脂质氢过氧化物氧化对核桃分离蛋白结构的影响
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摘要
采用脂肪氧合酶催化亚油酸构建脂质氢过氧化物——核桃分离蛋白氧化体系,研究脂质氧化反应中产生的氢过氧化物对核桃分离蛋白结构特性的影响。结果显示,向100 m L 0. 05 mg/m L蛋白溶液中添加9 m L亚油酸,核桃分离蛋白溶解度下降至6. 89%,游离巯基含量由2. 82μmol SH/g下降至1. 92μmol SH/g,羰基含量由2. 26 nmol/mg增加至4. 23 nmol/mg,表明氧化后核桃蛋白理化特性产生变化。圆二色谱分析表明,蛋白质氧化导致核桃分离蛋白α-螺旋和β-折叠含量下降,β-转角和无规则卷曲含量上升。随着亚油酸添加量的增加,蛋白质氧化程度加大,核桃分离蛋白表面疏水性从429. 66下降到405. 24。内源荧光最大荧光峰位红移,内源荧光强度下降,此结果表明,脂肪氧合酶(lipoxygenase,LOX)催化亚油酸氧化诱导核桃分离蛋白聚集,并进一步使蛋白质二级和三级结构发生变化。体积凝胶色谱法结果显示,核桃分离蛋白氧化后,小分子多肽含量下降,高分子量聚集体含量增加,说明脂质氢过氧化物氧化修饰导致核桃蛋白结构改变,形成氧化聚集体。
Walnut protein isolates were oxidized by lipid hydroperoxides,which were produced from different levels of linoleic acid oxidation catalysed by lipoxygenase. Effects of hydroperoxides on structural characteristics of walnut protein isolates were studied. The results showed that when 9 m L linoleic acid was added to 100 m L 0. 05 mg/m L protein solution,walnut protein solubility decreased to 6. 89%. The level of free sulfhydryl group decreased from 2. 82 umol SH/g to 1. 92 umol SH/g,and the carbonyl level increased from 2. 26 nmol/mg to 4. 23 nmol/mg. This indicated that the physicochemical properties of isolated walnut protein changed after oxidation. The CD spectrum analysis showed that protein oxidation caused reduction in the ɑ-helix and β-sheet contents,and the contents of β-turn and random-coil increased. With increasing amount of linoleic acid added,the degree of protein oxidation increased and protein surface hydrophobicity decreased from 429. 66 to 405. 24. Moreover,red shift happened to the highest fluorescent peak,and the intensity of endogenous fluorescence decreased. This suggested that lipid hydroperoxide oxidation could induce aggregation of isolated walnut protein,thus leading to further changes in its secondary and tertiary structures. Furthermore,oxidation caused the contents of small molecule polypeptides decreasing,and the levels of protein aggregates gradually increased in the molecular weight distribution of walnut protein. The results indicated that the structure of walnut protein changed by lipid hydroperoxide oxidation,which caused the formation of oxidative aggregates of walnut protein.
Walnut protein isolates were oxidized by lipid hydroperoxides,which were produced from different levels of linoleic acid oxidation catalysed by lipoxygenase. Effects of hydroperoxides on structural characteristics of walnut protein isolates were studied. The results showed that when 9 m L linoleic acid was added to 100 m L 0. 05 mg/m L protein solution,walnut protein solubility decreased to 6. 89%. The level of free sulfhydryl group decreased from 2. 82 umol SH/g to 1. 92 umol SH/g,and the carbonyl level increased from 2. 26 nmol/mg to 4. 23 nmol/mg. This indicated that the physicochemical properties of isolated walnut protein changed after oxidation. The CD spectrum analysis showed that protein oxidation caused reduction in the ɑ-helix and β-sheet contents,and the contents of β-turn and random-coil increased. With increasing amount of linoleic acid added,the degree of protein oxidation increased and protein surface hydrophobicity decreased from 429. 66 to 405. 24. Moreover,red shift happened to the highest fluorescent peak,and the intensity of endogenous fluorescence decreased. This suggested that lipid hydroperoxide oxidation could induce aggregation of isolated walnut protein,thus leading to further changes in its secondary and tertiary structures. Furthermore,oxidation caused the contents of small molecule polypeptides decreasing,and the levels of protein aggregates gradually increased in the molecular weight distribution of walnut protein. The results indicated that the structure of walnut protein changed by lipid hydroperoxide oxidation,which caused the formation of oxidative aggregates of walnut protein.
引文
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[30]吴伟,李彤,蔡勇建,等.过氧自由基氧化对大米蛋白结构的影响[J].现代食品科技,2016,32(11):111-116.
[31]叶林,廖钰,赵谋明.花生分离蛋白氧化过程中的结构变化[J].食品与机械,2015,31(2):3-6.
[32]王静,金征宇,江波,等. Aspergillus ficuum菊粉酶的荧光光谱研究[J].食品科学,2009,30(23):237-241.
[33] HUANG Y R,HUA Y F,QIU A Y. Soybean protein aggregation induced by lipoxygenase catalyzed linoleic acid oxidation[J]. Food Research International,2006,39:240-249.
[34] GIULIVI C,DAVIES K J A. Dityrosine and tyrosine oxidation products are endogenous markers for the selective proteolysis of oxidatively modified red blood cell hemoglobin by(the 19S)proteasome[J]. The Journal of Biological Chemistry,1993,268(12):8 752-8 759.
[35] YE L,LIAO Y,SUN W Z,et al. Effect of protein oxidation on the stability of peanut beverage[J]. Cy TA-Journal of Food,2015,13(1):49-55.
[36]袁德保.大豆蛋白热聚集行为及其机理研究[D].广州:华南理工大学,2010.
[2] GIESEG S P,PEARSON J,FIRTH C A. Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper,peroxyl radical and macrophagemediated oxidation[J]. Free Radical Research,2003,37(9):983-991.
[3] ROBINSON D S,WU Z,DOMONEY C,et al. Lipoxygenases and the quality of foods[J]. Food Chemistry,1995,54:33-43.
[4] STADTMAN ER,BERLETT BS. Reactive oxygen-mediated protein oxidation in aging and disease[J]. Drug Metabolism Re-views,1998,30(2):225-243.
[5] SAEED S,FAWTHROP S A,Howell N K. Electron spin resonance(ESR)study on free-radical transfer in fish lipid-protein interactions[J]. Journal of the Science of Food and Agriculture,1999,79:1 809-1 816.
[6] ROUBAL W T,TAPPEL A L. Polymerization of proteins induced by free-radical lipid peroxidation[J]. Archives of Biochemistry and Biophysics,1966,113:150-155.
[7] HUA Y F,HUANG Y R,QIU A Y,et al. Properties of soy protein isolate prepared from aqueous alcohol washed soy flakes[J]. Food Research International,2005,38(3):273-279.
[8]黄友如,裘爱泳,华欲飞,等.大豆分离蛋白风味物质的气相色谱-质谱分析[J].分析化学,2005,33(3):389-391.
[9] HUANG Y R,YU D,HUA Y F,et al. Detection of free radical transfer in lipoxygenase I-B-catalyzed linoleic acid soybean protein interaction by electron spin resonance spectroscopy(ESR)[J]. Journal of Agricultural and Food Chemistry,2006,54(24):9 216-9 220.
[10]刘云.核桃仁油脂及蛋白的综合利用研究[D].广州:华南理工大学,2011.
[11]何志平.浙江山核桃抗氧化与蛋白质特性研究[D].杭州:浙江大学,2011.
[12]徐华.气调贮藏对花生核桃仁及其加工品品质的影响[D].南京:南京农业大学,2011.
[13]毛晓英.核桃蛋白质的结构表征及其制品的改性研究[D].无锡:江南大学,2012.
[14]叶林.花生蛋白质氧化作用及其对花生乳饮料稳定性影响研究[D].广州:华南理工大学,2014.
[15] HUANG Y R,HUA Y F,QIU A Y. Soybean protein aggregation induced by lipoxygenase catalyzed linoleic acid oxidation[J]. Food Research International,2006,39(2),240-249.
[16]李艳青.蛋白质氧化对鲤鱼蛋白结构和功能性质的影响[D].哈尔滨:东北农业大学,2013.
[17] YE LIN,LIAO YU,ZHAO MOUMING,et al. Effect of protein oxidation on the conformational properties of peanut protein isolate[J]. Journal of Chemistry,2013,1-6.
[18]孙领鸽,王丹丹,毛晓英,等.丙烯醛氧化修饰对核桃蛋白结构和乳化特性的影响[J].食品科学,2018,39(20):43-48.
[19]吴伟,吴晓娟.过氧自由基氧化修饰对大米蛋白功能性质的影响[J].食品与机械,2016,32(6):5-8.
[20] ZIRLIN A,KAREL M. Oxidation effects in a freeze-dried gelatin-methyl linoleate system[J]. Journal of Food Science,1969,34:160-164.
[21]黄友如.脂肪氧合酶催化亚油酸诱导大豆蛋白聚集机理[D].无锡:江南大学,2006.
[22] ZAMORA R,ALAIZ M,HIDALGO F J. Modification of histidine residues by 4,5-epoxy-3-alkenals[J]. Chemical Research in Toxicology,1999,12:654-660.
[23]崔旭海,孔保华,熊幼翎.自由基氧化引起乳清蛋白理化性质变化的研究[J].中国乳品工业,2008,36(9):31-34.
[24] KALAPATHY U,HETTIARACHCHY N S,RHEE K C.Effect of drying methods on molecular properties and functionalities of disulfide bond-cleaved soy proteins[J].Journal of the American Oil Chemists Society,1997,74(3):195-199.
[25] GARDNER H W,KLEIMAN R,WEISLEDER D,et al.Cysteine adds to lipid hydroperoxide[J]. Lipids,1977,12(8):655-660.
[26] HOU D H,CHANG S K C. Structural characteristics of purified glycinin from soybeans stored under various conditions[J]. Journal of Agricultural and Chemistry,2004,52(12):3 792-3 800.
[27] GERBANOWSKI A,MALABAT C,RABILLER C,et al.Grafting of aliphatic and aromatic probes on rapeseed 2S and 12S proteins:influence on their structural and physicochemical[J]. Journal of Agricultural and Food Chemistry,1999,47:5 218-5 226.
[28]沈星灿,梁宏,何锡文,等.圆二色光谱分析蛋白质构象的方法及研究进展[J].分析化学,2004(3):388-394.
[29]吴明和.圆二色光谱在蛋白质结构研究中的应用[J].氨基酸和生物资源,2010,32(4):77-80.
[30]吴伟,李彤,蔡勇建,等.过氧自由基氧化对大米蛋白结构的影响[J].现代食品科技,2016,32(11):111-116.
[31]叶林,廖钰,赵谋明.花生分离蛋白氧化过程中的结构变化[J].食品与机械,2015,31(2):3-6.
[32]王静,金征宇,江波,等. Aspergillus ficuum菊粉酶的荧光光谱研究[J].食品科学,2009,30(23):237-241.
[33] HUANG Y R,HUA Y F,QIU A Y. Soybean protein aggregation induced by lipoxygenase catalyzed linoleic acid oxidation[J]. Food Research International,2006,39:240-249.
[34] GIULIVI C,DAVIES K J A. Dityrosine and tyrosine oxidation products are endogenous markers for the selective proteolysis of oxidatively modified red blood cell hemoglobin by(the 19S)proteasome[J]. The Journal of Biological Chemistry,1993,268(12):8 752-8 759.
[35] YE L,LIAO Y,SUN W Z,et al. Effect of protein oxidation on the stability of peanut beverage[J]. Cy TA-Journal of Food,2015,13(1):49-55.
[36]袁德保.大豆蛋白热聚集行为及其机理研究[D].广州:华南理工大学,2010.