黑米花青素对大豆7S/11S蛋白结构及界面功能特性的影响

采用紫外吸收光谱、荧光光谱、粒径电位、起泡性与乳化性及其稳定性测定等方法,探究黑米花青素(cyanidin-3-glucoside, C3G)对β-伴大豆球蛋白(β-conglycinin, 7S)和大豆球蛋白(glycinin, 11S)的相互作用对7S/11S蛋白结构及界面功能特性的影响。结果表明,C3G能够猝灭7S/11S蛋白的内源和外源荧光,使蛋白表面疏水性降低,并改变氨基酸微环境,诱导多肽链解折叠。在一定程度上,C3G使2种蛋白的平均粒径减小,ζ电位绝对值增大,起泡性和乳化性及其稳定性得到改善,但可能会引起7S(C3G>1 mg/mL)和11S(C3G>0.5 mg/mL)蛋白的广泛聚集。尽管C3G对11S蛋白改性效果更明显,但C3G-7S蛋白复合物的界面功能特性仍优于C3G-11S蛋白复合物。     

EGCG与β-伴大豆球蛋白/大豆球蛋白相互作用对蛋白质结构的影响

采用荧光光谱、紫外-可见光谱、傅里叶变换红外光谱和分子对接方法,研究中性条件下β-伴大豆球蛋白（β-conglycinin,7S）、大豆球蛋白（glycinin,11S）与表没食子儿茶素没食子酸酯（(–)-epigallocatechin-3-gallate,EGCG）的相互作用。结果表明,在中性条件下EGCG与7S/11S蛋白之间存在相互作用,并诱导了氨基酸残基微环境发生变化。EGCG通过动态和静态方式猝灭7S/11S蛋白内源荧光。与7S蛋白相比,EGCG对11S蛋白的亲和力更高。EGCG与7S/11S蛋白的反应自发进行,两者主要通过氢键和范德华力形成物质的量比1∶1的复合物。EGCG能够降低7S/11S蛋白的表面疏水性,并随着EGCG浓度的增加,11S蛋白变化更加明显。傅里叶变换红外光谱和分子对接研究表明除氢键外,疏水相互作用也参与了复合物的形成。EGCG结合导致7S/11S蛋白二级结构发生不同变化,使蛋白质发生解折叠。     

超声预处理对大豆蛋白酶解物结构及抗氧化活性的影响

为充分利用大豆蛋白资源,在大豆球蛋白（11S）和β-伴大豆球蛋白（7S）酶水解前进行超声预处理,通 过荧光光谱、傅里叶变换红外光谱和紫外-可见吸收光谱分析超声预处理后大豆球蛋白和β-伴大豆球蛋白酶解物的 空间构象变化。结果表明：大豆球蛋白和β-伴大豆球蛋白酶解物二级、三级结构发生改变,α-螺旋和β-转角结构含 量明显升高,无规卷曲和β-折叠结构含量明显降低,随着酶解效率提高,酶解物水解度显著增加（P＜0.05）。对 超声处理大豆球蛋白和β-伴大豆球蛋白酶解物进行还原能力、羟自由基清除率和Fe2＋螯合能力测定。结果表明：超 声预处理后大豆球蛋白和β-伴大豆球蛋白酶解物的还原能力、羟自由基清除率和Fe2＋螯合能力明显增强。综上,超 声预处理能够有效提高大豆球蛋白和β-伴大豆球蛋白酶解效率和抗氧化活性。本研究结果可为制备高品质改性大豆 蛋白提供技术参考和理论依据。     

碱性蛋白酶对β-伴大豆球蛋白抗原性的影响

选用碱性蛋白酶处理大豆分离蛋白,间接竞争ELISA法测定水解物中β-伴大豆球蛋白的抗原性,响应面法优化降低β-伴大豆球蛋白抗原抑制率的最佳工艺条件。结果表明,碱性蛋白酶可以显著降低β-伴大豆球蛋白的抗原性,在一定程度上,水解度与β-伴大豆球蛋白抗原抑制率呈负相关关系。碱性蛋白酶在酶解时间40 min、加酶量3 000 U/g、温度55℃、p H 8.5条件下,β-伴大豆球蛋白抗原抑制率为33.48%,比大豆蛋白降低了64.16%。SDSPAGE结果显示,β-伴大豆球蛋白基本被酶解成小分子量肽段。     

异黄酮与β-伴大豆球蛋白的相互作用及其对蛋白结构和潜在致敏性的影响

探究异黄酮和β-伴大豆球蛋白（β-conglycinin,BCG）的相互作用机理及其对复合物结构和潜在致敏性的影响。利用荧光光谱、圆二色光谱分析2种大豆异黄酮（染料木素（genistein,Gen）、大豆苷元（daidzein,Dai））与BCG相互作用的猝灭类型、结合位点数、作用力类型和蛋白二级结构含量的变化,表征不同条件下制备的异黄酮-BCG复合物的结构,并利用酶联免疫吸附测定（enzyme linked immunosorbent assay,ELISA）与消化产物免疫印迹检测其潜在致敏性。结果表明：活性异黄酮（Gen/Dai）对BCG的猝灭为静态猝灭,且相互作用力以疏水相互作用为主,结合位点数均接近1;并且与异黄酮相互作用诱导了BCG氨基酸微环境的极性增加,使蛋白肽链伸展,结构更为松散;ELISA与消化产物免疫印迹结果显示,与异黄酮结合后蛋白的潜在致敏性增强。本研究有助于科学认识异黄酮在食品复杂基质体系中对过敏蛋白致敏性影响机制,对其抗过敏的深度开发利用具有重要意义。     

大豆球蛋白和β-伴大豆球蛋白的富集分离研究

以低温脱脂豆粕为原料,采用优化Nagano法分离大豆球蛋白和β-伴大豆球蛋白。本文系统考察提取过程中多个单因素对分离大豆球蛋白和β-伴大豆球蛋白的蛋白质含量和提取率的影响。并根据单因素试验结果设计响应面试验,对浸提温度、浸提pH、沉淀剂用量进行优化,分别确定高提取率大豆球蛋白和β-伴大豆球蛋白的分离条件。试验结果表明:大豆球蛋白的最佳提取条件为浸提温度44.4℃,浸提pH 8.5,CaCl220 mmol/L,提取率64.14%,纯度83.6%;β-伴大豆球蛋白的最佳提取条件为浸提温度49.5℃,浸提pH 8.6,CaCl_2 0.00 mmol/L,提取率40.15%,纯度82.9%。     

脱脂豆粕预处理对大豆β-伴球蛋白结构的影响

采用新鲜低温脱脂豆粕、干热处理脱脂豆粕和溶剂浸提脱脂豆粕为原料制备大豆β-伴球蛋白,研究低温脱脂豆粕预处理对制备大豆β-伴球蛋白结构的影响。新鲜低温脱脂豆粕制备大豆β-伴球蛋白羰基、游离巯基和总巯基含量分别为2.93 nmol/mg、1.39nmol/mg和11.87 nmol/mg,干热处理脱脂豆粕制备大豆β-伴球蛋白羰基、游离巯基和总巯基含量分别为5.24 nmol/mg、0.41 nmol/mg和5.42 nmol/mg,溶剂浸提脱脂豆粕制备大豆β-伴球蛋白羰基、游离巯基和总巯基含量分别为1.85 nmol/mg、1.93 nmol/mg和15.64nmol/mg,表明干热处理脱脂豆粕增加制备大豆β-伴球蛋白氧化程度,溶剂浸提脱脂豆粕降低制备大豆β-伴球蛋白氧化程度。随着蛋白质氧化程度的增加,大豆β-伴球蛋白二级结构中α-螺旋和β-折叠含量、表面疏水性和内源荧光强度下降,内源荧光最大吸收峰发生蓝移,并且伴随着蛋白质聚集体的出现,表明蛋白质氧化使得大豆β-伴球蛋白聚集。     

矢车菊素-3-葡萄糖苷与四种乳蛋白相互作用的研究

采用荧光光谱法、傅里叶红外光谱法和圆二色谱法,研究矢车菊素-3-葡萄糖苷(cyanidin 3-O-glucoside,C3G)与α-酪蛋白、β-酪蛋白、乳清蛋白和β-乳球蛋白的相互作用。结果表明,C3G对上述四种乳蛋白都产生了荧光静态猝灭作用,在溶液中C3G与乳蛋白相互结合摩尔比约为1:1,且由热力学参数判定C3G与α-酪蛋白结合的分子间作用力为氢键与范德华力,而与β-酪蛋白、乳清蛋白和β-乳球蛋白结合主要靠静电引力。通过比较C3G与α-酪蛋白、β-酪蛋白、乳清蛋白和β-乳球蛋白相互作用的荧光猝灭率(84%、74%、77%、75%);温度分别为298、318、338 K时的结合常数(423.448、362.994、28.655×104 L/mol;9.524、8.056、8.308×104 L/mol;9.262、6.940、7.889×104 L/mol;30.440、11.830、17.262×104 L/mol);结合距离(2.17、2.66、2.18、2.19 nm),由此得出α-酪蛋白与C3G结合最紧密。傅里叶红外光谱和圆二色谱分析显示,C3G的加入使得α-酪蛋白的α-螺旋增加,β-折叠和转角降低;β-酪蛋白的α-螺旋、β-折叠和转角均增加;乳清蛋白的α-螺旋、β-折叠和转角均无明显变化;β-乳球蛋白的α-螺旋降低,β-折叠和转角增加。C3G对四种乳蛋白均有较强的结合能力,可以使其构象发生变化。     

β-伴大豆球蛋白的水解研究

采用Nagano法从豆粕中分离β-伴大豆球蛋白并酶解制备水解肽,以单因素试验和正交试验确定酶解最佳条件,通过高效液相法分析β-伴大豆球蛋白水解肽的分子量分布,比较并检测了β-伴大豆球蛋白水解肽和大豆分离蛋白水解肽的体外抗氧化效果。结果显示:在20g/L的底物浓度下的最佳条件为酶和底物比10000U/g,温度55℃,pH7.5,水解时间4h,水解度为72.7%,明显高于酶解大豆分离蛋白51.4%的水解度,且水解时间更短。β-伴大豆球蛋白水解肽主要为130～1000u的短肽,占肽总量的86.3%,均一性极高。β-伴大豆球蛋白水解肽对O2-.和.OH均有清除作用,清除.OH的能力明显高于大豆分离蛋白水解肽,即β-伴大豆球蛋白的水解肽对大豆肽清除.OH的作用贡献更大。     

pH值对大豆11S球蛋白结构和表面疏水性的影响

采用Lowry法、8-苯氨基萘-1-磺酸铵盐（8-anilinonaphthalene-1-sulfonic acid ammonium salt,ANS）荧光探针法研究pH值对大豆11S球蛋白的溶解性和表面疏水性的影响,并利用圆二色光谱和荧光光谱对不同pH值条件下11S球蛋白二级结构和三级结构进行分析,为研究大豆蛋白结构与表面疏水性之间的构效关系提供理论基础。结果表明：除等电点外,大豆11S球蛋白溶解性和表面疏水性呈负相关,并且随着pH值的升高,大豆球蛋白二级结构中发生β-折叠和无规卷曲向α-螺旋的转变,三级结构中色氨酸（Trp）残基微环境极性降低。大豆球蛋白的表面疏水性与α-螺旋结构含量呈负相关。     

Flavor Protein Interactions: Characteristics of 2-Nonanone Binding to Isolated Soy Protein Fractions

THE ABSORPTION COEFFICIENTS at 280 nm of 1% solutions of pure soy protein, β-conglycinin, glycinin, the acidic and basic sub-units of glycinin were 6.04, 4.4, 8.04, 7.18, and 8.8, respectively. Using equilibrium dialysis the binding affinities of these proteins for the model flavor compound 2-nonanone were determined. On an equivalent weight basis soy protein, β-conglycinin and glycinin had approximately 5,2 and 3 primary binding sites per 100,000 daltons and affinity constants (K) of 570, 3050 and 540 m^-1, respectively, i.e., β-conglycinin showed a fivefold greater affinity for nonanone than the other soy protein. The acidic and basic subunits showed binding behavior similar to that of glycinin.     

Denaturation of soy proteins in solution and at the oil–water interface: A fluorescence study

Structural changes ensuing from denaturation of soy proteins in solution or occurring at the oil–water interface were studied by fluorescence spectroscopy. Studies were carried out on solutions and emulsions stabilized with β-conglycinin or glycinin. Tryptophan fluorescence spectroscopy was used to evaluate tertiary structural changes. The binding of fluorescent dyes and the accessibility of reactive cysteine thiols were also used to better identify structural changes of these proteins in solution. Protein conformational changes after interaction with the hydrophobic oil surface were compared with those ensuing from physical (temperature) or chemical denaturation (chaotropes). Results from solution denaturation experiments indicate that structural changes of β-conglycinin by both temperature and chaotropes are reversible under appropriate conditions, and result in a rearrangement of the supramacromolecular assembly of the protein structure. On the other hand, glycinin treated under the same conditions undergoes irreversible denaturation in solution at temperatures well below 90 °C. Both proteins undergo partial denaturation after adsorption on the lipid surface, and no further denaturation occurs upon heating of the emulsions prepared with either protein.     

天然大豆蛋白的选择性酶解

研究了几种微生物蛋白酶水解天然大豆蛋白的选择性。用SDS-聚丙烯酰胺凝胶电泳分析酶解过程中大豆蛋白各组分的变化,电泳结果显示-βconglycinin比glycinin容易被蛋白酶水解,-βconglycinin的α’亚基比α亚基更容易水解,-βconglycinin的β亚基比α’亚基和α亚基更难水解;glycinin的酸性亚基比其碱性亚基更容易被水解。     

天然大豆蛋白的选择性酶解

研究了几种微生物蛋白酶水解天然大豆蛋白的选择性。用 DDD-聚丙烯酰胺凝胶电泳分析酶解过程中大豆蛋白各组分的变化,电泳结果显示β-conglycinin 比 glycinin 容易被蛋白酶水解,βconglycinin 的α’亚基比α亚基更容易水解,β-conglycinin的β亚基比α’亚基和α亚基更难水解；glycinin 的酸性亚基比其碱性亚基更容易被水解。     

Functional Properties of Improved Glycinin and β-nglycinin Fractions

ABSTRACT: Glycinin and β-conglycinin have unique functionality characteristics that contribute important properties in soy foods and soy ingredients. Limited functionality data have been published for glycinin and β-conglycinin fractions produced in pilot-scale quantities. Protein extraction conditions were previously optimized for our pilotscale fractionation process to maximize protein solubilization and subsequent product recovery. Glycinin, β-conglycinin, and intermediate (mixture of glycinin and β-conglycinin) fractions were prepared using optimized-process (OP) extraction conditions (10:1 water-to-flake ratio, 45°C) and previous conditions termed Wu process (WP) (15:1, 20°C). Viscosity, solubility, gelling, foaming, emulsification capacity, and emulsification activity and stability of the fractionated proteins, and soy protein isolate (SPI) produced from the same defatted soy white flakes were compared to evaluate functional properties of these different protein fractions. Differential scanning calorimetry, sodium dodecylsulfate-polyacrylamide gel electrophoresis, and surface hydrophobicity data were used to interpret functionality differences. OP β-conglycinin had more glycinin contamination than did the WP β-conglycinin. OP and WP solubility profiles were each similar for respective glycinin and β-conglycinin fractions. Emulsification activities and stabilities were higher for OP β-conglycinin and OP intermediate fractions compared with respective WP fractions. β-Conglycinin and SPI emulsification capacities (ECs) mirrored solubility profile, whereas glycinin ECs did not. OP glycinin had a higher foaming capacity than WP glycinin. OP and WP intermediate fraction apparent viscosities trended higher than those of other protein fractions. β-Conglycinin dispersions at pH 3 and 7 produced firm gels at 80°C, whereas glycinin dispersions formed weaker gels at 99°C and did not gel at 80°C.     

Formation and properties of glycinin-rich and β-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase

The gelation and gel properties of glycinin-rich and β-conglycinin-rich soy protein isolates (SPIs) induced by microbial transglutaminase (MTGase) were investigated. At the same enzyme and protein substrate concentrations, the on-set of gelation of native SPI and the viscoelasticity development of correspondingly formed gels depended upon the relative ratio of glycinin to β-conglycinin. The turbidity analysis showed that the glycinin components also contributed to the increase in the turbidity of SPI solutions incubated with MTGase (at 37 °C). Textural profile analysis indicated that the glycinin components of SPIs principally contributed to the hardness, fracturability, gumminess and chewiness values of corresponding gels, while the cohesiveness and springness were mainly associated with the β-conglycinin components. The strength of MTGase-induced gels of various kinds of SPIs could be significantly improved by the thermal treatment. The protein solubility analyses of MTGase-induced gels, indicated that besides the covalent cross-links, hydrophobic and H-bondings and disulfide bonds were involved in the formation and maintenance of the glycinin-rich SPI gels, while in β-conglycinin-rich SPI case, the hydrophobic and H-bondings were the principal forces responsible for the maintenance of the gel structure. The results suggested that various kinds of SPI gels with different properties could be induced by MTGase, through controlling the glycinin to β-conglycinin ratio.     

Structural modification of soy protein by the lipid peroxidation product acrolein

Acrolein was selected as a representative secondary byproduct of lipid peroxidation to investigate the effect of oxidative modification of reactive aldehyde on soy protein structure. Acrolein reacted with histidine, lysine and cysteine residues in soy protein to form covalent adducts, leading to protein carbonylation and degradation of sulfhydryl groups. Circular dichroism spectra showed that soy protein modification by acrolein was related to loss of α-helix and increase of β-sheet structure. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly implied that acrolein induced soy protein aggregation, and results obtained by size-exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of acrolein. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that acrolein caused soy protein cross-linking which non-disulphide covalent bonds were involved in the formation of cross-linking, and subunits of β-conglycinin were more vulnerable to acrolein than that of glycinin.     

大豆球蛋白自组装聚集及凝胶特性

研究了在低pH值、低离子强度下,分别加热诱导不同浓度11S(大豆球蛋白)和7S(大豆伴球蛋白)自组装纤维化聚集体的形成。通过平均粒径和Thioflavin T(硫磺素T)荧光光谱,对自组装纤维化聚集体的性质进行表征,并对其热致凝胶的流变学,硬度和微结构特性进行考察。结果表明:在低pH值、低离子强度的诱导条件下,蛋白浓度对自组装聚集的形成起着关键作用,随着诱导浓度的增大,蛋白的纤维化聚集越明显,7S比11S更容易形成纤维化聚集。在酸性环境下,大豆球蛋白的纤维化聚集程度越高,越有利于热致凝胶网络结构的形成。在相同的预处理条件下,11S自组装凝胶硬度强于7S。扫描电镜结果显示7S自组装凝胶的网络结构较11S致密,但有序性较11S低。     

Abundant proteins associated with lecithin in soy protein isolate

A new method for fractionating acid-precipitated soy proteins, so called soy protein isolate, involving three step acidification of a water extract of defatted soy flour was developed. Soy protein isolate was shown to be mostly composed of not only β-conglycinin and glycinin, but also a group of lipophilic proteins associated with lecithin (phospholipids). The proportions of three major proteins were 23%, 46%, and 31%, respectively. The proportions changed as the nitrogen solubility index (NSI) of defatted soy flour changed. The yield of lipophilic proteins depended on the NSI of defatted soy flour, different from those of β-conglycinin and glycinin. The variation in the proportions of the three major proteins may be due to the yield of lipophilic proteins.