Glycinin Subunits Research Articles

Characteristics of soy films as affected by transglutaminase cross-linking and inclusions of pectin and protein enhancers.

Previously, we showed that water extract (soymilk, except pH was increased to 8 from 6.5) of whole soybean could be used directly as a raw material for producing edible soy films by deposition of the film-forming solution (soy extract with enhancers). However, the strength of such soy films needed improvement because they were weak. The purpose of this study was to investigate how transglutaminase (TG) cross-linking reactions and film enhancers, including pectin (low- and high-methoxyl pectin), whey protein isolate (WPI), and soy protein isolate (SPI), improve the physical properties of soy films. Soy films prepared with TG had tensile strength (TS) of 3.01MPa and puncture strength (PS) of 0.78MPa, which were higher by as much as 51% and 30% than that of soy films without TG treatment, respectively. Pectin showed significant effects on the mechanical properties of TG-added soy films in terms of TS, PS, and % elongation. On the other hand, only TS and PS were increased by the addition of WPI or SPI. Heat curing had a significant effect on soy film's physical properties. TG treatment significantly reduced film solubility when soaked in water and various levels of acid (vinegar) and base (baking soda) solutions. Under the experimental conditions of 35 unit TG and 28min of reaction, the degrees of cross-linking were evidenced by the disappearance of individual protein subunits, except the basic subunit of glycinin, and the reduction of 21% of lysine residues of the proteins. HIGHLIGHTS: Edible soy films were made with transglutaminase and about 21% lysine cross-linked. The mechanical strength of soy films was increased by incorporating film enhancers. Transglutaminase enhanced the mechanical properties of soy films.

Simultaneous detection of glycinin and β-conglycinin in processed soybean products by high-performance liquid chromatography-tandem mass spectrometry with stable isotope-labeled standard peptides

Glycinin and β-conglycinin are the two main allergic proteins in soybean. Due to their complex structures and lack of protein standards, it is difficult to achieve quantitative determination of these proteins in soybeans. In this study, an HPLC-MS/MS method was developed for the simultaneous determination of five subunits of glycinin (G1, G2, G3, G4, and G5) and three subunits of β-conglycinin (α, α', and β) in processed soybean products based on 8 specific peptides and their stable isotope-labeled peptides. Here, each specific peptide was derived from one of the above 8 subunits. When soy protein was extracted and digested with trypsin, 8 specific peptides, and corresponding stable isotope-labeled peptides were analyzed by HPLC-MS/MS. The linear range for the specific peptides was between 3.2 and 1000 ng/mL (R2 > 0.9955). The recoveries of added peptides ranged from 83.4% to 117.8%, and the intra-day precisions (% CV) were below 17.4%. The limit of quantification of each subunit of glycinin and β-conglycinin in processed soybean products (in terms of protein amount) was between 15.1 and 156.1 g/g. This method was successfully applied to the analysis of 8 subunits of glycinin and β-conglycinin in 68 different processed soybean products, which provides technical support for processed product quality evaluation and monitoring soybean processing technology.

Interaction of mulberry anthocyanins with soybean protein isolate: effect on the stability of anthocyanins and protein in vitro digestion characteristics

SummaryThe interactions of anthocyanins and proteins might mutually influence each other on the physicochemical and functional properties. In this study, the interaction mechanism of soybean protein isolate (SPI) with mulberry anthocyanins (MA), and its effect on the thermal stability of anthocyanins and protein subunits’ digestibility were investigated through multiple spectroscopies and in vitro gastrointestinal models. Results showed that cyanidin‐3‐O‐glucoside (C3G), the main anthocyanin monomer in MA, could bind to SPI through hydrophobic interactions, resulting in static fluorescence quenching of SPI. The secondary structure of SPI changed by binding to C3G, with an increase of β‐sheet and a decrease of α‐helix and random coil. The formation of the SPI‐MA complexes improved the thermal stability of MA at 353 K, while, no significant protection occurred during heating at 368 K. Complexation with MA promoted the digestibility of SPI by pepsin, especially the α′ and α subunits of β‐conglycinin and the basic subunit of glycinin, and slightly delayed the digestibility of SPI under intestinal fluid. These results provide an in‐depth understanding of the influence of the protein‐anthocyanin interactions on the stability of anthocyanins and protein digestibility.

Effects of Using Soybean Protein Emulsion as a Meat Substitute for Chicken Breast on Physicochemical Properties of Vienna Sausage.

The aim of this study is to determine the effects of using emulsion manufactured with soybeans (ES) to substitute chicken breast in Vienna sausages. Four types of Vienna sausages (S1: 10% ES and 50% chicken, S2: 20% ES and 40% chicken, S3: 30% ES and 30% chicken, and S4: 40% ES and 20% chicken) for this study were made. The pH, color, proximate composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), microphotographs, cooking yields, and texture profile analysis of sausages were examined. The pH value of uncooked and cooked sausages increased significantly with increasing ES content (p<0.05). The crude protein contents of S2, S3, and S4 were significantly higher than that of the control (p<0.05). Furthermore, the SDS-PAGE results showed that α-conglycinin, β-conglycinin, and the acidic subunit of glycinin all increased with increasing ES content. Microphotographs revealed that increasing the ES content decreased the size of fat globules. The cooking yields of samples increased significantly with increasing ES content (p<0.05). The hardness values of ES treated samples were significantly lower than that of the control (p<0.05). Therefore, 30% substitute of chicken breast with ES can improve the quality and structure of Vienna sausage, without inducing critical defects.

Improvement of the protein quality and degradation of allergens in soybean meal by combination fermentation and enzymatic hydrolysis

In this study, we determined the effect of short-time fermentation-assisted enzymatic hydrolysis on the improvement of protein quality and reduction of potential antigenicity in soybean meal (SM). SM was fermented by using Lactobacillus plantarum, Lactobacillus casei, Bacillus subtilis, Bacillus licheniformis, or Aspergillus oryzae for 24 h, and then, each of the fermented SM was hydrolyzed with Alcalase for 15 min. The color, SDS-PAGE, degree of hydrolysis, molecular weight distribution, and amino acid profile of SM were evaluated. In addition, the potential antigenicity of SM was also assessed by ELISA. Results showed that fermentation-assisted with Alcalase hydrolysis could effectively decrease the antigenicity of β-conglycinin and glycinin in SM, exhibited higher degree of hydrolysis and loss of intensities in several major bands, especially those of α′ and α subunits of β-conglycinin and acidic subunit of glycinin, as indicated by SDS-PAGE. Increased peptide (<10 kDa) content was observed in the final fermented soybean meal. Simultaneously, most of the total amino acid increased significantly (p < 0.05) and only few of them suffered a decrease depending on the type of microorganisms. It was also found that Bacillus subtilis could be more favorable than other microorganisms for developing hypoallergenic soybean products.

The role of soy protein degradation caused by spoilage Bacillus amyloliquefaciens in texture deterioration of yuba, a soy product

Bacillus spp. are the dominant spoilage organisms in a rehydrated non-fermented soy product, Sichuan yuba in China. In this study, we used two spoilage Bacillus amyloliquefaciens strains DY1a and DY1b to evaluate their destructive capability on rehydrated Sichuan yuba at 25 °C. Compared with the uninoculated soy product, the texture profile of the inoculated samples showed that the hardness, springiness, chewiness, and cohesiveness decreased significantly. Scanning electron microscope (SEM) revealed that the surface of bacteria-inoculated soy product was scattered with deep holes and loose film network structure. The subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the α and α’ subunit of 7S globulin and the acid subunit of 11S glycinin were hydrolyzed by Bacillus during storage. In addition, disulfide bond contents in inoculated samples decreased by 38.7–27.5%. Protein secondary structure characterized using Fourier transform infrared spectroscopy (FTIR) revealed that spoilage Bacillus inoculation reduced the β-sheet by 13.02–14.11%, and increased the β-turn by 15.55–16.00%. Consequently, the texture deterioration of Sichuan yuba was associated with soy protein degradation caused by B. amyloliquefaciens.

Physicochemical properties of proteins extracted from four new Tennessee soybean lines

Physicochemical properties of soy proteins extracted from check soybean cultivar 5601T and four new Tennessee soybean lines TN03-349, TN04-5321, TN08-002, and TN08-015 were compared. The protein extracted from 5601T had the lowest content of total protein and the lowest denaturation temperature and enthalpy (P < 0.05). The lowest solubility was observed at pH 5.0 for 5601T, TN04-5321, and TN03-349, while it was at pH 6.0 for the other two, which agreed with zeta-potential data. TN04-5321 and TN08-002 extract powder had a higher content of total protein than the other three (P < 0.05). The TN04-5321 extract was absent in an acidic glycinin subunit which may be the cause of its best gelation properties. Despite difficulties in correlating structure and functionality, it was noted that the gel formed from TN04-5321 with CaCl2 at pH 7 was 6 times stronger than that of 5601T, which may be significant to bean curd products.

The separation and identification of the residual antigenic fragments in soy protein hydrolysates.

Soybean is one of the major food allergens. In this study, soy protein isolate was hydrolyzed by Neutrase and Flavourzyme. The hydrolysates were separated by ultrafiltration and ion-exchange chromatography. The antigenicity of proteins was determined by indirect competitive ELISA. The molecular weight distribution was characterized by SDS-PAGE. The amino acid sequence of chromatography fractions was analyzed by LC-MS. The results showed that proteins with >50kDa in hydrolysates had the highest antigenicity and were further separated into F1 -F5 fragments by ion-exchange chromatography. Fragment F4 , which was the most antigenic, was analyzed by LC-MS. The results of mass spectrometry showed that most of the peptides that contained antigen epitopes in chromatography fraction F4 belonged to glycinin subunits. The antigenicity of soy protein was reduced by enzymatic hydrolysis, but glycinin showed resistance to enzymatic hydrolysis. PRACTICAL APPLICATIONS: The identification of residual antigenicity in soy protein hydrolysates by LC-MS provides important information on the resistance mechanism of enzymatic hydrolysis of soybean protein allergens. In addition, the efficient separation of soy protein hydrolysates could be beneficial for developing low-allergenic soybean products.

Allergenicity reduction and rheology property of Lactobacillus-fermented soymilk.

This study aimed to investigate the reduction in the potential allergenicity of soymilk, and its rheological properties, after fermentation with Lactobacillus. Soymilk (SM) was fermented with Lactobacillus brevis and Lactobacillus sp. The molecular weight of fermented soymilk (FSM) was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the potential allergenicity of FSM was analyzed using enzyme-linked immunosorbent assay in vitro and the BALB/c mouse model to detect changes in histamine, mouse mast cell protease-1 (mMCP-1), allergen-specific IgG/IgE, and cytokine levels and histomorphology of jejunum in vivo. The SDS-PAGE and enzyme linked immunosorbent assay (ELISA) showed that allergens of soybean (β-conglycinin and acidic subunit of glycinin) were almost degraded, and their immunoreactivity was decreased. In the BALB/c mouse model, the FSM group did not show anaphylactic shock symptoms compared with the SM group. Moreover, a tendency toward decreased serum allergen-specific IgG/IgE levels, plasma histamine levels, and mMCP-1 concentrations was observed in the FSM group. Furthermore, Th2-related cytokines were decreased, while IFN-γ production increased in spleen cell cultures. The intestinal villus was slightly damaged after the challenge. All these findings indicated that the Th1/Th2 balance in the FSM group shifted toward a Th1 response, ultimately reducing the potential allergenicity of FSM. Rheological assessment suggested that FSM has good viscous and pseudoplastic properties. Fermentation might be a promising method for producing tasty, hypoallergenic soybean products. © 2019 Society of Chemical Industry.

A vacuolar sorting receptor-independent sorting mechanism for storage vacuoles in soybean seeds

The seed storage proteins of soybean (Glycine max) are composed mainly of glycinin (11S globulin) and β-conglycinin (7S globulin). The subunits of glycinin (A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3) are synthesized as a single polypeptide precursor. These precursors are assembled into trimers with a random combination of subunits in the endoplasmic reticulum, and are sorted to the protein storage vacuoles. Proteins destined for transport to protein storage vacuoles possess a vacuolar sorting determinant, and in this regard, the A1aB1b subunit contains a C-terminal peptide that is sufficient for its sorting to protein storage vacuoles. The A3B4 subunit, however, lacks a corresponding C-terminal sorting determinant. In this study, we found that, unlike the A1aB1b subunit, the A3B4 subunit does not bind to previously reported vacuolar sorting receptors. Despite this difference, we observed that the A3B4 subunit is sorted to protein storage vacuoles in a transgenic soybean line expressing the A3B4 subunit of glycinin. These results indicate that a protein storage vacuolar sorting mechanism that functions independently of the known vacuolar sorting receptors in seeds might be present in soybean seeds.

Preparation and characterisation of glyceollin‐enriched soya bean protein using solid‐state fermentation

SummaryGlyceollins, stress‐induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid‐state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin‐enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β‐conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient‐enhanced SPI as novel functional ingredients applied in food industry.

Insight into the gastro-duodenal digestion resistance of soybean proteins and potential implications for residual immunogenicity.

Soy is an important component of the human diet thanks to its nutritional value and high protein content; however, it also represents a risk for allergenic consumers due to its potential to trigger adverse reactions in sensitized individuals. The putative correlation between immunoreactivity and resistance to the human gastrointestinal (GI) digestion has drawn attention to investigating soybean proteins digestibility. In this work, we provided further insights into this field by performing in vitro simulated GI digestion experiments directly on ground soybean seeds, to provide more realistic results obtained from the digestion of the whole food matrix. Soybean digestion products were analyzed by SDS-PAGE followed by untargeted HPLC-MS/MS analysis and the final data were software treated to enable protein/peptide identification. The latter allowed monitoring the proteolytic degradation of the main soybean proteins during the gastric and duodenal phases. In particular, β-conglycinin and trypsin inhibitors showed the highest resistance to the combined activity of GI enzymes, showing a partial degradation at the end of the duodenal phase as ascertained by the strong electrophoretic bands displayed at 50 kDa and 20 kDa, respectively. Glycinin subunits also presented, even if to a lower extent, resistance to the complete proteolytic degradation, as demonstrated by polypeptide fragments with molecular weight lower than 20 kDa displayed in the gel at the end of duodenal digestion. In addition, by bioinformatics analysis it was demonstrated that the GI resistant fragments of the allergenic proteins, β-conglycinin and glycinin, retained in their primary structure linear epitopes potentially able to trigger an immunoreaction when exposed to the intestinal mucosa. Moreover, such resistant peptides also presented a structural homology with epitope sequences recognized in other legume species, presenting a potential risk of adverse cross-reaction for a larger category of allergic consumers.

Characterization of Seed Storage Proteins of Several Perennial Glycine Species.

Perennial Glycine species, distant relatives of soybean, have been recognized as a potential source of new genetic diversity for soybean improvement. The subgenus Glycine includes around 30 perennial species, which are well-adapted to drought conditions and possess resistance to a number of soybean pathogens. In spite of the potential of the perennial Glycine species for soybean improvement, very little is known about their storage proteins and their relationship with cultivated soybean seed proteins. We have examined the seed protein composition of nine perennial Glycine species by one- and two-dimensional (1-D and 2-D) gel electrophoresis. The relationship between cultivated soybean and perennial soybean seed proteins was examined by immunoblot analyses using antibodies raised against G. max β-conglycinin, glycinin A3 subunit, lipoxygenase, leginsulin, Kunitz trypsin inhibitor, and Bowman-Birk protease inhibitor. Additionally, we have measured the trypsin and chymotrypsin inhibitor activities from cultivated soybean and perennial Glycine species and have found marked differences between them. Our 2-D gel and immunoblot analyses demonstrate significant differences in the protein composition and size heterogeneities of the 7S and 11S seed storage proteins of soybean and perennial Glycine species. Perennial Glycine species accumulated a 45 kDa protein that was not detected in G. max and G. soja. This unique 45 kDa protein was immunologically related to the A3 glycinin subunit of G. max. The results of our studies suggest that even though the seed proteins of wild perennial Glycine species and G. max are immunologically related, their genes have diverged from each other during the course of evolution.

Composition of Proteins Extracted from Two Species of Leguminous Bauhinia Grains

There is a growing demand for alternative protein sources for industrial applications. Bauhinia are underutilized protein‐rich oilseed legumes primarily grown as ornamental plants. In this study, the composition and structure of proteins extracted from two Bauhinia species, B. galpinii and B. petersiana, were determined in comparison with soya bean. Albumin and globulin (75%) were the major storage proteins of Bauhinia. Albumin content of B. galpinii was substantially high (28%), about twice those of soya and B. petersiana. Bauhinia grains were good sources of lysine (approximately 4 g/100 g of protein). By SDS‐PAGE, Bauhinia proteins showed three major protein bands ranging from 17,000 to 76,000 for both species. Only one subunit (17,000) of the basic glycinin (11S) seemed to be present in Bauhinia compared with soya. Bauhinia proteins had more β‐sheet conformation (42%) than α‐helix (22%). Bauhinia grains may provide a new opportunity of protein for industrial applications.

Quantitative Trait Loci (QTL) Mapping for Glycinin and β-Conglycinin Contents in Soybean (Glycine max L. Merr.).

Compared to β-conglycinin, glycinin contains 3-4 times the methionine and cysteine (sulfur-containing amino acids), accounting for approximately 40 and 30%, respectively, of the total storage protein in soybean. Increasing the soybean storage protein content while improving the ratio of glycinin to β-conglycinin is of great significance for soybean breeding and soy food products. The objective of this study is to analyze the genetic mechanism regulating the glycinin and β-conglycinin contents of soybean by using a recombinant inbred line (RIL) population derived from a cross between Kefeng No. 1 and Nannong 1138-2. Two hundred and twenty-one markers were used to map quantitative trait loci (QTLs) for glycinin (11S) and β-conglycinin (7S) contents, the ratio of glycinin to β-conglycinin (RGC), and the sum of glycinin and β-conglycinin (SGC). A total of 35 QTLs, 3 pairs of epistatic QTLs, and 5 major regions encompassing multiple QTLs were detected. Genes encoding the subunits of β-conglycinin were localized to marker intervals sat_418-satt650 and sat_196-sat_303, which are linked to RGC and SGC; marker sat_318, associated with 11S, 7S, and SGC, was located near Glyma10g04280 (Gy4), which encodes a subunit of glycinin. These results, which take epistatic interactions into account, will improve our understanding of the genetic basis of 11S and 7S contents and will lay a foundation for marker-assisted selection (MAS) breeding of soybean and improving the quality of soybean products.

Rebalance between 7S and 11S globulins in soybean seeds of differing protein content and 11SA4

Protein content and globulin subunit composition of soybean seeds affect the quality of soy foods. In this proteomic study, the protein profile of soybean seeds with high (∼45.5%) or low (∼38.6%) protein content and with or without the glycinin (11S) subunit 11SA4 was examined. 44 unique proteins and their homologues were identified and showed that both protein content and 11SA4 influenced the abundance of a number of proteins. The absence of 11SA4 exerted a greater impact than the protein content, and led to a decreased abundance of glycinin G2/A2B1 and G5/A5A4B3 subunits, which resulted in lower total 11S with a concomitant higher total β-conglycinin (7S). Low protein content was associated with higher glycinin G3/A1aB1b and lower glycinin G4/A5A4B3. Using the proteomic approach, it was demonstrated that 11SA4 deficiency induced compensatory accumulation of 7S globulins and led to a similar total abundance for 7S+11S irrespective of protein content or 11SA4.

Soy Proteins Deficient in the α’ Subunit of β‐Conglycinin and Various Glycinin Subunits Maintain the Ability to Modulate Hepatic Lipid and Glucose Metabolism

Soy consumption has been associated with improved blood lipid profiles and decreased incidence of metabolic diseases. However, the involved molecular mechanism(s) and the bioactive components in soy remain unclear. This study examined the effect of soy proteins with depletion of different subunits of the two major storage proteins, β‐conglycinin and glycinin, on hepatic lipids and proteins involved in lipid and glucose metabolism as well as inflammation in rats and cultured human hepatocytes. Sprague‐Dawley rats (8 males and 8 females/group) were fed diets containing either 20% casein or 20% conventional soy proteins or soy proteins depleted in different β‐conglycinin and glycinin subunits. After 8 weeks, the rats were necropsied for collection of blood and tissue samples. Human HepG2 liver cells were treated with casein or test soy protein hydrolysates in the media containing low (5 mM) or high (25 mM) levels of glucose with or without insulin and tumour necrosis factor alpha (TNF‐α). Hepatic lipids were extracted and measured. Various modulators of lipid and glucose metabolisms in the liver and cultured human hepatocytes were determined by Western blot. Serum inflammation markers were analysed by a magnetic beads‐based multiplexing immunoassay. The results showed that the soy diets lacking glycinins reduced hepatic lipid droplet formation, total cholesterol, triglycerides, and non‐esterified fatty acids content in the male rats compared to rats on the casein control diets, and a similar trend was observed in the females. The soy diets lacking glycinins significantly attenuated serum inflammation markers and the abundance of mature hepatic sterol regulatory element binding protein ‐1 (SREBP‐1) and fatty acid synthase (FAS) in both genders and decreased hydroxymethylglutaryl‐CoA reductase (HMGCR) and low‐density lipoprotein receptor (LDL‐R) in female livers. Soy protein hydrolysates devoid of glycinins significantly increased Akt phosphorylation in high glucose concentrations. These results suggest that the remaining soy protein components other than α’ subunit of β‐conglycinin and glycinin may be associated with the health benefits of soy intake such as hypolipidemic function, improved insulin signalling and suppression of inflammation.Support or Funding InformationThis research was supported by Health Canada, Grain Farmers of Ontario Research Fund and the partnership of Agriculture and Agri‐Food Canada/Canadian Field Crops Research Alliance DIAP Fund. C.C. is supported by the National Science and Engineering Research Council (NSERC) Visiting Fellowship in Canadian Government Laboratories.

Changes in allergenic and antinutritional protein profiles of soybean meal during solid-state fermentation with Bacillus subtilis

Fermented soy-based products are believed to have improved nutritional qualities compared to non-fermented products; however, little is known about the biochemical changes that occur during fermentation. To investigate the changes in the protein profile during fermentation, soybean meal and Bacillus subtilis-fermented soybean meal were prepared. Soybean meal and fermented soybean meal were analyzed by two-dimensional electrophoresis and compared by image analyzer. Twelve major spots were selected and identified by Nano LC–MS/MS. The proteins identified were beta-conglycinin subunits, glycinin subunits, trypsin inhibitors, and sucrose-binding proteins. Seventy percent of the beta-conglycinin subunits were removed after 24 h of solid-state fermentation with B. subtilis. Fifty percent of trypsin inhibitors were removed after 24 h. Glycinin subunit degradation was the lowest, with 58% remaining after 24 h of fermentation. This study suggests that fermentation can improve the nutritional quality of soybean meal.

Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts.

Soybean consumption is increasing in many Western diets; however, recent reviews suggest that the prevalence of soy allergy can be as high as 0.5% for the general population and up to 13% for children. The immunoglobulin-E (IgE) binding of sera from six soy-sensitive adult human subjects to soybean proteins separated by 2D gel electrophoresis was studied. Synthetic peptide sets spanning the mature glycinin subunit A2 and A3 primary sequences were used to map the IgE-binding regions. Putative epitopes identified in this study were also localized on glycinin hexamer models using bioinformatics software. We identified linear IgE-binding epitopes of the major storage protein Gly m 6 by screening individual soy-sensitive patient sera. These epitopes were then further analysed by 3D in silico model localization and compared to other plant storage protein epitopes. Web-based software applications were also used to study the ability to accurately predict epitopes with mixed results. A total of nine putative IgE-binding epitopes were identified in the glycinin A3 (A3.1-A3.3) and A2 (A2.1-A2.6) subunits. Most patients' sera IgE bound to only one or two epitopes, except for one patient's serum which bound to four different A2 epitopes. Two epitopes (A3.2 and A2.4) overlapped with a previously identified epitope hot spot of 11S globulins from other plant species. Most epitopes were predicted to be exposed on the surface of the 3D model of the glycinin hexamer. Amino acid sequence alignments of soybean acidic glycinins and other plant globulins revealed one dominant epitope hot spot among the four reported hot spots. This study may be helpful for future development of soy allergy immunotherapy and diagnosis.

Analysis using fluorescence labeling and mass spectrometry of disulfide-mediated interactions of soy protein when heated.

It is well-known that disulfide-mediated interactions are important when soy protein is heated, in which soy proteins are dissociated and rearranged to some new forms. In this study, the disulfide bond (SS) linked polymer, which was composed of the acidic (A) polypeptides of glycinin, basic (B) polypeptides of glycinin, and a small amount of α' and α of β-conglycinin, was formed as the major product, accompanied by the formation of SS-linked dimer of B and monomer of A as minor products. The role of sulfhydryl (SH) of different subunits/polypeptides for forming intermolecular SS was investigated. The SH of B in glycinin (Cys298 of G1, Cys289 of G2, Cys440 of G4) was transformed into SS in polymer identified by mass spectrometry analysis. The SH content of polymer was lower than that of glycinin and β-conglycinin subunits when heated. The SH content of B in polymer was lower than that in glycinin subunit, and both of them were decreased by heating. The SH content of A in polymer was increased and higher than that of B in polymer and A in glycinin subunit when heated. These results indicated that the SH of B in glycinin subunit was subjected to not only SH oxidation but also SH-SS exchange (with SS of A) for forming intermolecular SS of polymer. The SH oxidation and SH-SS exchange were proven by the change of SH content for the first time. The SH of B was suggested to be reactive for forming intermolecular SS of polymer.