Effect of concentration,ionic strength and freeze-drying on the heat-induced aggregation of soy proteins

The effects of protein concentration, ionic strength, and lyophilization on heat-induced aggregation of soy proteins were analyzed by SDS-PAGE, SEC-HPLC and laser light scattering (LLS). SDS-PAGE profile suggested that the aggregates were formed via non-covalent forces and/or disulfide bonds. At ionic strength of zero, SEC-HPLC revealed that the samples were composed of three major fractions: aggregates, intermediate fractions and non-aggregated molecules. Furthermore, the relative proportion of the aggregate fraction increased as protein concentration increased. Similarly, LLS indicated that the average hydrodynamic radius (R_h) increased at higher protein concentration. In sample with an ionic strength of zero, the intermediate fraction decreased after freeze-drying with a concomitant increase of the aggregate fraction. When the sample was heated at elevated ionic strength, the SEC-HPLC and LLS profiles changed substantially, the intermediate fractions decreased, and lyophilization had effect on the fraction of aggregates. These experiments suggest novel strategies for producing soy protein aggregates with controlled properties.     

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     

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.     

大豆蛋白/葡聚糖混合体系相行为及流变性质的研究

研究了室温下,pH 7.0时不同尺寸大小的大豆蛋白热聚集体和不同分子质量的葡聚糖混合体系的相分离行为,并以天然大豆蛋白和葡聚糖混合体系作为对照体系。通过离心、化学分析和目测建立了相图,结果表明两种大分子的相分离是由于葡聚糖分子链的排空相互作用,使得蛋白质富集部分间产生了交联;蛋白质聚集体的尺寸和葡聚糖的分子质量大小同时影响了混合体系的相行为,随着蛋白质粒子增大或多糖分子质量增加,相边界发生了位移,混合体系的均相区域变窄,凝胶区域增大。流变研究进一步证明蛋白质聚集体的尺寸和葡聚糖的分子质量大小同时影响了相分离体系的微观结构。     

不同大豆原料形成蛋白纤维聚合物的比较

4种通过不同加工处理得到的大豆蛋白,在低pH条件下90℃加热10h所形成的聚合物形态存在很大不同。利用硫磺素T（Th T）荧光强度、差量扫描仪（DSC）和SDS-PAGE凝胶电泳分析了来自不同原料大豆蛋白的聚合动力学和组成差异。结果表明,4种大豆原料因其加工工艺不同,蛋白质组成存在差异,在本实验的条件下,11S的存在尤其是碱性亚基会抑制纤维聚合物的形成。此外,离子强度也是纤维形成的一个必要条件。      

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.     

Properties of soy protein isolate prepared from aqueous alcohol washed soy flakes

Defatted soy flakes were washed with aqueous alcohol and the resultant material was extracted with water to prepare soy protein isolate (ASPI) with improved functional properties and flavor profile in comparing with the conventional soy protein isolate (CSPI) extracted directly from the low-denatured soy flakes. Gelling property was determined by rheometer at the protein concentration of 12% (w/w) and the gel fracture force of ASPI was 385.4 g, substantially higher than that of CSPI (85.4 g). Emulsifying and foaming stability were also found to be improved through alcohol washing. High performance size exclusion chromatography showed that ASPI and CSPI was eluted around the same time, but laser light scattering analysis indicated that ASPI solution contained particles with larger hydrodynamic radius than CSPI, suggesting that ASPI formed larger protein aggregates with more flexible structure whereas CSPI formed smaller and more compacted aggregates. Off-flavor volatiles were collected by solid phase micro-extraction and analyzed by GC–MS. The results showed that hexanal, acetic ether, pentanal, decanal, 2-pentylfuran and 1-octen-3-ol contents of ASPI were 10 times lower than that of CSPI.     

不同浓度大豆分离蛋白热诱导聚集体的研究

采用体积排阻色谱(SEC-HPLC)和激光光散射(LLS)研究了由醇洗豆粕制备的不同浓度的大豆分离蛋白热诱导聚集体(100℃,15 min)的分子量分布和粒径分布。SEC-HPLC检测结果表明,经热处理的蛋白溶液主要由3部分组成,即聚集体、中间体及未聚集部分;蛋白浓度为1%时,聚集体的百分含量为18.70%;蛋白浓度增加到5%时,聚集体的百分含量增加到54.15%;同时LLS的测定结果表明,蛋白溶液有不均一的粒径分布且体系浓度增加时平均粒径(Rh)由56.5 nm增至144.9 nm。     

大豆蛋白纤维聚集体的体外消化特性研究

本研究以大豆分离蛋白为材料,通过在pH 2.0条件下调控大豆蛋白形成大豆蛋白纤维聚集体。采用标准的静态体外消化模型INFOGEST 2.0对大豆蛋白纤维聚集体进行体外模拟消化实验,并对不同消化时间的胃肠消化产物通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、动态激光散射仪（DLS）及液相色谱-质谱联用（LC-MS）对其分子质量、粒径及肽段组成进行鉴定。结果表明,大豆蛋白纤维聚集体通过消化后被水解为小分子的肽,并LC-MS证实消化过程中肽段数量随着消化时间的增加呈阶梯式水解。本实验探究了大豆蛋白在体外模拟消化过程中的消化特性,这将有助于更好地了解植物蛋白在人体中的消化方式及其对健康的影响。     

Effect of transglutaminase-induced cross-linking on gelation of myofibrillar/soy protein mixtures

Microbial transglutaminase (MTGase)-catalyzed interaction and gelation of mixed myofibrillar (MPI)/soy (SPI) protein isolates were investigated at varying ionic strengths and MPI:SPI ratios, with or without SPI being preheated (80?°C). MTGase treatments in deionized water converted myosin heavy chain and actin into lower molecular-weight polypeptides, which gradually diminished as the ionic strength increased up to 0.6 M NaCl. A reduced intensity in the electrophoretic bands of soy proteins (7S and 11S except the basic subunits) was observed in all treatments, suggesting cross-linking with MPI. The enzyme treatment slightly increased the thermal transition (denaturation) temperatures of MPI/SPI but greatly enhanced (P<0.05) the elasticity of the mixed protein gels when compared with untreated samples, independent of incubation time.     

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.     

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.     

Stabilization of acidic soy protein-based dispersions and emulsions by soy soluble polysaccharides

Soy soluble polysaccharides (SSPS) are shown to prevent destabilization of soy protein isolate (SPI) dispersions and SPI-based oil-in-water (O/W) emulsions under acidic conditions. Addition of SSPS above a critical concentration (0.25 wt%) increased the stability of 0.50 wt% SPI dispersions against aggregation and phase separation under conditions where SPI would normally precipitate (near its isoelectric point). Though SSPS neutralized SPI surface charge via electrostatic interaction, there was increased stability against aggregation due to steric repulsion. At acidic pH, addition of 1 wt% NaCl electrostatically screened protein–polysaccharide complexation which led to SPI precipitation and sedimentation. However, the order of salt addition had a significant impact on charge screening, with salt added before pH adjustment reducing SPI–SSPS complexation whereas it had less effect when added afterwards. Salt penetration efficacy diminished with decreasing pH. O/W emulsions (5 wt% oil) prepared with 0.50 wt% SPI destabilized at pH 4–5 due to protein aggregation, but addition of ≥0.25 wt% SSPS improved emulsion stability by inhibiting protein–protein interactions thus limiting increases in oil droplet diameter over time. Overall, both dispersion and emulsion stability greatly depended on pH, ionic strength and SSPS concentration. These results demonstrated that SSPS could effectively stabilize acidic SPI dispersions and that SPI–SSPS interactions may be used as a tool to improve the kinetic stability of SPI-based O/W emulsions.     

Heat induced gelling properties of soy protein isolates prepared from different defatted soybean flours

Three soy protein isolates were prepared from two commercial defatted soybean flours (SG and ADM) and flour indigenously prepared from soybeans grown in Ontario (ON). Denaturation degree and protein composition of three isolates as measured by DSC and SDS–PAGE were not different significantly, but remarkable differences in ANS surface hydrophobicity and turbidity were noticed. Heat induced gelling properties of these soy protein isolates were examined by small deformation oscillatory rheological test. The critical gelling concentrations of the two isolates from commercial soy flours (SG and ADM) in distilled water were 90 mg/ml, much lower than 110 mg/ml for ON. The effect of type and concentration of salts on the gelling properties of the three protein isolates were also examined. In 1 mol/L NaCl solution, ADM had the higher gel strength than SG. It was found that the increase in gel strength of ON by addition of NaSCN was much greater than that in NaCl solution. N-Ethylmaleimide (NEM) was observed to increased the gel strength for all soy protein isolates examined despite its sulfhydryl groups blocking function. It is suggested that a non-heat aggregation of protein which led to turbidity development was responsible for the inferior gelling property of ON while the different gelling behavior between SG and ADM could be attributed to their difference in ANS surface hydrophobicity.     

大豆蛋白组分/葡聚糖复合体系相行为及热性质研究

采用浊度、相图及DSC热性质分析研究了中性多糖葡聚糖(DT)对大豆7S蛋白及11S蛋白热稳定性、相行为及热性质的影响。结果表明增加葡聚糖分子质量或浓度可加速大豆7S蛋白热聚集,葡聚糖对大豆11S蛋白热聚集的影响趋势同7S蛋白。相图分析结果表明当葡聚糖分子质量由10 ku逐渐增加到67、100、500 ku,大豆蛋白组分/葡聚糖形成稳定混合物的区域逐渐减小,同时不稳定区域也呈降低趋势,但凝胶区域呈上升趋势,与大豆7S蛋白/葡聚糖相图相比大豆11S蛋白/葡聚糖相图中随葡聚糖分子质量增加凝胶区域进一步加大,相分离区域相对较大。大豆7S、11S蛋白体系中添加葡聚糖提高了7S、11S蛋白的热稳定性同时降低了热焓值,且此趋势随葡聚糖分子质量增加而增加。     

Improvement of functional properties of acid-precipitated soy protein by the attachment of dextran through Maillard reaction

The functional acid-precipitated soy protein (SAPP)–dextran conjugate was prepared by dry-heated storage at 60 °C under 79% relative humidity (RH) for 5 days through Maillard reaction between the ε-amino of lysine in soy proteins and the reducing-end carbonyl residue in the dextran. The covalent attachment of dextran to SAPP was confirmed by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography. Functional properties of soy protein depend on the structural and aggregation characteristics of their major components (storage globulins 7S and 11S). The conjugate seemed to be predominantly formed by 7S, and the acidic subunits of 11S in soy protein. The emulsifying properties of the SAPP–dextran conjugate were about four times higher than those of SAPP. The solubility of the protein was not enhanced as a result of preheating, but rather it was not decreased when the conjugated protein was heated at 90 °C for 20 min due to the presence of the polysaccharide. The excellent emulsifying properties of SAPP–dextran conjugate were maintained even at pH 3.0 and were further improved at pH 10.0. The object of Maillard reaction is to guarantee the suitable reaction degree, and the resulting soluble conjugate can have excellent emulsifying properties.     

预热变性程度对大豆蛋白凝胶性质的影响

以非还原SDS-PAGE表征大豆蛋白的预热变性程度,并采用质构分析、流变分析研究预热变性程度对大豆蛋白凝胶性质的影响。SDS-PAGE结果表明,随着加热温度的升高蛋白质变性速率逐渐增大,相同加热时间条件下,蛋白质变性程度随加热温度升高呈S型曲线上升。质构、流变分析结果表明:随着预热变性程度的增大,大豆蛋白凝胶硬度先增大后减小,变性程度为86.11%时凝胶硬度最大,是未经预热变性的大豆蛋白凝胶硬度的2.15倍;凝胶弹性则随预热变性程度的增大持续增大;变性程度在22.28%以上时大豆蛋白形成凝胶更快,但完全变性大豆蛋白在形成凝胶时的降温阶段不能形成很好的凝胶结构。粒径分析结果表明,预热变性程度对大豆蛋白凝胶性质的影响与蛋白质预热变性时形成的聚集体尺寸及形态有关。     

Roles of soluble and insoluble aggregates induced by soy protein processing in the gelation of myofibrillar protein

The role of soluble and insoluble aggregates induced by soy protein isolate (SPI) processing in the gelling properties of myofibrillar protein (MP) was studied. Incorporating soluble SPI aggregate could greatly improve (P < 0.05) the elastic modulus (G’) and water‐holding capacity (WHC) of MP gel, but had no notable effect on MP gel strength. In contrast, incorporating the insoluble SPI aggregate significantly enhanced the G’, strength and WHC of MP gel, although the improvement in WHC was smaller than that produced by the soluble aggregate. The results of environmental scanning electron microscopy showed that the soluble SPI aggregate induced a less randomly composite gel structure, which may explain its notable enhancement of WHC. However, the insoluble SPI aggregate appeared to be granules embedded in the continuous MP gel matrix, which may be related to the reinforcement of gel strength. Hence, the results of this study suggest further means of processing commercial SPI for use in meat products.     

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.     

Physicochemical properties of dry-heated soy protein isolate–dextran mixtures

Soy protein isolate–dextran mixtures were incubated for up to three weeks and an improvement of their ability to stabilize emulsions against creaming, giving emulsions with lower droplet size, at pH values near the protein isoelectric point, was observed. Analysis of the adsorbed protein fraction indicated that the protein–polysaccharide hybrid can adsorb, together with the other mixture constituents, at the interface, during emulsion formation and in this way may contribute to droplet stabilization by enhancing repulsive steric forces by interpenetrating neighbouring droplets and polysaccharide chains.