pH偏移结合温和热处理对蚕豆分离蛋白结构和功能的影响
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摘要
研究pH偏移结合温和热处理对蚕豆分离蛋白(broad bean protein isolate,BBPI)结构和功能的影响。BBPI在pH1. 5结合25、37和55℃条件下,分别处理0、1. 5、3. 5和5. 5 h后,逐渐恢复至中性,分析其溶解性、乳化性、巯基/总巯基含量、紫外、荧光光谱、表面疏水性、二级结构含量和微结构变化。结果表明:随时间延长,BBPI溶解性和乳化性均先降低后增加,37和55℃处理组巯基含量先增加后降低,25℃处理组则变化不大。随时间延长,37℃处理组总巯基含量先增加后迅速下降,25和55℃先平稳后降低。紫外和荧光光谱表明,随时间延长,25℃处理的大部分色氨酸残基包裹在分子内部疏水微环境,37℃处理组的色氨酸残基部分解折叠。25和37℃处理时,BBPI的表面疏水性先增加后降低,55℃处理则先增加后出现波动。随温度增加,BBPI的α-螺旋和β-折叠逐渐转化为β-转角和无规则卷曲,形成粒径约80～130 nm的球状体且存在团聚。pH偏移结合温和热处理在一定程度上可改变蚕豆分离蛋白的结构和功能。
Effects of pH-shifting combined with mild heating process on structural and functional properties of broad bean protein isolateS(BBPI) were investigated. The BBPI underwent heat treatment(25 ℃,37 ℃ and 55 ℃)at pH = 1. 5 for 0,1. 5 h,3. 5 h,and 5. 5 h,respectively,followed by refolding at neutral pH. Changes in its solubility,emulsification,sulfhydryl/total sulfhydryl content,UV and fluorescence spectrum,surface hydrophobicity,secondary structure content,and microstructure were explored. The results showed that both solubility and emulsification decreased first and then increased with time. The sulfhydryl content increased first and then decreased at 37 ℃and 55 ℃,respectively,and it remained almost unchanged at 25 ℃. At 37 ℃,total sulfhydryl content increased first and decreased rapidly. It remained almost stable at the beginning and reduced at 25 ℃ and 55 ℃,respectively. UV and fluorescence spectroscopy revealed that most of the tryptophan(Trp) residues were wrapped and located mainly in the hydrophobic area in the inner core of BBPI at 25 ℃. However,the Trp residues were found unfolded at 37 ℃.Surface hydrophobicity of BBPI increased first and then decreased at 25 ℃ and 37 ℃,respectively,but it increased and then fluctuated at 55 ℃. As temperature increasing,the α-helical and β-sheet of BBPI gradually transformed intoβ-turn and random coil,and the particles were spherical agglomerates with average diameter around 80-130 nm.Therefore,it was concluded that structural and functional properties of BBPI could be changed via pH-shifting combined with mild heating process to some extent.
Effects of pH-shifting combined with mild heating process on structural and functional properties of broad bean protein isolateS(BBPI) were investigated. The BBPI underwent heat treatment(25 ℃,37 ℃ and 55 ℃)at pH = 1. 5 for 0,1. 5 h,3. 5 h,and 5. 5 h,respectively,followed by refolding at neutral pH. Changes in its solubility,emulsification,sulfhydryl/total sulfhydryl content,UV and fluorescence spectrum,surface hydrophobicity,secondary structure content,and microstructure were explored. The results showed that both solubility and emulsification decreased first and then increased with time. The sulfhydryl content increased first and then decreased at 37 ℃and 55 ℃,respectively,and it remained almost unchanged at 25 ℃. At 37 ℃,total sulfhydryl content increased first and decreased rapidly. It remained almost stable at the beginning and reduced at 25 ℃ and 55 ℃,respectively. UV and fluorescence spectroscopy revealed that most of the tryptophan(Trp) residues were wrapped and located mainly in the hydrophobic area in the inner core of BBPI at 25 ℃. However,the Trp residues were found unfolded at 37 ℃.Surface hydrophobicity of BBPI increased first and then decreased at 25 ℃ and 37 ℃,respectively,but it increased and then fluctuated at 55 ℃. As temperature increasing,the α-helical and β-sheet of BBPI gradually transformed intoβ-turn and random coil,and the particles were spherical agglomerates with average diameter around 80-130 nm.Therefore,it was concluded that structural and functional properties of BBPI could be changed via pH-shifting combined with mild heating process to some extent.
引文
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[6] NIU Haili,XIA Xiufang,WANG Chao,et al. Thermal stability and gel quality of myofibrillar protein as affected by soy protein isolates subjected to an acidic p H and mild heating[J]. Food Chemistry,2018,242:188-195.
[7]苏海玲,张海英,李红卫,等.蚕豆蛋白酶解工艺及响应面法的优化[J].中国食品学报,2013,13(5):39-46.
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[10]李雪芬,韩涛,夏晓楠,等.铜螯合亲和层析分离抗氧化活性蚕豆蛋白酶解物[J].中国粮油学报,2017,32(1):119-124.
[11]杨希娟,党斌,刘玉皎,等.酶解蚕豆蛋白制备多肽酒及其抗氧化性研究[J].中国粮油学报,2012,27(12):22-27.
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[13] JIANG Jiang,XIONG Youling,NEWMAN M C,et al.Structure-modifying alkaline and acidic p H-shifting processes promote film formation of soy proteins[J]. Food Chemistry,2012,132(4):1 944-1 950.
[14] JIANG Jiang,CHEN Jie,XIONG You-ling. Structural and emulsifying properties of soy protein isolate subjected to acid and alkaline p H-shifting processes[J]. Journal of Agricultural Food Chemistry,2009,57(16):7 576-7 583.
[15] WANG Henan, WU Jianping, BETTI M. Chemical,rheological and surface morphologic characterization of spent hen proteins extracted by p H-shift processing with or without the presence of cryoprotectants[J]. Food Chemistry,2013,139(1-4):710-719.
[16] CHANG Cuihua,NIU Fuge,SU Yujie,et al. Characteristics and emulsifying properties of acid and acid-heat induced egg white protein[J]. Food Hydrocolloids,2016,54(part B):342-350.
[17] LIN Haixin,LI Zhengxin,LIN Hong,et al. Effect of p H shifts on Ig E-binding capacity and conformational structure of tropomyosin from short-neck clam(Ruditapes philippinarum)[J]. Food Chemsitry,2015,188:248-255.
[18]曾茂茂,曲刚,陈洁,等.糖基化反应对花生分离蛋白乳化性的影响[J].食品与发酵工业,2011,37(4):37-41.
[19]梁珊,高小丽,户月秀,等.芸豆蛋白的理化功能特性研究[J].中国粮油学报,2015,30(12):43-48.
[20] PEARCE,K N,KINSELLA J E. Emulsifying properties of protein:evaluation of a turbidimetric technique[J].Journal of Agricultural and Food Chemistry,1978,26(3):716-723.
[21]卢岩,刘骞,耿蕊,等.羟基自由基氧化对大豆分离蛋白结构特性的影响[J].中国食品学报,2015,15(10):73-79.
[22]李学鹏,陈杨,王金厢,等.中国对虾冷藏过程中肌肉组织结构与蛋白质生化性质的变化[J].中国食品学报,2014,14(5):72-79.
[23]潘明喆,李斌,孟宪军.木糖醇对大豆分离蛋白结构和起泡特性的影响[J].食品科学,2016,37(15):63-68.
[24]王艳萍,李双喜,ZAHEER A,等.蚕豆蛋白的提取及Na Cl浓度和p H值对其溶解性和乳化性的影响[J].农业工程学报,2010,26(1):380-384.
[25]耿蕊,卢岩,孔保华,等. p H偏移结合加热处理对大豆分离蛋白乳化特性的影响[J].中国食品学报,2016,16(1):173-180.
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[27] HWANG D C,DAMODARAN S. Metal-chelating properties and biodegradability of an ethylenediaminetetraacetic acid dianhydride modified soy protein hydrogel[J]. Journal of Applied Polymer Science,2015,64(5):891-901
[28] RAGONE R,COLONNA G,BALESTRIERI C,et al.Determination of tyrosine exposure in proteins by second derivative spectroscopy[J]. Biochemistry,1984,23(8):1 871-1 875.
[29] PADROS E,MORROS A,MANOSA J,et al. The state of tyrosine and phenylalanine residues in proteins analyzed by fourth derivative spectrophotometry Histone H1and ribonuclease A[J]. European Journal of Biochemistry,1982,127(1):117-122
[30] LANGE R,BALNY C. UV-visible derivative spectroscopy under high pressure[J]. Biochim Biophys Acta,2002,1595(1-2):80-93.
[31] HASKARD C A,LI-CHAN E C Y. Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged(PRODAN)and anionic(ANS-)fluorescent probes[J]. Journal of Agricultural and Food Chemistry,1998,46(7):2 671-2 677.
[32]余晶梅,林东强,童红飞,等.荧光探针法研究牛血清白蛋白和免疫球蛋白的表面疏水特性[J].高校化学工程学报,2014,28(4):771-776.
[33]付苇娅,周春霞,朱潘红,等. 3种离子强度下p H值对罗菲鱼肌球蛋白溶解度及分子构象的影响[J].食品与发酵工业,2017,43(9):64-70.
[34]魏冬旭,江连洲,王辰,等. p H值对大豆11S球蛋白结构和表面疏水性的影响[J].食品科学,2015,36(11):1-5.
[35]顾龙建,源博恩,赵强忠,等. p H调控-热处理改善大豆蛋白中间组分乳化特性研究[J].食品与发酵工业,2011,37(5):12-16.
[36]徐娟,刘刚,欧全宏,等.不同颜色蚕豆的红外光谱分析及元素含量测定[J].湖北农业科学,2015,54(13):3 244-3 247.
[37]岳鉴颖,王金枝,张春晖,等.提取时间对鸡骨蛋白凝胶特性和蛋白二级结构的影响[J].中国农业科学,2017,50(5):903-912.
[38] VOUTSINAS L P,CHEUNG E,NAKAI S. Relationships of hydrophobicity to emulsifying properties of heat denatured proteins[J]. Journal of Food Science,1983,48:26-32.
[39] BANACH J C,LIN Z,LAMSAL B P,et al. Enzymatic modification of milk protein concentrate and characterization of resulting functional properties[J]. LWT-Food Science and Technology,2013,54(2):397-403.
[2]叶茵.中国蚕豆学[M].北京:中国农业出版社,2003.
[3]白正晨,刘建福,胡娟,等.胰蛋白酶有限水解蚕豆盐溶性蛋白的研究[J].食品工业科技,2011,32(5):263-265.
[4] MULTARI S,STEWART D,RUSSELL W R. Potential of fava bean as future protein supply to partially replace meat intake in the human diet[J]. Comprehensive Reviews in Food Science and Food Safety,2015,14(5):511-522.
[5] LIU Qian,GENG Rui,ZHAO Juyang,et al. Structural and gel textural properties of soy protein isolate when subjected to extreme acid p H-shifting and mild heating processes[J]. Journal of Agricultural Food Chemistry,2015,63(19):4 853-4 861
[6] NIU Haili,XIA Xiufang,WANG Chao,et al. Thermal stability and gel quality of myofibrillar protein as affected by soy protein isolates subjected to an acidic p H and mild heating[J]. Food Chemistry,2018,242:188-195.
[7]苏海玲,张海英,李红卫,等.蚕豆蛋白酶解工艺及响应面法的优化[J].中国食品学报,2013,13(5):39-46.
[8]宋庆明.蚕豆肽制备及其生物活性的研究[D].天津:天津科技大学,2009:1-58.
[9]刘淳,张海英,韩涛,等. Alcalase碱性蛋白酶酶解蚕豆蛋白的研究[J].中国粮油学报,2011,26(12):29-33.
[10]李雪芬,韩涛,夏晓楠,等.铜螯合亲和层析分离抗氧化活性蚕豆蛋白酶解物[J].中国粮油学报,2017,32(1):119-124.
[11]杨希娟,党斌,刘玉皎,等.酶解蚕豆蛋白制备多肽酒及其抗氧化性研究[J].中国粮油学报,2012,27(12):22-27.
[12] JIANG Jiang,XIONG Youling,CHEN Jie. p H shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different p H,salt concentration,and temperature conditions[J]. Journal of Agricultural Food Chemistry,2010,58(13):8 035-8 042.
[13] JIANG Jiang,XIONG Youling,NEWMAN M C,et al.Structure-modifying alkaline and acidic p H-shifting processes promote film formation of soy proteins[J]. Food Chemistry,2012,132(4):1 944-1 950.
[14] JIANG Jiang,CHEN Jie,XIONG You-ling. Structural and emulsifying properties of soy protein isolate subjected to acid and alkaline p H-shifting processes[J]. Journal of Agricultural Food Chemistry,2009,57(16):7 576-7 583.
[15] WANG Henan, WU Jianping, BETTI M. Chemical,rheological and surface morphologic characterization of spent hen proteins extracted by p H-shift processing with or without the presence of cryoprotectants[J]. Food Chemistry,2013,139(1-4):710-719.
[16] CHANG Cuihua,NIU Fuge,SU Yujie,et al. Characteristics and emulsifying properties of acid and acid-heat induced egg white protein[J]. Food Hydrocolloids,2016,54(part B):342-350.
[17] LIN Haixin,LI Zhengxin,LIN Hong,et al. Effect of p H shifts on Ig E-binding capacity and conformational structure of tropomyosin from short-neck clam(Ruditapes philippinarum)[J]. Food Chemsitry,2015,188:248-255.
[18]曾茂茂,曲刚,陈洁,等.糖基化反应对花生分离蛋白乳化性的影响[J].食品与发酵工业,2011,37(4):37-41.
[19]梁珊,高小丽,户月秀,等.芸豆蛋白的理化功能特性研究[J].中国粮油学报,2015,30(12):43-48.
[20] PEARCE,K N,KINSELLA J E. Emulsifying properties of protein:evaluation of a turbidimetric technique[J].Journal of Agricultural and Food Chemistry,1978,26(3):716-723.
[21]卢岩,刘骞,耿蕊,等.羟基自由基氧化对大豆分离蛋白结构特性的影响[J].中国食品学报,2015,15(10):73-79.
[22]李学鹏,陈杨,王金厢,等.中国对虾冷藏过程中肌肉组织结构与蛋白质生化性质的变化[J].中国食品学报,2014,14(5):72-79.
[23]潘明喆,李斌,孟宪军.木糖醇对大豆分离蛋白结构和起泡特性的影响[J].食品科学,2016,37(15):63-68.
[24]王艳萍,李双喜,ZAHEER A,等.蚕豆蛋白的提取及Na Cl浓度和p H值对其溶解性和乳化性的影响[J].农业工程学报,2010,26(1):380-384.
[25]耿蕊,卢岩,孔保华,等. p H偏移结合加热处理对大豆分离蛋白乳化特性的影响[J].中国食品学报,2016,16(1):173-180.
[26]蒋将. p H偏移处理诱导熔球态大豆蛋白的结构变化及功能性质的改善[D].无锡:江南大学,2011:20-21.
[27] HWANG D C,DAMODARAN S. Metal-chelating properties and biodegradability of an ethylenediaminetetraacetic acid dianhydride modified soy protein hydrogel[J]. Journal of Applied Polymer Science,2015,64(5):891-901
[28] RAGONE R,COLONNA G,BALESTRIERI C,et al.Determination of tyrosine exposure in proteins by second derivative spectroscopy[J]. Biochemistry,1984,23(8):1 871-1 875.
[29] PADROS E,MORROS A,MANOSA J,et al. The state of tyrosine and phenylalanine residues in proteins analyzed by fourth derivative spectrophotometry Histone H1and ribonuclease A[J]. European Journal of Biochemistry,1982,127(1):117-122
[30] LANGE R,BALNY C. UV-visible derivative spectroscopy under high pressure[J]. Biochim Biophys Acta,2002,1595(1-2):80-93.
[31] HASKARD C A,LI-CHAN E C Y. Hydrophobicity of bovine serum albumin and ovalbumin determined using uncharged(PRODAN)and anionic(ANS-)fluorescent probes[J]. Journal of Agricultural and Food Chemistry,1998,46(7):2 671-2 677.
[32]余晶梅,林东强,童红飞,等.荧光探针法研究牛血清白蛋白和免疫球蛋白的表面疏水特性[J].高校化学工程学报,2014,28(4):771-776.
[33]付苇娅,周春霞,朱潘红,等. 3种离子强度下p H值对罗菲鱼肌球蛋白溶解度及分子构象的影响[J].食品与发酵工业,2017,43(9):64-70.
[34]魏冬旭,江连洲,王辰,等. p H值对大豆11S球蛋白结构和表面疏水性的影响[J].食品科学,2015,36(11):1-5.
[35]顾龙建,源博恩,赵强忠,等. p H调控-热处理改善大豆蛋白中间组分乳化特性研究[J].食品与发酵工业,2011,37(5):12-16.
[36]徐娟,刘刚,欧全宏,等.不同颜色蚕豆的红外光谱分析及元素含量测定[J].湖北农业科学,2015,54(13):3 244-3 247.
[37]岳鉴颖,王金枝,张春晖,等.提取时间对鸡骨蛋白凝胶特性和蛋白二级结构的影响[J].中国农业科学,2017,50(5):903-912.
[38] VOUTSINAS L P,CHEUNG E,NAKAI S. Relationships of hydrophobicity to emulsifying properties of heat denatured proteins[J]. Journal of Food Science,1983,48:26-32.
[39] BANACH J C,LIN Z,LAMSAL B P,et al. Enzymatic modification of milk protein concentrate and characterization of resulting functional properties[J]. LWT-Food Science and Technology,2013,54(2):397-403.