山核桃蛋白多肽制备方法的研究
摘要
蛋白质是人体所需的重要营养素之一，它在人体内以氨基酸或肽的形式被消化吸收。近年来研究发现非单一氨基酸形式的小肽类(2～7个氨基酸残基)，在人类的生命活动中具有重要的生理功能，且在体内的消化吸收优于游离氨基酸。
本研究以核桃仁为原料，经蛋白质提取、酶解、分离等过程制备有生物活性的核桃蛋白质多肽。
核桃仁经打浆、调PH、离心等一系列过程，分离出核桃蛋白质。
核桃蛋白质酶解所用的酶为木瓜蛋白酶，其作用范围较宽。
酶解工艺的确定：酶解工艺是制备蛋白水解物的重要步骤，不同的底物同一种酶有不同的最适条件，需经过实验找出其最适条件，因此做核桃蛋白的木瓜蛋白酶酶解的单因素分析，对PH、温度、底物浓度、酶量、反应时间这几个重要水解参数进行分析。综合多方面因素，将水解温度确定为40℃，反应时间为2h，作PH值、底物浓度、酶量的三因素的L_9(3~4)正交实验，经方差分析、直观分析，得到木瓜蛋白酶水解核桃蛋白的的最适条件为：温度40℃，PH值为9.0，底物浓度为40mg／ml，酶量为150.65u／g，反应时间为2h，水解度约为20％。
水解后的产物水浴加热使酶失活，调整溶液的PH，离心，得上清液，浓缩。此浓缩液中含大量的多肽混合物，需分离出分子量较小的多肽。
实验采用凝胶色谱法分离多肽，凝胶型号为SephadexG-25,0.1NHAC为洗脱液，洗脱速度为2.2ml／5min，测得外水体积为26ml，内水体积为61ml，加入一定量的浓缩液，洗脱，收集各管洗脱液，在280nm下检测各管吸光度，作洗脱曲线。根据测得内水体积、外水体积，排除大分子量的洗脱峰和分子量约为单个氨基酸的洗脱峰，则中间部分为分子量为5000左右的多肽混合物。
此多肽混合物再经过浓缩、干燥即成为成品。
由于短肽的吸收率、速度优于单一氨基酸，而且对人体蛋白质合成无任何不良影响，因此它对氨基酸吸收存在阻碍的人群的营养补充和手术后病人健康恢复是十分有益的。与氨基酸运输体系相比，肽吸收具有吸收快、能耗低、不容易饱和，且各种肽之间转运无竞争性与抑制性。因此，肽吸收具有效率高，速度快等优点。
由此可见以酶解法生产核桃蛋白多肽，可用做功能性食品基料，且具有生产成本低、产量大、速度快等优点，特别适合工业化生产。
Protein is one of the most important nutrients which human needs. They are absorbed by body in amino acids or peptides. In these years studies have shown that small peptides (2-7 amino acids) have important physiological functions in human body, and the absorption ratio of these peptides is much better than pure amino acid.
The process of this experiment is this: proteins derived from walnuts were hydrolyzed, and peptides were separated from the hydrolysate.
Walnuts proteins were obtained after the steps of beating, adjusting PH, and centrifugalizing.
The enzyme in this experiment is papain, for the active range of papain is much wider.
The hydrolysis of papain: This is an important step in the whole process of obtaining peptides from walnut protein. Because the same enzyme on the different proteins will be under different condition, to know the best condition, the analysis of single factor is necessary such as PH, temperature, substrate concentration, dose of enzyme, and reaction time. Considering other factors, temperature and time were determined. They are 40 癈 and 2h. Do experiment of orthogonal design including the other three factors: PH, substrate concentration, dose of enzyme. The best condition was made through analysis of variance on the results. They are temperature 40"C, PH is 9.0, substrate concentration is 40mg/ml, dose of enzyme is 150.65u/g, reaction time is 2h, the degree of hydrolysis is about 20%.
Heat the hydrolysate in 95 癈 hot water to make enzyme inactive, adjust PH, centrifugalize, and concentrate the clear liquid. There are mixtures of peptides in the concentration liquid, so the small peptides have to be separated from the mixture.
The gel filtration was adopted in this process of seperation. The gel is SephadexG-25. The eluate is 0.1NHCL, and the elution speed is 2.2ml/5min.The outside volume (Vo) of the gel is 26ml, and the inside volume (Vi) is 69ml. Concentration liquid was put on, eluted by the eluent, and then the eluates was collected. Detect the absorbance of the eluates under 280nm, and draw the elution curve. Remove the eluates of very large molecular weight and the eluates of molecular weight equal to the pure amino acid according to the Vo and Vi, and the left is the peptide mixture which the average molecular weight is about 5000.
These peptides product was obtained after concentration and dry.
Because the absorption ratio and speed of small peptides are much better than pure amino acid, and there will be no side effect on the synthesis of body protein, so it is beneficial to the people who are deficient in amino acid absorption and the patient who has experienced the operation.
Compared with amino acid transportation system, the absorption of peptides is quick, low consumption on energy, not easy to be saturated and there is no competition and inhibition between peptides. So the absorption of peptides has advantages of high efficiency and high speed.
The peptides derived from walnut protein by hydrolysis of papain can be used as functional food. And the method has so many advantages such a low cost, large production, high speed, that it is suitable for the industry production.
本研究以核桃仁为原料，经蛋白质提取、酶解、分离等过程制备有生物活性的核桃蛋白质多肽。
核桃仁经打浆、调PH、离心等一系列过程，分离出核桃蛋白质。
核桃蛋白质酶解所用的酶为木瓜蛋白酶，其作用范围较宽。
酶解工艺的确定：酶解工艺是制备蛋白水解物的重要步骤，不同的底物同一种酶有不同的最适条件，需经过实验找出其最适条件，因此做核桃蛋白的木瓜蛋白酶酶解的单因素分析，对PH、温度、底物浓度、酶量、反应时间这几个重要水解参数进行分析。综合多方面因素，将水解温度确定为40℃，反应时间为2h，作PH值、底物浓度、酶量的三因素的L_9(3~4)正交实验，经方差分析、直观分析，得到木瓜蛋白酶水解核桃蛋白的的最适条件为：温度40℃，PH值为9.0，底物浓度为40mg／ml，酶量为150.65u／g，反应时间为2h，水解度约为20％。
水解后的产物水浴加热使酶失活，调整溶液的PH，离心，得上清液，浓缩。此浓缩液中含大量的多肽混合物，需分离出分子量较小的多肽。
实验采用凝胶色谱法分离多肽，凝胶型号为SephadexG-25,0.1NHAC为洗脱液，洗脱速度为2.2ml／5min，测得外水体积为26ml，内水体积为61ml，加入一定量的浓缩液，洗脱，收集各管洗脱液，在280nm下检测各管吸光度，作洗脱曲线。根据测得内水体积、外水体积，排除大分子量的洗脱峰和分子量约为单个氨基酸的洗脱峰，则中间部分为分子量为5000左右的多肽混合物。
此多肽混合物再经过浓缩、干燥即成为成品。
由于短肽的吸收率、速度优于单一氨基酸，而且对人体蛋白质合成无任何不良影响，因此它对氨基酸吸收存在阻碍的人群的营养补充和手术后病人健康恢复是十分有益的。与氨基酸运输体系相比，肽吸收具有吸收快、能耗低、不容易饱和，且各种肽之间转运无竞争性与抑制性。因此，肽吸收具有效率高，速度快等优点。
由此可见以酶解法生产核桃蛋白多肽，可用做功能性食品基料，且具有生产成本低、产量大、速度快等优点，特别适合工业化生产。
Protein is one of the most important nutrients which human needs. They are absorbed by body in amino acids or peptides. In these years studies have shown that small peptides (2-7 amino acids) have important physiological functions in human body, and the absorption ratio of these peptides is much better than pure amino acid.
The process of this experiment is this: proteins derived from walnuts were hydrolyzed, and peptides were separated from the hydrolysate.
Walnuts proteins were obtained after the steps of beating, adjusting PH, and centrifugalizing.
The enzyme in this experiment is papain, for the active range of papain is much wider.
The hydrolysis of papain: This is an important step in the whole process of obtaining peptides from walnut protein. Because the same enzyme on the different proteins will be under different condition, to know the best condition, the analysis of single factor is necessary such as PH, temperature, substrate concentration, dose of enzyme, and reaction time. Considering other factors, temperature and time were determined. They are 40 癈 and 2h. Do experiment of orthogonal design including the other three factors: PH, substrate concentration, dose of enzyme. The best condition was made through analysis of variance on the results. They are temperature 40"C, PH is 9.0, substrate concentration is 40mg/ml, dose of enzyme is 150.65u/g, reaction time is 2h, the degree of hydrolysis is about 20%.
Heat the hydrolysate in 95 癈 hot water to make enzyme inactive, adjust PH, centrifugalize, and concentrate the clear liquid. There are mixtures of peptides in the concentration liquid, so the small peptides have to be separated from the mixture.
The gel filtration was adopted in this process of seperation. The gel is SephadexG-25. The eluate is 0.1NHCL, and the elution speed is 2.2ml/5min.The outside volume (Vo) of the gel is 26ml, and the inside volume (Vi) is 69ml. Concentration liquid was put on, eluted by the eluent, and then the eluates was collected. Detect the absorbance of the eluates under 280nm, and draw the elution curve. Remove the eluates of very large molecular weight and the eluates of molecular weight equal to the pure amino acid according to the Vo and Vi, and the left is the peptide mixture which the average molecular weight is about 5000.
These peptides product was obtained after concentration and dry.
Because the absorption ratio and speed of small peptides are much better than pure amino acid, and there will be no side effect on the synthesis of body protein, so it is beneficial to the people who are deficient in amino acid absorption and the patient who has experienced the operation.
Compared with amino acid transportation system, the absorption of peptides is quick, low consumption on energy, not easy to be saturated and there is no competition and inhibition between peptides. So the absorption of peptides has advantages of high efficiency and high speed.
The peptides derived from walnut protein by hydrolysis of papain can be used as functional food. And the method has so many advantages such a low cost, large production, high speed, that it is suitable for the industry production.
引文
[1] Neway, H.,Smith,P.h.Intercellutar hydrolysis of dipeptides during intestinal absorption.[J]Physiol. 1960,152:367～380
[2] 吴建平 生物活性肽的研究进展 [J]食品与机械 1998(1)：6～8
[3] 郭清泉，张兰威 食品中活性肽的研究 [J]食品与机械 1999，(6)：12～14
[4] Adibi,A.S Intestinal phase of protein assimilation in man.. [J]Amer. J.Clin.Nutr.1976 (29):205～215
[5] Hara, H. [J] Nutr. 1984,114:1122
[6] Silk, D.B.A. Uses of a peptide rather than amino acid nitrogen source in chemically defined elemental diets. [J]Parenter. Enter. Nutr. 1990,(4):548～553
[7] 李虹奇 活性多肽蛋白质研究近况 [J]中草药 1993(7)：373～378
[8] 贾素娟等 生物抗菌多肽 [J]氨基酸和生物资源 2000(4)：5～8
[9] Massaki Yoshikawa et al. Opioid peptides from milk proteins. [J]Agric.Biol.Chem. 1986 (9):2419～2421
[10] Chang. K.J etc Morphiceptin:a potent and specific agonistic for morphine(μ) receptors.[J]science 1981 (212):75～77
[11] Yatabe Tskuba.et al. Studies on the active site and antihypertensive activity of angiotensin I-converting enzyme inhibitors derived from casein. [J]Afric. Biol.Chem. 1987(6):1581～1586
[12] 盛国华 大豆多肽的功能及应刚 [J]食品工业科技 1993(6)：21～26
[13] 汤亚杰 吴思方 酪蛋白磷酸肽的研究进展 [J]食品科学1998(5)：3～6
[14] 尤新主编《功能性发酵制品》北京：中国轻工业出版社 2000，1，203～228
[15] Gill I.Lope-Fancino R..Jorba X. Biologically active peptides and enzymatic approaches to their production. [J]Enzyme Microb. Technol. 1996 (18): 162～183
[16] D.A.Clare and H.E.Swaisgood. Bioactive milk peptides:a prospectus. [J].Dairy Sci.2000 (83):1187～1195
[17] 保健食品功能因子-生物活性肽 中国食品报 2000．12．29
[18] Lubert Sryer. 魏光辉译，Biochem，北京：高等教育出版社，1995
[19] 寇文国，高洪庆 核桃产品的开发利用 [J]中国油脂2000(6)：112～113
[20] 富发斌，杨为燕，舒长青 核桃的综合加工与利用 [J]中国林副特产 2001(3)：36～37
[21] [美]A．H．恩斯明格等著 美国《食物与营养百科全书》选集(5)北京：农业出版社，1989，146～147
[22] [美]A．H．恩斯明格等著 美国《食物与营养百科全书》选集(1)北京：农业出版社，1989，322～324
[23] 李正明，王兰君编《植物蛋白生产工艺与配方》北京：中国轻工业出版社 1998，8
[24] 王者悦主编《中国药膳大辞典》 大连出版社，1992，6：89
[25] 王者悦主编《中华养生大辞典》 大连出版社，1990，1：433
[26] Yukio FuRUUCHI et al., Nippon Shokuhin Kogyo Cakkaishi. 1981, 10:548～553
[27] 崔莉 葛文光 核桃蛋白质功能性质的研究 [J]宁夏农学院学报 1999(4)：44～45
[28] 高福成主编《食品分离重组工程技术》北京：中国轻工业出版社，1998，787～807
[29] 赵新淮 冯志彪 蛋白质水解物水解度的测定 [J]食品科学 1994(11)：65～67
[30] 中山大学生物学生化微生物学教研室编《生化技术导论》人民教育出版社 1981，9
[31] 周顺伍主编《生物化学实验技术》北京农业大学出版社 1991，10
[32] 苏拔贤主编《生物化学制备技术》北京：科学出版社 1986，1
[33] 王尔惠编著《大豆蛋白质生产新技术》北京：中国轻工业出版社 1999，9
[34] 高雯等编著《食品酶学原理与分析方法》黑龙江科学技术出版社 1991，4
[35] [德]B．施特尔马赫著《酶的测定方法》中国轻工业出版社 1992，8
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[47] 冯凤琴，许时婴，王璋 酪蛋白磷酸肽(CPPs)的分子量及持钙功能的分析 [J]食品工业 1997(2)：18～21
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[52] 瞿瑞文等 玉米肽的制备、特性与应用 [J]食品工业1998(3)：11～14
[2] 吴建平 生物活性肽的研究进展 [J]食品与机械 1998(1)：6～8
[3] 郭清泉，张兰威 食品中活性肽的研究 [J]食品与机械 1999，(6)：12～14
[4] Adibi,A.S Intestinal phase of protein assimilation in man.. [J]Amer. J.Clin.Nutr.1976 (29):205～215
[5] Hara, H. [J] Nutr. 1984,114:1122
[6] Silk, D.B.A. Uses of a peptide rather than amino acid nitrogen source in chemically defined elemental diets. [J]Parenter. Enter. Nutr. 1990,(4):548～553
[7] 李虹奇 活性多肽蛋白质研究近况 [J]中草药 1993(7)：373～378
[8] 贾素娟等 生物抗菌多肽 [J]氨基酸和生物资源 2000(4)：5～8
[9] Massaki Yoshikawa et al. Opioid peptides from milk proteins. [J]Agric.Biol.Chem. 1986 (9):2419～2421
[10] Chang. K.J etc Morphiceptin:a potent and specific agonistic for morphine(μ) receptors.[J]science 1981 (212):75～77
[11] Yatabe Tskuba.et al. Studies on the active site and antihypertensive activity of angiotensin I-converting enzyme inhibitors derived from casein. [J]Afric. Biol.Chem. 1987(6):1581～1586
[12] 盛国华 大豆多肽的功能及应刚 [J]食品工业科技 1993(6)：21～26
[13] 汤亚杰 吴思方 酪蛋白磷酸肽的研究进展 [J]食品科学1998(5)：3～6
[14] 尤新主编《功能性发酵制品》北京：中国轻工业出版社 2000，1，203～228
[15] Gill I.Lope-Fancino R..Jorba X. Biologically active peptides and enzymatic approaches to their production. [J]Enzyme Microb. Technol. 1996 (18): 162～183
[16] D.A.Clare and H.E.Swaisgood. Bioactive milk peptides:a prospectus. [J].Dairy Sci.2000 (83):1187～1195
[17] 保健食品功能因子-生物活性肽 中国食品报 2000．12．29
[18] Lubert Sryer. 魏光辉译，Biochem，北京：高等教育出版社，1995
[19] 寇文国，高洪庆 核桃产品的开发利用 [J]中国油脂2000(6)：112～113
[20] 富发斌，杨为燕，舒长青 核桃的综合加工与利用 [J]中国林副特产 2001(3)：36～37
[21] [美]A．H．恩斯明格等著 美国《食物与营养百科全书》选集(5)北京：农业出版社，1989，146～147
[22] [美]A．H．恩斯明格等著 美国《食物与营养百科全书》选集(1)北京：农业出版社，1989，322～324
[23] 李正明，王兰君编《植物蛋白生产工艺与配方》北京：中国轻工业出版社 1998，8
[24] 王者悦主编《中国药膳大辞典》 大连出版社，1992，6：89
[25] 王者悦主编《中华养生大辞典》 大连出版社，1990，1：433
[26] Yukio FuRUUCHI et al., Nippon Shokuhin Kogyo Cakkaishi. 1981, 10:548～553
[27] 崔莉 葛文光 核桃蛋白质功能性质的研究 [J]宁夏农学院学报 1999(4)：44～45
[28] 高福成主编《食品分离重组工程技术》北京：中国轻工业出版社，1998，787～807
[29] 赵新淮 冯志彪 蛋白质水解物水解度的测定 [J]食品科学 1994(11)：65～67
[30] 中山大学生物学生化微生物学教研室编《生化技术导论》人民教育出版社 1981，9
[31] 周顺伍主编《生物化学实验技术》北京农业大学出版社 1991，10
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