, Sephadex G-75, Sephadex G-25 gel chromatography were selected for the M, Sephadex G-25 gel chromatography and Sephadex C-25 ion-exchange chromatography were selected for the W, the 12 components obtained are MG-75Ⅰ, MG-75Ⅱ, MG-25Ⅰ, MG-25Ⅱ, MG-25Ⅲ, MG-25Ⅳ, WG-25Ⅰ, WG-25Ⅱ, WC-25Ⅰ, WC-25Ⅱ, WC-25Ⅲ, WC-25Ⅳ, however none of these 12 compositions has serine protease activity.
引文
[1]Szetao KWC, Sathe SK. Walnut (Juglans regial):proximate composition, protein solubility, protein amino acid composition and protein in vitro digestibility. J Sci Food Agric,2000,80:1393-1401
[2]郑建仙.功能性食品(第三卷).中国轻工业出版社,2002：273-304
[3]周伯川,高洪庆.核桃的特性及其制油工艺的研究.中国油脂,1994,19(6)：3-5
[4]闫开明.核桃油、速溶核桃粉综合加工新工艺.中国油脂,2000,25(2)：25-26
[5]郭炎强.水代法生产葵花籽香油的研究.郑州工程学院学报,2003,24(1)：77-79
[6]刘涛,熊伟.野生核桃油的超临界萃取及其脂肪酸成分分析.林业科技开发,2008,22(1)：96-98
[7]赵声兰,李涛,蔡绍芬等.核桃油自氧化及其抗氧化的实验研究.食品工业科技,2001,22(2)：27-29
[8]李建科,张清安,沈杰等.核桃油对小鼠血脂及胆固醇的影响.食品与生物技术学报,2005,24(5)：77-79
[9]张清安,李建科,范学辉.核桃油对小鼠学习记忆能力的影响.陕西师范大学学报,2006,34(4)：89-91
[10]FAO/WHO. Protein Quality Evaluation.Rome:Food and Agricultura Organization of the United Nations,1990:66
[11]Payne T. California walnuts and light foods. Cereal Foods World,1985,30:215-218
[12]刘力,龚宁,夏国华等.山核桃种仁蛋白质及氨基酸成分含量的变异分析.林业科学研究,2006(3)：376-378
[13]Wallowitz M, Peterson WR, Uratsu S. A legumin group food allergen from walnut. J Agric Food Chem,2006 (21):8369-8375
[14]姜莉,徐怀德,何玉君等.核桃渣中蛋白质的提取工艺及其功能性研究.食品科技,2007(4)：237-240
[15]张红,姚舜,李国军等.核桃仁脱皮和蛋白提取研究.粮食与油脂,2003(12)：22-23
[16]刘金福,王浩田.核桃乳饮料生产工艺研究.天津农学院学报,2000,7(1)：8-11
[17]易建华,朱振宝,董文宾.核桃蛋白酶法水解工艺条件研究.食品科技,2007,(3)：76-79
[18]Szetao KWC, Schrimpf, Teuberss. Effect of processing and storage on walnut tannins. J Sci. Food Agric,2001,81(13):1215-1222
[19]李艳伏,徐怀德,陈金海等.木瓜蛋白酶酶解核桃粕蛋白产物抗氧化活性研究.中国食品学报,2008(5)：8-14
[20]刘昭明,黄翠姬,周远.核桃蛋白酶水解物的抑菌性能探讨.四川食品与发酵,2008(5)：20-23
[21]万政敏,郝艳宾,杨春梅等.核桃仁种皮中的多酚类物质高压液相色谱分析.食品工业科技,2007(7)：212-213
[22]Leonard Jurd,Plant Polyphenols.I.The Polyphenolie Constituents of the Pelliele of the Walnut. The Fruit Vegetable Chemistry Laboratory Agriculture Service, U.S. Department of Agriculture,1998(78):3445-3447
[23]Ngoc TM, Hung TM, Thuong PT, et al. Antioxidative activities of galloyl glucopyrano-sides from the stem-bark of Juglans mandshurica. Biosci Biotech Biochem,2008 (8): 2158-2163
[24]Spaccarotella KJ, Kris-Etherton PM, et al. The effect of walnut intake on factors related to prostate and vascular health in older men. Nutr J.,2008 (2)
[25]Brantl V,Teschemacher T,Henschen A.,et al. Novel opioid peptides derived from casein (Casomorphin) I:Isolation from bovine casein.Peptone. Hoppe Seyler's Zeitschrift fur Physiologische Chemic,1979,360:1211-1216
[26]Kitts D D. Bioactive substances in food:identification and potential Uses. Canadian J Phys Pharm,1994,72:423-434
[27]杨小军.灌胃大豆蛋白、面筋蛋白的胃蛋白酶酶解物对大鼠免疫功能的影响.南京农业大学硕士论文,2003
[28]侯传记,马春.肌肽在大鼠脑缺血再灌注损伤中的神经保护作用.中国老年学杂志,2001,21(7)：282-283
[29]江波,Mine Y.卵黄高磷蛋白磷酸的制备及钙给合性质的研究.无锡轻工业大学学报,2000(4)：325-330
[30]Meisel H. Bioactive peptides from milk proteins:A perspective for consumers and producers. Australian J Dairy Tech,2001,56:83-92
[31]王瑛瑶,夫俊刚,杜东平等.酪蛋白磷酸肽体外功能实验.无锡轻工大学学报,2001,20(3)：243-247
[32]Oshima G, Shimabukuro H, Nagasawa K. Peptide inhibitors of angioterisin I converting enzyme in digests of gelatin by bacterial collagenase.Biochim Biophys Acta,1979,566: 128-137
[33]Marina M, Stuart G, Dashper, et al. Kappacin, a novel antibacterial peptide from bovine milk. Antimicrobial Agents and Chem therapy,2001,45:2309-2315
[34]Ibrahim HR, Sugirnoto Y, Aoki T. Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism.Biochem Biophys Acta,2000, 1523(23):196-205
[35]Schagger H, Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa. Anal Biochem,1987,166: 368-379
[36]曹佐武.有效分离1kDa小肽的Tricine-SDS-PAGE方法.中国生物工程杂志,2004,24(1)：74-76
[37]Sommerer N,Salles C,et al.Isolation of oligopeptides from the water-soluble extract of goat cheese and their identification by mass spectrometry. J Agric Food Chem,2001,49: 402-408
[38]崔莉.核桃乳的研制及其稳定性的研究.无锡轻工大学硕士学位论文,2000
[39]张强,阐国仕等.玉米抗氧化肽的分离制备及其体外抗氧化活性的研究.中国粮油学报,2005,20(5)：36-39
[40]姜莉.核桃渣制备核桃蛋白和多肽的研究.西北农林科技大学学位论文,2007
[41]包怡红,盛和静.山核桃蛋白多肽的制备及对羟自由基的清除作用.食品科学,2005(9)：515-517.
[42]丁小雯,张庆祝,乔海鸥.核桃蛋白最佳酶解条件的研究.粮食与油脂,2005(10)：23-25.
[43]宁斌.胰蛋白酶酶解核桃的工艺改进.昆明理工大学学位论文,2009
[44]Taku Nagai, Shun-ichiro Kawabata. A Link between Blood Coagulation and Prophenol Oxidase Activation in Arthropod Host Defense. J Bio Che,2000,275 (38):29264-29267
[45]Zhang Y., Wiser A., Xiong Y. L., et al. A novel plasminogen activator from snake venom. J Biol Che,1995,270:10246-10255
[46]Rodrigues V.M.,Soare A.M.,Guerra-Sa R.,et al.Structure and function characterization of neuwiedase,a nonhemorrhagic fibrinolytic metalloproeinase from bothrops neuwidi snake venom.Arch.Biochem.Biophys,2000,381:213-224
[47]Chen R.Q.,Lin Y,Wu J.B.,et al.A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin. Toxicon,2004,44:281-387
[48]张雁林,朱运峰.线粒体中的丝氨酸蛋白酶在肿瘤发生中的作用.国外医学肿瘤学分册,2005(6)：423-426
[49]赵飞,王转花.丝氨酸蛋白酶抑制剂的结构特点及作用机制.山西大学学报(自然科学版),2008,31：166-170
[50]Laskowski M, Qasim MA. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes.Biochim Biophys Acta,2000,1477: 324
[51]许芳,冯建成,李洁.纳豆激酶基因在大肠杆菌中活性表达的比较研究.微生物学杂志,2004,24(2)：10-13
[52]武金霞,赵晓瑜.蚯蚓纤溶酶的研究进展及趋势.中国生化药物杂志,2004,25：3-5
[53]罗方,周建民.60例胃病患者胃液纤溶活性检测及其临床意义.交通医学,2002,16(4)：351-352
[54]Kishimura H,Hayashi K. Isolation and characteristics of phospholipase A2 from the pyloriceca of the starfish Asterina pectinifera. Comparative Bio Physiology Part B,1999, 124:483-488
[55]Anderson BO, Moore EE, BanerJee A, Phospholipase A2 regulates critical inflarnma tory mediators of multiple organ failure. J Surg Res,1994,56(2):199-205
[56]顾海峰,梁晋鄂,李春美等.柿子单宁对几种蛇毒中主要酶的抑制作用及其机理初探.中国农业科学,2008,41(3)：910-917
[57]Bignami G. S.A rapid and sensitive hemolysis neutralization assay for playtoxin. Toxin, 1993,31:817-820
[58]蔡玲斐,袁汇宁.红细胞凝集试验(HA)与红细胞凝集抑制试验(HI)的替代试验.中华现代医学与临床,2006,4(5)：61-62
[59]单良,姚惠源.麦胚凝集素红细胞凝集活性检测方法的改进.无锡轻工大学学报,2004,23(1)：49-53
[60]Mor A, Amiche M, Nicolas P. Structure, synthesis, and acticity of dermaseptin b, a novel vertebrate defensive peptide from frog skin:relationship with adenoregulin. Biochemistry, 1994,33(21):6642-6650
[61]何涛,张涛,吴昌英等.红芸豆红细胞凝集素单体的分离纯化和性质研究.成都医学院学报,2008,3(2)：107-110
[62]Huang T F, Holt J C, Lukasiewicz H, et al. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein Ⅱb/Ⅲa complex. J Biol Chem,1987,262(33):16157-16163
[63]周先果,雷丹青,周先丽等.尖吻蝮蛇毒活性肽K组分对血小板聚集和超微结构的影响.蛇志,2007,19(4)：253-256
[64]McLane M A, Marcinkiewicz C, Vijay-Kumar S, et al. Viper venom disintegrins and related molecules. Proc Soc Exp Biol Med,1998,219:109-119
[65]陈清勇,钱利生,郭勇等.茶多酚抗肿瘤的分子生物学研究进展.中国现代应用药学,2001,18(3)：175-178
[66]刘珊林,施冬云,潘喜华等.抗氧化干预对肝癌细胞增殖及Nras基因表达的作用.生物化学与生物物理学报,2001,33(4)：463-465
[2]郑建仙.功能性食品(第三卷).中国轻工业出版社,2002：273-304
[3]周伯川,高洪庆.核桃的特性及其制油工艺的研究.中国油脂,1994,19(6)：3-5
[4]闫开明.核桃油、速溶核桃粉综合加工新工艺.中国油脂,2000,25(2)：25-26
[5]郭炎强.水代法生产葵花籽香油的研究.郑州工程学院学报,2003,24(1)：77-79
[6]刘涛,熊伟.野生核桃油的超临界萃取及其脂肪酸成分分析.林业科技开发,2008,22(1)：96-98
[7]赵声兰,李涛,蔡绍芬等.核桃油自氧化及其抗氧化的实验研究.食品工业科技,2001,22(2)：27-29
[8]李建科,张清安,沈杰等.核桃油对小鼠血脂及胆固醇的影响.食品与生物技术学报,2005,24(5)：77-79
[9]张清安,李建科,范学辉.核桃油对小鼠学习记忆能力的影响.陕西师范大学学报,2006,34(4)：89-91
[10]FAO/WHO. Protein Quality Evaluation.Rome:Food and Agricultura Organization of the United Nations,1990:66
[11]Payne T. California walnuts and light foods. Cereal Foods World,1985,30:215-218
[12]刘力,龚宁,夏国华等.山核桃种仁蛋白质及氨基酸成分含量的变异分析.林业科学研究,2006(3)：376-378
[13]Wallowitz M, Peterson WR, Uratsu S. A legumin group food allergen from walnut. J Agric Food Chem,2006 (21):8369-8375
[14]姜莉,徐怀德,何玉君等.核桃渣中蛋白质的提取工艺及其功能性研究.食品科技,2007(4)：237-240
[15]张红,姚舜,李国军等.核桃仁脱皮和蛋白提取研究.粮食与油脂,2003(12)：22-23
[16]刘金福,王浩田.核桃乳饮料生产工艺研究.天津农学院学报,2000,7(1)：8-11
[17]易建华,朱振宝,董文宾.核桃蛋白酶法水解工艺条件研究.食品科技,2007,(3)：76-79
[18]Szetao KWC, Schrimpf, Teuberss. Effect of processing and storage on walnut tannins. J Sci. Food Agric,2001,81(13):1215-1222
[19]李艳伏,徐怀德,陈金海等.木瓜蛋白酶酶解核桃粕蛋白产物抗氧化活性研究.中国食品学报,2008(5)：8-14
[20]刘昭明,黄翠姬,周远.核桃蛋白酶水解物的抑菌性能探讨.四川食品与发酵,2008(5)：20-23
[21]万政敏,郝艳宾,杨春梅等.核桃仁种皮中的多酚类物质高压液相色谱分析.食品工业科技,2007(7)：212-213
[22]Leonard Jurd,Plant Polyphenols.I.The Polyphenolie Constituents of the Pelliele of the Walnut. The Fruit Vegetable Chemistry Laboratory Agriculture Service, U.S. Department of Agriculture,1998(78):3445-3447
[23]Ngoc TM, Hung TM, Thuong PT, et al. Antioxidative activities of galloyl glucopyrano-sides from the stem-bark of Juglans mandshurica. Biosci Biotech Biochem,2008 (8): 2158-2163
[24]Spaccarotella KJ, Kris-Etherton PM, et al. The effect of walnut intake on factors related to prostate and vascular health in older men. Nutr J.,2008 (2)
[25]Brantl V,Teschemacher T,Henschen A.,et al. Novel opioid peptides derived from casein (Casomorphin) I:Isolation from bovine casein.Peptone. Hoppe Seyler's Zeitschrift fur Physiologische Chemic,1979,360:1211-1216
[26]Kitts D D. Bioactive substances in food:identification and potential Uses. Canadian J Phys Pharm,1994,72:423-434
[27]杨小军.灌胃大豆蛋白、面筋蛋白的胃蛋白酶酶解物对大鼠免疫功能的影响.南京农业大学硕士论文,2003
[28]侯传记,马春.肌肽在大鼠脑缺血再灌注损伤中的神经保护作用.中国老年学杂志,2001,21(7)：282-283
[29]江波,Mine Y.卵黄高磷蛋白磷酸的制备及钙给合性质的研究.无锡轻工业大学学报,2000(4)：325-330
[30]Meisel H. Bioactive peptides from milk proteins:A perspective for consumers and producers. Australian J Dairy Tech,2001,56:83-92
[31]王瑛瑶,夫俊刚,杜东平等.酪蛋白磷酸肽体外功能实验.无锡轻工大学学报,2001,20(3)：243-247
[32]Oshima G, Shimabukuro H, Nagasawa K. Peptide inhibitors of angioterisin I converting enzyme in digests of gelatin by bacterial collagenase.Biochim Biophys Acta,1979,566: 128-137
[33]Marina M, Stuart G, Dashper, et al. Kappacin, a novel antibacterial peptide from bovine milk. Antimicrobial Agents and Chem therapy,2001,45:2309-2315
[34]Ibrahim HR, Sugirnoto Y, Aoki T. Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism.Biochem Biophys Acta,2000, 1523(23):196-205
[35]Schagger H, Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa. Anal Biochem,1987,166: 368-379
[36]曹佐武.有效分离1kDa小肽的Tricine-SDS-PAGE方法.中国生物工程杂志,2004,24(1)：74-76
[37]Sommerer N,Salles C,et al.Isolation of oligopeptides from the water-soluble extract of goat cheese and their identification by mass spectrometry. J Agric Food Chem,2001,49: 402-408
[38]崔莉.核桃乳的研制及其稳定性的研究.无锡轻工大学硕士学位论文,2000
[39]张强,阐国仕等.玉米抗氧化肽的分离制备及其体外抗氧化活性的研究.中国粮油学报,2005,20(5)：36-39
[40]姜莉.核桃渣制备核桃蛋白和多肽的研究.西北农林科技大学学位论文,2007
[41]包怡红,盛和静.山核桃蛋白多肽的制备及对羟自由基的清除作用.食品科学,2005(9)：515-517.
[42]丁小雯,张庆祝,乔海鸥.核桃蛋白最佳酶解条件的研究.粮食与油脂,2005(10)：23-25.
[43]宁斌.胰蛋白酶酶解核桃的工艺改进.昆明理工大学学位论文,2009
[44]Taku Nagai, Shun-ichiro Kawabata. A Link between Blood Coagulation and Prophenol Oxidase Activation in Arthropod Host Defense. J Bio Che,2000,275 (38):29264-29267
[45]Zhang Y., Wiser A., Xiong Y. L., et al. A novel plasminogen activator from snake venom. J Biol Che,1995,270:10246-10255
[46]Rodrigues V.M.,Soare A.M.,Guerra-Sa R.,et al.Structure and function characterization of neuwiedase,a nonhemorrhagic fibrinolytic metalloproeinase from bothrops neuwidi snake venom.Arch.Biochem.Biophys,2000,381:213-224
[47]Chen R.Q.,Lin Y,Wu J.B.,et al.A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin. Toxicon,2004,44:281-387
[48]张雁林,朱运峰.线粒体中的丝氨酸蛋白酶在肿瘤发生中的作用.国外医学肿瘤学分册,2005(6)：423-426
[49]赵飞,王转花.丝氨酸蛋白酶抑制剂的结构特点及作用机制.山西大学学报(自然科学版),2008,31：166-170
[50]Laskowski M, Qasim MA. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes.Biochim Biophys Acta,2000,1477: 324
[51]许芳,冯建成,李洁.纳豆激酶基因在大肠杆菌中活性表达的比较研究.微生物学杂志,2004,24(2)：10-13
[52]武金霞,赵晓瑜.蚯蚓纤溶酶的研究进展及趋势.中国生化药物杂志,2004,25：3-5
[53]罗方,周建民.60例胃病患者胃液纤溶活性检测及其临床意义.交通医学,2002,16(4)：351-352
[54]Kishimura H,Hayashi K. Isolation and characteristics of phospholipase A2 from the pyloriceca of the starfish Asterina pectinifera. Comparative Bio Physiology Part B,1999, 124:483-488
[55]Anderson BO, Moore EE, BanerJee A, Phospholipase A2 regulates critical inflarnma tory mediators of multiple organ failure. J Surg Res,1994,56(2):199-205
[56]顾海峰,梁晋鄂,李春美等.柿子单宁对几种蛇毒中主要酶的抑制作用及其机理初探.中国农业科学,2008,41(3)：910-917
[57]Bignami G. S.A rapid and sensitive hemolysis neutralization assay for playtoxin. Toxin, 1993,31:817-820
[58]蔡玲斐,袁汇宁.红细胞凝集试验(HA)与红细胞凝集抑制试验(HI)的替代试验.中华现代医学与临床,2006,4(5)：61-62
[59]单良,姚惠源.麦胚凝集素红细胞凝集活性检测方法的改进.无锡轻工大学学报,2004,23(1)：49-53
[60]Mor A, Amiche M, Nicolas P. Structure, synthesis, and acticity of dermaseptin b, a novel vertebrate defensive peptide from frog skin:relationship with adenoregulin. Biochemistry, 1994,33(21):6642-6650
[61]何涛,张涛,吴昌英等.红芸豆红细胞凝集素单体的分离纯化和性质研究.成都医学院学报,2008,3(2)：107-110
[62]Huang T F, Holt J C, Lukasiewicz H, et al. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein Ⅱb/Ⅲa complex. J Biol Chem,1987,262(33):16157-16163
[63]周先果,雷丹青,周先丽等.尖吻蝮蛇毒活性肽K组分对血小板聚集和超微结构的影响.蛇志,2007,19(4)：253-256
[64]McLane M A, Marcinkiewicz C, Vijay-Kumar S, et al. Viper venom disintegrins and related molecules. Proc Soc Exp Biol Med,1998,219:109-119
[65]陈清勇,钱利生,郭勇等.茶多酚抗肿瘤的分子生物学研究进展.中国现代应用药学,2001,18(3)：175-178
[66]刘珊林,施冬云,潘喜华等.抗氧化干预对肝癌细胞增殖及Nras基因表达的作用.生物化学与生物物理学报,2001,33(4)：463-465