反胶束萃取对大豆分离蛋白结构和特性的影响
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摘要
目的:大豆蛋白提取方法的深入研究能够在一定程度上为植物蛋白的开发利用提供新可能。本研究以常规碱溶酸沉法为对照,与反胶束法提取得到的蛋白质差异进行全面对比,进而探讨反胶束萃取环境的独特作用。方法:以全脂大豆粉为原料,分别采用反胶束法和碱溶酸沉法制备大豆分离蛋白,并进一步对比两种方法得到的大豆分离蛋白的结构、热力学和流变学特性。结果:反胶束法得到的大豆分离蛋白的β-折叠结构含量较低,β-转角结构含量较高,蛋白展开程度较低,具有较低的表面疏水性,相较于传统碱溶酸沉法能够较好地保持蛋白质的天然分子结构;反胶束法得到的蛋白质的热稳定性较差,更易于形成凝胶,但所形成的凝胶强度较低。结论:反胶束萃取环境独特的水核结构能够较好地保持大豆分离蛋白的天然分子结构。
More intensive research on the extraction of soybean protein isolate(SPI) may provide new possibilities for developing and utilizing plant proteins. In this study we made a comprehensive comparison between traditional alkali dissolution and acid precipitation method and reverse micellar extraction(RME) for soybean protein extraction and we investigated the unique properties of the reverse micellar environment. For this purpose, the structure, thermodynamic properties and rheological properties of SPI extracted from full-fat soybean powder by the two methods were studied. The results demonstrated that the SPI extracted by RME contained less β-sheet but more β-turn structure, representing a higher degree of unfolding, and therefore had a lower surface hydrophobicity. The reverse micelle environment could protect the protein from denaturation by organic solvents. The SPI extracted by RME had poor thermostability and was more prone to gelation, but the gel strength was lower when compared with the SPI extracted by the traditional method. Finally, we came to the conclusion that the unique water core structure of the reverse micelle could help to preserve the native structure of SPI.
More intensive research on the extraction of soybean protein isolate(SPI) may provide new possibilities for developing and utilizing plant proteins. In this study we made a comprehensive comparison between traditional alkali dissolution and acid precipitation method and reverse micellar extraction(RME) for soybean protein extraction and we investigated the unique properties of the reverse micellar environment. For this purpose, the structure, thermodynamic properties and rheological properties of SPI extracted from full-fat soybean powder by the two methods were studied. The results demonstrated that the SPI extracted by RME contained less β-sheet but more β-turn structure, representing a higher degree of unfolding, and therefore had a lower surface hydrophobicity. The reverse micelle environment could protect the protein from denaturation by organic solvents. The SPI extracted by RME had poor thermostability and was more prone to gelation, but the gel strength was lower when compared with the SPI extracted by the traditional method. Finally, we came to the conclusion that the unique water core structure of the reverse micelle could help to preserve the native structure of SPI.
引文
[1]WANG X S,TANG C H,LI B S,et al.Effects of high-pressure treatment on some physicochemical and functional properties of soy protein isolates[J].Food Hydrocolloids,2008,22(4):560-567.DOI:10.1016/j.foodhyd.2007.01.027.
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[4]BHATTARAI A,WILCZURA-WACHNIK H.Size and diffusion phenomena of AOT/alcohol/water system in the presence of morin by dynamic light scattering[J].International Journal of Pharmaceutics,2015,478(2):610-616.DOI:10.1016/j.ijpharm.2014.11.037.
[5]SHARMA S,YADAV N,CHOWDHURY P K,et al.Controlling the microstructure of reverse micelles and their templating effect on shaping nanostructures[J].The Journal of Physical Chemistry B,2015,119(34):11295-11306.DOI:10.1021/acs.jpcb.5b03063.
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[9]BU G H,YANG Y Y,CHEN F S,et al.Extraction and physicochemical properties of soya bean protein and oil by a new reverse micelle system compared with other extraction methods[J].International Journal of Food Science&Technology,2014,49(4):1079-1089.DOI:10.1111/ijfs.12403.
[10]MOHD-SETAPAR S H,MOHAMAD-AZIZ S N,HARUN N H,et al.Review on the extraction of biomolecules by biosurfactant reverse micelles[J].APCBEE Procedia,2012,3:78-83.DOI:10.1016/j.apcbee.2012.06.050.
[11]赵晓燕,薛文通,陈复生,等.影响反胶束体系萃取蛋白能力的因素及机理[J].农业工程学报,2009,25(11):354-360.DOI:10.3969/j.issn.1002-6819.2009.11.066.
[12]ZHAO X H,LI Y M,HE X W,et al.Study of the factors affecting the extraction of soybean protein by reverse micelles[J].Molecular Biology Reports,2010,37(2):669-675.DOI:10.1007/s11033-009-9515-5.
[13]ZHAO Xiaoyan,ZHU Haitao,CHEN Jun.Effects of sodium bis(2-ethylhexyl)sulfo succinate(AOT)reverse micelles on physicochemical properties of soy protein[J].Food and Bioproducts Processing,2015,94:500-506.DOI:10.1016/j.fbp.2014.07.009.
[14]ZHAO Xiaoyan,ZHANG Xiaowei,LIU Hongkai,et al.Functional,nutritional and flavor characteristic of soybean proteins obtained through reverse micelles[J].Food Hydrocolloids,2018,74:358-366.DOI:10.1016/j.foodhyd.2017.08.024.
[15]HAYAKAWA S,NAKAI S.Relationships of hydrophobicity and net charge to the solubility of milk and soy proteins[J].Journal of Food Science,1985,50(2):486-491.DOI:10.1111/j.1365-2621.1985.tb13433.x.
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[17]GUO Jian,YANG Xiaoquan,HE Xiuting,et al.Limited aggregation behavior ofβ-conglycinin and its terminating effect on glycinin aggregation during heating at pH 7.0[J].Journal of Agricultural and Food Chemistry,2012,60(14):3782-3791.DOI:10.1021/jf300409y.
[18]KEALLEY C S,ROUT M K,DEZFOULI M R,et al.Structure and molecular mobility of soy glycinin in the solid state[J].Biomacromolecules,2008,9(10):2937-2946.DOI:10.1021/bm800721d.
[19]YANG Y,WANG Z J,WANG R,et al.Secondary structure and subunit composition of soy protein in vitro digested by pepsin and its relation with digestibility[J].BioMed Research International,2016,2016:1-6.DOI:10.1155/2016/5498639.
[20]CHEN J,CHEN X Y J,ZHU Q,et al.Determination of the domain structure of the 7S and 11S globulins from soy proteins by XRD and FTIR[J].Journal of the Science of Food and Agriculture,2013,93(7):1687-1691.DOI:10.1002/jsfa.5950.
[21]BARTH A.Infrared spectroscopy of proteins[J].Biochimica et Biophysica Acta,2007,1767(9):1073-1101.DOI:10.1016/j.bbabio.2007.06.004.
[22]ZHAO X Y,CHEN F S,XUE W T,et al.FTIR spectra studies on the secondary structures of 7S and 11S globulins from soybean proteins using AOT reverse micellar extraction[J].Food Hydrocolloids,2008,22(4):568-575.DOI:10.1016/j.foodhyd.2007.01.019.
[23]惠特福德.蛋白质结构与功能[M].北京:科学出版社,2008:150-200.
[24]LEE H J,CHAN C,LEE S J.Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form[J].Journal of Biological Chemistry,2002,277(1):671-678.DOI:10.1074/jbc.M107045200.
[25]CARBONARO M,MASELLI P,NUCARA A.Relationship between digestibility and secondary structure of raw and thermally treated legume proteins:a Fourier transform infrared(FT-IR)spectroscopic study[J].Amino Acids,2012,43(2):911-921.DOI:10.1007/s00726-011-1151-4.
[26]WANG Z J,LI Y,JIANG L Z,et al.Relationship between secondary structure and surface hydrophobicity of soybean protein isolate subjected to heat treatment[J].Journal of Chemistry,2014(5):1-10.DOI:10.1155/2014/475389.
[27]MATZKE S F,CREAGH A L,HAYNES C A,et al.Mechanisms of protein solubilization in reverse micelles[J].Biotechnology&Bioengineering,1992,40(1):91-102.DOI:10.1002/bit.260400114.
[28]CHEN X Y,RU Y,CHEN F L,et al.FTIR spectroscopic characterization of soy proteins obtained through AOT reverse micelles[J].Food Hydrocolloids,2013,31(2):435-437.DOI:10.1016/j.foodhyd.2012.11.017.
[29]BOULET M,BRITTEN M,LAMARCHE F.Aggregation of some food proteins in aqueous dispersions:effects of concentration,pHand ionic strength[J].Food Hydrocolloids,2000,14(2):135-144.DOI:10.1016/S0268-005X(99)00059-4.
[30]MARUYAMA N,FUKUDA T,SAKA S,et al.Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins[J].Phytochemistry,2003,64(3):701-708.DOI:10.1016/S0031-9422(03)00385-6.
[31]SESSA D J.Thermal denaturation of glycinin as a function of hydration[J].Journal of the American Oil Chemists’Society,1993,70(12):1279-1284.DOI:10.1007/BF02564242.
[32]GEBHARD S.实用流变测量学(修订版)[M].北京:石油工业出版社,2009:8-26.
[33]KONG X Z,LI X H,WANG H J,et al.Effect of lipoxygenase activity in defatted soybean flour on the gelling properties of soybean protein isolate[J].Food Chemistry,2008,106(3):1093-1099.DOI:10.1016/j.foodchem.2007.07.050.
[34]SU Y J,DONG Y T,NIU F G,et al.Study on the gel properties and secondary structure of soybean protein isolate/egg white composite gels[J].European Food Research and Technology,2014,240(2):367-378.DOI:10.1007/s00217-014-2336-3.
[35]ARZENI C,MARTíNEZ K,ZEMA P,et al.Comparative study of high intensity ultrasound effects on food proteins functionality[J].Journal of Food Engineering,2012,108(3):463-472.DOI:10.1016/j.jfoodeng.2011.08.018.
[36]WINTER H H,CHAMBON F.Analysis of linear viscoelasticity of a crosslinking polymer at the gel point[J].Journal of Rheology,1986,30(2):367-382.DOI:10.1122/1.549853.
[37]WU C,HUA Y F,CHEN Y M,et al.Effect of 7S/11S ratio on the network structure of heat-induced soy protein gels:a study of probe release[J].Royal Society of Chemistry Advances,2016,6(104):101981-101987.DOI:10.1039/C6RA22388E.
[2]TANG C H,WU H,CHEN Z,et al.Formation and properties of glycinin-rich andβ-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase[J].Food Research International,2006,39(1):87-97.DOI:10.1016/j.foodres.2005.06.004.
[3]AFERNI A E,GUETTARI M,TAJOURI T.Determination of the water/AOT/isooctane reverse micelles size parameters from their refractive index data[J].Journal of Solution Chemistry,2017,46(1):89-102.DOI:10.1007/s10953-016-0563-x.
[4]BHATTARAI A,WILCZURA-WACHNIK H.Size and diffusion phenomena of AOT/alcohol/water system in the presence of morin by dynamic light scattering[J].International Journal of Pharmaceutics,2015,478(2):610-616.DOI:10.1016/j.ijpharm.2014.11.037.
[5]SHARMA S,YADAV N,CHOWDHURY P K,et al.Controlling the microstructure of reverse micelles and their templating effect on shaping nanostructures[J].The Journal of Physical Chemistry B,2015,119(34):11295-11306.DOI:10.1021/acs.jpcb.5b03063.
[6]SHRESTHA R G,SHRESTHA L K.Structure of nonionic reverse micelles in octylbenzene[J].Journal of Dispersion Science and Technology,2013,34(5):684-691.DOI:10.1080/01932691.2012.683977.
[7]LUISI P L,GIOMINI M,PILENI M P,et al.Reverse micelles as hosts for proteins and small molecules[J].Biochimica et Biophysica Acta,1988,947(1):209-246.DOI:10.1016/0304-4157(88)90025-1.
[8]NAOE K,YOSHIMOTO S,NAITO N,et al.Preparation of protein nanoparticles using AOT reverse micelles[J].Biochemical Engineering Journal,2011,55(2):140-143.DOI:10.1016/j.bej.2011.02.015.
[9]BU G H,YANG Y Y,CHEN F S,et al.Extraction and physicochemical properties of soya bean protein and oil by a new reverse micelle system compared with other extraction methods[J].International Journal of Food Science&Technology,2014,49(4):1079-1089.DOI:10.1111/ijfs.12403.
[10]MOHD-SETAPAR S H,MOHAMAD-AZIZ S N,HARUN N H,et al.Review on the extraction of biomolecules by biosurfactant reverse micelles[J].APCBEE Procedia,2012,3:78-83.DOI:10.1016/j.apcbee.2012.06.050.
[11]赵晓燕,薛文通,陈复生,等.影响反胶束体系萃取蛋白能力的因素及机理[J].农业工程学报,2009,25(11):354-360.DOI:10.3969/j.issn.1002-6819.2009.11.066.
[12]ZHAO X H,LI Y M,HE X W,et al.Study of the factors affecting the extraction of soybean protein by reverse micelles[J].Molecular Biology Reports,2010,37(2):669-675.DOI:10.1007/s11033-009-9515-5.
[13]ZHAO Xiaoyan,ZHU Haitao,CHEN Jun.Effects of sodium bis(2-ethylhexyl)sulfo succinate(AOT)reverse micelles on physicochemical properties of soy protein[J].Food and Bioproducts Processing,2015,94:500-506.DOI:10.1016/j.fbp.2014.07.009.
[14]ZHAO Xiaoyan,ZHANG Xiaowei,LIU Hongkai,et al.Functional,nutritional and flavor characteristic of soybean proteins obtained through reverse micelles[J].Food Hydrocolloids,2018,74:358-366.DOI:10.1016/j.foodhyd.2017.08.024.
[15]HAYAKAWA S,NAKAI S.Relationships of hydrophobicity and net charge to the solubility of milk and soy proteins[J].Journal of Food Science,1985,50(2):486-491.DOI:10.1111/j.1365-2621.1985.tb13433.x.
[16]SHIMADA K,CHEFTEL J C.Determination of sulfhydryl groups and disulfide bonds in heat-induced gels of soy protein isolate[J].Journal of Agricultural and Food Chemistry,1988,36(1):147-153.DOI:10.1021/jf00079a038.
[17]GUO Jian,YANG Xiaoquan,HE Xiuting,et al.Limited aggregation behavior ofβ-conglycinin and its terminating effect on glycinin aggregation during heating at pH 7.0[J].Journal of Agricultural and Food Chemistry,2012,60(14):3782-3791.DOI:10.1021/jf300409y.
[18]KEALLEY C S,ROUT M K,DEZFOULI M R,et al.Structure and molecular mobility of soy glycinin in the solid state[J].Biomacromolecules,2008,9(10):2937-2946.DOI:10.1021/bm800721d.
[19]YANG Y,WANG Z J,WANG R,et al.Secondary structure and subunit composition of soy protein in vitro digested by pepsin and its relation with digestibility[J].BioMed Research International,2016,2016:1-6.DOI:10.1155/2016/5498639.
[20]CHEN J,CHEN X Y J,ZHU Q,et al.Determination of the domain structure of the 7S and 11S globulins from soy proteins by XRD and FTIR[J].Journal of the Science of Food and Agriculture,2013,93(7):1687-1691.DOI:10.1002/jsfa.5950.
[21]BARTH A.Infrared spectroscopy of proteins[J].Biochimica et Biophysica Acta,2007,1767(9):1073-1101.DOI:10.1016/j.bbabio.2007.06.004.
[22]ZHAO X Y,CHEN F S,XUE W T,et al.FTIR spectra studies on the secondary structures of 7S and 11S globulins from soybean proteins using AOT reverse micellar extraction[J].Food Hydrocolloids,2008,22(4):568-575.DOI:10.1016/j.foodhyd.2007.01.019.
[23]惠特福德.蛋白质结构与功能[M].北京:科学出版社,2008:150-200.
[24]LEE H J,CHAN C,LEE S J.Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form[J].Journal of Biological Chemistry,2002,277(1):671-678.DOI:10.1074/jbc.M107045200.
[25]CARBONARO M,MASELLI P,NUCARA A.Relationship between digestibility and secondary structure of raw and thermally treated legume proteins:a Fourier transform infrared(FT-IR)spectroscopic study[J].Amino Acids,2012,43(2):911-921.DOI:10.1007/s00726-011-1151-4.
[26]WANG Z J,LI Y,JIANG L Z,et al.Relationship between secondary structure and surface hydrophobicity of soybean protein isolate subjected to heat treatment[J].Journal of Chemistry,2014(5):1-10.DOI:10.1155/2014/475389.
[27]MATZKE S F,CREAGH A L,HAYNES C A,et al.Mechanisms of protein solubilization in reverse micelles[J].Biotechnology&Bioengineering,1992,40(1):91-102.DOI:10.1002/bit.260400114.
[28]CHEN X Y,RU Y,CHEN F L,et al.FTIR spectroscopic characterization of soy proteins obtained through AOT reverse micelles[J].Food Hydrocolloids,2013,31(2):435-437.DOI:10.1016/j.foodhyd.2012.11.017.
[29]BOULET M,BRITTEN M,LAMARCHE F.Aggregation of some food proteins in aqueous dispersions:effects of concentration,pHand ionic strength[J].Food Hydrocolloids,2000,14(2):135-144.DOI:10.1016/S0268-005X(99)00059-4.
[30]MARUYAMA N,FUKUDA T,SAKA S,et al.Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins[J].Phytochemistry,2003,64(3):701-708.DOI:10.1016/S0031-9422(03)00385-6.
[31]SESSA D J.Thermal denaturation of glycinin as a function of hydration[J].Journal of the American Oil Chemists’Society,1993,70(12):1279-1284.DOI:10.1007/BF02564242.
[32]GEBHARD S.实用流变测量学(修订版)[M].北京:石油工业出版社,2009:8-26.
[33]KONG X Z,LI X H,WANG H J,et al.Effect of lipoxygenase activity in defatted soybean flour on the gelling properties of soybean protein isolate[J].Food Chemistry,2008,106(3):1093-1099.DOI:10.1016/j.foodchem.2007.07.050.
[34]SU Y J,DONG Y T,NIU F G,et al.Study on the gel properties and secondary structure of soybean protein isolate/egg white composite gels[J].European Food Research and Technology,2014,240(2):367-378.DOI:10.1007/s00217-014-2336-3.
[35]ARZENI C,MARTíNEZ K,ZEMA P,et al.Comparative study of high intensity ultrasound effects on food proteins functionality[J].Journal of Food Engineering,2012,108(3):463-472.DOI:10.1016/j.jfoodeng.2011.08.018.
[36]WINTER H H,CHAMBON F.Analysis of linear viscoelasticity of a crosslinking polymer at the gel point[J].Journal of Rheology,1986,30(2):367-382.DOI:10.1122/1.549853.
[37]WU C,HUA Y F,CHEN Y M,et al.Effect of 7S/11S ratio on the network structure of heat-induced soy protein gels:a study of probe release[J].Royal Society of Chemistry Advances,2016,6(104):101981-101987.DOI:10.1039/C6RA22388E.