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.     

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.     

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.     

Effect of soy milk characteristics and cooking conditions on coagulant requirements for making filled tofu

The amount of coagulant added to soy milk is a critical factor for tofu-making; particularly it affects the textural properties of tofu. Earlier research indicated that the critical point of coagulant concentration (CPCC) is a characteristic parameter of soy milk and could be used as an effective indicator of optimal coagulant concentration (OCC) for making filled tofu. The objective of this study was to investigate the possible correlations between CPCC and the characteristics of soy milk made from various soybean samples and the effect of soy milk cooking and dilution conditions on CPCC. CPCC was determined by a titration method. Calcium chloride and magnesium chloride were used as coagulants. Soy milk characteristics including solid, protein, phytate, pH, titratable acidity, mineral content, and 11S/7S protein and these characteristics as affected by heating rate, heating time, and sequence of dilution and heating were studied. The results showed that the CPCC was significantly (p < 0.05) positively correlated with phytate content (grams per gram of protein), pH, and 7S protein content but negatively correlated with protein content, 11S protein content, 11S/7S ratio, titratable acidity, and original calcium content. Within the same soybean material, more proteins required more coagulant, but higher protein concentration during cooking resulted in less coagulant required by each gram of protein during coagulation. The CPCC decreased with increasing soy milk heating time or decreasing heating rate. The sequence of heating and diluting for preparing soy milk also had an effect on CPCC.     

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。     

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.     

Composition of proteins in okara as a byproduct in hydrothermal processing of soy milk

Protein quality, based on its subunit composition, in okara obtained as a byproduct during hydrothermal cooking of soy milk was assessed. The composition of 7S and 11S protein fractions was correlated with the physicochemical properties of protein in okara produced from six soybean varieties. The basic 7S globulin (Bg7S) and 11S protein were two main proteins in okara. Investigated soybean genotypes produced okara with mainly acidic A(5) and basic B(1,2,4) polypeptides of 11S proteins. Soybean 11S content was not an indicator of okara protein recovery or extractability. Of all tested relationships, extractable soluble protein content of okara was influenced only by soybean Bg7S (r = 0.86; p < 0.05) and its light subunit contents (r = 0.93; p < 0.05). Okara protein recovery depended on Bg7S heavy subunit content in soybeans (r = 0.81; p < 0.05). The high quantity of vegetable protein in okara (around 35%) and very high protein extractability (around 85%) qualify this byproduct for potential application in food preparation as a functional ingredient.     

Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract

Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.     

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.     

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.     

晋豆24~(60)Coγ射线诱变后代种子贮藏蛋白的研究

选用晋豆24及其60Coγ射线辐照诱变后代为试验材料,利用SDS-PAGE聚丙烯酰胺凝胶电泳研究了诱变后代大豆贮藏蛋白亚基11S和7S的组分含量、亚基组成和各亚基的含量及其变异规律。结果表明:大豆贮藏蛋白种质资源中,同一品种的不同亚基和不同品种的同一亚基间均有较大变异;11S球蛋白与7S球蛋白呈极显著负相关,与11S/7S比值呈极显著正相关;聚类分析根据11S/7S比值将诱变后代群体分为比值较高(平均为1.70)、居中(平均为1.25)和较低(平均为0.79)3类,为诱变育种选育优质专用大豆新品种提供了依据。     

Effect of water-to-bean ratio on the contents and compositions of isoflavones in tofu

The present study investigated the changes of the bioavailable isoflavones, including daidzin, genistin, daidzein, and genistein, during the making of tofu. The amount of extracted daidzin and genistin in soy milk increased with increasing water-to-bean ratios from 5 to 9 and reached the maximum level at the ratios of 9-11. On the other hand, the amount of extracted free isoflavones (daidzein and genistein) was not affected by the water-to-bean ratio at the range of 5-11, and their extracted amounts in soy milk were 2-4-fold those in raw soybean. It is suggested that these free isoflavones are mainly derived from daidzin, genistin, malonyldaidzin, and malonylgenistin through enzymatic hydrolysis during the making of soy milk. Tofu made with water-to-bean ratios of 9:1 and 10:1 had the maximal retentions of daidzin and genistin, which were due to the fine homogeneous network microstructure that is supposed to be more effectively retained through hydrophilic interaction with protein. On the contrary, the retained amount of free isoflavones decreased significantly as the water-to-bean ratio increased from 7 to 11, due to their weakening hydrophobic interaction with protein. In this study it was found that the homogeneous microstructure of tofu improved the retention of hydrophilic daidzin and genistin and that the increased amount of drained water does not significantly reduce their retention in the final tofu products as generally imagined.     

Quantification of the group B soyasaponins by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins alphag, betag, and betaa, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 micromol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 micromol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 micromol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.     

Simplified process for soybean glycinin and beta-conglycinin fractionation

A simplification of the pilot-plant scale modified Nagano method yielding two protein fractions, glycinin and beta-conglycinin, by pH adjustment and ultrafiltration membrane separation was developed and compared with our pilot-plant-scale modified Nagano procedure and with a soy protein isolate pilot-plant procedure as our reference process. Two protein fractions, glycinin and beta-conglycinin, were produced from our simplified process and compared to the three protein fractions, glycinin, beta-conglycinin, and an intermediate protein mixture, produced with the modified Nagano method. The pilot-plant yields of glycinin, beta-conglycinin, and intermediate mixture fractions from the modified Nagano method were 9.4, 10.3, and 4.8% [dry basis (db)], respectively. The yield of glycinin fraction of the simplified method was 9.7% (db), and it had a protein content and purity similar to those obtained with the modified Nagano method. The yield of the beta-conglycinin fraction was 19.6% (db), which was twice that of the modified Nagano process. The protein content of beta-conglycinin was 91.6% (db), and the purity was 62.6% of the protein content, which was 9% lower in purity than the modified Nagano method. Process optimization of the simplified method suggested the best operating conditions for the membrane filtration system were 20-25 psi inlet pressure and 200-250 L/min ultrafiltration recirculation speeds.     

Changes of astringent sensation of soy milk during tofu curd formation

The effect of isoflavone on soy milk and tofu astringency was investigated, and no consistency was found between an undesirable astringent taste and isoflavone contents. Isoflavone-enriched extract (approximately 39% isoflavones) showed no astringency. Soybean foods having high amounts of isoflavones showed less astringency. About 80% of isoflavones exist freely in both soy milk and tofu, but 55% of phytates (which play an important role in the formation of the tofu curd network) exist freely in the soy milk, and 6-13%, on the basis of coagulation, existed freely in the tofu curds. A 1% potassium phytate solution at pH 7 showed the very same astringency as soy milk; however, calcium phytate at the same concentration and pH showed no undesirable sensation. Thus, it is assumed that the astringent characteristics caused by phytic ions in soy milk are lost upon conversion of phytic ions to their insoluble salt forms during soy milk coagulation.     

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.     

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.     

Incorporation of soy milk lipid into protein coagulum by addition of calcium chloride.

Lipid holding in tofu (soybean curd) has such stability that it is not released even when it is cooked. The lipid in soy milk is incorporated into the tofu with coagulation of proteins on processing by the addition of a calcium salt. In this study, the incorporation of lipid into the coagulum was examined from the association with protein. The lipid in soy milk was obtained as the floating fraction by centrifugation. The floating fraction decreased by the addition of calcium chloride prior to forming a protein coagulum. When half of the protein coagulated, all of the floating fraction became inseparable. The protein in soy milk, then, was separated into particulate and soluble fractions by centrifugation. The decrease of the floating fraction with added calcium chloride was parallel to the coagulation of the particulate protein. The association of the floating fraction and the soluble protein occurred after the new particles formed from the soluble proteins. These results indicated that the lipid incorporation took place by the conjugation of the lipid and protein particles.     

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.