超高压对大豆球蛋白抗原性及结构的影响
|
摘要
以脱脂大豆粉为原料,利用碱溶酸沉法分离提取大豆球蛋白,采用间接竞争酶联免疫吸附法测定不同压力、加压时间及不同质量浓度下大豆球蛋白的抗原性变化,并对超高压后产物的免疫原性及结构特性进行分析。结果表明:超高压能显著影响大豆球蛋白的抗原性;免疫印迹结果显示超高压处理后大豆球蛋白的免疫原性有一定程度的降低,但不能完全消除;傅里叶变换红外光谱结果表明超高压处理之后样品蛋白中α-螺旋和β-折叠的含量减少,β-转角和无规卷曲含量增加;非还原性电泳与荧光光谱结果表明,大豆球蛋白的空间结构解聚,疏水性氨基酸残基暴露在蛋白质表面,蛋白质的三、四级空间结构被破坏;蛋白质空间结构的改变可能会引起抗原表位的掩盖,从而使其抗原性降低。
Glycinin was isolated from defatted soybean meal by alkali solubilization and acid precipitation method in this study. The antigencity of glycinin was determined by indirect competitive enzyme-linked immunosorbent assay(ELISA) and investigated as a function of pressure, pressurization time and glycinin concentration. The immunogenicity and structural characteristics were analyzed after high hydrostatic pressure treatment. The indirect competitive ELISA showed that high hydrostatic pressure treatment could significantly affect the antigenicity of glycinin. The results of immunoblotting showed that the immunogenicity of glycinin was reduced but not completely eliminated after the treatment. Fourier transform infrared spectroscopy showed that the contents of α-helix and β-sheet in the treated sample decreased whereas β-turn and random coil increased. Non-reduced electrophoresis and fluorescence spectroscopy demonstrated that the spatial structure of glycinin was depolymerized and the hydrophobic amino acid residues were exposed on the surface of the protein. Moreover, its tertiary and quartenary structures were destroyed. The change in the spatial structure of the protein may mask its antigenic epitopes, thereby reducing its antigenicity.
Glycinin was isolated from defatted soybean meal by alkali solubilization and acid precipitation method in this study. The antigencity of glycinin was determined by indirect competitive enzyme-linked immunosorbent assay(ELISA) and investigated as a function of pressure, pressurization time and glycinin concentration. The immunogenicity and structural characteristics were analyzed after high hydrostatic pressure treatment. The indirect competitive ELISA showed that high hydrostatic pressure treatment could significantly affect the antigenicity of glycinin. The results of immunoblotting showed that the immunogenicity of glycinin was reduced but not completely eliminated after the treatment. Fourier transform infrared spectroscopy showed that the contents of α-helix and β-sheet in the treated sample decreased whereas β-turn and random coil increased. Non-reduced electrophoresis and fluorescence spectroscopy demonstrated that the spatial structure of glycinin was depolymerized and the hydrophobic amino acid residues were exposed on the surface of the protein. Moreover, its tertiary and quartenary structures were destroyed. The change in the spatial structure of the protein may mask its antigenic epitopes, thereby reducing its antigenicity.
引文
[1]陈新,顾和平,张红梅,等.江苏省大豆生产发展历史、现状与前景分析[J].江苏农业科学,2011(1):6-9.
[2]ZHU C Y,LIU H F,FU M,et al.Structure and property changes of soybean protein isolates resulted from the glycation and cross-linking by transglutaminase and a degraded chitosan[J].Cy TA-Journal of Food,2016,14(1):138-144.DOI:10.1080/19476337.2015.1067646.
[3]AMNUAYCHEEWA P,MEJIA E G D.Purification,characterisation,and quantification of the soy allergen profiling(Gly m 3)in soy products[J].Food Chemistry,2010,119(4):1671-1680.DOI:10.1016/j.foodchem.2009.09.034.
[4]HOLZHAUSER T,WACKERMANN O,BALLMER-WEBER B K,et al.Soybean(Glycine max)allergy in Europe:Gly m 5(betaconglycinin)and Gly m 6(glycinin)are potential diagnostic markers for severe allergic reactions to soy[J].Journal of Allergy and Clinical Immunology,2009,123(2):452-458.DOI:10.1016/j.jaci.2008.09.034.
[5]孙鹏.蒸汽处理对纯化大豆抗原的含量及免疫原性的影响[D].长春:吉林农业大学,2005:1.
[6]NATARAJAN S S,XU C,BAE H,et al.Characterization of storage proteins in wild(Glycine soja)and cultivated(Glycine max)soybean seeds using proteomic analysis[J].Journal of Agricultural and Food Chemistry,2006,54(8):3114-3120.DOI:10.1021/jf052954k.
[7]RIVALAIN N,ROQUAIN J,DEMAZEAU G.Development of high hydrostatic pressure in biosciences:pressure effect on biological structures and potential applications in biotechnologies[J].Biotechnology Advances,2010,28(6):659-672.DOI:10.1016/j.biotechadv.2010.04.001.
[8]HU Chunqiu,CHEN Hongbing,GAO Jinyan,et al.High-pressure microfluidisation-induced changes in the antigenicity and conformation of allergen Ara h 2 purified from Chinese peanut[J].Journal of the Science of Food&Agriculture,2011,91(7):1304-1309.DOI:10.1002/jsfa.4318.
[9]LI Huijing,ZHU Kexue,ZHOU Huiming,et al.Effects of high hydrostatic pressure treatment on allergenicity and structural properties of soybean protein isolate for infant formula[J].Food Chemistry,2012,132(2):808-814.DOI:10.1016/j.foodchem.2011.11.040.
[10]HILDEBRANDT S,SCHüTTEL,STOYANOV S,et al.I n vitro determination of the allergenic potential of egg white in processed meat[J].Journal of Allergy,2010,2010:1-5.DOI:10.1155/2010/238573.
[11]贺梦雪,席俊,皮江一.响应面法优化β-伴大豆球蛋白超高静压处理条件的研究[J].河南工业大学学报(自然科学版),2017,38(2):69-74.DOI:10.16433/j.cnki.issn1673-2383.2017.02.012.
[12]FERNANDES L,CASAL S,PEREIRA J A,et al.Effect of high hydrostatic pressure(HHP)treatment on edible flowers’properties[J].Food and Bioprocess Technology,2017,10(5):799-807.DOI:10.1007/s11947-017-1887-2.
[13]THANH V H,SHIBASAKI K.Major proteins of soybean seeds.a straightforward fractionation and their characterization[J].Journal of Agricultural and Food Chemistry,1976,24(6):1117-1121.DOI:10.1021/jf60208a030.
[14]LIU Chun,WANG Hongling,CUI Zhumei,et al.Optimization of extraction and isolation for 11S and 7S globulins of soybean seed storage protein[J].Food Chemistry,2007,102(4):1310-1316.DOI:10.1016/j.foodchem.2006.07.017.
[15]宋佳.大豆11S和7S球蛋白提取及其凝胶显微结构研究[D].天津:天津科技大学,2013:6.
[16]冯挺财,邵谱,胡珩,等.牛乳铁蛋白直接竞争ELISA检测法的建立[J].安徽农业科学,2008(18):7650-7652.DOI:10.3969%2fj.issn.0517-6611.2008.18.072.
[17]张楠,布冠好,朱婷伟,等.糖基化反应对大豆蛋白-乳糖复合物抗原性及结构的影响[J].现代食品科技,2015,31(8):117-121;272.DOI:10.13982/j.mfst.1673-9078.2015.8.020.
[18]DHAKAL S,LIU C Q,ZHANG Y,et al.Effect of high pressure processing on the immunoreactivity of almond milk[J].Food Research International,2014,62(1):215-222.DOI:10.1016/j.foodres.2014.02.021.
[19]LUO Y,LI M,ZHU K X,et al.Heat-induced interaction between egg white protein and wheat gluten[J].Food Chemistry,2016,197:699-708.DOI:10.1016/j.foodchem.2015.09.088.
[20]方佳茂,刘偲琪,庄楚周,等.复合酶水解蚕蛹蛋白制备功能性寡肽的工艺研究[J].现代食品科技,2012,28(3):323-328.DOI:10.13982/j.mfst.1673-9078.2012.03.020.
[21]郭丽萍.超高压结合热处理对猪肉蛋白质氧化、结构及特性的影响[D].绵阳:?西南科技大学,2016:13.
[22]ZHOU H,WANG C Z,YE J Z,et al.Effects of high hydrostatic pressure treatment on structural,allergenicity,and functional properties of proteins from ginkgo seeds[J].Innovative Food Science and Emerging Technologies,2016,34:187-195.DOI:10.1016/j.ifset.2016.02.001.
[23]SHRIVER S K,YANG W W.Thermal and nonthermal methods for food allergen control[J].Food Engineering Reviews,2011,3(1):26-43.DOI:10.1007/s12393-011-9033-9.
[24]王章存,徐贤.超高压处理对蛋白质结构及功能性质影响[J].粮食与油脂,2007(11):10-12.DOI:10.3969/j.issn.1008-9578.2007.11.003.
[25]薛路舟.超高压处理对生物大分子的影响研究[D].大连:大连理工大学,2010:48.
[26]ZHANG X,QI J R,LI K K,et al.Characterization of soy β-conglycinin-dextran conjugate prepared by Maillard reaction in crowded liquid system[J].Food Research International,2012,49(2):648-654.DOI:10.1016/j.foodres.2012.09.001.
[27]王治平.菜籽分离蛋白糖接枝改性及其功能、结构和消化吸收特性研究[D].镇江:江苏大学,2016:32.
[28]BU G H,ZHANG N,CHEN F S.The influence of glycosylation on the antigenicity,allergenicity,and structural properties of 11S-lactose conjugates[J].Food Research International,2015,76(Pt 3):511-517.DOI:10.1016/j.foodres.2015.08.004.
[29]BU G H,ZHU T W,CHEN F S,et al.Effects of saccharide on the structure and antigenicity ofβ-conglycinin in soybean protein isolate by glycation[J].European Food Research and Technology,2015,240(2):285-293.DOI:10.1007/s00217-014-2326-5.
[30]TANG C H,MA C Y.Effect of high pressure treatment on aggregation and structural properties of soy protein isolate[J].LWT-Food Science and Technology,2009,42(2):606-611.DOI:10.1016/j.lwt.2008.07.012.
[31]苏丹,李树君,赵凤敏,等.超高压处理对大豆分离蛋白结构的影响[J].食品科技,2009,34(12):51-55.DOI:10.13684/j.cnki.spkj.2009.12.059.
[32]罗春萍.热加工、辐照及超高压微射流对花生过敏原Ara h 6结构与抗原性的影响[D].南昌:南昌大学,2011:49.
[2]ZHU C Y,LIU H F,FU M,et al.Structure and property changes of soybean protein isolates resulted from the glycation and cross-linking by transglutaminase and a degraded chitosan[J].Cy TA-Journal of Food,2016,14(1):138-144.DOI:10.1080/19476337.2015.1067646.
[3]AMNUAYCHEEWA P,MEJIA E G D.Purification,characterisation,and quantification of the soy allergen profiling(Gly m 3)in soy products[J].Food Chemistry,2010,119(4):1671-1680.DOI:10.1016/j.foodchem.2009.09.034.
[4]HOLZHAUSER T,WACKERMANN O,BALLMER-WEBER B K,et al.Soybean(Glycine max)allergy in Europe:Gly m 5(betaconglycinin)and Gly m 6(glycinin)are potential diagnostic markers for severe allergic reactions to soy[J].Journal of Allergy and Clinical Immunology,2009,123(2):452-458.DOI:10.1016/j.jaci.2008.09.034.
[5]孙鹏.蒸汽处理对纯化大豆抗原的含量及免疫原性的影响[D].长春:吉林农业大学,2005:1.
[6]NATARAJAN S S,XU C,BAE H,et al.Characterization of storage proteins in wild(Glycine soja)and cultivated(Glycine max)soybean seeds using proteomic analysis[J].Journal of Agricultural and Food Chemistry,2006,54(8):3114-3120.DOI:10.1021/jf052954k.
[7]RIVALAIN N,ROQUAIN J,DEMAZEAU G.Development of high hydrostatic pressure in biosciences:pressure effect on biological structures and potential applications in biotechnologies[J].Biotechnology Advances,2010,28(6):659-672.DOI:10.1016/j.biotechadv.2010.04.001.
[8]HU Chunqiu,CHEN Hongbing,GAO Jinyan,et al.High-pressure microfluidisation-induced changes in the antigenicity and conformation of allergen Ara h 2 purified from Chinese peanut[J].Journal of the Science of Food&Agriculture,2011,91(7):1304-1309.DOI:10.1002/jsfa.4318.
[9]LI Huijing,ZHU Kexue,ZHOU Huiming,et al.Effects of high hydrostatic pressure treatment on allergenicity and structural properties of soybean protein isolate for infant formula[J].Food Chemistry,2012,132(2):808-814.DOI:10.1016/j.foodchem.2011.11.040.
[10]HILDEBRANDT S,SCHüTTEL,STOYANOV S,et al.I n vitro determination of the allergenic potential of egg white in processed meat[J].Journal of Allergy,2010,2010:1-5.DOI:10.1155/2010/238573.
[11]贺梦雪,席俊,皮江一.响应面法优化β-伴大豆球蛋白超高静压处理条件的研究[J].河南工业大学学报(自然科学版),2017,38(2):69-74.DOI:10.16433/j.cnki.issn1673-2383.2017.02.012.
[12]FERNANDES L,CASAL S,PEREIRA J A,et al.Effect of high hydrostatic pressure(HHP)treatment on edible flowers’properties[J].Food and Bioprocess Technology,2017,10(5):799-807.DOI:10.1007/s11947-017-1887-2.
[13]THANH V H,SHIBASAKI K.Major proteins of soybean seeds.a straightforward fractionation and their characterization[J].Journal of Agricultural and Food Chemistry,1976,24(6):1117-1121.DOI:10.1021/jf60208a030.
[14]LIU Chun,WANG Hongling,CUI Zhumei,et al.Optimization of extraction and isolation for 11S and 7S globulins of soybean seed storage protein[J].Food Chemistry,2007,102(4):1310-1316.DOI:10.1016/j.foodchem.2006.07.017.
[15]宋佳.大豆11S和7S球蛋白提取及其凝胶显微结构研究[D].天津:天津科技大学,2013:6.
[16]冯挺财,邵谱,胡珩,等.牛乳铁蛋白直接竞争ELISA检测法的建立[J].安徽农业科学,2008(18):7650-7652.DOI:10.3969%2fj.issn.0517-6611.2008.18.072.
[17]张楠,布冠好,朱婷伟,等.糖基化反应对大豆蛋白-乳糖复合物抗原性及结构的影响[J].现代食品科技,2015,31(8):117-121;272.DOI:10.13982/j.mfst.1673-9078.2015.8.020.
[18]DHAKAL S,LIU C Q,ZHANG Y,et al.Effect of high pressure processing on the immunoreactivity of almond milk[J].Food Research International,2014,62(1):215-222.DOI:10.1016/j.foodres.2014.02.021.
[19]LUO Y,LI M,ZHU K X,et al.Heat-induced interaction between egg white protein and wheat gluten[J].Food Chemistry,2016,197:699-708.DOI:10.1016/j.foodchem.2015.09.088.
[20]方佳茂,刘偲琪,庄楚周,等.复合酶水解蚕蛹蛋白制备功能性寡肽的工艺研究[J].现代食品科技,2012,28(3):323-328.DOI:10.13982/j.mfst.1673-9078.2012.03.020.
[21]郭丽萍.超高压结合热处理对猪肉蛋白质氧化、结构及特性的影响[D].绵阳:?西南科技大学,2016:13.
[22]ZHOU H,WANG C Z,YE J Z,et al.Effects of high hydrostatic pressure treatment on structural,allergenicity,and functional properties of proteins from ginkgo seeds[J].Innovative Food Science and Emerging Technologies,2016,34:187-195.DOI:10.1016/j.ifset.2016.02.001.
[23]SHRIVER S K,YANG W W.Thermal and nonthermal methods for food allergen control[J].Food Engineering Reviews,2011,3(1):26-43.DOI:10.1007/s12393-011-9033-9.
[24]王章存,徐贤.超高压处理对蛋白质结构及功能性质影响[J].粮食与油脂,2007(11):10-12.DOI:10.3969/j.issn.1008-9578.2007.11.003.
[25]薛路舟.超高压处理对生物大分子的影响研究[D].大连:大连理工大学,2010:48.
[26]ZHANG X,QI J R,LI K K,et al.Characterization of soy β-conglycinin-dextran conjugate prepared by Maillard reaction in crowded liquid system[J].Food Research International,2012,49(2):648-654.DOI:10.1016/j.foodres.2012.09.001.
[27]王治平.菜籽分离蛋白糖接枝改性及其功能、结构和消化吸收特性研究[D].镇江:江苏大学,2016:32.
[28]BU G H,ZHANG N,CHEN F S.The influence of glycosylation on the antigenicity,allergenicity,and structural properties of 11S-lactose conjugates[J].Food Research International,2015,76(Pt 3):511-517.DOI:10.1016/j.foodres.2015.08.004.
[29]BU G H,ZHU T W,CHEN F S,et al.Effects of saccharide on the structure and antigenicity ofβ-conglycinin in soybean protein isolate by glycation[J].European Food Research and Technology,2015,240(2):285-293.DOI:10.1007/s00217-014-2326-5.
[30]TANG C H,MA C Y.Effect of high pressure treatment on aggregation and structural properties of soy protein isolate[J].LWT-Food Science and Technology,2009,42(2):606-611.DOI:10.1016/j.lwt.2008.07.012.
[31]苏丹,李树君,赵凤敏,等.超高压处理对大豆分离蛋白结构的影响[J].食品科技,2009,34(12):51-55.DOI:10.13684/j.cnki.spkj.2009.12.059.
[32]罗春萍.热加工、辐照及超高压微射流对花生过敏原Ara h 6结构与抗原性的影响[D].南昌:南昌大学,2011:49.