Soy Glycinin Research Articles

Soy glycinin as food-grade Pickering stabilizers: Part. III. Fabrication of gel-like emulsions and their potential as sustained-release delivery systems for β-carotene

Formulation of food-grade Pickering emulsions has attracted growing attention in the food and drug fields, due to their promising potential as novel delivery systems for lipophilic bioactive ingredients. The present work reported that a kind of gel-like Pickering emulsions stabilized by heat (at 100 °C)-treated soy glycinin (SG; a major globulin in soy proteins), fabricated by modulating protein concentration (c) and/or oil fraction (ϕ), can perform as sustained release delivery systems for β-carotene. Rheological measurements and microstructural observations indicated that increasing c and/or ϕ resulted in a progressive strengthening of gel-like network in the emulsions stabilized by heated SG, which was closely related to a high percentage of proteins adsorbed at the interface, or entrapped within the network. In contrast, no distinct gel formation was observed for the emulsions stabilized by unheated SG. In vitro intestinal digestion experiments showed that for the emulsions (stabilized by unheated or heated SG) without distinct gel formation, the release behavior of β-carotene was mainly determined by droplet size, while in the case of gel-like emulsions by heated SG, the gel-like network formation greatly slowed down the β-carotene release (irrespective of the applied c and/or ϕ). All the test emulsions did not suffer a distinct degradation of β-carotene over the whole digestion process. The findings are of great interest for the development of novel sustained delivery systems for lipophilic components, based on Pickering emulsions stabilized by a kind of protein-based particle stabilizers.

Impeding <i>Bacillus</i> spore germination <i>in vitro</i> and in milk by soy glycinin during long cold storage

This paper presents a study of the magnitude and mechanism of impeding the emergent spore germination of Bacillus subtilis (non-hemolytic strain) and Bacillus licheniformis (hemolytic strain) under the action of soybean glycinin and its basic subunit (BS). Incubating B. licheniformis spores with 100 μg/ml of glycinin or BS at 35°C for 24 h totally prevented the hemolytic activity associated with the emergent vegetative cells on blood agar during 3-5 days of incubation at 35°C in contrast to the control. Glycinin and BS (100 μg/ml) also resulted in the leakage of 70 and 73% of the initially germinating spore contents of A260 absorbing materials of the two bacterial species after 2 h of incubation at 35°C, respectively. Increasing the concentration of glycinin and BS up to 400 μg/ml increased the germinating spore leakage to 83 and 88%, respectively. Spore germination in Muller Hinton Broth containing glycinin and BS (100 μg/ml) was practically nil after 4 days of incubation at 35°C. Supplementing milk preheated at 80°C/5 min with both glycinin and BS kept the final vegetative cell counts down to about 30 and 20% of the corresponding control, respectively, after 50 days of storage at 4°C. Both light, and transmission electron, microscopy images revealed significant morphological and structural distorting changes in BS-treated spores of B. licheniformis.

Three Peptides from Soy Glycinin Modulate Glucose Metabolism in Human Hepatic HepG2 Cells.

Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA), Ile-Ala-Val-Pro-Thr-Gly-Val-Ala (IAVPTGVA) and Leu-Pro-Tyr-Pro (LPYP), three peptides deriving from soy glycinin hydrolysis, are known to regulate cholesterol metabolism in human hepatic HepG2 cells. We have recently demonstrated that the mechanism of action involves the activation of adenosine monophosphate-activated protein kinase (AMPK). This fact suggested a potential activity of the same peptides on glucose metabolism that prompted us to also investigate this aspect in the same cells. After treatment with IAVPGEVA, IAVPTGVA and LPYP, HepG2 cells were analyzed using a combination of molecular techniques, including western blot analysis, glucose uptake experiments and fluorescence microscopy evaluation. The results showed that these peptides are indeed able to enhance the capacity of HepG2 cells to uptake glucose, via glucose transporter 1 GLUT1 and glucose transporter 4 GLUT4 activation, through the stimulation of protein kinase B Akt and adenosine monophosphate-activated protein kinase AMPK pathways, both involved in glucose metabolism.

Soy glycinin as food-grade Pickering stabilizers: Part. II. Improvement of emulsification and interfacial adsorption by electrostatic screening

The development of food-grade particles as Pickering stabilizers has recently attracted increasing attention, and for protein-based particles, the role of surface charge in their emulsifying and interfacial properties is still little understood. The work reported that for the nanoparticles of heated (at 100 °C) soy glycinin (SG), the electrostatic screening by addition of increasing concentrations (0–500 mM) of NaCl was remarkably favorable for their emulsification performance and interfacial packing at oil-water interface. The influence of increasing ionic strength (μ) on the ability of these nanoparticles to form a gel-like emulsion was also investigated. In general, increasing the μ resulted in a progressive decrease in emulsion size and surface coverage at the interface, indicating improvement of the emulsification performance and interfacial packing. The improvement of emulsification and interfacial packing was largely associated with the enhanced diffusion and/or adsorption at the interface. If a high energy input of emulsification (microfluidization) was applied, a kind of gel-like Pickering emulsions stabilized by these nanoparticles would be produced. At protein concentrations of 0.5–2.0% (w/v), increasing the μ progressively increased the stiffness of the resultant gel-like emulsions. The results confirmed the importance of surface charge to the emulsification and interfacial behavior of protein-based (nano)particles, as well as formulation of Pickering emulsions stabilized by charged particles.

Impact of extraction and elution media on non-size effects in size exclusion chromatography of proteins

Size exclusion chromatography is extensively used to separate proteins and to determine their apparent molecular weights. It separates proteins based on hydrodynamic volume, but interactions between the chromatography resin and proteins lead to non-size effects. This report discusses the impact of co-solvents [salt, urea, sodium dodecyl sulfate (SDS), dithiothreitol] in extraction media when separating wheat gluten proteins, soy glycinin, bovine serum albumin and ovalbumin on a Biosep-SEC-S4000 column. With acetonitrile/water (1:1, v/v) containing 0.05% (v/v) trifluoroacetic acid as eluent, salts and SDS in the extraction media increase while urea decreases non-size effects. Most gluten and globular proteins are extractable in sodium phosphate buffer (0.050M; pH 6.8) containing 2.0% (w/v) SDS. This chromatographic medium allows analyzing mixtures of various proteins without any non-size effects.

Soy glycinin as food-grade Pickering stabilizers: Part. I. Structural characteristics, emulsifying properties and adsorption/arrangement at interface

The development of food-grade Pickering stabilizers has recently attracted growing interests, due to their potential applications in food and pharmaceutical formulations. We report that glycinin (SG), one of major globulins in soy proteins, exhibits a very promising potential to perform as a kind of novel food-grade Pickering stabilizers for oil-in-water emulsions. The unheated SG at pH 7.0 was mainly present in the nanoparticle form (with a z-average diameter of 57 nm), and the heating at 90 or 100 °C considerably increased the particle size and surface hydrophobicity (by 5–6 fold) of the nanoparticles. The heating also remarkably changed the pattern of intra-particle interactive forces, and for the heated SG nanoparticles, their internal structure was mainly maintained by both hydrophobic interactions and disulfide bonds. The heating greatly improved the emulsification performance of the nanoparticles, especially at high concentrations (e.g. > 0.5%, w/v), as well as the stability of the corresponding emulsions against coalescence and creaming. As compared with the SG preparation heated at 90 °C, that at 100 °C exhibited a higher effectiveness to pack at the interface. The better emulsification performance for the heated SG preparations was closely associated with their more efficient packing and higher diffusion (or initial adsorption) at the interface. The results indicated that SG nanoparticles, unheated or heated, could be applied as a kind of Pickering stabilizers for oil-in-water emulsions; as compared to the unheated SG, the heated SG nanoparticles exhibited much better interfacial and emulsifying properties, thus imparting a better Pickering stabilization for the emulsions.

IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin, modulate cholesterol metabolism in HepG2 cells through the activation of the LDLR-SREBP2 pathway

Although the hypocholesterolaemic activity of soy foods has been well known for decades, its molecular mechanism is still poorly understood. The aim of this study was a detailed molecular characterization of the activity of IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin hydrolysis, which in the past had been shown to be competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR). HepG2 cells were treated with these peptides and the samples were analysed with a combination of molecular techniques. The experiments demonstrated that IAVPGEVA, IAVPTGVA, and LPYP are able to interfere with the catalytic activity of HMGCoAR and to modulate the cholesterol metabolism, through the activation of the LDLR-SREBP2 pathway, increasing the ability of HepG2 cells to uptake the LDL. This also involves the activation of AMPK and ERK 1/2. For the first time, this study provides a characterization of the molecular mechanism through which peptides derived from soy glycinin exert their cholesterol-lowering activity.

Characterization of Thermal Denaturation Structure and Morphology of Soy Glycinin by FTIR and SEM

The conformation and microstructure of soy glycinin were investigated by Fourier transform infrared spectroscopy and scanning electron microscopy after dry heating and autoclaving thermal treatments. The changes in frequency and signal intensity of infrared bands revealed the thermal denaturation on the solid-state structure of glycinin. The Fourier transform infrared spectral changes were subsequently assessed using the second derivative spectroscopy in the amide I region (1700–1600 cm−1). The bands at 1618 cm−1 and 1682 cm−1 were considered to reflect the formation of intermolecular and intramolecular aggregates (A1 and A2), and the contents of β -sheet indicated the degree of denaturation. In autoclaved samples, the contents of the glycinin α -helix, turn, random coil, and A2 significantly increased (P < 0.05), while the contents of the glycinin β -sheet and A1 significantly decreased (P < 0.05). The dry heating slightly affected the secondary structures of glycinin. The scanning electron microscopy results showed that the autoclaving treatment glycinin had denser and more uniform gel with homogeneous pores and heterogeneous granular structure, and the dry heating glycinin had crumbled multi-layered sheet like structure. The data suggested that the conformations of the autoclaved glycinins had more changes than dry thermally treated glycinins. While both dry thermally treated and autoclaved treated samples showed a high content in β -sheet structures which may adversely affect protein utilization.

Stable and pH-sensitive protein nanogels made by self-assembly of heat denatured soy protein.

In this study, we examined the possibility of preparing stable soy protein nanogels by simply heating homogeneous soy protein dispersion. The protein nanogels formed were characterized by z-average hydrodynamic diameter, polydispersity index, turbidity, ζ-potential, morphology, and their stability to pH and ionic strength change. Soy protein dispersion (1% w/v) was homogeneous around pH 5.9 where it had the lowest polydispersity index (∼0.1). Stable and spherical nanogels were formed by heating soy protein dispersion at pH 5.9 under 95 °C. They sustained constantly low polydispersity index (∼0.1) in the investigated pH range of 6.06-7.0 and 2.6-3.0. The nanogels were pH-sensitive and would swell with pH change. They were stable at 0-200 mM NaCl concentration. Denaturation of soy glycinin was the prerequisite for the formation of stable nanogels. Soy protein nanogels had a core-shell structure with basic polypeptides and β subunits interacting together as the hydrophobic core; and acid polypeptides, α', and α subunits locating outside the core as hydrophilic shell. The inner structure of soy protein nanogels was mainly stabilized by disulfide bonds cross-linked network and hydrophobic interaction. Soy protein nanogels made in this study would be useful as functional ingredients in biotechnological, pharmaceutical, and food industries.

Structural modification in condensed soy glycinin systems following application of high pressure

The structural behaviour of soy glycinin systems following application of industrially relevant high hydrostatic pressure (600 MPa for 15 min at ambient temperature) was investigated throughout the experimental range up to 80% (w/w) solids content, and results were compared to conventional thermal treatment. Using small-deformation dynamic oscillation in shear, modulated differential scanning calorimetry, infrared spectroscopy and X-ray scattering, it was demonstrated that soy glycinin with twelve disulphide linkages displays extensive unfolding at low to intermediate solid levels (30–60%, w/w). In contrast, it largely maintains native conformation at 70 and 80% (w/w) solids showing about 20% denaturation, as compared to the thermal transition of native counterparts. Experimental data from infrared spectroscopy also argue for retention of the native conformation in condensed soy glycinin systems comprising mainly beta sheets in the secondary structure. At subzero temperatures, condensed glycinin at atmospheric and pressurised conditions undergoes vitrification phenomena recording experimental glass transition temperatures. Experimental data were successfully modelled using theoretical frameworks for mechanical studies on amorphous synthetic polymers.

Characterization of oil-in-water emulsions stabilized by tyrosinase-crosslinked soy glycinin

The effect of crosslinked soy glycinin with tyrosinase from Bacillus megaterium (TyrBm) on o/w emulsion properties was studied. The ability of TyrBm to crosslink soy glycinin was evaluated in the presence or absence of three phenolic mediators. It was observed that crosslinking of glycinin is facilitated by a phenolic mediator and is negligible in its absence. Subsequently, the glycinin-stabilized emulsions were evaluated in two systems: (i) homogenization after crosslinking in the presence of a mediator, caffeic acid, and (ii) homogenization prior to crosslinking in the absence of caffeic acid. Emulsions were prepared using a high-pressure homogenizer and their particle size, creaming resistance, viscosity and microstructure were measured. Results indicate that the method of emulsion preparation affected the emulsion physical stability, thus, the first system led to a decrease in emulsion stability against creaming while the second system resulted with improved properties after the enzymatic treatment; Crosslinking after homogenization eliminated the need for a phenolic mediator and led to a lower creaming velocity and higher viscosity. In addition, fluorescence microscopy observations demonstrated that the crosslinking reaction of TyrBm after homogenization led to the formation of cold-set gel-like structures of small droplets linked by covalent bonds.

Synergistic foaming and surface properties of a weakly interacting mixture of soy glycinin and biosurfactant stevioside.

The adsorption of the mixtures of soy glycinin (11S) with a biosurfactant stevioside (STE) at the air-water interface was studied to understand its relation with foaming properties. A combination of several techniques such as dynamic surface tension, dilatational rheology, fluorescence spectroscopy, and isothermal titration calorimetry (ITC) was used. In the presence of intermediate STE concentrations (0.25-0.5%), the weak binding of STE with 11S in bulk occurred by hydrophobic interactions, which could induce conformational changes of 11S, as evidenced by fluorescence and ITC. Accordingly, the strong synergy in reducing surface tension and the plateau in surface elasticity for mixed 11S-STE layers formed from the weakly interacting mixtures were clearly observed. This effect could be explained by the complexation with STE, which might facilitate the partial dissociation and further unfolding of 11S upon adsorption, thus enhancing the protein-protein and protein-STE interfacial interactions. These surface properties were positively reflected in foams produced by the weakly interacting system, which exhibited good foaming capacity and considerable stability probably due to better response to external stresses. However, at high STE concentrations (1-2%), as a consequence of the interface dominated by STE due to the preferential adsorption of STE molecules, the surface elasticity of layers dramatically decreased, and the resultant foams became less stable.

Binding Interactions of β-Conglycinin and Glycinin with Vitamin B12

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.

Inactivation of bacterial proteases and foodborne pathogens in condensed globular proteins following application of high pressure

The present investigation deals with the effectiveness of high pressure processing as a preservation technique for protein based materials. Increasing protein concentration up to 80% (w/w) in bovine serum albumin (BSA), soy glycinin and ovalbumin systems results in overall reduction in foodborne pathogens (i.e. Staphylococcus aureus, Bacillus cereus and Escherichia coli) for atmospheric samples due to reduced water activity. Application of high pressure (600 MPa for 15 min) reduces further microbial counts in these systems at comparable levels of solids. The effect of high hydrostatic pressure on proteolytic activity of proteases from Pseudomonas fluorescens strains 73 and 113 with globular proteins as substrates throughout the experimental range of protein concentration (10–80% w/w) has also been observed. Enzymatic activity in samples pressurized at 600 MPa for 15 min declined considerably, as compared to atmospheric counterparts. In addition, glycinin with the highest water holding capacity exhibits increased protease and microbiological activity at a given level of solids and treatment. Our work demonstrates that high pressure processing can act as an effective means of reducing microbial and enzymatic activity in condensed systems of globular proteins.

Characterization and formation mechanism of proteins in the secondary precipitate of soy sauce

To reveal the characteristics and formation mechanism of proteins in the secondary precipitate of soy sauce (SPSS), proteins in the supernatant of soy sauce were used as control, and SPSS was prepared by centrifugation in combination with lyophilization. The proteins in SPSS were isolated and identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. Acidic polypeptide A1a of soy glycinin G1 (32–35 kDa) and basic polypeptide B3 of soy glycinin G4 (23 kDa) were verified as the predominant proteins (95.2 %) in SPSS. The amino acid composition, average hydrophobicity (HΦavg), and the secondary structure of the proteins in SPSS were investigated by high-performance liquid chromatography and Fourier transform infrared spectroscopy. Results revealed that SPSS contained not only proteins but also free amino acids; the significantly higher average hydrophobicity and much lower contents of β-sheet and random coil were the main characteristics and reasons for the formation of proteins in SPSS.

Water-Wettable Polypropylene Fibers by Facile Surface Treatment Based on Soy Proteins

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.

Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.

Molecular modelling for investigating structure–function relationships of soy glycinin

Increasing global food protein demand drives research to improve existing sources for efficient use or to convert unconventional sources to mainstream protein ingredients. The structure at all levels is the most important intrinsic property that dictates suitability of a protein for food use. High throughput sequencing has facilitated genome mapping of food plants at an accelerated pace, however, the information is poorly utilized in food protein research. Use of bioinformatics for data mining and molecular modelling in revealing structure–function relations is discussed using soybean glycinin as a model protein. This in silico approach is complementary to the process of understanding food protein molecule structure, and linking molecule physico-chemical properties with the functionalities that protein provides in food.

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.

Soybean glycinin (11S) increases HDL‐cholesterol in hypercholesterolemic rats

PurposeThe purpose of this paper is to determine the effects of isolated soy glycinin (11S) on lipid metabolism in animals subjected to a hypercholesterolemic diet.Design/methodology/approachMale Wistar rats were kept in individual cages under appropriate conditions. The animals were divided into three groups (n=9): normal diet (STD) given a diet containing casein as protein source, recommended in AIN‐93M; hypercholesterolemic (HC) fed a normal diet with 1 per cent cholesterol and 0.5 per cent cholic acid; and hypercholesterolemic+glycinin (HC+11S), fed a hypercholesterolemic diet, plus 11S soy protein (300 mg/kg/day), dissolved in saline and administered by gavage. After 28 days, the animals were sacrificed and blood and liver removed for biochemical analysis of total cholesterol (TC), HDL‐cholesterol (HDL‐C) and triglycerides (TG) in the plasma, hepatic TC and TG.FindingsA single daily dose of glycinin given to the hypercholesterolemic group demonstrated its functional role, particularly in raising HDL‐C and reducing triglycerides in the liver.Originality/valueThis study demonstrates the action of the 11S globulin in soybean as a serum lipid lowering agent, in addition to its nutritional properties, especially in raising the HDL‐C.