Search

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max [L.] Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins (HPs). We isolated 2 high-seed protein soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes β-conglycinin (GmCG-1) and HP2 encodes sucrose-binding protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Soy 11S globulin has high thermal stability, limiting its application in the production of low-temperature gel foods. In this study, the low-frequency magnetic field (LF-MF, 5 mT) treatment (time, 30, 60, 90, and 120 min) was used to improve the solubility, conformation, physicochemical properties, surface characteristics, and gel properties of soy 11S globulin., Results: Compared with the native soy 11S globulin, the sulfhydryl content, emulsifying capacity, gel strength, water-holding capacity, and absolute zeta potential values significantly increased (P < 0.05) after LF-MF treatment. The LF-MF treatment induced the unfolding of the protein structure and the fracture of disulfide bonds. The variations in solubility, foaming properties, viscosity, surface hydrophobicity, and rheological properties were closely related to the conformational changes of soy 11S globulin, with the optimum LF-MF modification time being 90 min., Conclusion: LF-MF treatment is an effective method to improve various functional properties of native soy 11S globulin, and this study provides a reference for the development of plant-based proteins in the food industry. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans- acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1 , and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1 -gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T 1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Understanding the transport mechanism is crucial for developing inhibitors that block allergen absorption and transport and prevent allergic reactions. However, the process of how beta-conglycinin, the primary allergen in soybeans, crosses the intestinal mucosal barrier remains unclear. The present study indicated that the transport of beta-conglycinin hydrolysates by IPEC-J2 monolayers occurred in a time- and quantity-dependent manner. The beta-conglycinin hydrolysates were absorbed into the cytoplasm of IPEC-J2 monolayers, while none were detected in the intercellular spaces. Furthermore, inhibitors such as methyl-beta-cyclodextrin (MβCD) and chlorpromazine (CPZ) significantly suppressed the absorption and transport of beta-conglycinin hydrolysates. Of particular interest, sodium cromoglycate (SCG) exhibited a quantity-dependent nonlinear suppression model on the absorption and transport of beta-conglycinin hydrolysates. In conclusion, beta-conglycinin crossed the IPEC-J2 monolayers through a transcellular pathway, involving both clathrin-mediated and caveolae-dependent endocytosis mechanisms. SCG suppressed the absorption and transport of beta-conglycinin hydrolysates by the IPEC-J2 monolayers by a quantity-dependent nonlinear model via clathrin-mediated and caveolae-dependent endocytosis. These findings provide promising targets for both the prevention and treatment of soybean allergies.

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

Background: The two major storage proteins of soymilk are the globulins 7S and 11S. Freeze-thaw fractionation is a simple method for separating these proteins in raw soymilk. In this study, we assessed the freeze-thaw fractionation ability of raw soymilk under various pH (4.3-11.6) conditions and added salt (sodium chloride) concentrations (0.00-0.67 mol L -1 )., Results: We successfully achieved fractionation within a pH range of 5.8-6.7 and when the salt concentration was 0.22 mol L -1 or lower. Analysis of particle size distribution and microscopic examination of soymilk revealed no direct correlation between particle size and freeze-thaw fractionation ability. Interestingly, it was confirmed that the ranges of zeta potential values associated with successful freeze-thaw fractionation in raw soymilk remained consistent across different pH and salt concentration conditions. These ranges were between -23 and -28 mV at pH levels ranging from 5.8 to 6.7 and between -18 and -29 mV at added salt concentrations ranging from 0 to 0.22 mol L -1 ., Conclusion: The pH and salt concentration in raw soymilk markedly influence the freeze-thaw fractionation process. We confirmed that the range of zeta potential values where fractionation was possible remained consistent under various pH and salt concentration conditions. These findings suggest that the zeta potential value might serve as an indicator for evaluating the freeze-thaw fractionation ability of raw soymilk. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus , a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

To date, it still remains unknown how β-conglycinin, a major soybean allergen, crosses intestinal epithelial barrier to reach immune cells. The purpose of this study was to elucidate the pathway and molecular mechanism of β-conglycinin absorption and transport across intestinal mucosal epithelium using a β-conglycinin allergic piglet model. Ten-day old piglets were orally sensitized with diets containing 2% and 4% β-conglycinin. The digestion, absorption and transport of β-conglycinin in gastrointestinal tract was investigated. The results showed that β-conglycinin had a certain resistance to gastrointestinal digestion, and the digestion-resistant subunits and fragments were absorbed into the intestinal mucosa and then induced an anaphylaxis in early weaned piglets. The absorption occurred in the form of IgE-allergen immune complex through transcellular pathway with CD23 as the receptor. These results provided important clues for using the pathway and molecule as inhibitor target to prevent and alleviate soybean β-conglycinin allergy in infants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Soybean glycinin (11S) and β-conglycinin (7S) are major antigenic proteins in soybean and can induce a variety of allergic reactions in the young animals. This study aimed to investigate the effect of 7S and 11S allergens on the intestine of piglets., Methods: Thirty healthy 21-day-old weaned "Duroc × Long White × Yorkshire" piglets were randomly divided into three groups fed with the basic diet, the 7S supplemented basic diet, or the 11S supplemented basic diet for 1 week. Allergy markers, intestinal permeability, oxidative stress, and inflammatory reactions were detected, and we observed different sections of intestinal tissue. The expressions of genes and proteins related to NOD-like receptor thermal protein domain associated protein 3 (NLRP-3) signaling pathway were detected by IHC, RT-qPCR, and WB., Results: Severe diarrhea and decreased growth rate were observed in the 7S and 11S groups. Typical allergy markers include IgE production and significant elevations of histamine and 5-hydroxytryptamine (5-HT). More aggressive intestinal inflammation and barrier dysfunction were observed in the experimental weaned piglets. In addition, 7S and 11S supplementation increased the levels of 8-hydroxy-2 deoxyguanosine (8-OHdG) and nitrotyrosine, triggering oxidative stress. Furthermore, higher expression levels of NLRP-3 inflammasome ASC, caspase-1, IL-1β, and IL-18 were observed in the duodenum, jejunum, and ileum., Conclusion: We confirmed that 7S and 11S damaged the intestinal barrier of weaned piglets and may be associated with the onset of oxidative stress and inflammatory response. However, the molecular mechanism underlying these reactions deserves further study., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Key Message: We characterize GFP expression driven by a soybean glycinin promoter in transgenic soybean. We demonstrate specific amino acid-mediated induction of this promoter in developing soybean seeds in vitro. In plants, gene expression is primarily regulated by promoter regions which are located upstream of gene coding sequences. Promoters allow transcription in certain tissues and respond to environmental stimuli as well as other inductive phenomena. In soybean, seed storage proteins (SSPs) accumulate during seed development and account for most of the monetary and nutritional value of this crop. To better study the regulatory functions of a SSP promoter, we developed a cotyledon culture system where media and media addenda were evaluated for their effects on cotyledon development and promoter activity. Stably transformed soybean events containing a glycinin SSP promoter regulating the green fluorescent protein (GFP) were generated. Promoter activity, as visualized by GFP expression, was only observed in developing in planta seeds and in vitro-cultured isolated embryos and cotyledons from developing seeds when specific media addenda were included. Asparagine, proline, and especially glutamine induced glycinin promoter activity in cultured cotyledons from developing seeds. Other amino acids did not induce the glycinin promoter. Here, we report, for the first time, induction of a reintroduced glycinin SSP promoter by specific amino acids in cotyledon tissues during seed development., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study explores utilization of a sustainable soybean by-product (okara) based on in silico approach. In silico approaches, as well as the BIOPEP database, PeptideRanker database, Peptide Calculator database (Pepcalc), ToxinPred database, and AllerTop database, were employed to evaluate the potential of glycinin and conglycinin derived peptides as a potential source of bioactive peptides. These major protein precursors have been found as protein in okara as a soybean by-product. Furthermore, primary structure, biological potential, and physicochemical, sensory, and allergenic characteristics of the theoretically released antioxidant peptides were predicted in this research. Glycinin and α subunits of β-conglycinin were selected as potential precursors of bioactive peptides based on in silico analysis. The most notable among these are antioxidant peptides. First, the potential of protein precursors for releasing bioactive peptides was evaluated by determining the frequency of occurrence of fragments with a given activity. Through the BIOPEP database analysis, there are several antioxidant bioactive peptides in glycinin and β and α subunits of β-conglycinin sequences. Then, an in silico proteolysis using selected enzymes (papain, bromelain) to obtain antioxidant peptides was investigated and then analyzed using PeptideRanker and Pepcalc. Allergenic analysis using the AllerTop revealed that all in silico proteolysis-derived antioxidant peptides are probably nonallergenic peptides. We also performed molecular docking against MPO (myeloperoxidases) for this peptide. Overall, the present study highlights that glycinin and β and α subunits of β-conglycinin could be promising precursors of bioactive peptides that have an antioxidant peptide for developing several applications.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

We investigated the effects of low and high doses of β-conglycinin and the ameliorative effects of sodium butyrate (based on high-dose β-conglycinin) on the growth performance, serum immunity, distal intestinal histopathology, and gene, protein expression related to intestinal health in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). The results revealed that the instantaneous growth rate (IGR) of grouper significantly increased, decreased, and increased in the low-dose β-conglycinin (bL), high-level β-conglycinin (bH) and high-level β-conglycinin plus sodium butyrate (bH-NaB), respectively. The feed coefficient ratio (FCR) was significantly increased in the bH and bH-NaB, serum levels of IFN-γ, IL-1β, and TNF-α were upregulated in the bH. The intestinal diameter/fold height ratio was significantly increased in the bH. Furthermore, there were increases in nitric oxide (NO), total nitric oxide synthase (total NOS), and peroxynitrite anion (ONOO - ) in the bH, and decreases in total NOS and ONOO - in the bH-NaB. In the distal intestine, IL-1β and TGF-β1 mRNA levels were downregulated and upregulated, respective in the bL. The mRNA levels of TNF-α and IL-6 were upregulated in the bH, and downregulated in the bH-NaB, respectively. Occludin, claudin3 and ZO-3 mRNA levels were upregulated in the bL, downregulated in the bH and then upregulated in the bH-NaB. No significant differences were observed in the mRNA levels of IFN-γ and jam4. And the p-PI3K p85 Tyr458 /total PI3K p85 value was significantly increased in the bH and then decreased in the bH-NaB, and the total Akt value was significantly increased in the bH. These indicate β-conglycinin has a regulatory effect on serum immunity and affect distal intestinal development by modulating distal intestinal injury-related parameters. Within the distal intestinal tract, low- and high-dose β-conglycinin differentially affect immune responses and tight junctions in the distal intestine, which eventually manifests as a reduction in growth performance. Supplementing feed with sodium butyrate might represent an effective approach for enhancing serum immunity, and protects the intestines from damage caused by high-dose β-conglycinin., (© 2021. The Author(s).)

The effect of the reverse micelle extraction method (RMEM) on the physicochemical properties of soy 11S globulin was studied and compared with that of the traditional alkali solution-acid precipitation method (ASAPM). The results showed that the β-sheet structure content of soy 11S globulin obtained by RMEM was lower, while the β-turn structure content was higher compared with that obtained by ASAPM. Furthermore, the protein unfolding degree and surface hydrophobicity were lower than those observed using ASAPM. Therefore, RMEM better maintained the natural molecular structure of soy 11S globulin. The thermodynamic and rheological properties of soy 11S globulin obtained by these two methods were further compared, showing that the highest denaturation temperature and transition heat of soy 11S globulin extracted using ASAPM were different from those obtained using RMEM. Furthermore, soy 11S globulin extracted by RMEM showed stronger heat resistance and a higher denaturation temperature than that extracted by ASAPM. The final storage modulus and frequency sweep results showed that the gel formed by soy 11S globulin obtained using RMEM had high storage modulus and loss modulus. PRACTICAL APPLICATION: In this study, the effects of two different extraction methods on structural and functional properties of soy 11S globulin, such as thermodynamics and rheology, were investigated. We can know the 11S globulin extracted using the reverse micelle environment was more heat-resistant and heat-induced gel quality of 11S globulin was improved by the reverse micelle environment. These results will provide theoretical basis that would help determine the potential applications of soy 11S globulin in the food system., (© 2021 Institute of Food Technologists®.)

Hyperoside (HYP) is an important natural product that is widely distributed in fruits and whole grasses of various plants. It is also used by consumers as a healthy ingredient. This work explored the interaction mechanisms between HYP and two main soy proteins, namely, β-conglycinin (7S) and glycinin (11S), using computational simulation and multi-spectroscopic technology. In this study, the docking and dynamic simulation showed that HYP was stable in the hydrophobic pockets of the proteins. The conformation and microenvironment of 7S/11S also changed after binding to HYP. The binding of HYP to 7S/11S was a state quenching with a good affinity at 4 °C. This result was determined from the binding constant values of (1.995 ± 0.170) × 10 7 M -1 and (2.951 ± 0.109) × 10 7 M -1 , respectively. The 7S/11S-HYP complex delineated here will provide a novel idea to construct an embedding and delivery system in improving the benefits of HYP for the development of high value-added food products., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this study, the effects of heat treatment on antigenicity, antigen epitopes, and structural changes in β-conglycinin were investigated. Results showed that the IgG (Immunoglobulin G) binding capacity of heated protein was inhibited with increased temperature, although IgE (Immunoglobulin E) binding capacity increased. Linear antigen epitopes generally remained intact during heat treatment. After heat treatment, β-conglycinin was more easily hydrolyzed by digestive enzymes, and a large number of linear epitopes was destroyed. In addition, heat denaturation of β-conglycinin led to the formation of protein aggregates and reduction of disulfide bonds. The contents of random coils and β-sheet of heated β-conglycinin decreased, but the contents of β-turn and α-helix increased. Moreover, the protein structure of heated β-conglycinin unfolded, more hydrophobic regions were exposed, and the tertiary structure of β-conglycinin was destroyed. Heat treatment affected the antigenicity and potential sensitization of β-conglycinin by changing its structure., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Key Message: Linkage mapping and GWAS identified 67 QTLs related to soybean glycinin, β-conglycinin and relevant traits. Polymorphisms of the candidate gene Gy1 promoter were associated with the glycinin content in soybean. The major components of storage proteins in soybean seeds are glycinin and β-conglycinin, which play important roles in determining protein nutrition and soy food processing properties. Increasing the protein content while improving the ratio of glycinin to β-conglycinin is substantially important for soybean protein improvement. To investigate the genetic mechanism of storage protein subunits, 184 recombinant inbred lines (RILs) derived from a cross of Kefeng No. 1 and Nannong 1138-2 and 211 diverse soybean cultivars were used to detect loci related to glycinin (11S), β-conglycinin (7S), the sum of glycinin and β-conglycinin (SGC), and the ratio of glycinin to β-conglycinin (RGC). Sixty-seven QTLs and 11 hot genomic regions were identified as affecting the four traits. One genetic region (q10-1) on chromosome 10 was associated with multiple traits by both linkage and association analysis. Eight genes in 11 hot genomic regions might be related to soybean protein subunit. The candidate gene analysis showed that polymorphisms in Gy1 promoters were significantly correlated with the 11S content. The QTLs and candidate genes identified in the present study allow for further understanding the genetic basis of 11S and 7S regulation and provide useful information for marker-assisted selection (MAS) in soybean quality improvement.

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

The current research aimed to estimate the effect of dietary supplementation with glycinin isolated from soybeans on the growth performance, carcass traits, and selected blood metabolites of broiler chicks. A total of 200 1-wk-old broiler chicks were administered diets without glycinin (control treatment) or diets supplemented with 3 concentrations of soy glycinin (0.5, 1.0, or 1.5 g/kg of feed) for 6 wk. At the end of the feeding period, body weight was significantly higher in broiler chicks with glycinin supplementation (P < 0.05 or 0.01). The best values for body weight and body weight gain were recorded in the groups fed diets supplemented with 0.5 and 1.0 g glycinin/kg feed. Feed conversion was significantly (P < 0.05) improved in broilers in the glycinin-supplemented groups during the 1 to 6 and 3 to 6 wk growth periods. The highest value of breast yield was observed in broiler chicks supplemented with glycinin at a concentration of 1.0 g/kg of feed. Water-holding capacity increased with increasing concentrations of glycinin in the feed, up to 1.0%. Serum creatinine and urea concentrations decreased gradually (P < 0.01) as the concentration of glycinin in the feed increased. Broiler chicks receiving increasing concentrations of glycinin exhibited significantly (P < 0.01) lower levels of serum triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. All meat samples from broiler chicks supplemented with glycinin had significantly higher catalase activities. These data suggest that feeding broiler chicks diets supplemented with soy glycinin (0.5 to 1.5 g/kg of feed) can improve feed conversion, enhance body weight gain, and lower abdominal fat., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The noncovalent binding mechanisms between cyanidin-3-O-glucoside (C3G) and two main soy protein fractions: β-conglycinin (7S) and glycinin (11S) at pH 3.0 were investigated and compared. C3G modified the secondary structure of two fractions by increasing α-helix and random coil content while decreasing β-sheet content. The binding of C3G also altered the tertiary structure and microenvironment of two fractions, demonstrated by synchronous and three-dimensional fluorescence spectra. Additionally, C3G binding reduced the surface hydrophobicity and thermostability of both 7S and 11S. Moreover, the fluorescence quenching results showed that the binding of C3G to two fractions were spontaneous complexation processes driven by electrostatic forces. The number of C3G bound per protein molecule (n) was near 1. The binding constant (K a ) was 2.41 (±0.42) × 10 4  M -1 for 11S and 0.81 (±0.01) × 10 4  M -1 for 7S at 298 K. 11S showed a stronger binding ability for C3G than 7S. These findings contribute to the knowledge of interactions between soy protein fractions and dietary polyphenols under acidic condition, and are beneficial for the application of soy protein-based products in foods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of the present study was to enhance oxidative stability and bioaccessibility of β-conglycinin (7S) prepared from low denatured defatted soybean flours with residual lipids and high lipoxygenase (LOX) activity. The model system consisting of linoleic acid (LA), LOX and unheated 7S (UH-7S)/heated 7S (H-7S) or UH-7S-catechin/H-7S-catechin complex, and in vitro gastrointestinal (GI) digestion model were used to investigate the effect of complexation with catechin on protein oxidation and characterisation of GI digest. The interaction of UH-7S/H-7S with catechin dramatically inhibited LOX-catalyzed LA peroxidation-induced protein oxidation. The interaction also promoted the degree of proteolysis in GI digestion and intestinal absorption for oxidized UH-7S/H-7S, increasing the antioxidant activity of oxidized UH-7S/H-7S, bioaccessibility for catechin and release of di-/tripeptides with dipeptidyl peptidase-IV/angiotensin converting enzyme inhibitory effects or antioxidant activities during GI digestion. The complexation with catechin is a potential strategy to enhance the oxidative stability, GI digestibility and bioaccessibility of 7S., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Soybean protein exhibits nutritional significance for the control of metabolic syndrome, and evidence suggests that gut microbiota are implicated in the control of metabolic disorders. This study aimed to investigate the modulation of pepsin-released peptides of soybean 7S globulin on gut microbiota and possible association between changes of gut microbiota composition and lipopolysaccharide (LPS)-peptide interaction. In vitro fermentation experiments showed that the extension region (ER) fragments of soybean 7S globulin selectively suppressed proinflammatory Gram-negative bacteria. ER peptides also promoted the highest production of short-chain fatty acids (SCFAs), which were associated with increase of the relative abundance of Lachnospiraceae and Lactobacillaceae. Isothermal titration calorimetry (ITC) and Langmuir monolayer studies demonstrated that ER peptides exhibited high affinity to LPS in the presence of Ca 2+ and developed into β-sheet-rich aggregate structures, thus weakening the stability of LPS monolayers. This finding supplies a possible explanation for improvement of the effects of soybean 7S globulin on metabolic disease.

This study investigates the effects of two soybean antigens (glycinin and β-conglycinin) as an antinutritional substance in the diet on the growth, digestive ability, intestinal health and microbiota of juvenile Chinese mitten crabs (Eriocheir sinensis). The isonitrogenous and isolipidic diets contained two soybean antigens at two levels each (70 and 140 g/kg β-conglycinin, 80 and 160 g/kg glycinin) and a control diet without β-conglycinin or glycinin supplementation, and were used respectively to feed juvenile E. sinensis for seven weeks. Dietary inclusion of either glycinin or β-conglycinin significantly reduced crab survival and weight gain. The crabs fed diets containing soybean antigens had higher malondialdehyde concentrations and lower catalase activities in the intestine than those in the control. The activities of trypsin and amylase in the intestine were suppressed by dietary β-conglycinin and glycinin. Dietary glycinin or β-conglycinin impaired the immunity and morphological structure of intestine, especially the peritrophic membrane. The mRNA expression of constitutive and inducible immune responsive genes (lipopolysaccharide-induced TNF-α factor and interleukin-2 enhancer-binding factor 2) increased while the mRNA expression of the main genes related to the structural integrity peritrophic membrane (peritrophin-like gene and peritrophic 2) significantly decreased in the groups with soybean antigen addition. Soybean antigen could also change the intestinal microbial community. The abundance of pathogenic bacteria (Ochrobactrum, Burkholderia and Pseudomonas) increased significantly in both soybean antigen groups. Although pathogenic bacteria Vibrio were up-regulated in the glycinin group, the abundance of Dysgonomonas that degraded lignocellulose and ameliorated the gut environment decreased in the glycinin group. This study indicates that existence of soybean antigens (glycinin or β-conglycinin) could induce gut inflammation, reshape the community of gut microbiota, and cause digestive dysfunction, ultimately leading to impaired growth in crabs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The study aimed at improving the antioxidant activity of β-conglycinin to enhance the oxidative and physical stabilities of safflower oil-in-water emulsion stabilized by β-conglycinin. Heating promoted binding affinity and antioxidant activity of β-conglycinin. Catechin and chlorogenic acid showed higher binding affinities towards unheated (or heated) β-conglycinin than caffeic acid and quercetin. The enhancement efficiencies of the phenolics on the antioxidant activity of unheated (or heated) β-conglycinin decreased in the order of catechin > quercetin > chlorogenic acid > caffeic acid. Hydrophobic force and hydrogen bonding were the important binding forces for the selected phenolics to β-conglycinin. The complexation with catechin has no side effect on interfacial behavior and emulsifying property of β-conglycinin. The use of heated β-conglycinin-catechin complex as an emulsifier for preparing safflower oil emulsion effectively improved the oxidative and physical stabilities of the emulsion treated with lipoxygenase through inhibition of lipid oxidation, protein carbonyl formation and sulfhydryl loss., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To understand the underlying molecular mechanism of the cholesterol-lowering effect of soybean 7S globulins, the interactions of their pepsin-released peptides (7S-peptides) with cell membrane models consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL) were systematically studied. The results showed that 7S-peptides were bound to DPPC/DOPC/CHOL liposomes mainly through van der Waals forces and hydrogen bonds, and the presence of higher CHOL concentrations enhanced the binding affinity (e.g., DPPC/DOPC/CHOL = 1:1:0, binding ratio = 0.114; DPPC/DOPC/CHOL = 1:1:1, binding ratio = 2.02). Compression isotherms indicated that the incorporation of 7S-peptides increased the DPPC/DOPC/CHOL monolayer fluidity and the lipid raft size. The presence of CHOL accelerated the 7S-peptide accumulation on lipid rafts, which could serve as platforms for peptides to develop into β-sheet rich structures. These results allow us to hypothesize that 7S-peptides may indirectly influence membrane protein functions via altering the membrane organization in the enterocytes.

Purpose: 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 metabolism in rats, since the bioactive component of soy responsible for lipid-lowering is unclear., Methods: Weanling Sprague Dawley rats were fed diets containing either 20% casein protein in the absence (casein) or presence (casein + ISF) of isoflavones or 20% alcohol-washed soy protein isolate (SPI) or 20% soy protein concentrates derived from a conventional (Haro) or 2 soybean lines lacking the α' subunit of β-conglycinin and the A1-3 (1TF) or A1-5 (1a) subunits of glycinin. After 8 weeks, the rats were necropsied and liver proteins and lipids were extracted and analysed., Results: The results showed that soy protein diets reduced lipid droplet accumulation and content in the liver compared to casein diets. The soy protein diets also decreased the level of hepatic mature SREBP-1 and FAS in males, with significant decreases in diets 1TF and 1a compared to the casein diets. The effect of the soy protein diets on female hepatic mature SREBP-1, FAS, and HMGCR was confounded since casein + ISF decreased these levels compared to casein alone perhaps muting the decrease by soy protein. A reduction in both phosphorylated and total STAT3 in female livers by ISF may account for the gender difference in mechanism in the regulation and protein expression of the lipid modulators., Conclusions: Overall, soy protein deficient in the α' subunit of β-conglycinin and A1-5 subunits of glycinin maintain similar hypolipidemic function compared to the conventional soy protein. The exact bioactive component(s) warrant identification.

This study evaluated the effects of Bacillus fermentation on soybean meal protein (SBMP) microstructure and major anti-nutritional factors (ANFs) in soybean meal (SBM). The Bacillus siamensis isolate JL8 producing high yield of protease at 519·1 U g -1 was selected for the laboratory production of fermented soybean meal (FSBM). After 24 h fermentation, the FSBM showed better properties compared with those of SBM, the ANFs such as glycinin, β-conglycinin and trypsin inhibitor significantly decreased by 86·0, 70·3 and 95·01%, while in vitro digestibility and absorbability increased by 8·7 and 18·9% respectively. Scanning electron microscopy (SEM) image of fermented soybean meal protein showed smaller aggregates and looser network than that of SBMP. Secondary structure examination of proteins revealed fermentation significantly decreased the content of β-sheet structure by 43·2% and increased the random coil structure by 59·9%. It is demonstrated that Bacillus fermentation improved the nutritional quality of SBM through degrading ANFs and changing the microstructure of SBMP., Significance and Impact of the Study: There is limited information about the structural property changes of soybean protein during fermentation. In this study, physicochemical analysis of soybean meal protein showed evidence that the increase in in vitro digestibility and absorbability of fermented soybean meal reflected the decrease in β-conformation and destruction of original structure in soybean meal protein. The results directly gained the understanding of nutritional quality improvement of soybean meal by Bacillus fermentation, and supply the potential use of Bacillus siamensis for fermented soybean meal production., (© 2017 The Society for Applied Microbiology.)

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the β-subunit from β-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

p -Coumalic acid (PCA), caffeic acid (CA), gallic acid (GA) and chlorogenic acid (CGA) are the major phenolic acids that co-exist with soy protein components in foodstuffs. Surprisingly, there are only a handful of reports that describe their interaction with β-Conglycinin (7S), a major soy protein. In this report, we investigated the interaction between phenolic acids and soy protein 7S and observed an interaction between each of these phenolic acids and soy protein 7S, which was carried out by binding. Further analysis revealed that the binding activity of the phenolic acids was structure dependent. Here, the binding affinity of CA and GA towards 7S was found to be stronger than that of PCA, because CA and GA have one more hydroxyl group. Interestingly, the binding of phenolic acids with soy protein 7S did not affect protein digestion by pepsin and trypsin. These findings aid our understanding of the relationship between different phenolic acids and proteins in complex food systems., Competing Interests: The authors declare no conflict of interest.

The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

The coagulation of β-conglycinin (7S), glycinin (11S) and isoflavones induced by calcium chloride was investigated. Approximately 92.6% of the soymilk proteins were coagulated into the soymilk pellet fraction (SPF) after the addition of 5 mM calcium chloride. SDS-PAGE and two-dimensional electrophoresis analysis indicated that most of the 7S (α', α and β), 11S acidic (A1a, A1b, A2, A3 and A4) and 11S basic (B1a) proteins in the SSF were coagulated into the SPF after treatment with 5 mM calcium chloride. Isoflavones, including daidzein and genistein, were also coagulated into the SPF after the addition of 5 mM calcium chloride. The amounts of daidzein and genistein in the SSF decreased to 39.4 ± 1.6 and 11.8 ± 7.0%, respectively. HPLC analysis suggested that daidzein and genistein were bound with 7S and 11S proteins and then were coprecipitated into the SPF by 5 mM calcium chloride.

β-Conglycinin, a major component of seed storage protein in soybean, comprises three subunits: α, α' and β. The expression of genes for these subunits is strictly controlled during embryogenesis. The proximal promoter region up to 245 bp upstream of the transcription start site of the α subunit gene sufficiently confers spatial and temporal control of transcription in embryos. Here, the binding profile of nuclear proteins in the proximal promoter region of the α subunit gene was analysed. DNase I footprinting analysis indicated binding of proteins to the RY element and DNA regions including box I, a region conserved in cognate gene promoters. An electrophoretic mobility shift assay (EMSA) using different portions of box I as a probe revealed that multiple portions of box I bind to nuclear proteins. In addition, an EMSA using nuclear proteins extracted from embryos at different developmental stages indicated that the levels of major DNA-protein complexes on box I increased during embryo maturation. These results are consistent with the notion that box I is important for the transcriptional control of seed storage protein genes. Furthermore, the present data suggest that nuclear proteins bind to novel motifs in box I including 5'-TCAATT-3' rather than to predicted cis-regulatory elements., (© 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.)

By now, the digestive stability experiments provided by most authoritative organizations are in vitro tests. Evaluating the protein digestive stability with in vivo models should be more objective. The present study aimed to verify the in vivo digestibility of soybean β-conglycinin β-subunit in Wuzhishan (WZS) minipigs. Three minipigs were surgically fitted with O-stomach and T-ileum cannulae and fed with soybean meals. According to SDS-PAGE, the 50 kD fraction of soybean β-conglycinin β-subunit persisted in the gastric fluid until 6 h after feeding, which was detected at 3 h and clearly visible at 4-6 h in the intestinal fluid. Western blot with anti-β-conglycinin β-subunit McAb confirmed it., (Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Soybean 7S globulin, known as β-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of β-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of β-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the β-conglycinin α' subunit.

Basic 7S globulin (Bg7S), which accumulates in mature soybean (Glycine max) seeds, is an extracellular matrix protein. A large amount of Bg7S is synthesized de novo and is eluted from soybean seeds when immersed in 50-60°C water (hot water treatment, HWT). However, the Bg7S elution mechanism remains unclear. Under HWT, the seeds probably undergo heat stress and flooding stress. To obtain fundamental knowledge related to how Bg7S is eluted from hot-water-treated seeds, this study compared Bg7S elution among soybean cultivars having different flooding tolerance during pre-germination. The amounts of Bg7S eluted from seeds varied significantly among cultivars. Elution was suppressed by seed coats regarded as preventing the leakage of seed contents by rapid water imbibition. Furthermore, Bg7S expression levels differed among cultivars, although the difference did not result from any variation in Bg7S promoter sequences. However, the expression levels of Bg7S under HWT were not associated with the flooding tolerance level. Immunoelectron microscopy revealed that the Bg7S accumulated in the intercellular space of hot-water-treated seeds. Plasma membrane shrinkage was observed. The main proteins eluted from seeds under HWT were located in the extracellular space. This study clarified the mechanism of Bg7S elution from seeds under HWT., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

¹³C metabolic flux analysis (MFA) has become the experimental method of choice to investigate the cellular metabolism of microbes, cell cultures and plant seeds. Conventional steady-state MFA utilizes isotopic labeling measurements of amino acids obtained from protein hydrolysates. To retain spatial information in conventional steady-state MFA, tissues or subcellular fractions must be dissected or biochemically purified. In contrast, peptides retain their identity in complex protein extracts, and may therefore be associated with a specific time of expression, tissue type and subcellular compartment. To enable 'single-sample' spatially and temporally resolved steady-state flux analysis, we investigated the suitability of peptide mass distributions (PMDs) as an alternative to amino acid label measurements. PMDs are the discrete convolution of the mass distributions of the constituent amino acids of a peptide. We investigated the requirements for the unique deconvolution of PMDs into amino acid mass distributions (AAMDs), the influence of peptide sequence length on parameter sensitivity, and how AAMD and flux estimates that are determined through deconvolution compare to estimates from a conventional GC-MS measurement-based approach. Deconvolution of PMDs of the storage protein β-conglycinin of soybean (Glycine max) resulted in good AAMD and flux estimates if fluxes were directly fitted to PMDs. Unconstrained deconvolution resulted in inferior AAMD and flux estimates. PMD measurements do not include amino acid backbone fragments, which increase the information content in GC-MS-derived analyses. Nonetheless, the resulting flux maps were of comparable quality due to the precision of Orbitrap quantification and the larger number of peptide measurements., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Vitamin B12 deficiency is a problem for many elderly people around the world that is caused by food-cobalamin malabsorption syndrome due to gastrointestinal problems. In this study, the molecular interactions of VB12 with two major soy protein fractions, β-conglycinin (7S) and glycinin (11S), were studied using fluorescence and far-UV circular dichroism spectroscopic techniques. The results show that the fluorescence of 7S and 11S was quenched by VB12 through binding-related quenching after correcting for the inner-filter effect. Both 7S and 11S had a good affinity to VB12 as indicated by their high binding constant 1.252 (±0.085) × 10(4) M(-1) for 7S and 0.952 (±0.04) × 10(4) M(-1) for 11S at 292 K, respectively. Such binding induced a more organized protein confirmation with increased β-sheet and β-turn structure components and a more folded tertiary structure. It is deduced that VB12 was bound in the interior of protein three-dimensional network mainly via hydrophobic interactions to form 7S-VB12 and 11S-VB12 complexes. The results suggest that soy protein has potential to be used as carrier of VB12.

Modification of the wetting behavior of hydrophobic surfaces is essential in a variety of materials, including textiles and membranes that require control of fluid interactions, adhesion, transport processes, sensing, etc. This investigation examines the enhancement of wettability of an important class of textile materials, viz., polypropylene (PP) fibers, by surface adsorption of different proteins from soybeans, including soy flour, isolate,glycinin, and β-conglycinin. Detailed investigations of soy adsorption from aqueous solution (pH 7.4, 25 °C) on polypropylene thin films is carried out using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). A significant amount of protein adsorbs onto the PP surfaces primarily due to hydrophobic interactions. We establish that adsorption of a cationic surfactant, dioctadecyldimethylammonium bromide (DODA) onto PP surfaces prior to the protein deposition dramatically enhances its adsorption. The adsorption of proteins from native (PBS buffer, pH 7.4, 25 °C) and denatured conditions (PBS buffer, pH 7.4, 95 °C) onto DODA-treated PP leads to a high coverage of the proteins on the PP surface as confirmed by a significant improvement in water wettability. A shift in the contact angle from 128° to completely wettable surfaces (≈0°) is observed and confirmed by imaging experiments conducted with fluorescence tags. Furthermore, the results from wicking tests indicate that hydrophobic PP nonwovens absorb a significant amount of water after protein treatment, i.e., the PP-modified surfaces become completely hydrophilic.

Limited and extensive proteolysis occur when β-conglycinin β homo-trimer (β(3)-conglycinin) from soybeans is attacked by papain. Slow limited proteolysis is restricted to cleavage of β(3)-conglycinin polypeptides into subunit halves (N- and C-terminal domains) that are further slightly truncated. The kinetics of limited and extensive proteolyses analyzed separately indicates that the two processes occur independently from the very beginning of the reaction. In contrast, limited proteolysis of phaseolin from common beans has been found to be prerequisite for the onset of its extensive proteolysis. The dramatic distinction between the degradation patterns of β(3)-conglycinin and phaseolin, homologous storage 7S globulins, suggests the existence of intrinsic differences in their structures. This hypothesis is supported by comparative analysis of the accessibilities to the solvent of amino acid residues in phaseolin and β(3)-conglycinin structures, which indicated the relatively low packing density of the latter, resulting in enhanced susceptibility of it to extensive proteolysis.

Lignins are used often in formulations involving proteins but little is known about the surface interactions between these important biomacromolecules. In this work, we investigate the interactions at the solid-liquid interface of lignin with the two main proteins in soy, glycinin (11S) and β-conglycinin (7S). The extent of adsorption of 11S and 7S onto lignin films and the degree of hydration of the interfacial layers is quantified via Quartz crystal microgravimetry (QCM) and surface plasmon resonance (SPR). Solution ionic strength and protein denaturation (2-mercaptoethanol and urea) critically affect the adsorption process as protein molecules undergo conformational changes and their hydrophobic or hydrophilic amino acid residues interact with the surrounding medium. In general, the adsorption of the undenatured proteins onto lignin is more extensive compared to that of the denatured biomolecules and a large amount of water is coupled to the adsorbed molecules. The reduction in water contact angle after protein adsorption (by ~40° and 35° for undenatured 11S and 7S, respectively) is explained by strong nonspecific interactions between soy proteins and lignin.

The time course of glycinin hydrolysis by papain was followed using densitometry of SDS-PAGE patterns, quantification of the residual protein and determination of its molecular mass by gel filtration, and by other appropriate methods. The hydrolysis occurs in two steps. In the first step, a limited proteolysis was observed consisting of a gradual detachment of the α-chain C-terminal sequence region, leading to the formation of glycinin-P, a relatively stable proteolysis product retaining the primordial hexameric structure. Glycinin-P was found to be composed of the intact β-chains covalently bound with the C-terminally truncated α-chains lacking the helix domain, strand J', and the C-terminal disordered region. Glycinin-P is further hydrolyzed in the second step exclusively by a one-by-one mechanism. Comparison of the kinetics of the limited and one-by-one proteolyses analyzed separately indicated that the decrease of protein concentration by 24-25% in the first step occurs almost exclusively due to the decrease of the molecular mass of the residual protein. Thus, the onset of the one-by-one proteolysis is delayed, suggesting a regulatory role of the preceding limited proteolysis in the subsequent massive degradation of glycinin. Probable structural alterations of glycinin generated by this limited proteolysis are discussed., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

A subtilisin-like enzyme, soybean protease C1 (EC 3.4.21.25), initiates the degradation of the β-conglycinin storage proteins in early seedling growth. Previous kinetic studies revealed a nine-residue (P5-P4') length requirement for substrate peptides to attain optimum cleavage rates. This modeling study used the crystal structure of tomato subtilase (SBT3) as a starting model to explain the length requirement. The study also correlates structure to kinetic studies that elucidated the amino acid preferences of soybean protease C1 for P1, P1' and P4' locations of the cleavage sequence. The interactions of a number of protease C1 residues with P5, P4 and P4' residues of its substrate elucidated by this analysis can explain why the enzyme only hydrolyzes peptide bonds outside of soybean storage protein's core double β-barrel cupin domains. The findings further correlate with the literature-reported hypothesis for the subtilisin-specific protease-associated (PA) domain to play a critical role. Residues of the SBT3 PA domain also interact with the P2' residue on the substrate's carboxyl side of the scissile bond, while those on protease C1 interact with its substrate's P4' residue. This stands in contrast with the subtilisin BPN' that has no PA domain, and where the enzyme makes stronger interaction with residues on the amino side of the cleaved bond. The variable patterns of interactions between the substrate models and PA domains of tomato SBT3 and soybean protease C1 illustrate a crucial role for the PA domain in molecular recognition of their substrates., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.

β-conglycinin, a major seed protein in soybean, is composed of α, α', and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F₂ population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

The 7S and 11S globulins from soybean proteins using reverse micelle and aqueous buffer extraction methods were characterized by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscope (SEM), and their amino acid compositions were also evaluated. SDS-PAGE did not show electrophoretic differences between 7S and 11S globulin subunits with two extraction methods. SEM analysis showed that the AOT reverse micelle processing of 7S and 11S globulins induced a reduction of droplet size. Some individual amino acid contents of 7S and 11S globulins using two extraction methods were different, some were similar. In all the samples, the glutamic acid, aspartic acid, and leucine were the dominant amino acids while the cystine and methionine were the first-limiting amino acids. The proportion of essential amino acids to the total amino acids (E/T) of the 7S globulin from aqueous buffer and reverse micelles was similar. While significant differences were obtained in the proportion of E/T of the 11S globulin.

The effects of moisture and thermal denaturation on the solid-state structure and molecular mobility of soy glycinin powder were investigated using multiple techniques that probe over a range of length and time scales. In native glycinin, increased moisture resulted in a decrease in both the glass transition temperature and the denaturation temperature. The sensitivity of the glass transition temperature to moisture is shown to follow the Gordon-Taylor equation, while the sensitivity of the denaturation temperature to moisture is modeled using Flory's melting point depression theory. While denaturation resulted in a loss of long-range order, the principal conformational structures as detected by infrared are maintained. The temperature range over which the glass to rubber transition occurred was extended on the high temperature side, leading to an increase in the midpoint glass transition temperature and suggesting that the amorphous regions of the newly disordered protein are less mobile. (13)C NMR results supported this hypothesis.

Limited biochemical information is available on soybean accessions that have seed protein content greater than 45% of the seed dry weight. SDS-PAGE analysis of seed proteins from nine soybean accessions revealed significantly higher amount of seed storage proteins in these accessions when compared with that of soybean cultivar Williams 82. High-resolution two-dimensional gel electrophoretic analysis of seed proteins revealed significant differences among several seed storage protein components in these accessions. A total of 51 protein spots were identified using peptide mass fingerprinting (MALDI-TOF MS). The contribution of these proteins to the overall protein content of the accessions was quantified using Delta2D image analysis software. Results showed that among the majority of the nine accessions, the largest difference in higher protein quantity was within the seed 11S storage globulins. The high protein trait from PI407788A was successfully transferred to an experimental line, LG99-469, demonstrating that this trait was transferable and robust.

A peptic digest of soybean β-conglycinin (BconP) suppresses the appetite in rats through cholecystokinin (CCK) secretion by enteroendocrine cells. We investigate in this study more appetite-suppressing hydrolysates. β-Conglycinin hydrolyzed with food-processing proteases thermolysin (BconT), bromelain (BconB), chymotrypsin, protease S, and protease M was examined for CCK-secreting activity in a CCK-producing cell line for comparison with BconP. The potent CCK-releasing hydrolysates were then tested for their suppression of the food intake by rats. BconB, BconT, and BconP stimulated high CCK secretion, with the highest by BconB. Orogastric preloading by BconB, but not by BconT, suppressed the 60-min food intake. A meal-feeding trial twice a day in the morning (a.m.) and evening (p.m.) for 10 d showed that BconB preloading before every meal attenuated the p.m. meal size, but not that a.m., resulting in an overall reduction of the daily meal size. These results demonstrate that the bromelain hydrolysate of β-conglycinin having potent CCK-releasing activity suppressed the appetite of rats under meal-feeding conditions.

A truncated form of alpha' chain (talpha'), the soybean 7S globulin subunit previously demonstrated to be active in controlling the cholesterol and triglyceride homeostasis in in vitro and in vivo models, was cloned and expressed in the yeast Pichia pastoris. The recombinant polypeptide spanned 216 amino acid residues from the N-terminal side and included the N-terminal extension region of the soybean subunit. The talpha' polypeptide was purified by conventional biochemical techniques, and its potential to modulate the activity of low-density lipoprotein (LDL) receptor was evaluated in a human hepatoma cell line (Hep G2) by monitoring the uptake and degradation of labeled LDL. The LDL uptake (+192%) and degradation (+143%) by cells tested at the highest talpha' dose (8 microM) were similar to those found in cells incubated with 1 microM simvastatin, a potent inhibitor of cholesterol biosynthesis. The cell response to talpha' was found to be dose dependent. The use of a recombinant polypeptide ruled out the involvement of any other soybean component. These findings open new prospects in the studies aimed at identifying soybean regulatory (poly)peptide(s) and the mechanism involved in this biological response, as a gateway to their utilization for the management of human health., (Copyright 2010 Elsevier Inc. All rights reserved.)