Effects of high hydrostatic pressure on some functional and nutritional properties of soy protein isolate for infant formula

The effects and mechanism of high hydrostatic pressure (HHP) on some functional and nutritional properties of soy protein isolate (SPI) for infant formula were investigated. Results indicated that solubility, water holding capacity, emulsification activity index, and foaming capacity were improved at lower pressure and time levels, whereas these properties declined at higher levels. However, the emulsification stability index dropped when the pressure increased and the foaming stability decreased with pressure and time levels rising. HHP-treated SPI gave better swallowing properties and in vitro digestibility than control. The hardness, adhesive force, and springiness of SPI gels increased with increaded pressure and elongated time, being lower than those of the control. Near UV circular dichroism spectra confirmed the alteration of tertiary and/or quaternary conformations caused by HHP. Sodiumdoecyl sulfate polyacrylamide gel electrophoresis results indicated that β-conglycinin was more pressure labile than glycinin, and high molecular weight subunits formed via disulfide linkage at higher treatment levels.     

Limited aggregation behavior of β-conglycinin and its terminating effect on glycinin aggregation during heating at pH 7.0

In this work, different thermal aggregation behaviors of soy β-conglycinin and glycinin at pH 7.0 were characterized with size exclusion chromatography and low-angle light scattering. Limited aggregation that grew via the consumption of "monomers" was detected in β-conglycinin, forming soluble aggregates. For glycinin, the association between the aggregates that led to the appearance of insoluble materials was observed. Heated with β-conglycinin, the assembly between the glycinin aggregates was terminated and its solubility was recovered. The structure of the soluble and insoluble aggregates was analyzed by small-angle X-ray scattering and dynamic light scattering. Unlike the β-conglycinin soluble aggregates that possessed limited size and less compact conformation, particles with a denser core and a less dense outer shell were found in the glycinin insoluble aggregates. Evidence is presented to reveal the transition between the soluble and insoluble aggregates and the role of β-conglycinin in the solubilization of the soy protein aggregates during heating.     

Physicochemical properties of β and α'α subunits isolated from soybean β-conglycinin

Soy protein has shown great potential for use in biobased adhesives. β-Conglycinin is a major component of soy protein; it accounts for 30% of the total storage protein in soybean seeds. β-Conglycinin was isolated and purified, and its subunits' (β, α'α) physicochemical and adhesive properties were characterized. Crude β-conglycinin was isolated from soy flour and then purified by the ammonium sulfate precipitation method. The α'α and β subunits were isolated from the purified β-conglycinin by anion exchange chromatography. Yields of α'α subunits and β subunits from 140 g of soy flour were 1.86 g (1.3%) and 0.95 g (0.67%), respectively. The minimum solubility for α'α subunits, β subunits, and β-conglycinin occurred in pH ranges of 4.1-5.4, 3.5-7.0, and 4.8-5.3, respectively. Transmission electron microscopy showed that the β subunits existed as spherical hydrophobic clusters, whereas α'α subunits existed as uniformly discrete particles at pH 5.0. Differential scanning calorimetry showed that β subunits had higher thermal stability than α'α subunits. The pH had a lesser effect on adhesion strength of the β subunits than on that of the α'α subunits. The adhesives made from β subunits also showed greater water resistance than those from α'α subunits and β-conglycinin. Soy protein rich in β subunits is likely a good candidate for developing water-resistant adhesives.     

Hydrophobic probe binding of beta-lactoglobulin in the native and molten globule state induced by high pressure as affected by pH,KIO(3) and N-ethylmaleimide

High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.     

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.     

超高压处理对绵羊肉嫩化机理的研究

实验研究了超高压处理条件下绵羊肌肉感官特性、显微结构、钙激活酶(Calpains)粗酶活性和剪切力值的变化，并探讨了超高压处理对绵羊肌肉的嫩化机理。超高压处理后绵羊肌肉的感官特性发生变化，随处理压力升高，绵羊肌肉颜色变淡，出现轻微的类似蒸煮的成熟风味。在压力为400 MPa，保压时间为10 min的处理条件下，绵羊肌肉显微组织结构变化明显：肌节收缩，肌原纤维的Z线断裂，M线降解，I带变白。当压力水平在100～400 MPa范围变动时，随压力升高，Calpains粗酶活性显著下降(P＜0.01)；当压力达到400 MPa时，Calpains粗酶活性几乎失活。超高压处理后绵羊肉的剪切力值显著下降(P＜0.05)。实验结果表明，超高压处理促进了绵羊肉的嫩化。     

Soy glycinin: influence of pH and ionic strength on solubility and molecular structure at ambient temperatures

This study describes the relationship between the solubility of glycinin, a major soy protein, and its structural properties at a quaternary, tertiary, and secondary folding level under conditions representative for food products. When the ionic strength is lowered from 0.5 to 0.2 or 0.03, the basic polypeptides shift more to the exterior of the glycinin complex, as determined at pH 7.6 by labeling solvent-exposed lysines, supported by the study of the proteolytic action of clostripain on glycinin. This structural reorganization caused the pH of minimal solubility to shift to higher values. Ultracentrifugational analysis shows that at pH 7.6 and an ionic strength of 0.5 glycinin forms hexameric complexes (11S), whereas at pH 3.8 and at an ionic strength of 0.03 glycinin exists as trimers (7S). Intermediate situations are obtained by modulation of pH and ionic strength. The observed quaternary dissociation correlates with an increased amount of nonstructured protein at a secondary level and with changes in tertiary folding as determined using circular dichroism. Tryptophan fluorescence shows no significant structural changes for different ionic strengths but demonstrates a more tightly packed fluorophore environment when the pH is lowered from 7.6 to 3.8.     

Regulation of accumulation of β-subunit of β-conglycinin in soybean seeds by nitrogen

Extract

The storage protein of soybean [Glycine max (L.)] seed mainly consists of glycinin, composed of acidic (38 and 45 kDa) and basic (22 kDa) subunits (Kitamura et al. 1976), and β-conglycinin composed of α′- (75 kDa), α- (72 kDa), and β-(52 kDa) subunits (Thanh and Shibasaki 1978).     

Characterization of beta-conglycinin and glycinin soy protein fractions from four selected soybean genotypes

The beta-conglycinin and glycinin fractions of soy protein were isolated from Macon, Ohio FG1, Enrei, and IL2 genotypes that were grown under the same environmental conditions. The soy protein fractions were evaluated to determine whether chemical composition and gel-forming properties were related. Amino acid analyses suggested that the hydrophobic residues may be the primary cause of differences in soy protein gel characteristics as the storage moduli increased with higher percentages of hydrophobic residues. Reversed-phase high-performance liquid chromatography profiles revealed variations in the composition of each fraction that corresponded to differences observed among the storage moduli. The gel-forming properties may be related to more than just protein content, such as the amount and type of amino acid in the fraction.     

Beta-lactoglobulin molten globule induced by high pressure.

Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.     

高静压处理改善白果蛋白致敏性和功能特性

为了研究高静压处理对白果蛋白结构、抗原性及功能特性的影响,分别采用100,200,300,400,500,600和700 MPa的压力对白果蛋白进行处理,采用酶联免疫吸附检测法测定蛋白的致敏性,分别采用聚丙烯酰胺凝胶电泳,圆二色谱,荧光光谱和紫外吸收光谱检测白果蛋白分子量和构象的改变,功能特性的检测包括热稳定性和乳化特性。结果表明,高静压处理在300~700 MPa范围内可显著降低白果蛋白的致敏性(P0.05),同时高压处理后,白果蛋白能被分解为分子量为4~30 k Da范围内的小分子蛋白,此外,其二级结构中的α-螺旋和β-折叠结构被大量破坏形成无规则卷曲结构,其紫外吸收强度,表面疏水性和游离巯基含量明显提高(P0.05),高压对白果蛋白的致敏性影响与其结构变化密切相关,另外高压处理(300~700 MPa)可明显改善白果蛋白的热稳定性和乳化性能(P0.05)。因此,高静压技术可以作为一种降低白果蛋白致敏性和改善其功能特性的有效手段。     

大豆蛋白热变性程度对速溶豆腐花粉凝胶成型的影响

针对速溶豆腐花粉的制备工艺中需要对大豆进行热处理,热处理过程中大豆蛋白的热变性程度对豆腐花粉凝结的凝胶强度与凝结所需的时间具有显著影响,而现在速溶豆腐花粉的工业生产中还没有对豆浆热处理程度较为合适的标准。该文以大豆、大豆分离蛋白(soybean protein isolate,SPI)、大豆球蛋白(glycinin,11S)、β-伴大豆球蛋白(beta-conglycinin,7S)为原料,研究大豆蛋白热变性程度对速溶豆腐花粉凝胶成型的影响。研究表明11S比7S更难发生完全变性,SPI中的7S和11S比单独存在的7S、11S更难发生完全变性。传统制备方式、前热处理后喷雾干燥或冷冻干燥制备方式、先喷雾干燥或冷冻干燥后热处理制备方式对豆腐花凝结成型影响不同,其中传统方式制备的豆腐花凝胶效果最好,先干燥后热处理制备的豆粉凝胶效果比前热处理后干燥的豆粉好,引起豆腐花凝胶强度差异的主要原因是大豆蛋白中7S和11S热变性程度不同。制备同一凝胶强度的豆腐花,热处理温度越低,所需的热处理时间越长;制备高凝胶强度的豆腐花比制备低凝胶强度的豆腐花所能进行的热处理温度与时间范围小。大豆蛋白的7S处于完全变性而11S处于未完全变性的状态时,适合制备速溶豆腐花粉的大豆蛋白变性程度应控制热处理温度与时间范围为80℃时热处理20~65 min,85℃时热处理15~50 min,90℃时热处理10~35 min,95℃时热处理5~20 min。该研究结果为调控速溶豆腐花粉的凝胶特性提供理论依据。     

Properties of tofus and soy milks prepared from soybeans having different subunits of glycinin

The contribution of soybean protein to the physical properties of tofu, a product manufactured by curdling soy milk with coagulants such as calcium or magnesium chloride, was studied by comparing the properties of soy milk prepared from soybeans with different subunits (I, IIa, and IIb) of glycinin with amino acid residues deleted. The breaking stress value of the tofu curds prepared from soybeans having group I was higher than those without group I. The soy milks having group I contained more protein particles and showed more sensitivity to calcium and magnesium ions than those without group I. The amounts of glycinin and protein particles were higher in the soy milks having group I than those in the soy milks without group I. To elucidate the influence of each group on the breaking stress, the glycinin content was adjusted to an identical level in soy milks having each group. Among the tofu curds from three groups, their order of hardness according to their breaking stress was IIa, IIb, and I. The order of particle content among these soy milks was also IIa, IIb, and I. Therefore, the results suggested that the breaking stress value of the tofu curd is dependent upon the number of protein particles in the soy milk and that the number of the particles is determined by the proportion and structure of glycinin in the soybean.     

Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins

Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.     

Relation between solubility and surface hydrophobicity as an indicator of modifications during preparation processes of commercial and laboratory-prepared soy protein isolates

Because water solubility is the main hydration property of proteins, solubility values of commercial and laboratory soy protein isolates, prepared under different conditions, were comparatively analyzed. In contrast, the surface hydrophobicity manifested by proteins is a physicochemical property that determines, to a great extent, the tendency of protein molecules to aggregate and so to lose solubility. On these grounds, the solubility of isolates was analyzed as a function of the surface hydrophobicity of their proteins, and, as a result, three well-defined groups of laboratory isolates were identified: (A) native, (B) partially or totally denatured with high solubility and surface hydrophobicity, and (C) totally denatured with low solubility and surface hydrophobicity. Commercial isolates could not be included in any of these groups; they were grouped as (A') partially native and (C') totally denatured. Solubility values in these two groups were similar to those of group C, but the surface hydrophobicity levels were much lower. The different processes leading to the groups mentioned above are discussed, along with the way the soy proteins are influenced by the specific preparation conditions, namely, protein concentration, chemical or thermal treatments, presence of salts, drying, and phospholipid addition, among others.     

Growth kinetics of amyloid-like fibrils derived from individual subunits of soy β-conglycinin

The amyloid-like fibrillation of soy β-conglycinin subunits (α, α', and β) upon heating (0-20 h) at 85 °C and pH 2.0 was characterized using dynamic light scattering, circular dichroism (CD), binding to amyloid dyes (Thioflavin T and Congo red), and atomic force microscopy. The fibrillation of all three subunits was accompanied by progressive polypeptide hydrolysis. The hydrolysis behaviors, fibrillation kinetics, and morphologies of amyloid-like fibrils considerably varied among α, α', and β subunits. Faster hydrolysis rates and special fragments were observed for the α and α' subunits compared to the β subunit. However, the order of the fibrillation rate and capacity to form β-sheets was α' > β > α, as evidenced by CD and Thioflavin T data. Moreover, sequential growth of twisted screw-structure fibrils, leading to macroscopic fibrils with distinct morphological characteristics, was observed for β-conglycinin and individual subunits. The different fibrillation kinetics and morphologies of α, α', and β subunits appear to be associated with the differences in the amino acid composition and typical sequence of peptides. Besides, the disruption of ordered structure of fibrils occurred upon further heating (6-20 h) due to extensive hydrolysis. These results would suggest that all subunits are involved in the fibrillation of β-conglycinin, following multiple steps including polypeptide hydrolysis, assembly to amyloid structure, and growth into macroscopic fibrils with a fibril shaving process.     

高压微射流压力对大豆蛋白-大豆多糖体系功能性质的影响

为提升大豆分离蛋白（soy protein isolate,SPI）的功能性质,该文引入大豆可溶性多糖（soybean soluble polysaccharides,SSPS）,构建大豆分离蛋白-大豆可溶性多糖体系（SPI-SSPS）,研究动态高压微射流（dynamic high-pressure microfluidization,DHPM）处理对SPI-SSPS功能特性的影响。分别采用0,60,100,140和180 MPa的 DHPM压力处理SPI-SSPS,探究不同压力对SPI-SSPS起泡特性、乳化特性、溶解性、粒度分布和表面疏水性的影响。结果表明,DHPM处理能提高SPI的溶解性和起泡特性,且SSPS的存在能显著提高DHPM对SPI功能性质的改善效果（P<0.05）。100和60 MPa的DHPM处理能使SPI-SSPS呈现较高的起泡能力和起泡稳定性,分别为未处理样品的1.2和2.4倍。140 MPa的DHPM处理使SPI-SSPS溶解性较强,为未处理样品的1.8倍。然而,DHPM处理会显著降低SPI-SSPS的乳化特性、粒径和表面疏水性（P<0.05）。随着处理压力的增加,SPI-SSPS的粒度和表面疏水性逐渐降低,在180MPa的DHPM处理下SPI-SSPS具有较小的粒径和较低的荧光强度。综上所述,DHPM结合SSPS改性技术可用于改善SPI的功能性质（如溶解性、起泡性）,促进SPI在食品工业的应用。该文的研究结果可为SPI的功能性质改性提供参考。     

Novel method for the production of pure glycinin from soybeans

A novel method for the purification of glycinin from soy meal is presented. The method is based on the isoelectric precipitation of glycinin by using carbon dioxide as a volatile precipitant. Gaseous CO(2) was pressurized into the protein solution, thus lowering the pH and initiating glycinin precipitation. Pressurization and, consequently, acidification were done in a slow and controlled manner, with the end point of pH 6.4. The acidity of the protein solution was well controlled via the pressure of gaseous CO(2). In this way simultaneous precipitation of other soybean proteins was prevented and very pure glycinin was obtained. Approximately 40% of the glycinin present in the protein solution was recovered with purity as high as 98%. The purification process was successfully performed on both small and large scales, without affecting glycinin purity.     

Assessment of soy genotype and processing method on quality of soybean tofu

Protein quality in six soybean varieties, based on subunit composition of their protein, was correlated with quality of the produced tofu. Also, protein changes due to a pilot plant processing method involving high temperature/pressure and commercial rennet as coagulant were assessed. In each soybean variety, glycinin (11S) and β-conglycinin (7S) as well as 11S/7S ratio significantly changed from beans to tofu. Between varieties, the 11S/7S protein ratio in seed indicated genotypic influence on tofu yield and gel hardness (r = 0.91 and r = 0.99, respectively; p < 0.05). Also, the 11S/7S ratio correlated with soymilk pH (r = 0.89, p < 0.05), leading to a relationship between soymilk pH with protein recovery and yield of tofu (r = 0.94 and r = 0.91, respectively; p < 0.05). The soybean β'-subunit of 7S protein negatively influenced tofu hardness (r = -0.91, p < 0.05). Seed protein composition and proportion of 7S protein subunits under the applied production method had an important role in defining tofu quality.     

Physicochemical modifications of high-pressure-treated soybean protein isolates

Changes induced by high pressure (HP) treatment (200-600 MPa) on soybean protein isolates (SPI) at pH 3 (SPI3) and pH 8 (SPI8) were analyzed. Changes in protein solubility, surface hydrophobicity (Ho), and free sulfhydryl content (SH(F)) were determined. Protein aggregation and denaturation and changes in secondary structure were also studied. An increase in protein Ho and aggregation, a reduction of free SH, and a partial unfolding of 7S and 11S fractions were observed in HP-treated SPI8. Changes in secondary structure were also detected, which led to a more disordered structure. HP-treated SPI3 was partially denatured and presented insoluble aggregates. A major molecular unfolding, a decrease of thermal stability, and an increase of protein solubility and Ho were also detected. At 400 and 600 MPa, a decrease of the SH(F) and a total denaturation were observed.