Structural modification of soy protein by the lipid peroxidation product malondialdehyde

BACKGROUND: Protein oxidation results in covalent modification of structure and deterioration of functional properties of target protein. Oxidation extent of soy protein was affected by the content and type of lipid peroxidation (LPO) products in defatted soybean flours during storage and processing. Malondialdehyde (MDA) was selected as a secondary byproduct of LPO to investigate the effects of oxidative modification of LPO‐derived reactive aldehyde on soy protein structure. RESULTS: MDA reacted with ε‐amino and sulfhydryl groups of soy protein, and resulted in an increase in protein carbonyl groups but a decrease in sulfhydryl/disulfide, free amines and lysine. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly indicated that MDA induced soy protein aggregation, and results of high‐performance size‐exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of MDA. Results of electrophoresis indicated that MDA caused soy protein aggregation, and non‐disulfide covalent bonds were involved in aggregate formation. CONCLUSION: The results showed that sensitivity of soy protein was related to MDA concentration. Soy protein gradually aggregated with increase of MDA concentration; β‐conglycinin was more sensitive to MDA modification than glycinin. Copyright © 2009 Society of Chemical Industry     

Oxidative modification of soy protein by peroxyl radicals

Oxidative modification of soy protein by peroxyl radicals generated in a solution containing 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) under aerobic condition was investigated. Incubation of soy protein with increasing concentration of AAPH resulted in gradual generation of protein carbonyl derivatives and loss of protein sulphydryl groups. Circular dichroism spectra indicated that exposure of soy protein to AAPH led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity steadily decreased, accompanied by loss and burial of some tryptophan residues, indicating that soy protein gradually aggregated. The results of the size exclusion chromatogram (SEC) implied that incubation caused an AAPH-dose-dependent increase of fragmentation and aggregation of oxidised soy protein. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that non-disulphide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to peroxyl radicals than glycinin.     

Box-Behnken模型优化大豆分离蛋白共价改性

以提高产品凝胶强度为目的,利用大豆分离蛋白作为原料,通过添加葡萄糖进行共价改性处理。单因素实验初步得到优化共价改性的工艺条件。在此基础上,采用Box-Behnken模型对大豆分离蛋白共价改性工艺条件进行优化,测定并分析了改性复合物在各个条件下的凝胶强度。结果表明:适宜反应条件为,葡萄糖添加量1.0%,反应温度为60℃,反应时间为50min,此条件下凝胶强度可以达到1865.02g,较未改性大豆分离蛋白提高20%。试验证明优化工艺能有效且显著提高大豆分离蛋白的凝胶强度。     

Structural modification of soy protein by 13-hydroperoxyoctadecadienoic acid

To reveal the role of primary products of lipid peroxidation during soy protein oxidation process, oxidative modification of soy protein by 13-hydroperoxyoctadecadienoic acid (13-HPODE) generated by lipoxygenase-catalyzed oxidation of linoleic acid was investigated in this article. Incubation of soy protein with increasing concentration of 13-HPODE resulted in generation of protein carbonyl derivatives and loss of protein sulfhydryl groups. Circular dichroism spectra indicated that exposure of soy protein to 13-HPODE led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity gradually decreased, accompanied by loss and burial of some tryptophan residues. The results of surface hydrophobicity and tryptophan fluorescence implied that aggregation was induced by oxidation. Size exclusion chromatogram indicated that the extent of aggregation was increased in a 13-HPODE dose-dependent manner. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that non-disulfide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to 13-HPODE than glycinin.     

丙烯醛氧化修饰对核桃蛋白结构和乳化特性的影响

采用不同浓度（0～1?mmol/L）的丙烯醛代表多不饱和脂肪酸在脂肪氧合酶诱导下发生脂质过氧化反应过程中产生的小分子醛类物质,研究脂质活性氧化产物丙烯醛氧化对核桃蛋白结构和乳化特性的影响。结果表明,随着丙烯醛浓度的增加,核桃蛋白羰基含量从4.287?nmol/mg显著增加到11.078?nmol/mg。游离巯基和总巯基含量分别下降了55.18%和31.13%,说明丙烯醛使核桃蛋白发生了显著氧化。随着核桃蛋白氧化程度的增加,溶解度下降了84%;表面疏水指数从469.47下降到412.22;β-折叠、α-螺旋（有序）结构相对含量减少,β-转角、无规则卷曲（无序）结构相对含量增加。同时,核桃蛋白的乳化性和乳化稳定性分别从46.87?m2/g和25.93?min下降至9.86?m2/g和17.63?min。研究结果说明丙烯醛氧化能够导致核桃蛋白发生结构性氧化修饰,从而引起其乳化特性的变化。     

Change in optical properties of stratum corneum induced by protein carbonylation in vitro

The skin is the frontier against the external environment and continuously exposed to the environmental oxidative stress such as ultraviolet (UV) irradiation. Protein carbonyls are the major oxidative products of protein and may be introduced by reaction with aldehydes derived from lipid peroxide. Acrolein is one of the most reactive aldehydes generated during degradation of lipid peroxides and protein-acrolein adducts have been found in the oxidatively damaged lesion including UV-damaged skin. Recent studies revealed that protein carbonyls are also detected in thin outermost layer of the skin, the stratum corneum (SC). However, the effect of protein carbonylation on the function of SC was still unclear. In this study, we treated the SC sheets of reconstructed human epidermis and porcine epidermis with acrolein in the experimental conditions to explore the influence of protein carbonylation on the SC. Human and porcine SC sheets treated with acrolein showed less transmission at visible light than untreated SC sheets. Attenuated total reflection-infrared spectroscopy with curve fitting analysis of amide I region showed that acrolein induced alterations in protein secondary structure of the porcine SC sheets, which were accompanied by diminished fibrous keratin structure observed by transmission electron microscopy. These results show the possibility that carbonylation of the SC caused by environmental factors is one of factors altering the fibrous structure of keratin and decreasing the light transmission of SC, which changes the quality of the skin appearance.     

Reduction of acrolein by elemental iron: kinetics, pH effect, and detoxification

Acrolein is a highly toxic alpha,beta-unsaturated aldehyde that is widely used as a biocide, a cross-linking agent, and an intermediate in the chemical industry, among other applications. In this study we investigated the reductive transformation of acrolein by elemental iron and evaluated the feasibility of using iron to detoxify acrolein. At acidic and neutral pH, acrolein was transformed by iron through reduction of the C=C double bond to propionaldehyde. The reduction appeared to involve the chemisorption of acrolein to the iron surface followed by reduction of adsorbed acrolein. Both the adsorption and reduction rate constants decreased with increasing pH. Between pH 7.0 and 7.4, the acrolein adsorption rate constant decreased precipitously, resulting in a sharp decline in its removal rate. At higher pH, acrolein disappeared rapidly in control without iron, presumably due to reversible, base-catalyzed hydration. At equilibrium, approximately 93% of acrolein was hydrated, corresponding to an equilibrium constant of 13. Acrolein at 25 mg/L completely inhibited aerobic respiration; in contrast, its reduction product propionaldehyde was biodegradable. This suggests that elemental iron may be used to pretreat acrolein-containing wastes prior to aerobic biodegradation. To our knowledge, this is the first report of reduction and detoxification of an alpha,beta-unsaturated aldehyde by elemental iron.     

氧化对大豆蛋白结构、乳液稳定性及消化特性的影响

以大豆分离蛋白为原料,以脂质过氧化产物丙二醛（malondialdehyde,MDA）为氧化引发剂,逐级研究氧化对大豆蛋白结构、乳液稳定性及乳液消化特性的影响。结果发现：随着MDA浓度的升高,蛋白羰基及席夫碱含量明显升高而巯基含量显著降低。同时,MDA可促进蛋白聚集并诱导β-伴大豆球蛋白（7S）组分形成二硫键和非二硫键诱导的共价交联。进一步制备O/W型乳液,发现不同浓度MDA处理蛋白对乳液的形成影响较小,但可以显著改变界面蛋白组成。其中经中高浓度（2.5～10 mmol/L）MDA氧化后,更多7S组分以聚集状态参与界面组成。体外模拟胃肠道消化实验进一步表明,乳液消化主要在肠道进行,氧化诱导的蛋白交联/聚集可延缓或降低胆盐在界面的替代,进而减缓乳液消化并降低脂质释放率。     

PROTEIN-CARBOHYDRATE INTERACTIONS AT ELEVATED TEMPERATURES*

A series of protein types have been characterized with respect to their interaction with low dextrose equivalent corn starch hydrolysates. From an assessment of the chemical modification of selected functional groups, classical denaturation studies, gel filtration and precipitation with tri-chloro-acetic acid, the partial basis for molecular interaction has been elucidated. Although all proteins under investigation were prominent components of a variety of food systems, particular emphasis was placed on the behavior of β-lactoglobulin and selected soy fractions. Results from the modification and denaturation studies indicated that several factors are required to account for the extent of interaction, but that the predominant effect appears to be non-covalent in nature. In the case of commercial soy proteins, considerable polydispereity was detected prior to the interaction - much of which was doubtless induced during isolation. Many such fragments of this preparation showed self-interacting properties, which could provide a basis for texture in the reported system. The starch hydrolysates appear to exhibit a protective effect against excessive protein aggregation, which could have significance in retention of nutritional value during food processing.     

大豆分离蛋白与染料木素共价交联对蛋白表征和结构的影响

探究大豆分离蛋白和染料木素的共价交联对蛋白表征和结构的影响。制备大豆分离蛋白与不同质量浓度染料木素（0、1.2、1.5、2.0 mg/mL）的共价复合物,通过探究粒径、Zeta电位、浊度、表面疏水性分析蛋白体系的表征变化,并采用紫外分光光度计、荧光分光光度计、傅里叶变换红外光谱仪分析蛋白体系的结构变化。结果表明：大豆分离蛋白与染料木素共价复合后,蛋白的中位径由135.6 μm最低减小至98.0 μm,Zeta电位绝对值由15.0 mV最高增大至21.4 mV,表面疏水性由216.0最低减小至115.5,总巯基含量由31.5 μmol/g最低减小至20.4 μmol/g。与对照组相比,共价复合物的浊度增加,并且实验组中SPI-Ge-1.2组低于SPI-Ge-1.5组和SPI-Ge-2.0组。光谱分析表明染料木素对大豆分离蛋白有猝灭效果,二者共价交联后蛋白质的色氨酸与酪氨酸残基所处的微环境疏水性减少,蛋白质二级结构中α-螺旋含量增多、β-折叠含量减少、β-转角含量增多、无规卷曲含量减少,并且加入1.2 mg/mL的染料木素对大豆分离蛋白的表征特征和结构影响效果更好。本研究结果表明在大豆分离蛋白中加入染料木素后,二者的共价交联能够影响蛋白的表征与结构。     

花青素与大豆分离蛋白的不同共价交联法对蛋白结构的影响

分别运用生物酶法和化学碱法,研究不同质量浓度花青素与大豆分离蛋白（soy protein isolate,SPI）发生共价相互作用后对蛋白结构的影响。采用共价结合率测定和游离巯基含量测定方法对花青素和SPI在不同共价交联方式下的结合强度进行比较,后采用傅里叶变换红外光谱、紫外-可见吸收光谱和荧光光谱对不同共价交联方式下的花青素-SPI共价聚合物的结构及构象进行解析。结果表明：随着花青素添加量的升高,花青素与SPI的结合率逐渐提升,游离巯基含量持续下降。光谱测定显示花青素对SPI的共价交联可以改变蛋白的二级结构,使蛋白多肽链解折叠,并且改变蛋白芳香族氨基酸残基所处的微环境,进而使蛋白的构象发生改变。此外,相较于化学碱法,生物酶法共价交联的花青素-SPI聚合物结合率更高,巯基含量下降更显著,结构及构象的变化更为明显,这表明花青素与SPI在生物酶法处理下,共价结合能力强于化学碱法处理。     

大豆分离蛋白与花青素非共价及共价作用对蛋白构象变化的影响

为对比解析大豆分离蛋白-花青素复合体系中非共价/共价作用对蛋白质构象变化规律的影响,以非共价结合（pH?7.4、2?h）和共价交联（pH?9、24?h）2?种作用机制为处理手段,以大豆分离蛋白-花青素复合物为研究对象,采用浊度测定、结合度测定、凝胶电泳分析、荧光光谱和红外光谱法研究复合体系中蛋白质结构变化。结果表明：在大豆分离蛋白-花青素复合体系中,pH?9、24?h条件下样品4～6（20∶1、10∶1、5∶1,m/m）电泳图谱中有大分子衍生物生成,由此说明其形成共价复合物。共价交联处理（pH?9、24?h）样品4～6（20∶1、10∶1、5∶1,m/m）的浊度值低于非共价结合处理（pH?7.4、2?h）样品1～3（20∶1、10∶1、5∶1,m/m）,且复合物4～6中花青素对大豆分离蛋白的亲和能力较强;随复合物中花青素含量的增加,花青素会降低蛋白的荧光强度使色氨酸残基暴露于较为亲水环境中,表明复合样品4～6的荧光猝灭效果明显,共价交联作用强度大于非共价结合作用;在低比例（20∶1,m/m）中,复合物1、4中的蛋白红外光谱吸收强度明显下降,表明复合物中蛋白质二级结构发生改变,且样品4中β-转角及无规则卷曲结构相对含量较高,这表明复合物中花青素共价交联机制对蛋白的解折叠能力较强,结构易展开。     

Associative interactions between chitosan and soy protein fractions: Effects of pH,mixing ratio,heat treatment and ionic strength

The objective of this paper is to explore the complexation between the soy protein fractions (glycinin and β-conglycinin) and chitosan (CS) and to investigate the influence of pH, mixing ratio, heat treatment and ionic strength. Phase behavior and microstructure showed that soluble complex and coacervate were obtained in glycinin/CS and β-conglycinin/CS mixtures at specific pHs, following a nucleation and growth mechanism. Moreover, the coacervates showed higher thermal stability than protein alone. Specially, the glycinin/CS mixture displayed a gel-like network structure at pH 5.5 and 6.0, and this structure kept the mixture soluble at a long pH region. The turbidity versus ζ-potential pattern showed that, independent of protein, the self aggregation of soy protein fractions and the coacervation of glycinin/CS and β-conglycinin/CS mixtures were all obtained at charge neutralization pH, indicating that the ζ-potential is the most critical parameter to understand the stability of soy protein/chitosan mixture. This predictive parameter was less affected by mixing ratio and heating but was significantly affected by ionic strength because mixing ratio and heating only changed the equilibrium between repulsive and attractive forces in colloid system while sodium chloride destroyed the predictability of colloidal stability via shielding charged reactive sites on both biopolymers to disrupt electrostatic interactions.     

Effect of ascorbic acid and protein concentration on the molecular weight profile of bovine serum albumin and β-lactoglobulin by γ-irradiation

The effects of ascorbic acid and protein concentration on the molecular weight size distribution of BSA and β-lactoglobulin were examined after irradiation of proteins at various doses. Gamma-irradiation of protein solutions caused disruption of the ordered structure of protein molecules resulting in degradation, cross-linking, and aggregation of the polypeptide chains. SDS–PAGE and gel permeation chromatography study showed that ascorbic acid protected the aggregation and degradation of proteins by scavenging oxygen radicals produced by irradiation and the effect of irradiation on protein conformation was more significant at lower concentrations of proteins.     

丙烯醛氧化修饰对大豆蛋白凝胶性质的影响

以丙烯醛代表脂质次生氧化产物,研究脂质次生氧化产物氧化修饰对大豆蛋白凝胶性质的影响.采用质构仪、流变仪、持水性测定以及扫描电镜等方法表征大豆蛋白的凝胶性质,结果发现蛋白质氧化造成大豆蛋白的凝胶硬度、凝胶形成温度、凝胶强度以及凝胶持水性下降.随着大豆蛋白氧化程度的增加,大豆蛋白凝胶的粗糙度增加,内部空隙变大,并且分布不均匀.蛋白质氧化对大豆蛋白凝胶性质的影响与丙烯醛浓度有关,当丙烯醛浓度低于0.01 mmol/L时,蛋白质氧化对大豆蛋白凝胶性质的影响程度很小.     

谷氨酰胺转移酶对大豆分离蛋白膜性能的影响

研究注模前酶作用时间对谷氨酰胺转移酶(TGase)改性大豆分离蛋白(SPI)膜性能的影响。在注模之前,将TGase(8U/g SPI)加入到成膜溶液中,分别在磁力搅拌下作用0、30、60、120min,然后注模成膜。利用质构仪检测蛋白膜的机械性能,结合哈克流变仪的动态黏弹实验及SDS-PAGE 实验进一步分析。注模前适度的酶作用(≤60min)在一定程度上有利于TGase 改性的SPI 膜机械强度的提高,特别是抗拉强度(TS)值;但是,时间不宜过长,因为注模前的酶作用也会诱导SPI 蛋白组分的聚沉反应,从而降低成膜溶液中可溶解蛋白的含量。结果表明,TGase改性SPI 膜时,一方面会诱导蛋白交联;另一方面,交联过多又会导致沉淀;在利用TGase 提高SPI 膜的机械性能时如何把握两者之间的关系,在交联的同时抑制酶促聚沉非常重要。     

Effects of oxidative modification on thermal aggregation and gel properties of soy protein by peroxyl radicals

Effects of protein oxidation on thermal aggregation and gel properties of soy protein by 2,2′‐azobis (2‐amidinopropane) dihydrochloride (AAPH)‐derived peroxyl radicals were investigated in this article. Incubation of soy protein to increase concentration of AAPH resulted in a decrease in particle size and content of thermal aggregates during thermal‐induced denaturation. Protein oxidation resulted in a decrease in water‐holding capacity (WHC), gel hardness and gel strength of soy protein gel. An increase in coarseness and interstice of the gel network was accompanied by uneven distribution of interstice as extent of oxidation of soy protein increased. A decrease in disulphide content and formation of oxidation aggregates in the process of oxidative modification were contributed to the decline of particle size and content of thermal aggregates during thermal‐induced denaturation, leading to a decrease in WHC, gel hardness and gel strength of soy protein gel.     

花青素共价交联大豆蛋白对其结构及营养吸收特性的影响

研究不同质量浓度的花青素与大豆分离蛋白（soy protein isolate,SPI）在漆酶条件和碱性条件下的共价交联机制,以及两者在共价交联后对蛋白结构及营养吸收特性的影响。采用三维荧光光谱研究花青素与SPI在不同共价交联条件下的复合程度,并揭示被花青素交联后的SPI的结构构象变化;后采用胃蛋白酶、胰蛋白酶及Caco-2细胞对SPI与花青素-SPI共价复合物进行模拟人体胃肠消化及吸收,探究消化过程中水解度、抗氧化性、多肽渗透率等指标。结果表明：花青素对SPI的交联可以降低蛋白荧光值,使蛋白多肽链解折叠从而改变蛋白的三级结构。同时,蛋白的消化率和抗氧化性因花青素的交联而升高,花青素质量浓度越高,这种改善现象越明显,尤其是在漆酶条件下。值得注意的是,花青素对蛋白多肽的渗透率有抑制作用,且花青素含量越高,抑制作用越强烈。添加同质量浓度花青素时,漆酶条件下的花青素对多肽渗透率的抑制作用比碱性条件下的花青素更强。     

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.     

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。