Soy Β-conglycinin Research Articles

Limited Aggregation Behavior of β-Conglycinin and Its Terminating Effect on Glycinin Aggregation during Heating at pH 7.0

In this work, different thermal aggregation behaviors of soy β-conglycinin and glycinin at pH 7.0 were characterized with size exclusion chromatography and low-angle light scattering. Limited aggregation that grew via the consumption of "monomers" was detected in β-conglycinin, forming soluble aggregates. For glycinin, the association between the aggregates that led to the appearance of insoluble materials was observed. Heated with β-conglycinin, the assembly between the glycinin aggregates was terminated and its solubility was recovered. The structure of the soluble and insoluble aggregates was analyzed by small-angle X-ray scattering and dynamic light scattering. Unlike the β-conglycinin soluble aggregates that possessed limited size and less compact conformation, particles with a denser core and a less dense outer shell were found in the glycinin insoluble aggregates. Evidence is presented to reveal the transition between the soluble and insoluble aggregates and the role of β-conglycinin in the solubilization of the soy protein aggregates during heating.

Improvement of emulsifying properties of Maillard reaction products from β-conglycinin and dextran using controlled enzymatic hydrolysis

A novel emulsifier was prepared by conjugating soy β-conglycinin and dextran (MW 67 kDa) under dry-heated Maillard reaction followed by trypsin hydrolysis with the degree of hydrolysis (DH) at 2.2% and 6.5%. The emulsifying properties of β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates (DH 2.2% and DH 6.5%) were investigated using zeta-potential, droplet size and creaming index of the emulsions. The results showed that hydrolysates of β-conglycinin–dextran conjugates (DH 2.2%) were capable of forming a fine emulsion (d43 = 0.62 ± 0.04 μm, pH 7.0) which remained stable during 4 weeks of storage. A variety of physicochemical and interfacial properties of β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates were investigated. Hydrolysates of β-conglycinin–dextran conjugates (DH 2.2%) had a much higher fraction of protein adsorption (Fads) and a significantly lower saturation surface load (Γsat) compared with β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates (DH 6.5%). This might be due to its higher molecular flexibility, which benefited the adsorption and unfolding of peptide molecules at the droplet interface. These might explain its markedly improved emulsifying capability. The conjugation of β-conglycinin and dextran effectively enhanced the hydrophilicity of the oil droplets surfaces and improved the steric repulsion between the oil droplets. Therefore the emulsions were still stable after 4 weeks of storage against pH, ionic strength and thermal treatment. This study demonstrated that controlled enzymatic hydrolysis of protein–polysaccharide conjugates could be an effective method for preparing favourable emulsifiers.

Improvement of heat-induced fibril assembly of soy β-conglycinin (7S Globulins) at pH 2.0 through electrostatic screening

Protein fibril assembly at acidic conditions has recently attracted increasing interests in the fields of biochemical and food science, however, the obtained knowledge about the assembly of the proteins with complex structure (e.g. plant 7S/11S globulins) is still limited. This study investigated the influence of NaCl addition (0–300mM) on the fibril assembly of soy 7S globulins (β-conglycinin), induced by heating at 80°C and pH 2.0, with the aim of confirming the improvement of their fibril assembly by electrostatic screening. The formed fibril formation was investigated by using Th T fluorescence and atomic force microscopy (AFM) techniques. Heat-induced structure changes were traced by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectroscopy (CD). The results indicated that the whole fibril assembly experienced protein dissociation/hydrolysis and subsequent fibrillization process, and increasing NaCl concentration progressively increased the amount of formed fibrils, and even fibril length. AFM analyses confirmed that the increase in NaCl concentration shortened the time at which the short fibrils were visually observed, and the morphology of formed fibrils, including height of fibrils, width at half-height, and coil periodicity, slightly varied with the added NaCl concentration. The CD analyses indicated that increasing NaCl concentration greatly favored formation of β-type secondary structure, as well as extensive disruption of tertiary and/or quaternary conformations. These results confirmed that the electrostatic screening greatly improved the heat-induced fibril assembly process of soy β-conglycinin at pH 2.0. The improvement of fibril formation was largely attributed to increased extent of conformational changes at higher ionic strength. These findings would provide important information about the fibril assembly of plant oligomeric globulins at acidic pH that have important implications for the development of protein protein-based fibrillar gels.

Rice α-globulin decreases serum cholesterol concentrations in rats fed a hypercholesterolemic diet and ameliorates atherosclerotic lesions in apolipoprotein E-deficient mice

The hypocholesterolemic and antiatherogenic effects of rice α-globulin remain unclear. We investigated the hypocholesterolemic effect of rice α-globulin in rats fed a hypercholesterolemic diet. The rats were divided into 4 groups and were orally administrated the following three proteins or a vehicle for 4weeks: rice protein, rice α-globulin, or soy β-conglycinin at a dose of 100mg/kg body weight or carboxymethylcellulose to the control rats. In the rice α-globulin group, serum cholesterol concentrations were 28% lower than the control group and fecal neutral steroid excretion was increased by 30%. The hypocholesterolemic effect of rice α-globulin was equal to soy β-conglycinin in SD rats fed the hypercholesterolemic diet. However, the serum cholesterol concentrations in the rice protein group did not change compared to the control group. To investigate the antiatherogenic effects of rice α-globulin, male apolipoprotein E-deficient mice were orally administered the same dose of rice α-globulin for 9weeks. The en face lesion area in the aorta was 46% lower than in the control group. In conclusion, administration of rice α-globulin improves hypercholesterolemia in rats fed a hypercholesterolemic diet by increasing the fecal excretion of neutral sterols, and inhibits atherosclerosis development in apolipoprotein E-deficient mice. The anti-atherosclerotic effect exerts by mechanism(s) other than the regulation of serum MCP-1 and NO concentrations.

Growth Kinetics of Amyloid-like Fibrils Derived from Individual Subunits of Soy β-Conglycinin

The amyloid-like fibrillation of soy β-conglycinin subunits (α, α', and β) upon heating (0-20 h) at 85 °C and pH 2.0 was characterized using dynamic light scattering, circular dichroism (CD), binding to amyloid dyes (Thioflavin T and Congo red), and atomic force microscopy. The fibrillation of all three subunits was accompanied by progressive polypeptide hydrolysis. The hydrolysis behaviors, fibrillation kinetics, and morphologies of amyloid-like fibrils considerably varied among α, α', and β subunits. Faster hydrolysis rates and special fragments were observed for the α and α' subunits compared to the β subunit. However, the order of the fibrillation rate and capacity to form β-sheets was α' > β > α, as evidenced by CD and Thioflavin T data. Moreover, sequential growth of twisted screw-structure fibrils, leading to macroscopic fibrils with distinct morphological characteristics, was observed for β-conglycinin and individual subunits. The different fibrillation kinetics and morphologies of α, α', and β subunits appear to be associated with the differences in the amino acid composition and typical sequence of peptides. Besides, the disruption of ordered structure of fibrils occurred upon further heating (6-20 h) due to extensive hydrolysis. These results would suggest that all subunits are involved in the fibrillation of β-conglycinin, following multiple steps including polypeptide hydrolysis, assembly to amyloid structure, and growth into macroscopic fibrils with a fibril shaving process.

Enhanced Antiviral Activity of Soybean β‐Conglycinin‐Derived Peptides by Acylation with Saturated Fatty Acids

Peptide mixtures prepared from soybean β-conglycinin (7S-peptides) were acylated with saturated fatty acids of different chain length (6C-18C) in order to improve their antiviral activity against Feline calicivirus (FCV) strain F9 which is a typical norovirus surrogate. Among the fatty acids varieties, it was revealed that 7S-peptides acylated with myristic and palmitic acids potently inhibited FCV replication. Myristorylation and palmitoylation of 7S-peptides kept host cells viability at 91.51% and 98.90%, respectively. The infectivity of FCV on Crandell-Reese feline kidney cells was further determined after exposure of initial titer of 10(6.47) TCID₅₀/mL. Myristoylated and palmitoylated 7S-peptides significantly (P < 0.006) reduced FCV infectivity as compared to native 7S-peptides. Native 7S-peptides showed 25% FCV inhibitory activity while myristoylated and palmitoylated 7S-peptides exhibited 98.59% and 99.98% reduction in FCV infectivity, respectively. Myristoylated and palmitoylated 7S-peptides demonstrated higher anti-FCV activity in a wide range of concentration with complete reduction at 25 μg/mL. Surface hydrophobicity was significantly (P < 0.05) increased after attachment of long hydrocarbon fatty acids to 7S-peptides as supported by changes in fluorescence intensity. Enzymatic hydrolysis together with acylation will give an insight into surface and physiological functional lipopeptides derived from soy β-conglycinin.

Structural Rearrangement of Ethanol-Denatured Soy Proteins by High Hydrostatic Pressure Treatment

The effects of high hydrostatic pressure (HHP) treatment (100-500 MPa) on solubility and structural properties of ethanol (EtOH)-denatured soy β-conglycinin and glycinin were investigated using differential scanning calorimetry, Fourier transform infrared and ultraviolet spectroscopy. HHP treatment above 200 MPa, especially at neutral and alkaline pH as well as low ionic strength, significantly improved the solubility of denatured soy proteins. Structural rearrangements of denatured β-conglycinin subjected to high pressure were confirmed, as evidenced by the increase in enthalpy value (ΔH) and the formation of the ordered supramolecular structure with stronger intramolecular hydrogen bond. HHP treatment (200-400 MPa) caused an increase in surface hydrophobicity (F(max)) of β-conglycinin, partially attributable to the exposure of the Tyr and Phe residues, whereas higher pressure (500 MPa) induced the decrease in F(max) due to hydrophobic rearrangements. The Trp residues in β-conglycinin gradually transferred into a hydrophobic environment, which might further support the finding of structural rearrangements. In contrast, increasing pressure induced the progressive unfolding of denatured glycinin, accompanied by the movement of the Tyr and Phe residues to the molecular surface of protein. These results suggested that EtOH-denatured β-conglycinin and glycinin were involved in different pathways of structural changes during HHP treatment.

Role of β‐Conglycinin and Glycinin Subunits in the pH‐Shifting‐Induced Structural and Physicochemical Changes of Soy Protein Isolate

Soy β-conglycinin (7S) and glycinin (11S) were incubated up to 4 h in acidic (pH 1.5 to 3.5) or alkaline (pH 10 to 12) solutions to induce protein structural unfolding followed by refolding 1 h at pH 7.0, a process known as pH-shifting. The pH-shifting markedly increased (P < 0.05) emulsifying activity of 11S and to a lesser extent 7S; the former also produced more uniform oil droplets. The emulsifying activity improvements were accompanied by a significant rise in protein surface hydrophobicity, slight loss of the secondary structure (circular dichroism), and substantial dissociation of disulfide-linked basic and acidic 11S subunits. The findings suggested that 11S globulins of soy protein isolate (SPI) were more responsive to pH-shifting treatments than were 7S globulins, and the resulting emulsifying activity enhancements of 11S, in parallel with that of SPI, were indicative of its determinant role in the overall emulsifying properties of pH-shifting-treated SPI. Extreme alkaline (pH 12) and acidic (pH 1.5) medium treatments can significantly modify the structure and enhance the emulsifying properties of both β-conglycinin and glycinin components of SPI. The functionality improvement by the pH processes is more remarkable for the glycinin protein fraction. Therefore, SPI enriched with glycinin seems to be particularly suitable for extreme acidic or alkaline processes to produce surface-active functional ingredients for food applications.

Formation and Characterization of Amyloid-like Fibrils from Soy β-Conglycinin and Glycinin

The fibrillar aggregation at pH 2.0 of soy β-conglycinin, glycinin, and the 1:1 mixture thereof, induced by heating at 80 °C for various periods of time, was investigated using Th T and Congo Red spectroscopic techniques. The morphology of the formed fibrillar aggregates was characterized using atomic force microscopy (AFM), whereas the conformational changes and the polypeptide hydrolysis of the proteins upon heating were also evaluated. Th T fluorescence analysis indicated that β-conglycinin had a much higher potential to form heat-induced amyloid-like aggregates than glycinin. AFM analyses showed that all of the soy globulins could form twisted screw-structure fibrils with heights of 1.4-2.2 nm, but the morphology of the amyloid-like fibrils considerably varied among various soy proteins. Significantly lower width at half-heights and higher coil periodicity values were observed for the β-conglycinin fibrils than the glycinin counterpart. Far-UV CD spectral analysis indicated that upon heating, the secondary conformations of the proteins changed considerably, especially during initial heating (e.g., <4 h), and the changes were much more distinct in the β-conglycinin case than in the glycinin case. Furthermore, reducing electrophoresis analyses indicated that progressive polypeptide hydrolysis occurred upon heating, but the polypeptide hydrolysis for the β-conglycinin was much more severe than that of glycinin. The data suggest that soy β-conglycinin exhibited a much higher potential to form thermally fibrillar aggregates than glycinin, and the differences seem to be mainly associated with the differences in their conformational changes and extent of polypeptide hydrolysis by the heating. The results would be of vital importance for the utilization of soy proteins to produce thermally induced fibrillar gels with excellent properties.

Orally administered soymorphins, soy-derived opioid peptides, suppress feeding and intestinal transit via gut μ1-receptor coupled to 5-HT1A, D2, and GABABsystems

We previously reported that soymorphins, mu-opioid agonist peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic-like activity. The aim of this study was to investigate the effects of soymorphins on food intake and gut motility, along with their mechanism. We found that soymorphins decreases food intake after oral administration in fasted mice. Orally administered soymorphins suppressed small intestinal transit at lower dose than that of anorexigenic activity. Suppression of food intake and small intestinal transit after oral administration of soymorphins was inhibited by naloxone or naloxonazine, antagonists of mu- or mu(1)-opioid receptor, respectively, after oral but not intraperitoneal administration. The inhibitory activities of small intestinal transit by soymorphins were also inhibited by WAY100135, raclopride, or saclofen, antagonists for serotonin 5-HT(1A), dopamine D(2), or GABA(B) receptor, respectively. We then examined the order of activation of 5-HT(1A), D(2), and GABA(B) receptors, using their agonists and antagonists. The inhibitory effect of 8-hydroxy-2-dipropylaminotetralin hydrobromide, a 5-HT(1A) agonist, after oral administration on small intestinal transit was blocked by raclopride or saclofen. Bromocriptine, a D(2) agonist-induced small intestinal transit suppression, was inhibited by saclofen, but not by WAY100135. Baclofen, a GABA(B) agonist-induced small intestinal transit suppression, was not blocked by WAY100135 or raclopride. These results suggest that 5-HT(1A) activation elicits D(2) followed by GABA(B) activations in small intestinal motility. We conclude that orally administered soymorphins suppress food intake and small intestinal transit via mu(1)-opioid receptor coupled to 5-HT(1A), D(2), and GABA(B) systems.

Discovery of soy peptides from enzymatic hydrolysis of purified β‐conglycinin with potent fatty acid synthase inhibitory activity

Current evidence indicates that fatty acid metabolism pathway represents a potential target for obesity prevention. Inhibition of fatty acid synthase (FAS) results in decrease in food intake and body weight in animal models. In addition, FAS is over‐expressed in several tumor types; therefore, its inhibition is an important therapeutic opportunity inducing tumor cell‐specific apoptosis. The objectives were the discovery and characterization of FAS inhibitory peptides derived from purified soy β‐conglycinin. FAS inhibitory peptides were produced by enzymatic hydrolysis of pure soy β‐conglycinin (BCH) using alcalase (from Bacillus licheniformis), isolated by co‐immunoprecipitation and identified on a Q‐Tof API‐US nanoAcquity UPLC tandem ESI mass spectrometer. Inhibitory activity of BCH was assayed against purified FAS from chicken liver. Soybean BCH showed a potent FAS inhibitory activity (IC50 = 35 µM) which was higher than the positive control 4‐methylene ‐2‐ octyl‐ 5‐ oxotetrahydrofuran ‐3 ‐ carboxylic acid (IC50 = 76 µM). Three hydrophilic peptides KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE were identified. The molecular interactions of these peptides with FAS catalytic sites were evaluated by computational molecular docking. In conclusion, soy peptides were discovered for the first time with a potent FAS inhibitory activity.Supported by European Commission (PIOF‐GA‐2008‐219860), Cargill Co.

Anti-alopecia mechanisms of soymetide-4, an immunostimulating peptide derived from soy β-conglycinin

Previously, we found that orally administered soymetide-4 (MITL), an immunostimulating peptide derived from soybean β-conglycinin α′ subunit, suppressed alopecia induced by the anti-cancer drug etoposide in neonatal rats. Soymetide-4 has weak affinity for N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor. fMLP showed an anti-alopecia effect after intraperitoneal administration, though it was inactive after oral administration. Anti-alopecia effect of fMLP was blocked by pyrilamine or cimetidine, antagonists for histamine H 1 or H 2 receptor, respectively. However, the anti-alopecia effect of soymetide-4 was not inhibited by the histamine antagonists but by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP 4 receptor for PGE 2. Anti-alopecia effect of soymetide-4 was also blocked by pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-κB (NF-κB). These results suggest that PGE 2, which is produced after activation of COX by soymetide-4, might suppress apoptosis of hair matrix cells and etoposide-induced alopecia by activating NF-κB.

Characterization and Functional Properties of Soy β‐Conglycinin and Glycinin of Selected Genotypes

ABSTRACT: The principal soy storage proteins, β‐conglycinin and glycinin, may provide valuable functionality as food ingredients. These soy protein fractions were isolated from K1430, Hutcheson, K93‐90‐29, and KS4997 and characterized for solubility, emulsion and surface properties, water holding capacity, and gel rheology. There were significant differences among genotypes regarding all functional properties. The K93‐90‐29 β‐conglycinin exhibited significantly better functional properties compared to the other genotypes except for foam stability. Regarding glycinin, Hutcheson exhibited significantly better functional properties compared to the other genotypes except for foam stability. Continuous evaluation of soy genotypes in various food systems coupled with genetic manipulation should continue to better assess the potential of soy proteins as food ingredients.

The phosphorylation and characterisation of soybean isolate and its ?-conglycinin component by casein kinase II

A commercial soybean isolate was phosphorylated using casein kinase II purified from the yeast Yarrowia lipolytica. Both major reserve proteins, β-conglycinin and glycinin, were phosphorylated in a sequential way. The soybean isolate incorporated up to 0.7mol phosphate per mole in 2h. It was found that the phosphoester bonds were stable over time. The solubility of the phosphorylated isolate with respect to pH was not dramatically increased in comparison with the native one. However, counting the radioactivity of 32 P incorporated into the proteins (only the solubility of the phosphorylated proteins was measured in this case) showed that the solubility of the proteins was dramatically improved (up to 90% solubility for phosphorylated β-conglycinin at pH 4). β-Conglycinin became more soluble in the presence of CaCl 2 upon phosphorylation; this was not the case for the isolate. The iron-binding capacity of the soy isolate and β-conglycinin was significantly improved after phosphorylation (two and six times respectively).

Enzymatic phosphorylation of food proteins by purified and recombinant protein kinase CK2.

Protein kinase CK2 formerly called casein kinase II is a protein kinase able to phosphorylate more than 100 proteic substrates. We have purified protein kinase CK2 from the yeast Y. lipolytica to phosphorylate milk and plant reserve proteins to a significant extent. In the case of plant reserve proteins, which are polymeric substrates, not all subunits are substrate for protein kinase CK2, even if non phosphorylated subunits contain significant potent phosphorylations sites. Best substrates were soy beta-conglycinin (0.72 P/mol) and dephosphorylated caseins (0.5 P/mol). We have studied some functional properties of phosphorylated caseins. Solubility was improved for all pH values but pI. Sensitivity to calcium has also been assessed, and it is slightly improved upon phosphorylation. We have cloned the catalytic subunit of protein kinase CK2 from yeast Y. lipolytica. The recombinant catalytic subunit expressed in E. coli was active and displayed kinetic properties similar to those of the purified enzyme. The recombinant catalytic subunit was able to phosphorylate plant reserve proteins and milk proteins to a significant extent. Best substrates were soy beta-conglycinin (1.0 P/mol), and glycinin (0.59 P/mol).