Adhesion and Physicochemical Properties of Soy Protein Modified by Sodium Bisulfite

Soy protein adhesives with a high solid content (28–39 %) were extracted from soy flour slurry modified with sodium bisulfite (NaHSO₃) at different concentrations. 11S‐dominated soy protein fractions (SP 5.4) and 7S‐dominated soy protein fractions (SP 4.5) were precipitated at pH 5.4 and pH 4.5, respectively. The objective of this work was to study the effects of NaHSO₃ on adhesion and physicochemical properties of soy protein. The adhesion performance of NaHSO₃‐modified SP 4.5 was better than SP 5.4; the wet strength of these two fractions was from 2.5 to 3.2 MPa compared with 1.6 MPa of control soy protein isolate. SDS‐PAGE results revealed the reducing effects of NaHSO₃ on soy protein. The isoelectric pH of soy protein decreased as NaHSO₃ increased due to the induced extra negative charges (RS‐SO₃^?) on the protein surface. The rheological properties of soy protein adhesives were improved significantly. Unmodified samples SP 5.4 and SP 4.5 had clay‐like properties and extremely high viscosity, respectively; with 2–8 g/L NaHSO₃ modification, both SP 5.4 and SP 4.5 had a viscous cohesive phase with good flowability. Overall, NaHSO₃‐modified soy protein adhesives in our study have many advantages over the traditional soy protein isolate adhesive such as better adhesion performance, higher solid content but with good flowability and longer shelf life.     

Characterization of Fractionated Soy Proteins Produced by a New Simplified Procedure

It was possible to fractionate soy protein into two soy protein isolate fractions (>90% protein) enriched in either glycinin or β-conglycinin by using a new simplified procedure (referred to as the Deak procedure) employing CaCl₂ and NaHSO₃. The Deak procedure produced fractions with higher yields of solids, protein, and isoflavones, and similar protein purities as well as improved functional properties compared to fractions recovered by established, more complex soy protein fractionation procedures. The Deak glycinin-rich fraction comprised 15.5% of the solids, 24.4% of the protein, and 20.5% of the isoflavones in the starting soy flour, whereas the glycinin-rich fraction of the established procedure (Wu procedure) comprised only 11.6% of the solids, 22.3% of the protein, and 9.6% of the isoflavones. The Deak β-conglycinin-rich fraction comprised 23.1% of the solids, 37.1% of the protein, and 37.5% of the isoflavones in the starting soy flour, whereas the Wu β-conglycinin-rich fraction comprised only 11.5% of the solids, 18.5% of the protein, and 3.3% of the isoflavones. Protein purities were >80% for both fractions when using both procedures. The Wu procedure produced protein fractions with slightly higher solubilities and similar surface hydrophobicities; whereas, the fractions produced by the Deak procedure had superior emulsification and foaming properties and similar dynamic viscosity behaviors.     

Identification and Validation of Soy Peptides with In-vitro Hemagglutination Activity

We previously demonstrated that different enzyme hydrolysates of soybean agglutinin (SBA), β-conglycinin and glycinin-rich fractions had in-vitro hemagglutination activities. In the present study, the three proteins were subjected to trypsin hydrolysis, and N-acetyl-d-galactosamine (GalNAc)-agarose beads were used to isolate the active peptides. Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI−TOF) Mass Spectrometry (MS) was used to determine the mass of the peptides and the molecular weights were compared to the peptide profiles given by theoretical cleavage of the proteins, so that the peptide sequence could be identified. Two peptides from SBA, 24 peptides from β-conglycinin and 16 peptides from glycinin were identified from the active peptide extracts. The two SBA peptides, three peptides from β-conglycinin having strong MS signal intensity and three peptides from glycinin with strong MS intensity were synthesized and their activities were assessed by using the in-vitro hemagglutination assay. These peptides were proven to have hemagglutination activity, whereas a synthesized control peptide from SBA did not. These results validated our previous observation of various soy protein fractions having hemagglutination activities.     

Urea-Modified Soy Globulin Proteins (7S and 11S): Effect of Wettability and Secondary Structure on Adhesion

This investigation characterized wettability and adhesive properties of the major soy protein components conglycinin (7S) and glycinin (11S) after urea modification. Modified 7S and 11S soy proteins were evaluated for gluing strength with pine, walnut, and cherry plywood and for wettability using a bubble shape analyzer. The results showed that different adhesives had varying degrees of wettability on the wood specimens. The 7S soy protein modified with urea had better wettability on cherry and walnut. The 11S soy protein modified with 1M urea had better wettability on pine. The 1M urea modification gave 11S soy protein the greatest bonding strength in all the wood specimens. The 3M urea modification gave 7S soy protein stronger adhesion on cherry and walnut than did 11S protein; but with pine, 11S soy protein had greater adhesion strength than 7S soy protein. Measurement of protein secondary structures indicated that the β-sheet played an important role in the adhesion strength of 3M urea-modified soy protein in cherry and walnut, while random coil was the major factor reducing adhesion strength of 7S soy protein modified with 1M urea.     

The influence of genotypic variation in protein composition on emulsifying properties of soy proteins

This study describes the relationship between the emulsifying properties of soybean proteins and their composition, i.e., glycinin (11S) and β-conglycinin (7S). Twelve investigated soybean genotypes showed significant differences in storage protein composition. The β-conglycinin concentration positively correlated with extractable soluble protein content, which was positively correlated with protein extractability. These data suggest that the level of β-conglycinin has a positive influence on protein extractability. The emulsion activity index (EAI) was strongly and positively correlated with the 11S∶7S ratio and strongly and negatively correlated with the concentration of β-conglycinin. The emulsion stability index (ESI) showed a moderate positive correlation with the monomeric form of glycinin and a strong positive correlation with the ratio of the monomeric to dimeric form of glycinin. No association was evident between ESI and EAI. Also, no relationship was found between ESI or EAI and extractability. Based on these data, it appears that the 11S∶7S ratio strongly reflects the ability of soybean proteins to form emulsions, whereas the ratio of the two different forms of glycinin may be crucial factors for the stability of soybean protein emulsions. Thus, understanding the relationship between protein composition and functionality could be useful for further improvement of functional behavior of soy proteins in food systems.     

Functional properties of soy protein fractions produced using a pilot plant-scale process

Soy proteins fractionated by the modified Nagano process (Nagano method) and a simplified pilot-plant process (CCUR method) were studied for their functional properties, including solubility, viscosity, emulsification, and foaming. The functional properties of the three fractions produced by the Nagano method—glycinin (11S), β-conglycinin (7S), and an intermediate fraction (IM)—were studied under a selected range of pH, ionic strengths, and protein concentrations. The 11S fraction was more soluble than the 7S at pH 2–3, whereas the 7S was more soluble than 11S at pH 5–6. Adding NaCl changed the solubility of both fractions at pH 4–5 compared to a neutral pH. Other functional properties were related to solubility in the 7S and 11S fractions. The CCUR method yielded only two fractions, 11S and 7S, and the functionality of those fractions was tested at a neutral pH. The solubility of the CCUR samples was slightly higher at extreme pH levels compared to 11S and 7S fractions from the Nagano method at a neutral pH. The relationship between solubility and other functional properties was clearer in CCUR samples. These results indicate that the simplified pilot-scale CCUR fractionation process can influence the functional properties of the protein fractions.     

Pilot-plant fractionation of soybean glycinin and β-conglycinin

A laboratory process for separating glycinin and β-conglycinin from soybean flakes was successfully scaled up to the pilot-plant scale (15 kg soy flakes). Average yields of the glycinin and β-conglycinin fractions were both 9.4% on a dry basis (db). The protein contents of glycinin and β-conglycinin fractions were 92.8 and 97.7% db, respectively. The glycinin and β-conglycinin purities were 90.4 and 72.7% of the protein content, respectively, which were very comparable to those of the laboratory-scale process. The total sulfhydryl plus half cystine content of the glycinin fraction was 37.8 mol/mol protein and 14.8 mol/mol protein for the β-conglycinin fraction. The native glycinin structure loss in the glycinin fraction was negligible. The native β-conglycinin loss in the β-conglycinin fraction was 10%, as estimated by rocket immunoelectrophoresis analysis. Hydrophobicity index value showed that hydrophobic properties of the pilot-plant protein fraction were ordered, from high to low: β-conglycinin fraction > glycinin fraction > intermediate mixture fraction.     

Fate of Phytic Acid in Producing Soy Protein Ingredients

Phytic acid (myo-inositol hexaphosphate) is present in soybeans and soy protein products at 1–2% dry matter. Phytate causes poor absorption of essential electrolytes and minerals, and binds to proteins and co-precipitates with isoelectric soy protein isolates. We determined how phytic acid partitioned during different procedures to prepare soy protein ingredients. Procedure and soybean variety significantly affected phytic acid content and recovery. High-sucrose/low-stachyose (HS/LS) soybeans contained significantly (P < 0.05) less phytate than did a typical variety of commodity soybeans (IA2020). In addition, phytate was more readily extracted from the commodity soybeans than from HS/LS soybeans. Among all procedures studied, ethanol-washed soy protein concentrate had the highest phytate contents and yields in the protein products for both soybean varieties (~80 mg/g and 99%, respectively). When protein extraction was carried out at room temperature the protein products had significantly lower phytate yields (60–78%) than when extraction was at 60 °C (80–99%). The protein products obtained from normal soybeans had significantly higher phytate contents than the same products made from HS/LS soybeans. When fractionating soy proteins, the glycinin-rich fraction contained significantly less phytate than the β-conglycinin fraction except for the fractionation procedure performed at room temperature instead of 4 °C.     

Soy Protein Adhesive Blends with Synthetic Latex on Wood Veneer

Environmental pollution has prompted an interest in and a need for bio-based wood adhesives. Modified soy protein has shown adhesion properties similar to those of formaldehyde based adhesives. The objective of this research was to investigate the compatibility of a modified soy protein (MSP) with six commercial synthetic latex adhesives (SLAs). Four different blending ratios of MSP and SLAs were studied. Adhesion; structural change; and rheological, thermal, and morphological properties of the MSP/SLAs blends were characterized. Dry adhesion strength of MSP, SLAs and their blends were all similar with 100% wood cohesive failure. Water resistance of all six SLAs was improved by blending with MSP in terms of the wet adhesion strength. The wet adhesion strength of MSP/PBG (40/60) blends was 6.416 MPa, as compared to 4.66 MPa of pure PBG (press bond glue, urea formaldehyde based resin). Viscosity of MSP/SLAs blends was reduced significantly and reached the lowest value at 40–60% MSP. Infrared spectra, thermal properties, and morphological images indicated that chemical reactions occurred between soy protein and PBG molecules. The MSP provided some functional groups, such as carboxylic (–COOH), hydroxyl (–OH) and amino groups (–NH₂), that cross-linked with hydroxymethyl groups (–CH₂–OH) of PBG, and also acted as an acidic catalyst for the self-polymerization of urea formaldehyde based resin.     

Current aspects of soy protein fractionation and nomenclature

Significant advances have been made recently in studying the basic physico-chemical properties of the major soy storage proteins and their subunits using such techniques as differential solubility, ion exchange chromatography, gel filtration chromatography, immunoelectrophoresis, isoelectric focusing and dissociating gel electrophoresis. This paper reviews and discusses the principal findings from this work and summarizes the current status of the nomenclature of the major soy storage proteins, e.g., 11S glycinin, 7S β-conglycinin and γ-conglycinin and their subunits.     

In Vitro Hemagglutination Activity of β-Conglycinin and Glycinin Fractions and Feeding Study of Non-Thermal Treated Soy Protein

The purpose of this study was to use non-thermal means to reduce hemagglutination activities of soy proteins and thereby improve the feeding quality of the resulting treated proteins. Two storage proteins in soybeans, β-conglycinin- and glycinin-rich fractions, were shown to have in vitro hemagglutination activity. The activity of the β-conglycinin fraction was not reduced by hydrolysis with single proteases, but the hemagglutination activity was fully eliminated by multiple enzyme treatments if it was first heated. However, the activity of glycinin fraction was not fully eliminated by either single or multiple enzyme hydrolysis. Pepsin and pancreatin hydrolysis, which was effective in eliminating hemagglutination activity of soybean agglutinin (SBA), was used to generate a feed material for in vivo evaluation of nutritional quality of soy white flake (SWF). SBA in reducing agent-treated then pepsin–pancreatin treated SWF was deactivated on analytical scale experiment, but not in the feed material. The treated SWF feed material did not improve chick growth performance compared with the raw SWF. However, chicks did not show enlargement of the pancreas or the intestines compared to the raw SWF feed, indicating deactivation of anti-nutritional factors. The trypsin inhibitors seemed to play a more important role than the hemagglutination activity of soy proteins in this nutritional test.     

Development of High-Strength Soy Protein Adhesives Modified with Sodium Montmorillonite Clay

This study investigated the high strength of a soy protein adhesive system with good flowability at high protein concentration. Sodium montmorillonite (Na MMT), the most widely used silicate clay, was incorporated into viscous, cohesive soy protein adhesives at concentrations ranging from 1 to 11 % (dry basis, w/w). Hydroxyethyl cellulose was used as a suspension agent to stabilize the soy protein and nano clay to be the dispersion system. The interaction between soy protein and Na MMT was characterized by XRD, FTIR, Zeta potential and DSC. Results indicated that soy protein molecules were adsorbed on the surface of the interlayer of Na MMT through hydrogen bonding and electrostatic interaction. The soy protein/Na MMT adhesives had the intercalation structure with Na MMT contents ranging from 1 to 11 %. Adhesion strength, specifically wet adhesion strength, of soy protein adhesives at isoelectric point (pI) was significantly improved by the addition of Na MMT. It is believed that the physical cross‐linking reactions between soy protein and Na MMT mainly contribute to the improved adhesion performance of soy protein adhesives. Wet adhesion strength increased from 2.9 MPa of control soy protein adhesive to 4.3 MPa at 8 % Na MMT. An increase of pH beyond pI value resulted in decreased adhesion strength due to increased surface charges of soy protein and slightly reduced affinity of soy protein on the nano clay surface.     

Sodium Bisulfite-Induced Changes in the Physicochemical,Surface and Adhesive Properties of Soy β-Conglycinin

The effects of sodium bisulfite on the electrophoresis profile; turbidity; and thermal, surface, and adhesive properties of soy β-conglycinin protein were studied. Sodium bisulfite dissociated high-molecular-weight aggregates in the protein, and the aggregate percentage decreased with increasing sodium bisulfite concentration. Denaturation temperature of sodium-bisulfite-treated β-conglycinin increased as sodium bisulfite increased. However, at high sodium bisulfite concentration (i.e. 36 g/L), denaturation enthalpy decreased significantly. Sodium bisulfite caused changes in the β-conglycinin secondary structure and promoted ionization of lysine residues as indicated by FT-IR results. A sudden drop in turbidity at pH 4.8 was observed at the same salt level. The contact angle of β-conglycinin on cherry wood reached its minimum at 6 g/L sodium bisulfite and 24 g/L on glass. Water resistance of β-conglycinin was improved but not significantly by 6 g/L sodium bisulfite at pH 9.5. An obvious increase in adhesion strength of the protein occurred at 3 and 6 g/L sodium bisulfite at pH 4.8. A high sodium bisulfite concentration at 36 g/L sharply reduced the adhesive performance of β-conglycinin.     

Quantitative trait loci for β-conglycinin (7S) and glycinin (11S) fractions of soybean storage protein

Glycinin (11S) and β-conglycinin (7S) are important seed storage proteins in soybean [Glycine max (L.) Merr.]. A major limitation of soybean seed storage proteins is their low levels of the sulfur-containing amino acids, methionine and cysteine, which are important nutritional components of protein mea. Glycinin contains significantly more S-containing amino acids than does β-conglycinin. Thus, detection of quantitative trait loci (QTL) that govern 11S may provide marker-assisted selection (MAS) opportunities to improve soybean total S-containing amino acids. The objective of this study was to detect and map QTL governing 7S and 11S fractions of soybean seed storage proteins. To achieve this objective, 101 F₆-derived recombinant inbred lines (RIL) developed from a cross of N87-984-16 ×TN93-99 were used. Storage proteins were extracted from all RIL and separated in 10–20% linear gradient polyacrylamide gels. Dried gels were scanned for individual subunits of storage protein with a densitometer equipped with a He−Ne laser light source. Data were converted to concentration for each subunit component and analyzed using SAS software. A significant (P<0.05) difference among genotypes was found for glycinin and β-conglycinin. A total of 94 polymorphic simple sequence repeat molecular genetic markers were used in screening all RIL. Three QTL for glycinin (Satt461, Satt292, and Satt156) were distributed on linkage group (LG) D2, I, and L, respectively, whereas two QTL for conglycinin (Satt461 and Satt249) were distributed on LG D2 and J. Phenotypic variation explained by individual QTL ranged from 9.5 to 22%. These QTL may provide useful MAS opportunities for improvement of nutritional quality in soybean.     

Protein Subunit Composition Effects on the Thermal Denaturation at Different Stages During the Soy Protein Isolate Processing and Gelation Profiles of Soy Protein Isolates

This study focussed on the evaluation of thermal denaturation at three different stages during soy protein isolation and the effect of subunit composition on the formation of heat-induced soy protein gels. Soy protein isolates (SPI) were prepared from 12 high protein lines, Harovinton variety and 11 derived null-lines which lacked specific glycinin (11S) and β-conglycinin (7S) protein subunits. Protein denaturation during SPI processing was monitored by differential scanning calorimetry (DSC). The results showed that hexane extraction of oil from soybean flours at 23 °C or 105 °C did cause changes in protein conformation. Rheological measurements showed that lines with different subunit compositions and 11S:7S ratio had distinctive gelation temperatures and resulted in gels with different network structures. All lines formed particulate gels at 11% protein. The 11S:7S ratio was not correlated to final stiffness, measured as the storage modulus G′, of SPI gels. Lower gelation temperatures were usually observed for 7S-rich lines. The absence of A3 and the combination of A1, A2 and A4 subunits of 11S fraction may suggest the formation of stiffer gels. A more detailed study of the frequency dependence of G′ for the various networks formed also indicated that differences in subunit composition influenced the network structures.     

Potential β-sheet surfaces of soybean seed proteins

Volume and amphiphilicity profiles computed for β-sheet conformations of soybean seed proteins (the acidic and basic subunits of glycinin, the α subunit of β-conglycinin, an extensin-like high-proline protein, and a lipid body oleosin) were compared to profiles for selected nonseed proteins and random-sequence polypeptides. The major soy proteins resemble fibrinogen more than silk or collagen but are differentiated from each other by surface polarity. Polarity in acidic glycinin fluctuates somewhat regularly and symmetrically along both sides of the β-sheet, but in basic glycinin, polarity on one side is fourfold that on the other throughout 70% of the protein. Polar residues distribute equally on either side of the β-conglycinin subunit, but half of the acidic and basic residues concentrate in the N-terminal third of the molecule. The remainder of the β-conglycinin contains 90% of the molecule’s tyrosine, of which 70% is along one side. In the high-proline protein, 90% of the tyrosine distributes to one side of a β-sheet; 95% of the acidic residues to the other. Only soy oleosin approximates the per-residue volume, polarity, and uniformity of silk or collagen. Its 85-residue central lipophilic domain is much less polar than silk and nearly as uniform.     

A new approach to genetic alteration of soybean protein composition and quality

Although soybeans produce high-quality meal, modern animal and fish production systems often require synthetic essential amino acid supplements to fortify feed rations. However, biotechnology may enable development of soybeans with naturally adequate levels of certain essential amino acids for advanced feed formulations. One approach involves genetic manipulation of glycinin (11S) and β-conglycinin (7S) contents, the principal components of soybean storage proteins. Because 11S contains more cysteine and methionine than 7S protein, a higher 11S:7S ratio could lead to beneficial changes in the nutritional quality of soybean meal. Although genotypic variation for 11S:7S may be low among soybean [Glycine max (L.) Merr.] germplasm, ratios ranging from 1.7–4.9 were observed among accessions of the wild ancestor of cultivated soybean (Glycine soja Sieb, and Zucc.). Thus, wild soybean germplasm was evaluated as a potential source of genes that govern protein synthesis that may have been lost during the domestication of G. max. Change in the amount of 11S protein accounts for a significant portion of the genotypic variation in protein concentration and composition among wild soybeans. Strong positive correlation exists between the 11S:7S ratio and methionine or cysteine concentration of total protein. Moderate positive associations were found for threonine or tyrosine. A moderate negative correlation was found between lysine and 11S:7S. No association was found for leucine and phenylalanine or for total essential amino acid concentration. Based on these data, G. soja may contain a different complement of genes that influence expression of 11S and 7S proteins than G. max germplasm. Thus, through interspecific hybridization, wild soybeans may be a useful genetic resource for the further improvement of protein quality in cultivated soybeans.     

Assessing Glycinin (11S) and β-Conglycinin (7S) Fractions of Soybean Storage Protein by Near-Infrared Spectroscopy

Soybean breeding programs underway today are addressing the goal of improving the protein profile to benefit the human diet as well as that of livestock. Glycinin, a globulin storage protein of the meal and designated as the 11S size fraction by ultracentrifugation, is desirable because of its relative abundance of sulfur-containing amino acids, such as methionine and cysteine. The current study examined the feasibility of near-infrared (NIR) measurement of glycinin and the other prevalent protein fraction, β-conglycinin (7S size fraction), as well as the electrophoretically separable sub fractions that comprise these two components. From a population of 101 F₆-derived recombinant inbred lines in a field replicated trial, single whole soybeans were scanned in transmittance (800–1,798 nm, 24 beans/sample × 197 samples total). Additional scanning of the ground meal was performed in reflectance (1,100–2,498 nm). Partial least squares (PLS) calibrations were developed, using the 24-bean average log(1/T) spectrum for each sample, as well as the average spectrum from duplicate packs of log(1/R) spectra of the meal. The results indicate that NIR prediction of 11S and 7S, as well as the sub fractions thereof, is at best limited to screening purposes in soybean breeding programs for probable reasons of an inherent lack of spectral specificity of the protein fractions and a non-constant proportion of soluble-to-total protein.

Functionality of soy protein produced by enzyme-assisted extraction

This study investigated the potential of enzymes to increase soy protein extractability without causing protein degradation. The aqueous extraction of protein was performed from defatted soy flakes on a laboratory-and pilot-plant scale. Yields of protein and reducing sugars were determined in the alkali extracts obtained with cellulases and pectinase, added alone or as cocktails. Using 5% (wt/g of protein) Multifect pectinase resulted in the best improvement of protein yields, which were 50 and 17% greater than the controls in laboratory- and pilot-plant-scale trials, respectively. This enhanced protein extraction was accompanied by an increased reducing sugar content in the aqueous extract compared with the control. Under the conditions tested, no enzyme cocktail markedly increased the protein yield compared with the use of single enzymes. The solubility curve for Multifect pectinase-treated soy protein isolate (SPI) was typical of SPI at pH 2–10. Its foam stability significantly improved, but the emulsification properties declined. Multifect pectinase markedly reduced the viscosity of SPI. SDS-PAGE showed that the α’ and α subunits of β-conglycinin were modified, and glycoprotein staining showed that these modifications were probably due to a protease secondary activity in the pectinase preparation. One cellulase and one pectinase were identified as effective in modifying the protein and reducing sugar extractablity from the defatted soy flakes.     

Influence of drying conditions on the gelling properties of the 7S and 11S soy protein fractions

Soy protein fractions rich in β-conglycinin (7S) or glycinin (11S) were freeze dried or spray dried at temperatures of 120, 150 or 180 °C. The fractions were characterized for their particle size distribution, sorption isotherms and by scanning differential calorimetry. The gelling capacity of the protein fractions was studied at pH values of 3 and 7 using oscillatory measurements, mechanical properties and water holding capacity. The rheological measurements showed that viscous modulus (G″) predominated at low temperatures and the elastic modulus (G′) at high temperatures. At pH 3, the G′–G″ crossover occurred at lower temperatures when compared to pH 7. This behaviour was more accentuated for the 11S fractions due to its capacity to form stronger gels. An increase of drying temperature led to a displacement of the gel point to higher temperatures and decreased the elasticity modulus or gelling capacity of protein fractions. These results were confirmed by the mechanical properties, since at higher temperatures the gels were more fragile and brittle, especially when formed at pH 7.