11S Soy Research Articles

SOY PEPTIDE AND CHOLESTEROL HOMEOSTASIS

The aim of the present investigation was to evaluate the effect of a synthetic peptide, corresponding to a sequence which differs between the alfa and alfa’ subunits from 7S soy globulin, on cell cholesterol homeostasis both in HepG2 cells and in rats fed casein-cholesterol diet. Hep G2 cells were incubated for 24 hr in minimum essential medium (MEM) + 5% LPDS in the presence/absence of whole 7S globulin (0.5 mg/mL) or its alfa’ subunit (0.125 mg/mL) or the synthetic peptide (10-5 M and 10-6 M). Cell preincubation with whole 7S soy globulin caused an increase in the mature forms of SREBP-1, both in membranes (+ 90%) and in nuclei (+ 130%), vs that found in the control Hep G2 cells (MEM + 5% LPDS). Moreover, a lower level of mature nuclear SREBP-1 was detected in Hep G2 cells exposed to the alfa’ subunit compared to that found after exposure to whole 7S globulin. Hep G2 cells exposed to the synthetic peptide, at concentration of 10-5 M and 10-6 M, showed an increased 125I-LDL uptake (+ 38% and + 42%, respectively) and degradation (+46% and +53%, respectively) vs values recorded in non exposed cells; a dose-dependent increase in the SREBP2 mRNA and LDL-receptor mRNA was detected by RT-PCR in the same cell line exposed to the synthetic peptide (10-5 M and 10-6 M). The oral administration (14 days) of synthetic peptide (5 mg/Kg b.w.) to casein-cholesterol fed rats resulted in lower plasma lipid levels (cholesterol, -25%; triglycerides, −28%) vs values recorded in rats fed casein-cholesterol diet alone. These results represent the first in vivo evidence of the plasma lipid-lowering properties induced by the synthetic peptide in an animal model of human hypercholesterolemia. Supported in part by a grant from MIUR of Italy

Determination of Allergenic Cross-Reactivity between 11S-Globulins (Seed Storage Proteins) in Peanut, Tree-Nut and Sesame Allergic Patients Using Peptide Microarray Immunoassay

RATIONALE: The 11S-globulins of peanut and tree-nuts are highly homologous. Structural similarity among proteins is believed to be the molecular basis for immunological and clinical cross-sensitization, which is common in peanut and tree-nut allergic patients.METHODS: Commercially synthesized peptides covering the 11S-globulin acidic subunit of peanut and the acidic and basic subunits of hazelnut, Brazil nut, cashew, walnut and sesame seed were site-specifically bound to epoxy-derivatized glass slides at two locations in triplicate. Peptides were 20 residues in length with an offset of four. A total of 102 sera from patients allergic to at least one tree-nut or peanut were assayed. Sera from 10 non-atopic volunteers and 13 atopic but not peanut/tree-nut allergic individuals served as negative controls. Specific IgE- and IgG4- binding was detected using fluorochrome-labeled monoclonal secondary antibodies.RESULTS: We mapped IgE and IgG4 epitopes of multiple 11S allergens in parallel by microarray immunoassay. Seven cross-reactive antigenic regions were defined by shared recognition between at least two of the six homologues. Unexpectedly, these regions did not coincide with regions of highest identity. Extrapolating from the soy 11S structure, the majority of these sites are expected to be on the surface of the native protein.CONCLUSIONS: Clinical cross-reactivity between peanut and nut allergic individuals is not explained by primary sequence homology of 11S-globulins, as determined by peptide microarray analysis. RATIONALE: The 11S-globulins of peanut and tree-nuts are highly homologous. Structural similarity among proteins is believed to be the molecular basis for immunological and clinical cross-sensitization, which is common in peanut and tree-nut allergic patients. METHODS: Commercially synthesized peptides covering the 11S-globulin acidic subunit of peanut and the acidic and basic subunits of hazelnut, Brazil nut, cashew, walnut and sesame seed were site-specifically bound to epoxy-derivatized glass slides at two locations in triplicate. Peptides were 20 residues in length with an offset of four. A total of 102 sera from patients allergic to at least one tree-nut or peanut were assayed. Sera from 10 non-atopic volunteers and 13 atopic but not peanut/tree-nut allergic individuals served as negative controls. Specific IgE- and IgG4- binding was detected using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: We mapped IgE and IgG4 epitopes of multiple 11S allergens in parallel by microarray immunoassay. Seven cross-reactive antigenic regions were defined by shared recognition between at least two of the six homologues. Unexpectedly, these regions did not coincide with regions of highest identity. Extrapolating from the soy 11S structure, the majority of these sites are expected to be on the surface of the native protein. CONCLUSIONS: Clinical cross-reactivity between peanut and nut allergic individuals is not explained by primary sequence homology of 11S-globulins, as determined by peptide microarray analysis.

Detection of 7S and 11S soy protein fractions by monoclonal antibody-based immunoassays in food

A panel of four monoclonal antibodies (MAbs) recognizing antigenic determinants on soy protein was produced and partially characterized. The protein fractions 7S and 11S were extracted and purified from the soy protein isolate and defatted flour of soy, respectively. To obtain hybridomas, popliteal lymph nodes of BALB/c immunized mice (10,0 µg protein/footpad) were excised and fused to SP2-0 myeloma cells. The hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) using peroxidase-labelled rat anti-mouse IgG MAb and cloned under limiting dilution conditions. The specificity of these antibodies was evaluated by Western blotting. Three antibodies produced by stable clones (1H4, 2A8 and 1F9) recognized the subunits α, α′ and β of the 7S soy protein fraction and one monoclonal antibody (3F2) recognized the basic subunit of the 11S fraction. ELISA assays with the MAbs anti-7S (IgG2b) and 11S (IgG1) fractions were able to detect the presence of these fractions in soy-containing commercial products as yogurt, milk and juice. The present study showed that these MAbs specifically detect the subunits of the 7S fraction and the basic subunits of the 11S fraction of soy protein and may be used in immunoassays to detect these protein fractions in soy-based products.

Relationship between structural changes and functional properties of soy protein isolates–carrageenan systems

The aim of this work was to study the change in the structural and functional properties of a native soy protein isolate (I) induced by the incorporation of carrageenan (C). Two types of samples were used: samples lyophilized from mixtures of I and C dispersions (IC ratio: 20:1 to 8:1), and samples prepared in situ with I and C powders (IC ratio: 5:1 to 1:1). In both kinds of I:C samples a new endotherm (DSC), which may be attributed to the IC-complex, and an increase in the thermal stability of 7S and 11S soy globulins was observed. In in situ samples, at a high ionic strength and C content, an increase in the temperature of this new endotherm was observed. 7S fraction was thought to be further involved in the formation of the complex; result confirmed through experiments carried out with 7S-C and 11S-C mixtures. The complex formation modified the pH-solubility profile of soy proteins. As the amount of C increased, a decrease in solubility—to a greater extent in the acidic zone—was observed. The loss of solubility was accompanied by a decrease in the surface hydrophobicity of soluble fractions. Electrostatic as well as hydrophobic interactions would be involved in the complex formation, depending on environmental conditions. Concerning the rheological behavior of such systems, the addition of C allowed the formation of gels of a greater gelation velocity and higher viscoelasticity compared to I dispersions. Results showed that lyophilized I:C mixtures can be employed as gelling agents with improved properties regarding independent ingredients. On the other hand, gelation of I dispersions of very low protein concentration can be performed through the addition of carrageenan in appropriate proportions.

Ameliorative effects of soy 11S protein on liver damage and hyperlipidemia in alcohol-fed rats.

This study sought to investigate the ameliorating effects of soy 11S protein on the impacts of alcohol consumption in rat hepatocytes and in reducing total cholesterol levels and total lipid levels in the serum. Liver histology and the clinically important enzyme markers (Aspartate Aminotransferase: AST and Alanine Aminotransferase: ALT) of rats, administered with both alcohol and soy 11S protein treatments, were compared with those in the control group. The treatment regimen (11S soy protein extract) significantly reduced serum ALT and AST levels, indicating the hepato-protective effects of soy 11S protein. Furthermore, total cholesterol and total lipid levels were significantly reduced. In addition to preventing the presence of lipid droplets and secondary lysosomes, electron microscopy indicated that the administration of the soy 11S protein treatment preserved important hepatocyte structures. These results indicate that soy 11S protein can positively mediate the effects of alcohol on hepatocytes and general liver functions.

Functional properties of the acidic and basic subunits of the glycinin (11S) soy protein fraction

AbstractThe functional properties of low‐ and high‐M.W. (LMW and HMW, respectively) acidic subunits and the basic subunit separated from the 11S soy protein fraction were studied and compared with the functional properties of the 11S fraction. Among the functional properties investigated were solubility, emulsification, and viscosity. The results showed that the LMW acidic subunit had higher solubility than the HMW acidic subunit. Among all the samples, the LMW subunit separated by using β‐mercaptoethanol (ME) was the most soluble, with a solubility of 98–100% at a pH of 6–12. The solubility profile of the HMW subunit followed a pattern similar to the solubility of 11S. The lowest solubility was observed around pH values in the range close to the isoelectric point for both the LMW and HMW subunit. The basic subunit was not soluble in the pH range 3–10; however, the solubility increased more than 50% at pH 13 compared to the solubility at pH 10. The emulsification capacity of all subunits was higher than 11S in the following descending order: LMW, basic, HMW, 11S. Emulsification activity and stability of the subunits were greater than those of the 11S samples at room temperature and 95°C. With the exception of the LMW subunit separated with ME, the subunits had a higher viscosity than 11S. The basic subunit separated with sodium bisulfite had the highest viscosity of all the samples tested.

Interaction of Myofibrillar and Preheated Soy Proteins

ABSTRACT: Soy protein isolate (SPI) was preheated at 90 °C and 95 °C for 3 min to obtain preheated samples, SPI90 and SPI95, respectively. The preheat treatment increased protein hydrophobicity and decreased the aggregation of 11S acidic and basic subunits. The 7S and 11S soy proteins exhibited a decreased thermal stability when mixed with pork myofibrillar protein isolate (MPI). The presence of preheated SPI accelerated the disappearance of myosin heavy chain in the gelling process. Incorporation of preheated SPI significantly increased the MPI gel elasticity and hardness while native SPI showed negative effects.

Comparative study of foaming properties of whey and isolate soybean proteins

Whey soy proteins (WSP) is obtained from the soluble fraction separated in the isoelectric precipitation of the major components of native soy isolate (NSI), the 7S and 11S fractions. WSP is composed mainly by Kunitz Trypsin Inhibitor and Lectin. Water solubility of WSP was less affected by pH than that of NSI. Therefore, WSP has good foaming capacity in all pH range (pH 2–10). According to the rate of liquid incorporation into the foam, WSPs have higher tensioctive behavior, and in consequence, they are rapidly adsorbed into the water–air interface. At extreme pHs, with low or high ionic strength, good foams were obtained with WSP and NSI. Mixtures of both in different relations, showed better stabilization and foam formation than that expected for the simple additivity of both preparations. This effect was more evident at high ionic strength. These results indicate that there is a synergistic effect between these proteins in relation to foaming properties.

Emulsifying properties of high pressure treated soy protein isolate and 7S and 11S globulins

The influence of high pressure (HP) treatment (200–600 MPa) on the emulsifying activity index (EAI) and emulsifying stability index (ESI) on the 7S and 11S globulins and soya protein isolate (SPI) at pHs 7.5 and 6.5, at different concentrations (0.25–0.75%) was studied. Solubility and surface hydrophobicity were used as indices of the degree of denaturation caused by HP. 7S showed the highest EAI and surface hydrophobicity after treatment at 400 MPa, whereas 11S showed its highest EAI and surface hydrophobicity after treatment at 200 MPa. No significant correlation ( P>0.05) was found between solubility and EAI or hydrophobicity. SPI showed the optimum value of EAI after treatment at 400 MPa although its surface hydrophobicity was low. It is suggested that pressure at 400 MPa dissociated the 7S of the SPI into partially or totally denatured monomers that enhanced the surface activity but at the same time, the unfolding of the polypeptides of the 11S within the hexamer led to aggregation, negatively affecting the surface hydrophobicity of the SPI. The ESI values for the non-treated samples of SPI, 7S and 11S were higher at lower concentrations. At the same pH and concentration, the ESI decreased with increasing HP, except for the 7S at pH 7.5 and a protein concentration of 0.75%.

Thermal and mechanical properties and water absorption of sodium dodecyl sulfate‐modified soy protein (11S)

AbstractThe thermal and mechanical properties and water absorption of sodium dodecyl sulfate (SDS)‐modified 11S soy protein and molded plastics made from it were studied using differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), mechanical tests, and scanning electron microscopy (SEM). The DSC results showed that both the temperature and enthalpy of thermal denaturation of modified 11S solutions decreased as the SDS concentration increased. Nonfreezing water of the modified 11S solution had a minimum value at 1.0% SDS. The ordered structure of SDS‐modified 11S protein was recovered and/or newly formed during the freeze‐drying process. Both DSC and DMA results showed that SDS was a plasticizer of 11S, and the glass transition temperature of modified 11S plastics decreased with increasing SDS concentration. Both the tensile strength and elongation of modified 11S plastics first decreased and then increased as the SDS concentration increased, and 5.0% SDS‐modified 11S plastic had the highest tensile strength and elongation. The SEM observations supported these results. A water‐absorption test showed a reduction in the water resistance of 11S plastics after SDS modification. The rate of water absorption increased with increasing SDS concentration. The hydrophobic interaction between SDS molecules and 11S protein was found to have important effects on the thermal and mechanical properties and the water absorption of 11S plastics. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 166–175, 2001

Soy Protein Peptides Regulate Cholesterol Homeostasis in Hep G2 Cells

The activation of LDL receptors was described recently in a human hepatoma cell line (Hep G2) exposed both to alpha + alpha' subunits from 7S soy globulin and to Croksoy(R)70, a commercial isoflavone-poor soy concentrate. To assess the final identity of the peptide(s) putatively responsible for the biochemical effect, experiments were performed in Hep G2 cells, exposed either to synthetic peptides corresponding to specific sequences of 7S soy globulin or to peptides from the in vitro digestion of Croksoy(R)70. Moreover, the ability of the whole 7S globulin, its subunits and whole Croksoy(R)70 to interfere in the apolipoprotein B (apo B) secretion in the medium as well as in sterol biosynthesis was evaluated in the same model. Increased (125)I-LDL uptake and degradation vs. controls were shown after Hep G2 incubation with a synthetic peptide (10(-)(4) mol/L, MW 2271 Da) corresponding to positions 127-150 of the 7S globulin. Cells exposed to Croksoy(R)70 enzyme digestion products showed a more marked up-regulation of LDL receptors vs. controls, compared with vs. Hep G2 cells incubated with undigested Croksoy(R)70. Among soy-derived products, only the 7S globulin inhibited apo B secretion and (14)C-acetate incorporation when tested in Hep G2 cells at a concentration of 1.0 g/L. These findings support the hypothesis that if one or more peptides can reach the liver after intestinal digestion, they may elicit a cholesterol-lowering effect. Moreover, the protein moiety, devoid of isoflavone components, is likely to be responsible for this major biochemical effect of soy protein.

Thermal transitions and dynamic gelling properties of oxidatively modified myosin, β‐lactoglobulin, soy 7S globulin and their mixtures

Thermal transitions and dynamic gelling properties of oxidatively modified myosin, β‐lactoglobulin, soy 7S globulin and their mixtures

Thermal and mechanical properties and water absorption of guanidine hydrochloride-modified soy protein (11s)

Thermal and mechanical properties and water absorption of guanidine hydrochloride (GuHCl)-modified 11S soy protein and molded plastics made from it were studied using differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), mechanical tests, and scanning electron microscopy (SEM). The DSC results showed that the denaturation temperature of GuHCl-modified 11S solutions was higher than that of the control sample and the high concentration GuHCl completely denatured 11S. Nonfreezing water of the modified 11S solution exhibited a minimum value at 0.9M GuHCl. Both DSC and DMA results showed that GuHCl was a plasticizer of 11S and the glass transition temperature of modified 11S plastics decreased with increasing GuHCl concentration. Both the stress and strain of modified 11S plastics reached their highest values at a 0.9 GuHCl concentration. The SEM observations supported these results. A water-absorption test showed an improvement in the water resistance of 11S plastics with GuHCl modification. The water absorption had a minimum value at 0.9M GuHCl. The interaction between GuHCl molecules and 11S protein was found to have important effects on the thermal and mechanical properties and the water absorption of 11S plastics. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 1063–1070, 2000

Oxidatively induced chemical changes and interactions of mixed myosin, ?-lactoglobulin and soy 7S globulin

The effects of oxidation on the chemical characteristics of myosin, β-lactoglobulin and soy 7S globulin were investigated in a free radical-generating system (FeCl 3 /H 2 O 2 /ascorbate). Oxidised myosin exhibited a marked increase (> 10-fold) in carbonyls, a small increase in amines and conversion of some free sulphhydryls to disulphide bonds. Oxidised β-lactoglobulin and 7S globulin also showed a major increase in the carbonyl content (five- and threefold respectively), but other chemical changes were relatively minor. In the oxidised myosin/β-lactoglobulin composites, some cross-linked aggregates were formed. Aggregation was also evidenced in the myosin/7S globulin composites exposed to the oxidising agents. The results indicated that oxidation enhanced interactions of the non-muscle proteins with myosin apparently through the modification of amino acid side chains. These physicochemical changes may influence the functionality of processed muscle foods.

Thermal transitions and dynamic gelling properties of oxidatively modified myosin, ?-lactoglobulin, soy 7S globulin and their mixtures

Oxidative effects on myosin, β-lactoglobulin and soy 7S globulin as well as their composites were studied by differential scanning calorimetry and dynamic rheological analysis. Myosin, oxidised with a free radical-generating system (FeCl3/H2O2/ascorbate), was destabilised, showing a marked decrease (>50%) in storage modulus during thermal gelation. On the other hand, oxidised β-lactoglobulin and 7S globulin exhibited only minor changes in their thermal stability and gelation properties. In the non-oxidised myosin/β-lactoglobulin composite, β-lactoglobulin affected the thermal denaturation and gelation properties of myosin through non-covalent interactions. The nature and extent of the interactions were altered by oxidation. 7S globulin did not change the thermal stability but slightly enhanced the rheological properties of myosin, regardless of oxidative conditions. Hence, in muscle foods that contain whey or soy protein ingredients, all the protein components can be oxidatively modified, and the extent of the functionality changes in myosin will be influenced by the presence of the non-muscle proteins. © 2000 Society of Chemical Industry

Emulsifying properties of acidic subunits of soy 11S globulin.

The emulsifying properties of the acidic subunits (AS11S) isolated from soy glycinin (11S) have been studied. The isolated AS11S existed in solution mainly as a dimer species. Circular dichroic analysis indicated only a slight increase in aperiodic structure and no significant difference in beta-sheet structure when compared with those of soy 11S. At similar experimental conditions, the emulsifying properties of AS11S were superior to those of soy 11S and heat-denatured 11S. Emulsions prepared with 1% AS11S remained very stable without any visible oil separation for more than a month under gentle agitating conditions, whereas those prepared with 1% 11S collapsed and separated into phases within 2-3 days. The AS11S-stabilized emulsions were very stable below 0.15 M ionic strength. Studies on the rate of adsorption and surface tension reduction at the air-water interface showed that AS11S was significantly more surface active than soy 11S. It is proposed that, because the mass fraction of acidic subunits in soy 11S is approximately 60% and it is relatively easy to separate the acidic subunits from soy 11S, it may be industrially feasible to develop an economical process to isolate functional acidic subunits for use in emulsion-based food products.

State diagrams of soy globulins

State transitions of 7S and 11S soy globulins were studied as a function of moisture by monitoring their rheological and calorimetric properties. Small-amplitude oscillatory rheometry and differential scanning calorimetry (DSC) were used to characterize their denaturation and complexing reactions within a temperature range of 40–200 °C and moisture contents of approximately 20%, 30%, and 40%. Major increases in rheological properties showed that the 7S and 11S fractions with 40% moisture underwent structure forming reactions above 70 and 100 °C, respectively, while they reacted at higher temperatures for lower moisture. DSC helped to identify the individual complexing behavior of each fraction. The above, along with the information from their glass transition, previously reported, allowed us to develop state diagrams for each protein, where glassy, rubbery, entangled polymer, reaction, and free-flow states were identified.

Glass Transition of Soy Globulins Using Differential Scanning Calorimetry and Mechanical Spectrometry

The glass transition of soy 7S and 11S globulin fractions, isolated from defatted soy flour by the method of Thanh and Shibasaki (J. Agric. Food Chem. 1976, 24, 6), was studied as a function of moisture content, using differential scanning calorimetry (DSC) and mechanical spectrometry. Both proteins showed to be plasticizable by water, suggesting that their hydrophilic domains play an important role in determining their calorimetric and rheological properties. Due to the presence of a contaminating 7S fraction in the 11S fraction, and vice versa, the DSC and mechanical spectrometry scans from both fractions showed two different glass transitions which correspond to the 7S and 11S globulins, respectively. The glass transition temperature (Tg) of the 7S fraction ranged from 114 to −67 °C for moisture contents from 0 to 35%, while the Tg of the 11S fraction ranged from 160 to −17 °C for moisture contents from 0 to 40%. Results from both methods superimposed each other very well in the overlapping moisture range. Similar results have been found in other amorphous polymers.

Hydrodynamic Characterization of Lupin Proteins: Solubility, Intrinsic Viscosity, and Molar Mass

The solubility, intrinsic viscosity ([η]), and sedimentation behavior of a lupin protein system extracted from Lupinus luteus seeds have been determined. The results were compared with those published in the literature for soy proteins. The relationship between solubility and pH for the lupin protein isolate was similar to that reported in literature for soy isolates. The [η] of the lupin protein isolate was ∼7 mL/g. These values are comparable to those obtained by other authors for soy proteins. Sedimentation velocity studies suggested that there are three main globulins in lupin protein with sedimentation coefficients of 13, 7, and 2 S. The molar mass of the major lupin globulin (390 000 g/mol) was slightly higher than reported for the soy 11S globulin (340 000 g/mol), but the lupin 7S component gave a much smaller value (105 000 g/mol) than the soy 7S (180 000 g/mol). The consequences in thickening and thermal gelation properties of the lupin protein system are discussed. Keywords: Lupin proteins; solubility; intrinsic viscosity; molar mass

Chemical Modification Strategies for Synthesis of Protein-Based Hydrogel

A soy protein-based hydrogel capable of imbibing a large amount of water was synthesized by first modifying the lysyl residues of soy protein isolate (SPI) with ethylenediaminetetraacetic dianhydride (EDTAD) followed by cross-linking with glutaraldehyde. Modification of SPI with EDTAD at pH 9 resulted in polymerization involving intramolecular cross-linking of soy 11S and 7S globulin, which decreased the number of carboxyl moieties introduced per lysyl residue and impaired the swellability of the hydrogel. Pretreatment of SPI involving heating for 30 min at 65 °C and pH 12.0 followed by modification with EDTAD at 25 °C/pH 12 did not result in polymerization and, under these conditions, about 3.3 carboxyl moieties/lysyl residue were incorporated into the protein. The hydrogel prepared under these conditions was able to absorb more than 105 g of water/g of dry gel. The structural changes in SPI caused by the modification and its relevance to swelling properties of the gel are discussed. Keywords: Chemical modification; protein; hydrogel; soy protein