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.     

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.     

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.     

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.     

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.     

A circular dichroism and fluorescence spectrometric assessment of effects of selected chemical denaturants on soybean (Glycine max L.) storage proteins glycinin (11S) and beta-conglycinin (7S)

Soybean glycinin (11S) and beta-conglycinin (7S) were subjected to select chemical treatments at various concentrations and resulting changes in protein structures were investigated by circular dichroism (CD) and fluorescence spectrometry. Fluorescence quenching results indicated that urea >/=3 M caused significant unfolding of 11S, but not that of 7S. GuHCl was more effective than urea in denaturation of 11S. A two-step transition in 11S structure was observed with a possible existence of a folding intermediate at 2.5 M GuHCl. Sodium dodecyl sulfate (SDS) measurably altered secondary and tertiary structures of 11S and 7S below SDS critical micellar concentration (CMC), possibly due to formation of mixed peptide-SDS micelles. SDS treatment increased alpha-helical and unordered structures of both proteins at the expense of beta-sheet structure. NaCl and CaCl 2 caused a significant decrease in fluorescence intensity without shifting emission lambda max. Exposure of 7S and 11S to NaSCN respectively at >/=0.3 and >/=0.6 M NaSCN caused a significant increase in fluorescence intensity measured at the corresponding lambda max of the protein. beta-Mercaptoethanol (beta-ME), N-ethylmaleimide (NEM), and phytic acid caused variable red shifts, 2.5-4 nm, in the emission lambda max.     

Inhibitory action of soybean beta-conglycinin hydrolysates on Salmonella typhimurium translocation in Caco-2 epithelial cell monolayers

Soybean protein hydrolysates are widely used as functional foods as they have antioxidative properties able to enhance immune responses in humans. The alcalase enzymatic hydrolysates of beta-conglycinin were fractionated by ultrafiltration, and two main fractions, SP1 (<10 kDa) and SP2 (10-20 kDa), were obtained. The effects of these two fractions on the growth, development of epithelial cells, and formation of intercellular tight junctions were tested on an in vitro Caco-2 cell culture system. The inhibitory effects of SP1 and SP2 on the penetration of Salmonella typhimurium into Caco-2 epithelial cells were also examined. The results showed that the addition of >0.05 g/L of SP2 improved epithelial cell growth and that a concentration of 0.5 g/L of SP2 increased intercellular tight junction formation, which resulted in increased of transepithelial monolayer resistance (TER) values. Moreover, a lower S. typhimurium count compared to control was obtained when Caco-2 cells were grown in 0.05 and 0.5 g/L of SP2. These results show that beta-conglycinin hydrolysates play an important role in resisting S. typhimurium penetration into intestinal epithelial cells and that high molecular mass peptides (10-20 kDa) were more effective overall than low molecular mass peptides.     

Changes in characters of soybean glycinin groups I, IIa, and IIb caused by heating

Soybean glycinin groups I, IIa, and IIb were purified from soybeans composed of only glycinin groups I, IIa, and IIb, respectively. When these protein solutions were heated, the amount of the particulate protein formed in these solutions was greatest in the order of groups IIa, IIb, and I. The protein solubilities decreased upon the addition of magnesium chloride in the order of groups IIa, IIb, and I. It was determined by differential scanning calorimetry analysis that the denaturation temperatures of groups I, IIa, and IIb were 92.8, 96.0, and 97.9 degrees C, and that the enthalpies of their transitions were 24.2, 27.4, and 28.1 J g(-)(1), respectively. The alpha-helix rates of groups I, IIa, and IIb in aqueous solution were analyzed by circular dichroism and were 19, 16, and 15%, respectively. The beta-sheet rates of groups I, IIa, and IIb were 44, 38, and 39%, respectively. In all group proteins, the alpha-helix rates were decreased by heating and the beta-sheet rates were increased. The surface hydrophobicity of these group proteins increased as a result of heating, and those of groups IIa and IIb were larger than that of group I. The surface hydrophobicity of these protein groups increased by heating, and those of groups IIa and IIb were larger than that of group I and beta-conglycinin. Breaking stress of curds prepared from these group proteins containing more than 1 of beta-conglycinin ration showed similar values, but the order of those containing less than 1 in strength was groups I, IIb and IIa. These results suggest that the increase of particulate contents and the curd formation are related to the increase of surface hydrophobicity by heating.     

Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.     

Isolation and enzymatic digestion of body complex of soybean seed

The body complex of the soybean seed (BCSS) was isolated from the single cells (27.2%) by a sequential procedure of autoclaving with water, cellulase digestion for the primary cell wall, pectinase digestion for the secondary cell wall, and defatting with hexane washing. Its characteristics were then investigated. The defatted BCSS (DBCSS) consisted of protein (76.5%) and mannose-rich carbohydrates (3.2%). Screening of the food-processing protease for the digestion of DBCSS was carried out, and a kind of alkaline protease was selected. The inner protein of DBCSS was easily extracted with 0.1 M sodium carbonate buffer, pH 10, and the insoluble shell of the body complex (SDBCSS) was left. SDBCSS consisted of hydrophobic amino acid-rich protein. SDBCSS was easily digested by the selected alkaline protease. SDBCSS was dissolved by boiling with sodium dodecyl sulfate-mercaptoethanol, and it was found to consist of a protein of approximately 3 kDa. The high enzymatic digestion including the selected protease for soybean seed and defatted soybean meal was carried out; both were extracted and digested with a yield of >99.5%. The final indigestible residue was found as paired hexagonal and filamentous organs of the soybean cells.     

Ionic strength and pH-induced changes in the immunoreactivity of purified soybean glycinin and its relation to protein molecular structure

This study examined the immunogenic response of glycinin under varying conditions of pH and ionic strength using enzyme-linked immunosorbent assay. Differential scanning calorimetric (DSC) analysis and Fourier transform infrared spectroscopy (FTIR) were used to investigate the conformational changes induced as a result of these conditions, and the correlation with the changes observed in glycinin immunoreactivity were determined. A highly purified glycinin obtained by isoelectric precipitation followed by native preparative continuous flow electrophoresis was used for these studies. Purity was confirmed by two-dimensional-polyacrylamide gel electrophoresis and mass spectroscopy. DSC and FTIR results suggest that glycinin immunoreactivity is affected by changes in the tertiary and secondary packing of the protein, when flexibility, stability, and accessibility of certain substructures are modified. Aggregation and/or increased compactness of glycinin subcomponents could have potentially prevented epitopes from reacting with the IgG antibodies.     

Effects of Ca2+ and sulfhydryl reductant on the polymerization of soybean glycinin catalyzed by mammalian and microbial transglutaminases

Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin). The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated. The conformational change of glycinin was probed by spectral methods. The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively. Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation. It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations. In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.     

Rapid gastric fluid digestion and biochemical characterization of engineered proteins enriched in essential amino acids.

The barley high lysine (BHL) proteins are nutritionally enhanced derivatives of barley chymotrypsin inhibitor-2 (CI-2). A compactly folded new CI-2 derivative, BHL9, was engineered with the highest content of threonine, tryptophan, and isoleucine yet achieved in this protein family (15.1, 9.4, and 12.1 wt %, respectively). BHL9 had an unfolding midpoint of 5.5 M guanidinium chloride, significantly greater than values for wild type (3.9 M) or for the previously most stable BHL protein, BHL8 (3.6 M). BHL9 and all other derivatives were digested within 15 s in simulated gastric fluid (SGF), suggesting nutritional availability upon ingestion. Denaturation of the proteins in SGF minus pepsin was revealed by changes in their fluorescence emission spectra and/or far UV circular dichroism spectra. The proteins lack homology to known allergens. Significantly, the BHL8 and BHL9 proteins were stable to proteases at pH 7.5 or 8.0, attesting to their potential for high expression in plants.     

Electron spin resonance studies of free radicals in gamma-irradiated soybean paste.

Free radicals in gamma-irradiated soybean paste were investigated by electron spin resonance (ESR) spectroscopy to determine the effect of temperature (77-296 K) and moisture content (1-54%) of samples irradiated at high dose (1-40 kGy). The samples were kept in liquid nitrogen (77 K) during irradiation and subsequent ESR measurements. The spectra shown at 77 K consisted of the hydrogen atom lines at low and high field and complicated symmetric spectrum. By increasing the microwave power, the line shape of ESR spectra altered, which indicated the detection of different paramagnetic centers at different microwave powers. In saturation curves, it was possible to select four types of spectra components which were different in their relaxation times. By the different irradiation doses, the change in free radical concentration showed a curvilinearly increasing relationship with irradiation dose in wet samples, whereas a proportional relationship was observed with dried samples. This might indicate that the indirect process of free radical formation was involved with the existence of free water radicals in the wet samples.     

Quantification and sensory studies of character impact odorants of different soybean lecithins.

Fifty-four potent odorants in standardized, hydrolyzed, and deoiled and hydrolyzed soybean lecithins were quantified by high-resolution gas chromatography/mass spectrometry (HRGC/MS). The characterization of their aroma impact was performed by calculation of nasal (n) and retronasal (r) odor activity values (OAVs). For this, the nasal and retronasal recognition thresholds of 18 odor-active compounds were determined in vegetable oil. The following compounds showed the highest nOAVs: 2,3-diethyl-5-methylpyrazine, methylpropanal, acetic acid, pentanoic acid, 2-ethyl-3,5-dimethylpyrazine, pentylpyridine, (Z)-1,5-octadien-3-one, 2-methylbutanal, and beta-damascenone. In addition to the compounds above, 1-octen-3-one, 1-nonen-3-one, and 3-methyl-2,4-nonandione showed potent rOAVs. The results of quantification and OAV calculation were confirmed by a model mixture of 25 impact odorants, which yielded a highly similar sensory profile to that of the original soybean lecithin. The sensory importance of pyrazines and free acids increased through enzymatic hydrolysis and decreased by the process of deoiling. The impact of unsaturated ketones on the lecithin aroma was not changed by either process.     

Effect of some phenolic compounds and beverages on pepsin activity during simulated gastric digestion

The effect of some polyphenols (resveratrol, catechin, epigallocatechin-3-gallate, and quercetin) and beverages (red wine and green tea) on the enzymatic activity of pepsin during the digestion of three different substrates (pork meat, insoluble azocasein, and denatured hemoglobin) has been investigated. The tested polyphenols and beverages increase the initial velocity of the reaction, and the activating effect is concentration dependent. The order of effectiveness of polyphenols in increasing the initial velocity of the reaction is resveratrol > or = quercetin > epigallocatechin-3-gallate > catechin. The kinetic data obtained with soluble denatured hemoglobin show that the K(m) for the substrate is not changed, whereas the V(max) of the reaction is increased. Pepsin activity follows a simple Michaelis-Menten kinetic suggesting that k(3) is increased by polyphenols. To the authors' knowledge, the present study is the first demonstration that some polyphenols and related beverages are able to enhance the enzymatic activity of pepsin.     

Identification, characterization, epitope mapping, and three-dimensional modeling of the alpha-subunit of beta-conglycinin of soybean, a potential allergen for young pigs

Soybean meal (SBM), the major byproduct of soybean oil extraction, is the main protein source for swine diets globally. In the United States, 8.6 million metric tons of SBM was used in swine rations in 2004. The pathological effect and immunological response of SBM feeding have been demonstrated in swine. In this study, we have utilized plasma collected from piglet feed with SBM in immunoblot analysis to detect proteins that elicited antigenic responses. We have identified soybean beta-conglycinin alpha-subunit as being a potential allergen for young piglets. Characterization of this protein indicated that deglycosylation and pepsin digestion did not eliminate immunoreactivity of this protein. Epitope mapping utilizing planar cellulose supports technology (SPOT) showed that three peptides spanning amino acids S185-R231 were critical for the allergenicity. A computer-generated three-dimensional structure model of the alpha-subunit of beta-conglycinin indicated that the antigenic epitopes were located on the surface of the protein. Information from this study may assist in the construction of recombinant nonallergenic soybean protein useable for both immunotherapy and the potential production of hypoallergenic soybean plants.     

Antioxidant properties of soybean isoflavone extract and tofu in vitro and in vivo

Isoflavones in soybean were extracted in the crude form using 80% food-grade ethanol at 80 degrees C for 6 h and followed by concentration and dehydration. The soy extract contained isoflavones primarily in the forms of glucosides. In vitro antioxidant activities of the soy extract containing 20-500 ppm isoflavones were conducted using a Rancimat method. The results showed that soy isoflavone extract had strong in vitro antioxidant activity. There was a dose-dependent response for the in vitro antioxidant activity at the lower concentrations but not at the higher concentrations. In vivo antioxidant property was determined by measuring the antioxidant enzymes, superoxide dismutase, and catalase in various organs of rats that were fed with diets containing partially oxidized oil and various levels of isoflavones for up to 24 weeks. Neither short-term (8 weeks) feeding nor low isoflavone content (50 ppm) induced changes in superoxide dismutase or catalase activities in rats. Only diets containing high isoflavone contents (150 and 250 ppm) showed obvious elevated enzymatic levels in various organs. In addition, a laboratory-prepared tofu containing approximately 50 ppm isoflavones had better effects than the soy extract with the 250 ppm isoflavone group, which indicated that molecules other than isoflavones may have a synergistic effect on in vivo antioxidant enzyme inductions of tofu.