羟自由基氧化对草鱼肌原纤维蛋白结构和凝胶性质的影响
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摘要
以草鱼肉为研究对象,采用芬顿反应体系(H_2O_2浓度分别为0.1、1.0、5.0、10.0 mmol/L)产生不同浓度·OH对其肌原纤维蛋白进行模拟氧化,研究·OH氧化对草鱼肌原纤维蛋白结构及凝胶性质的影响。结果表明,氧化使肌原纤维蛋白羰基含量显著增加(P<0.05),当氧化剂H_2O_2的浓度为10.0 mmol/L时,羰基含量比对照组增加了111.35%;随着H_2O_2浓度的提高,肌原纤维蛋白总巯基、活性巯基、游离氨基含量显著下降(P<0.05);二聚酪氨酸水平和表面疏水性显著增加(P<0.05)。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明氧化使蛋白质分子间发生交联和聚集,产生了分子质量大于200 ku的蛋白聚集体。可见,·OH氧化使草鱼肌原纤维蛋白中巯基、酪氨酸、色氨酸等氨基酸残基发生了显著氧化修饰,蛋白质分子产生了交联和聚集,蛋白质构象发生了显著变化。此外,氧化可进一步影响肌原纤维蛋白的凝胶性质,使其凝胶网络结构变疏松、凝胶孔隙增大,凝胶中部分不易流动水转变为自由水,最终导致凝胶强度和凝胶持水性显著降低。
In this paper,myofibrillar protein from grass carp was artificially oxidized in hydroxyl radical-generating system with H_2O_2 concentrations of 0.1,1.0,5.0 and 10.0 mmol/L,and the effect of oxidization on the structural characteristics and gel properties of myofibrillar protein was investigated.The results showed that the carbonyl content increased significantly(P < 0.05) after oxidization by 111.35% compared to the initial value when the concentration of H_2O_2 was 10.0 mmol/L.With the increase in H_2O_2 concentration,the total thiol group level,free thiol group level,and free amine content decreased significantly(P < 0.05),while bityrosine content and surface hydrophobicity increased significantly(P < 0.05).The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) result showed that hydroxyl radical oxidation could induce the crosslinking and aggregation of protein molecules,thereby forming aggregates with molecular mass higher than 200 ku.The above results indicated that the thiol group,tyrosine and tryptophane residues of myofibrilar protein were modified after hydroxyl radical oxidation,thus leading to a significant change in protein structure.In addition,the gel properties of myofibrillar protein were also influenced by oxidation,the network structure of the gel was damaged,and the portable water in the gel was converted to free water.These changes finally induced apparent decreases in the gel strength and water-holding capacity.
In this paper,myofibrillar protein from grass carp was artificially oxidized in hydroxyl radical-generating system with H_2O_2 concentrations of 0.1,1.0,5.0 and 10.0 mmol/L,and the effect of oxidization on the structural characteristics and gel properties of myofibrillar protein was investigated.The results showed that the carbonyl content increased significantly(P < 0.05) after oxidization by 111.35% compared to the initial value when the concentration of H_2O_2 was 10.0 mmol/L.With the increase in H_2O_2 concentration,the total thiol group level,free thiol group level,and free amine content decreased significantly(P < 0.05),while bityrosine content and surface hydrophobicity increased significantly(P < 0.05).The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) result showed that hydroxyl radical oxidation could induce the crosslinking and aggregation of protein molecules,thereby forming aggregates with molecular mass higher than 200 ku.The above results indicated that the thiol group,tyrosine and tryptophane residues of myofibrilar protein were modified after hydroxyl radical oxidation,thus leading to a significant change in protein structure.In addition,the gel properties of myofibrillar protein were also influenced by oxidation,the network structure of the gel was damaged,and the portable water in the gel was converted to free water.These changes finally induced apparent decreases in the gel strength and water-holding capacity.
引文
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[2]LIU D S,LIANG L,XIA W S,et al.Biochemical and physical changes of grass carp(Ctenopharyngodon idella)fillets stored at-3and 0℃[J].Food Chemistry,2013,140(1):105-114.DOI:10.1016/j.foodchem.2013.02.034.
[3]农业部渔业渔政管理局.2015中国渔业统计年鉴[M].北京:中国农业出版社,2015:1-2.
[4]PARK J W.Surimi and surimi seafood[M]//LANIER T C,YONGSAWATDIGUL J,CARVAJAL-RONDANELLI P.Surimi gelation chemistry.New York:CRC Press Inc.,2013:101-140.
[5]TIMM-HEINRICH M,EYMARD S,BARON C P,et al.Oxidative changes during ice storage of rainbow trout(Oncorhynchus mykiss)fed different ratios of marine and vegetable feed ingredients[J].Food Chemistry,2013,136(3/4):1220-1230.DOI:10.1016/j.foodchem.2012.09.019.
[6]KRISTINSSON H G.Antioxidants and functional components in aquatic foods[M]//BARON C P.Protein oxidation in aquatic foods.New York:John Wiley&Sons Inc.,2014:23-42.
[7]LIU Q,CHEN Q,KONG B H.The influence of superchilling and cryoprotectants on protein oxidation and structural changes in the myofibrillar proteins of common carp(Cyprinus carpio)surimi[J].Food Science and Technology,2014,57(2):603-611.DOI:10.1016/j.lwt.2014.02.023.
[8]李艳青.蛋白质氧化对鲤鱼蛋白结构和功能性的影响及其控制技术[D].哈尔滨:东北农业大学,2013:35-38.
[9]PAZOS M,DA ROCHA A P,ROEPSTORFF P,et al.Fish proteins as targets of ferrous-catalyzed oxidation:identification of protein carbonyls by fluorescent labeling on two-dimensional gels and MALDI-TOF/TOF mass spectrometry[J].Journal of Agricultural and Food Chemistry,2011,59(14):7962-7977.DOI:10.1021/jf201080t.
[10]ZHANG W G,XIAO S,AHN D U.Protein oxidation:basic principles and implications for meat quality[J].Critical Reviews in Food Science and Nutrition,2013,53(11):1191-1201.DOI:10.1080/10408398.2011.577540.
[11]DELLES R M,XIONG Youling L..The effect of protein oxidation on hydration and water-binding in pork packaged in an oxygen-enriched atmosphere[J].Meat Science,2014,97(2):181-188.DOI:10.1016/j.meatsci.2014.01.022.
[12]OLIVER C N,AHN B W,MOERMAN E J,et al.Age-related changes in oxidized proteins[J].Journal of Biological Chemistry,1987,262(12):5488-5491.
[13]YONGSAWATDIGUL J,PARK J W.Thermal denaturation and aggregation of threadfin bream actomyosine[J].Food of Biological Chemistry,2003,83(3):409-416.DOI:10.1016/S0308-8146(03)00105-5.
[14]HABEEB A S A.Determination of free amino groups in proteins by trinitrobenzenesulfonic acid[J].Analytical Biochemistry,1966,14:328-336.DOI:10.1016/0003-2697(66)90275-2.
[15]SAEED S,HOWELL N K.Rheological and differential scanning calorimetry studies on structural and textural changes in frozen Atlantic mackerel(Scomber scombrus)[J].Journal of Science of Food and Agriculture,2004,84:1216-1222.DOI:10.1002/jsfa.1807.
[16]DAVIES K J A,DELSIGNORE M E,LIN S W.Protein damage and degradation by oxygen radicals.Ⅱ:modification of amino acids[J].Journal of Biological Chemistry,1987,262(20):9902-9907.
[17]XIONG Youling L.,PARK D,OOIZUMI T.Variation in the cross-linking pattern of porcine myofibrillar protein exposed to three oxidative environments[J].Journal of Agricultural and Food Chemistry,2008,57(1):153-159.DOI:10.1021/jf8024453.
[18]SμNCHEZ-GONZALEZ I,CARMONA P,MORENO P,et al.Protein and water structural changes in fish surimi during gelation as revealed by isotopic H/D exchange and raman spectroscopy[J].Food Chemistry,2008,106(1):56-64.DOI:10.1016/j.foodchem.2007.05.067.
[19]DEBUSCA A,TAHERGORABI R,BEAMER S K,et al.Interactions of dietary fibre and omega-3-rich oil with protein in surimi gels developed with salt substitute[J].Food Chemistry,2013,141(1):201-208.DOI:10.1016/j.foodchem.2013.02.111.
[20]OUJIFARD A,BENJAKUL S,AHMAD M,et al.Effect of bambara groundnut protein isolate on autolysis and gel properties of surimi from threadfin bream(Nemipterus bleekeri)[J].LWT-Food Science and Technology,2012,47(2):261-266.DOI:10.1016/j.lwt.2012.01.016.
[21]李睿智,王嵬,仪淑敏,等.白鲢鱼鱼糜凝胶过程中水分及凝胶特性的变化[J].现代食品科技,2016,32(5):91-97;198.DOI:10.13982/j.mfst.1673-9078.2016.5.015.
[22]李艳青,孔保华,杨赫鸿,等.自由基氧化引起鲤鱼肌原纤维蛋白结构的变化[J].食品科学,2012,33(13):70-74.
[23]BUTTERFIELD D A,STADTMAN E R.Protein oxidation processes in aging brain[J].Advances in Cell Aging and Gerontology,1997,2:161-191.DOI:10.1016/S1566-3124(08)60057-7.
[24]崔旭海,孔保华,熊幼翎,等.羟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;64.
[25]LUND M N,HEINONEN M,BARON C P,et al.Protein oxidation in muscle foods:a review[J].Molecular Nutrition&Food Research,2011,55(1):83-95.DOI:10.1002/mnfr.201000453.
[26]LUND M N,LAMETSCH R,HVIIDM S,et al.High-oxygen packaging atmosphere influences protein oxidation and tenderness of porcine longinessimus dorsi during chill storage[J].Meat Science,2007,77(3):295-303.DOI:10.1016/j.meatsci.2007.03.016.
[27]LIU G,XIONG Youling L.,BUTTERFIELD D A.Chemical,physical,and gel-forming properties of oxidized myofibrils and whey-and soyprotein isolates[J].Journal of Food Science,2000,65(5):811-818.DOI:10.1111/j.1365-2621.2000.tb13592.x.
[28]刘泽龙.蛋白质氧化对肉及肉制品持水与水合特性的影响机理研究[D].无锡:江南大学,2012:38-39.
[29]孙妍,孔保华,刘骞.羟基自由基氧化体系对乳清蛋白、β-乳球蛋白化学结构的影响[J].食品科学,2009,30(11):17-21.DOI:10.3321/j.issn:1002-6630.2009.11.002.
[30]孔保华,孙妍,熊幼翎.抗氧化剂对羟自由基引起的乳清分离蛋白氧化抑制效果的研究[J].食品科学,2010,31(3):5-10.
[31]吴伟.蛋白质氧化对大豆蛋白结构和凝胶性质的影响[D].无锡:江南大学,2010:27-28.
[32]蔡联辉,曾虹燕,蔡西玲,等.莲子蛋白组分二级结构的研究[J].光谱学与光谱分析,2011,31(9):2394-2398.DOI:10.3964/j.issn.1000-0593(2011)09-2394-05.
[33]黄曼,卞科.理化因子对大豆蛋白疏水性的影响[J].郑州工程学院学报,2002,23(3):5-9.DOI:10.16433/j.cnki.issn1673-2383.2002.03.002
[34]SUN Weizheng,ZHAO Mouming,YANG Bao,et al.Oxidation of sarcoplasmic proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitrodigestibility[J].Meat Science,2011,88(3):462-467.DOI:10.1016/j.meatsci.2011.01.027
[35]LIU Qian,CHEN Qian,KONG Baohua,et al.The influence of superchilling and cryoprotectants on protein oxidation and structural changes in the myofibrillar proteins of common carp(Cyprinus carpio)surimi[J].LWT-Food Science and Technology,2014,57(2):603-611.DOI:10.1016/j.lwt.2014.02.023.
[36]李学鹏,王祺,周凯,等.丙烯醛氧化对大黄鱼肌肉组织和肌原纤维蛋白结构性质的影响[J].现代食品科技,2014(10):1-7.DOI:10.13982/j.mfst.1673-9078.2014.10.001.
[37]王耀松.共价交联对乳清蛋白成膜的影响及作用机理[D].无锡:江南大学,2012:24-25.
[38]XIONG Youling L.,BLANCHARD S P,OOIZUMI T,et al.Hydroxyl radical and ferryl-generating systems promote gel network formation of myofibrillar protein[J].Journal of Food Science,2010,75(2):215-221.DOI:10.1111/j.1750-3841.2009.01511.x.
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