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.     

Protein recovery in soymilk and various soluble fractions as a function of genotype differences, changes during heating, and homogenization

Harovinton, a variety of tofu type soybean, and 11 derived null soybean genotypes lacking specific glycinin (11S) and beta-conglycinin (7S) protein subunits were investigated to determine whether changes in protein composition affected the protein recovery in soymilk and its soluble fractions after various centrifugation steps. As both heating and homogenization have a marked effect on the increase in protein solubility, the changes occurring during these processing steps were studied for each soybean genotype. Harovinton and 11S-null genotypes showed significantly higher protein yields than the other genotypes evaluated. Subunits of group I (A(1), A(2)) of glycinin had a negative impact on protein solubility in all treatments, but this effect was the greatest in unheated soymilk samples. Samples containing a high beta-conglycinin to glycinin ratio showed an effect of heating on the solubility of the protein, as beta-conglycinin subunits aggregate with heating. The presence of the alpha' subunit of beta-conglycinin aids in the recovery of protein in the supernatant prepared from lines containing group I (A(1,4) A(2)) glycinin. The results of this study will help determine which specific protein composition will confer an increased stability in soymilk and soymilk-derived products.     

Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines

Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the high-protein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of beta-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of beta-conglycinin and glycinin, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing beta-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin, beta-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin, beta-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of beta-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.     

Improved fractionation of glycinin and beta-conglycinin and partitioning of phytochemicals

Glycinin-rich and beta-conglycinin-rich products are prepared by soy protein fractionation. Physicochemical characteristics of these proteins affect their unique, important functionality in food systems and industrial applications. Soybean isoflavones and saponins are phytochemicals with potential health benefits. Objectives of this protein fractionation research were to (1) improve protein and phytochemical extraction from defatted soy flakes and recovery in product fractions and (2) evaluate phytochemical partitioning and profile changes during fractionation. Extraction environments (pH, ethanol concentration, temperature, and water-to-flake ratio) were each varied during bench-scale optimization. Optimized conditions of 45 degrees C and 10:1 water-to-flake ratio were compared with previous conditions of 20 degrees C and 15:1 water-to-flake ratio and a soy protein isolate process at pilot scale. Optimized conditions yielded more beta-conglycinin with higher isoflavone and saponin concentrations, but fraction purity was diminished by glycinin contamination. Bench-scale data demonstrated that increased phytochemical extraction did not translate into increased concentrations in product fractions.     

beta-Conglycinin and glycinin in high-protein soybean seeds

The agronomic performance and storage proteins of high seed protein lines of soybeans [Glycine max L. (Merr.)] were investigated to determine if the two major storage proteins, beta-conglycinin and glycinin, contribute to the increased protein content of high seed protein lines. Subunits of these two major storage proteins were estimated by scanning SDS-PAGE gels by scanning densitometry. The relative rankings of the lines with respect to seed size and protein content were not different between years in one environment over 5 years, but oil and total protein and oil contents and the ratio of protein to oil differed. The alpha', alpha, and beta subunits of beta-conglycinin were significantly higher in the high-protein lines except CX797-115, CX804-108, CX804-3, D81-8498, and NC-2-62. The acidic A(3) polypeptide of glycinin was significantly higher in high-protein lines except 76-48773, CX804-108, CX804-3, D81-8498, and NC-2-62, whereas the acidic polypeptides A(1,2,4) of glycinin were significantly higher in all of the high-protein lines. The basic polypeptides of glycinin were significantly higher in all high seed protein lines except D81-8259. In conclusion, high-protein lines appear to contain more beta-conglycinin and glycinin than normal-protein soybean lines, and the amounts of subunits and polypeptides differ among lines.     

Immunochemical studies on gastric and intestinal digestion of soybean glycinin and beta-conglycinin in vivo.

Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and beta-conglycinin in preruminant calves fed milk replacers containing a mixture of skim milk powder and antigenic heated soybean flour. In experiment 1, duodenal passage of immunoreactive beta-conglycinin lasted for a much longer time after the morning meal than that of glycinin. Western blotting revealed the early abomasal outflow of glycinin subunits that associated nearly intact basic polypeptides to partially degraded acidic polypeptides. Intact beta-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (M(r) = 21000) associated with partially digested acidic glycinin (7000 < M(r) < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. beta-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.     

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。     

Processing effect on soybean storage proteins and their relationship with tofu quality

Tofu was prepared from 13 soybean varieties according to three different methods (bench, pilot, and production methods). Different soybean varieties showed significant differences in storage protein composition (glycinin and beta-conglycinin). The beta-conglycinin (7S) and glycinin (11S) contents were 7.3-9.9 and 14.1-22.9% on the dry matter basis, respectively. The 11S/7S protein ratio varied from 1.64 to 2.51 among the varieties. Glycinin content and 11S/7S protein ratio of the 13 varieties did not change significantly from soy milk to tofu for the production and pilot methods. Soybean 11S/7S protein ratio positively correlated with the 11S/7S ratio of soy milk and tofu (0.57 < or = r < or = 0.83, p < or = 0.01). The correlation coefficient depended on the processing method. Processing method affected 7S and 11S protein contents of tofu and their contribution to tofu hardness, yield, and sensory quality. This may explain in part the contradictory findings of the relationships between storage proteins and tofu quality because processing methods differed in various studies.     

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 indigenous Nepalese soybean as a potential germplasm resource for improvement of protein in North American cultivars

Soybeans contain approximately 40% protein and 20% oil and represents an important source of protein in animal rations and human diets. Attempts are being made to increase further the overall protein content of soybeans by utilization of exotic germplasms. In this study, soybean cultivars from Nepal have been characterized and their potential as a germplasm resource for improvement of the protein content and quality of North American cultivars assessed. Soybean cultivars 'Sathia', 'Seti', 'Kavre', and 'Soida Chiny', indigenous to various regions of Nepal, contained 42-45% protein, which is significantly higher in comparison to that of the North American cultivar 'Williams 82' (39%). Fractionation of seed protein by high-resolution two-dimensional gel electrophoresis revealed differences in the protein profiles of these cultivars. Various isoelectric forms of glycinin and beta-conglycinin were identified by comparing the matrix-assisted laser desorption ionization time-of-flight mass fingerprinting data against the National Center for Biotechnology Information nonredundant database. Nepalese cultivar Sathia was distinct, lacking some isoelectric forms of acidic and basic glycinin subunits while expressing other unique forms. The contribution of these unique protein spots present in either Sathia or Williams 82 to the total protein content was quantified using scanning laser densitometry. Distinct restriction fragment length polymorphisms (RFLP) for group 1 glycinin genes were observed among the tested Nepalese genotypes, indicating sequence variation among the cultivars. Conversely, evaluation of RFLP for the genes encoding group 2 glycinins, beta-conglycinin, and Bowman-Birk proteinase inhibitors indicated a high degree of conservation in these genes. Determination of amino acid composition, a reflection of protein quality, indicated that the arginine content of the Nepalese soybeans ranged from 7.7 to 8.1%, which was 5-10% higher than the 7.4% expressed in Williams 82. Additionally, Karve and Seti contained significantly more cysteine than Williams 82. Nepalese high-protein soybeans having a desirable amino acid composition hold potential to increase the protein quality and diversity of North American cultivars.     

Pilot-Plant Wet-Milling Process for Producing Corn Gluten Meal

A pilot-plant wet-milling process was specially used to produce corn gluten meal (CGM) with a relatively high protein content (49.8% from Pioneer 3394 and 53.7% from Wilson D110). The protein content of the CGM obtained from a starch table in our pilot-plant procedure was similar to the results obtained by using the hydrocyclone method. Wilson D110 has a higher protein content in its corn kernels, producing higher yield and protein content gluten than the Pioneer 3394, which has lower protein content in the corn. The effects of drying method and temperature on the color of CGM were also discussed.     

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.     

Comparison of Laboratory and Pilot-Plant Corn Wet-Milling Procedures

One waxy and three regular yellow dent corn hybrids were wet milled by using two scales of laboratory procedures (modified 100-g and 1-kg) and a pilot-plant procedure (10-kg). The modified 100-g and 1-kg laboratory procedures gave similar yields of wet-milling fractions. Starch yields and recoveries were significantly lower for the pilot-plant procedure, whereas gluten and fiber yields were greater because of their high contents of unrecovered starch. Protein contents of the starches obtained by all three procedures were within commercially acceptable limits (<0.50% db for normal dent corn and <0.30% for waxy corn). Rankings for starch yields and starch recoveries for the four hybrids, having very different physical and compositional properties, were the same for all three procedures. The harder the grain, the lower the yield and recovery of starch. Least significant differences (P < 0.05) for starch yield were 0.8% for the modified 100-g procedure, 1.2% for the 1-kg procedure, and 2.0% for the pilot-plant procedure.     

Dissolution behavior of soy proteins and effect of initial concentration

The solution concentration profiles of soy protein and its components, glycinin and beta-conglycinin, as a function of pH and initial concentration have been measured. The concentration profiles generally followed a U-shaped trend with pH, with a minimum at around pH 4-5. Dissolution concentration unexpectedly increased with the initial concentration of the solution, with the increase being approximately proportional to the increase in initial concentration. The reasons for this are not clear. For the initial concentrations studied, beta-conglycinin is undersaturated between pH 5 and 7 and remains in solution, while glycinin becomes supersaturated in the same pH range and precipitates. Therefore separation of the two proteins can be achieved.     

Yield of Starch and By-Products in the Processing of Different Varieties of Wrinkled Peas on a Pilot Scale

A recently developed pilot-scale process for the extraction of starch and by-products from wrinkled peas was used to determine the attainable yield and purity of starch and by-products from different varieties of these peas. It was shown that the starch yield mainly depends on the removal of the starch from the fibers. From the pilot scale experiments, it could be concluded that, for an industrial process, the optimum values of the total starch yield of wrinkled peas and the purity of the starch in relation to the starch content of the starch fraction would be ~80 and 98%, respectively. The best possible value that can be achieved for mechanical separation of the protein from the starch is in the range of 1.5% protein content of the starch fraction. The protein content can be adjusted to values ≤0.5% by alkali extraction of the starch fraction. Similar positive results cannot be achieved for the other fractions. In addition, the water flow balance in the pilot plant demonstrated that the process water flows can only be limited to an economically feasible size when applying membrane filter technology.     

Improved storage stability of model infant formula by whey peptides fractions

The purpose of this study was to evaluate the shelf-life stability (6 months) of model infant formula with whey protein hydrolysates or peptidic fractions as carrageenan replacers. Whey protein hydrolysates were prepared with trypsin and followed by ultrafiltration of the hydrolyzed mixture, and peptidic fractions were isolated from the ultrafiltered tryptic hydrolysate by anion- or cation-exchange chromatography. The stability of the model infant formula was evaluated using a stratification method based on fat content differences between the top and bottom strata of the samples. With protein hydrolysate-based formulations, the creaming rate of the fat in the product was slightly higher than in the standard formulation (with carrageenan), which is indicative of lower storage stability. The addition of cationic fractions to model infant formula also resulted in lower product stability, whereas the fat creaming rate was retarded in anionic fraction based formulations. The physicochemical characteristics of certain peptides combined with the reported high emulsifying properties of peptidic sequences found within these fractions may account for their ability to act as carrageenan replacers.     

Positional effect on protein and oil content and composition of soybeans

Soybean (Glycine max [L.] Merr.) protein and oil qualities, with respect to monogastric nutrition, have been linked to the relative abundance of specific protein subunits and fatty acids, respectively. An analysis of field-grown soybean seeds by near-infrared spectroscopy revealed significant differences in their protein and oil contents as a function of nodal position. Seed proteins from the plant apex were high in protein and low in oil content, while those from the basal region exhibited an opposite pattern of accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total seed proteins revealed that the beta-subunit of beta-conglycinin content was 4-fold higher in seeds from the apical nodes than in seeds from basal nodes. The glycinin A3 polypeptide content gradually increased in successively lower nodes from the top of the plant. Its accumulation was drastically reduced when nitrogen was applied at specific growth stages. Exogenous nitrogen did not alter the pattern of beta-subunit accumulation, but accrual of the acidic and basic polypeptides of glycinin was diminished. The remaining seed storage protein components were not influenced by nodal position or nitrogen application. Gas chromatographic analysis of fatty acids indicated that only oleic (18:0) and linoleic (18:2) acids showed variability in accumulation at different nodes. Neither the abundance nor the distribution of the fatty acids was altered by nitrogen application.     

超滤膜分离纯化花生壳中水溶性膳食纤维

为探索花生壳中水溶性膳食纤维(SDF)的有效分离纯化方法和效果,选择适宜的膜组件,采用不同截留分子量的超滤膜以及优化膜分离过程的操作条件,对SDF提取液进行超滤分离,试验结果表明:选用PS-30聚砜膜,在压力为0.08MPa、料液比为1:75g/mL、温度为30℃时,分离纯化效果最为显著。其中,膜通量达到127.2L/(m2·h)、SDF的得率达到67.56%,非淀粉多糖(NSP)的质量分数由49.85%提高到92.36%,蛋白质量分数从5.53%降到0.92%。与传统的提取法比较,超滤膜分离纯化花生壳水溶性膳食纤维具有生产周期短,成本低,产品纯度高等优点。     

Beta-conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro

Obesity is a worldwide health concern because it is a well-recognized predictor of premature mortality. The objective was to identify soybean varieties that have improved potential to inhibit fat accumulation in adipocytes by testing the effects of soy hydrolysates having a range of protein subunit compositions on lipid accumulation and adiponectin expression in 3T3-L1 adipocytes. The results showed that differences in the protein distribution of 15 soy genotypes led to different potentials for the reduction of fat accumulation. The inhibition of lipid accumulation of soy alcalase hydrolysates in 3T3-L1 adipocytes ranged from 29 to 46%. Soy hydrolysates made from genotypes with 45.3 +/- 3.3% of total protein as beta-conglycinin, on average, showed significantly higher inhibition of lipid accumulation compared to those with 24.7 +/- 1.5% of extracted total protein as beta-conglycinin. Moreover, after in vitro simulated digestion with pepsin-pancreatin of the soy alcalase hydrolysates, 86% of the original activity remained. Adiponectin expression was induced in 3T3-L1 adipocytes treated with 15 soy hydrolysates up to 2.49- and 2.63-fold for high and low molecular weight adiponectin, respectively. The inhibition of lipid accumulation calculated from a partial least squares (PLS) analysis model correlated well with experimental data (R(2) = 0.91). In conclusion, it was feasible to differentiate soy varieties on the basis of the potential of their proteins to reduce fat accumulation using a statistical model and a cell-based assay in vitro. Furthermore, beta-conglycinin embeds more peptides than glycinin subunits that inhibit lipid accumulation and induce adiponectin in 3T3-L1 adipocytes. Therefore, soy ingredients containing beta-conglycinin may be important food components for the control of lipid accumulation in adipose tissue.     

Wet Fractionation of Garbanzo Bean and Pea Flours

Garbanzo bean and pea flours originating from the central part of the cotyledons were higher in starch but lower in protein and fiber than those from the outer layer of the cotyledon. These flours were fractionated by the wet process into prime starch, tailing starch, and solubles. The patented wet-fractionation method was successfully modified to reduce the total amount of water during the process. The modified process produced comparable yield and purity of separated fractions. Under the recycling water method, the yields of prime starch were 46.7, 33.6, and 41.1%, respectively, in garbanzo bean and two smooth pea cultivars (Latah and SS Alaska). Isolated prime starches were <0.4% in protein and <0.19% in ash, indicating high purity.