蛋清抗氧化肽分离纯化及结构鉴定
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摘要
为了获得高活性、高纯度的蛋清抗氧化肽,以蛋清酶解物为原料,依次釆用超滤、离子交换色谱、凝胶色谱分离纯化抗氧化活性较强的肽段,运用基质辅助激光解吸离子化质谱解析肽链的氨基酸序列。结果表明:超滤法分离纯化EWPH所得的三个组分中,EWPH-Ⅲ(M_W<3 kDa)组分的DPPH自由基清除率最高,达到79.62%。离子交换层析分离纯化EWPH-III所得到的碱性组分B的DPPH自由基清除率最高,达到82.05%。凝胶过滤色谱分离EWPH-III-B所得到4组分中E组分的DPPH自由基清除率最高,为88.49%。高活性高纯度EWPH-III-B-E组分的相对分子质量为237.575,该二肽的氨基酸序列为丙氨酸-甲硫氨酸。
In order to obtain high activity and high purity egg white antioxidant peptides,the egg white proteolytics were used as raw materials,and the peptides with strong antioxidant activity were separated and purified by ultrafiltration,ion exchange chromatography and gel chromatography.The amino acid sequence of the peptide chain was analyzed by matrix-assisted laser desorption ionization mass spectrometry.The results showed that the DPPH free radical scavenging rate of EWPH-III( MW <3 kDa) was up to 79.62% among the three components obtained by ultrafiltration separation and purification of EWPH.Among the two components obtained by ion exchange chromatography separation and purification of EWPH-III,the DPPH free radical scavenging rate of alkaline component B was up to 82.05%.The 4 components of EWPH-III-B were separated by gel filtration chromatography.The DPPH free radical scavenging rate of EWPH-III-B-E component was 88.49%. The relative molecular mass of EWPH-III-B-E was 237.575 and the amino acid sequence was Ala-Met.
In order to obtain high activity and high purity egg white antioxidant peptides,the egg white proteolytics were used as raw materials,and the peptides with strong antioxidant activity were separated and purified by ultrafiltration,ion exchange chromatography and gel chromatography.The amino acid sequence of the peptide chain was analyzed by matrix-assisted laser desorption ionization mass spectrometry.The results showed that the DPPH free radical scavenging rate of EWPH-III( MW <3 kDa) was up to 79.62% among the three components obtained by ultrafiltration separation and purification of EWPH.Among the two components obtained by ion exchange chromatography separation and purification of EWPH-III,the DPPH free radical scavenging rate of alkaline component B was up to 82.05%.The 4 components of EWPH-III-B were separated by gel filtration chromatography.The DPPH free radical scavenging rate of EWPH-III-B-E component was 88.49%. The relative molecular mass of EWPH-III-B-E was 237.575 and the amino acid sequence was Ala-Met.
引文
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[22]Chen H M,Muramoto K,Yamauchi F.Structural analysis of antioxidative peptides from soybean beta-conglycinin[J].Journal of Agricultural and Food Chemistry,1995,43(3):574-578.
[23]Stadtman E R,Berlett B S,Chock P B.Manganesedependent disproportionation of hydrogen peroxide in bicarbonate buffer[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(1):384-388.
[2]刘建涛,赵利,苏伟.鸡蛋中的生物活性蛋白质和肽[J].中国酿造,2008,24:27-29.
[3]张铁华.蛋清活性蛋白高压脉冲电场提取及功能特性研究[D].长春:吉林大学,2008.
[4]李勇.生物活性肽研究现况和进展[J].食品与发酵工业,2007,33(1):3-9.
[5]杜永盛.酶解咸鸭蛋蛋清制取白蛋白肽的研究[D].杭州:浙江大学,2006.
[6]Bodnar R J,Klein G E.Endogenous opiates and behavior:2003[J].Peptides,2004,25(12):2205-2256.
[7]Zambrowicz A,Timmer M,Eckert E,et al.Evaluation of the ACE-inhibitory activity of egg-white proteins degraded with pepsin.[J].Polish Journal of Food&Nutrition Sciences,2013,63(2):103-108.
[8]Mills S,Ross R P,Hill C,et al.Milk intelligence:Mining milk for bioactive substances associated with human health[J].International Dairy Journal,2011,21(6):377-401.
[9]Murakami M,Tonouchi H,Takahashi R,et al.Structural analysis of a new anti-hypertensive peptide(lactosin B)isolated from a commercial whey product[J].Journal of Dairy Science,2004,87(7):1967-1974.
[10]Liu J,Jin Y,Lin S,et al.Purification and identification of novel antioxidant peptides from egg white protein and their antioxidant activities[J].Food Chemistry,2015,175(9):258-266.
[11]Stuknyte M,De Noni I,Gugliemetti S,et al.Potential immunomodulatory activity of bovine casein hydrolysates produced after digestion with proteinase of lactic acid bacteria[J].Int Dairy J,2011,21:763-769.
[12]刘长江,王雪,曹向宇.麦麸多肽稳定性的研究[J].食品科技,2009(5):164-167.
[13]Ali B,Yousra T E,Moncef N,et al.Biochemical and functional properties of Sardinella(Sardinella aurita)by-product hydrolysates[J].Food Technology&Biotechnology,2007,45(2):187-194.
[14]Ruxton C H S,Derbyshire E,Gibson S.The nutritional properties and health benefits of eggs[J].Nutrition&Food Science,2010,40(3):263-279.
[15]陈日春.鲢鱼鱼鳞胶原蛋白肽的制备及其抗氧化活性的研究[D].福州:福建农林大学,2013.
[16]程云辉,文新华,王璋.超滤分离纯化麦胚蛋白酶解物的研究[J].湖南科技大学学报:自然科学版,2007,22(2):102-105.
[17]Ye M,Zou H,Liu Z,et al.Separation of peptides by strong cation-exchange capillary electrochromatography[J].Journal of Chromatography A,2000,869(1):385-394.
[18]钟芳.大豆肽的离子交换色谱分离及其活性评价[J].食品与机械,2006,22(5):16-19.
[19]Shazly A B,He Z,El-Aziz M A,et al.Fractionation and identification of novel antioxidant peptides from buffalo and bovine casein hydrolysates[J].Food Chemistry,2017,232:753-762.
[20]Wu H C,Chen H M,Shiau C Y.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Food Research International,2003,36(9):949-957.
[21]You L,Zhao M,Regenstein J M,et al.Purification and identification of antioxidative peptides from loach(Misgurnus anguillicaudatus)protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry[J].Food Research International,2010,43(4):1167-1173.
[22]Chen H M,Muramoto K,Yamauchi F.Structural analysis of antioxidative peptides from soybean beta-conglycinin[J].Journal of Agricultural and Food Chemistry,1995,43(3):574-578.
[23]Stadtman E R,Berlett B S,Chock P B.Manganesedependent disproportionation of hydrogen peroxide in bicarbonate buffer[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(1):384-388.