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.     

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.     

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.     

Cultivar differences in proteins of oriental mustard (Brassica juncea [L.] coss.)

The variability in the amino acid composition of the lipid-free meals from the seeds of five cultivars ofBrassica juncea, Burgonde, Ekla, Lethbridge 22A, Primus and Stoke has been studied. Arginine, tyrosine and histidine were the more variable amino acids. The meals were extracted with 0.5 M NaCl and 64–68% of the meal nitrogen was released. The extracts were fractionated by molecular exclusion chromatography to produce (a) purified preparations of the major storage protein, a globulin having a sedimentation coefficient of 12S; and (b) a fraction containing low molecular weight proteins. No difference in the relative proportions of these two components was observed. The amino acid composition of the low molecular weight protein fraction varied more than that of the 12S protein. One of six papers presented in the symposium “Rapeseed Marketing and Breeding,” AOCS Meeting, Ottawa, September 1972. NRCC No. 13392.     

Evaluation of Variation in Protein Composition on Solubility,Emulsifying and Gelling Properties of Soybean Genotypes Synthesizing β' Subunit

The effects of subunit composition of two major proteins of soybean: glycinin (11S) and β‐conglycinin (7S) in nine different genotypes, on solubility and emulsifying and gelling properties at different pH (3, 5, 6, and 8) were examined. High‐protein genotypes (more than 40%) contained low amounts of the β′ subunit. The main factors influencing solubility at pH 6 were the content of α′, α (r = 0.89 and r = 0.91 at P < 0.05, respectively) and β′ subunit contents (r = ?0.71 at P < 0.05) of β‐conglycinin, while at pH 3 acidic subunits in glycinin had a positive correlation with solubility (r = 0.69 at P < 0.05). Emulsion activity at pH 6 was higher for genotypes synthesizing β′ subunit (r = 0.57 at P < 0.05). Genotypes synthesizing higher amounts of α′ and α subunit had higher emulsion stability at pH 6 (r = 0.85 and r = 0.92 at P < 0.05, respectively) and pH 8 (r = 0.91 and r = 0.97 at P < 0.05, respectively). The rheological measurements showed that genotypes with 11S/7S ratio higher than 2.2 formed gels with enhanced storage moduli. This influence was largely due to the high content of SH groups in glycinin acidic polypeptides resulting in stabilization of gels via disulfide bonding. Gels prepared from genotypes containing higher amounts of β′ subunit of β‐conglycinin exhibited reduced elastic properties. Genotypes showing better solubility also had higher emulsion stability, but formed weaker gels and had lower emulsion activity near neutral pH.     

A Study on Subunit Groups of Soybean Protein Extracts under SDS-PAGE

Protein extracts of 640 soybean cultivars and landraces, mainly from China and a few from the US, were analyzed for their components and subunits based on distribution patterns of bands with varying molecular weights (MW) under SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The number and molecular weight of the bands in SDS-PAGE varied among materials and showed a tendency of continuous distribution. Accordingly, the SDS-PAGE patterns of the soybean protein extracts were divided into two regions: the region of bands with MW < 44 KDa and that with MW ≥ 44 KDa. The first region containing mainly 11S proteins was divided into four parts, called subunit groups, i.e. 11S-1 (14.4–22 KDa), 11S-2 (22–26 KDa), 11S-3 (26–34 KDa) and 11S-4 (34–44 KDa). The second region containing mainly 7S protein was divided into six subunit groups, i.e. 7S-1 (44–49 KDa), 7S-2 (49–55 KDa), 7S-3 (55–67 KDa), 7S-4 (67–73 KDa), 7S-5 (73–82 KDa) and 7S-6 (82–91 KDa). The sum of relative contents of 11S-1–11S-4 was obtained as the relative content of 11S protein, those of 7S-1–7S-6 as that of 7S protein, and therefore, the 11S/7S ratio obtained. The proposed criteria were demonstrated to be simple, stable and feasible. Among the 640 tested materials, 39 lacked 11S-1 but none lacked the other 11S subunit groups, while deficiencies existed in all the six subunit groups of 7S, indicating a great potential for the genetic variation of protein components and subunits for breeding for the improvement of protein qualities.     

Plastic performance of soybean protein components

Soybean proteins recently have been considered as petroleum polymer alternatives in the manufacture of adhesives, plastics, and various binders. The objective of this work was to characterize the plastic performance of soybean protein components during molding processes. Two major soybean protein fractions, 7S-rich globulin (7S-RG) and 11S-rich globulin (11S-RG) were separated from defatted soybean flour, and their purity was examined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and high-performance liquid chromatography. The thermal transition properties of the two fractions at 10% moisture content were 137.6°C for 7S and 163°C for 11S, as analyzed using differential scanning calorimetry (DSC). Plastics were prepared using a hot press at various molding temperatures that were selected based on the proteins’ thermal transition temperatures obtained by DSC. The plastics were evaluated for mechanical properties, water absorption, and microstructure. The plastics prepared with temperatures at or close to the thermal transition temperature showed a smooth, uniform, and complex structure. Results showed that the plastics made from 11S-RG at its thermal transition temperature were stronger (35 MPa) and had lower water absorption than those made from 7S-RG at 145°C (26 MPa). The plastics made from the 7S- and 11S-RG mixture had the highest tensile strength (39 MPa) and medium water absorption compared to those made from 7S- and 11S-RG alone. These mechanical properties and water absorption behaviors were significantly affected by molding temperatures. The results obtained from this research indicated that interaction between 7S- and 11S-RG could occur during molding and that thermal transition temperature played an important role in thermal processing of soybean proteins.     

Characterization of Soybean Proteins–Fatty Acid Systems

This study investigated, with the aim of obtaining more flexible and hydrophobic proteins, the attachment of a low chain fatty acid, decanoic acid, to alkaline and acid soybean proteins; and the effect on their conformational and functional properties. The extent of esterification was high at acid pH and also increased with heating. Protein solubility decreased, mainly at the highest temperature (60 °C). Increasing levels of fatty acid formed a complex with a slightly more soluble protein with less surface hydrophobicity. Esterified proteins exhibited aggregation/dissociation and were stabilized by different protein subunits belonging to 7S and 11S globulins. Denaturation of these soybean protein fractions (7S and 11S) were also detected in these complexes. The highest level of fatty acids favored formation of a more ordered protein structure.     

Molecular mapping and identification of soybean fatty acid modifier quantitative trait loci

Altering FA content in soybean [Glycine max (L.) Merr.] oil for improved functionality is a research goal of many soybean breeders. Several of the genes that alter palmitic, stearic, oleic, linoleic, and linolenic acids are modifier genes with small effects, causing these FA traits to act as quantitative traits. The objective of this study was to identify modifier FA quantitative trait loci (QTL) in soybean. A recombinant inbred line population was created from two prominent ancestors of currently available U.S. cultivars (Essex and Williams) and grown in five environments. One hundred simple sequence repeat markers spaced throughout the genome were mapped in this population. QTL were found for all five FA traits on the soybean linkage groups C2, D2, D1b, F, K, and L. A single marker interval on linkage group L contained the largest QTL for palmitic (r ²=13.1%), oleic (r ²=35.3%), linoleic (r ²=50.5%), and linolenic acids (r ²=24.8%); however, this interval also contained the gene for growth habit (Dt1) and was significantly associated with maturity. Other modifier QTL found in this study may be of use in marker-assisted selection to enable breeders to increase genetic gains for desirable FA composition of soybean.     

Characterization of corn proteins of the cultivars Venezuela-1, Arichuna, Obregon and Venezuela-1 Opaque-2

A study is presented on the protein composition of the Venezuelan corn cultivars Venezuela-1, Arichuna, Obregón and Venezuela-1 Opaque-2. The proteins were isolated and analyzed for their molecular weight and lysine and tryptophan content. Protein fractionation showed significant differences between normal corn cultivars and Opaque-2. The latter showed a low level of zein and a high content of alcohol-insoluble reduced glutelins (AIG), albumins and globulins in relation to the normal kernel. The relative increase of these protein fractions, rich in lysine and tryptophan, results in a higher concentration of these amino acids in the Opaque kernel, as revealed by the analysis of these compounds carried out in different cultivars. Electrophoretic analysis showed only small differences in the number and intensity of bands from the protein fractions of the maize cultivars under study.     

Fractionation and Some Chemical Studies on Ailanthus excelsa Roxb. Seed Protein

The unavailability of protein foods, particularly in the context of population growth, has been an important factor in the protein malnutrition encountered in developing countries. The fractionation, gel filtration and polyacrylamide gel electrophoresis (PAGE) of Ailanthus excelsa seed (a nontraditional source containing 15.81% protein) proteins were carried out in the present study, and their solubility profiles, surface topographies and amino acid compositions were evaluated. The globulin fraction dominated the seed protein composition, accounting for 51.31% (w/w) of the total soluble proteins in the seeds. Protein isolate and protein fractions of A. excelsa seeds showed similar topographical structures to those of other plant seed proteins. Analysis of the isolated proteins identified 17 amino acids, of which nine were essential. Gel filtration on Sephadex G-200 revealed the presence of seven components. PAGE detected different polypeptide bands in the range of 28.8−154.9 kDa in the protein isolate as well as in protein fractions for A. excelsa. The amino acid compositions, the solubility patterns and the high abundances of low molecular weight proteins indicate that the isolated seed protein of A. excelsa may be a potential food protein.     

Characterization of soybean protein isolates The effect of calcium presence

Results of a study of the effect of calcium addition on polypeptide composition, hydrophobicity, sulfhydryl content, thermal stability, enthalpy of denaturation, and water solubility of soybean protein isolates suggest that the addition of small amounts of calcium (1.23–5.0 mg/g protein) induced the formation of α, α′ soluble aggregates, whereas large amounts (5.0–9.73 mg/g protein) induced the selective insolubilization of the glycinin fraction. The addition of this ion also produced thermal stabilization of the soybean proteins, mainly the glycinin fraction, and increased the enthalpy of denaturation. A decrease in the surface hydrophobicity of proteins with increasing calcium content was also observed. The results obtained suggested the existence of specific calcium-soy protein interactions, especially with the glycinin fraction.     

Physicochemical Properties and ACE-I Inhibitory Activity of Protein Hydrolysates from a Non-Genetically Modified Soy Cultivar

Arkansas‐grown non‐genetically modified soybean cultivar, R08‐4004, was selected to prepare a protein isolate, which was treated with Alcalase for limited enzymatic hydrolysis. The objective was to optimize the Alcalase hydrolysis condition to produce soy protein hydrolysate (SPH) with high protein yield, low bitterness, and clarity for beverage applications. The degree of hydrolysis ranged between 14 and 52 % during the study at varying incubation times using two different concentrations of Alcalase enzyme. Recovery of soluble protein, between 21 and 53 %, was achieved with a decrease in turbidity. There was an increase in surface hydrophobicity (S₀) which is correlated with bitterness of SPH treated with 1.0 AU (3.2 µL/g) of Alcalase 2.4 L. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis showed a distinct hydrolysis pattern in which 7S globulin and the two acidic sub‐units of 11S globulin were hydrolyzed extensively in comparison to the two basic sub‐units of 11S globulin. Limited enzymatic hydrolysis produced low molecular weight peptides <17 kDa. Among these SPHs, the one derived after 120 min incubation had the highest soluble protein yield (43 %), low S₀ value (35.4), low turbidity (0.88), and highest angiotensin‐I converting enzyme (ACE‐I) inhibition activity (66.6 %). This hydrolysate has potential use as protein rich nutraceutical for developing many non‐genetically modified food product applications.     

Effect of Extraction and Precipitation Conditions During Soybean Protein Isolate Production on the Genistein Series Content

The influence of the conditions for isolation of soy protein on the content of genistein and its conjugated forms was studied. The major components of the genistein series isolated from soybean flour were malonyl genistin (54.3%), genistin (36.9%), and equal amounts (4.4%) of genistein and acetyl genistin. A modification in the conjugation profile of genistein between pH 4.5 and 8.0 and above pH 10 was attributed to the action of β-glucosidase and the saponification reaction, respectively. A decrease in the content of total genistein in the insoluble flour residue and in the soy protein isolate (SPI) with increasing extraction pH was detected, while in the whey, the total genistein content was not affected by pH. The effect of pH variation during acid precipitation on the content of genistein and its conjugated forms, at a constant extraction pH (8.0), was also studied. Under these conditions, the highest absolute content of total genistein in the SPI was obtained at pH 3.5 and the lowest was obtained at pH 5.6. The total genistein content in the whey followed an inverse trend compared with that of the protein yield. A temperature increase did not substantially affect the distribution of the different genistein forms or their total contents. The content of total genistein was higher in the glycinin than in the β-conglycinin protein fraction. The effect of the pH during the alcohol/water extraction of the isoflavones was also analyzed. The efficiency of the extraction was lower at pH values between 3.25 and 3.5 than at other pH values.     

Chemical and functional characterization of major protein fractions extracted from nontoxic Jatropha curcas byproduct meals

Jatropha curcas seeds are a suitable source of oil for biofuel, among other use. A protein-rich meal is obtained after oilseed extraction. The goals of this study were to determine the physicochemical and functional properties of a nontoxic genotype of J. curcas defatted meal (JCDM) and the seed storage protein fractions to identify future applications. Both glutelin and globulin were the predominant protein fractions obtained from JCDM (42.03 and 20.17 g/100 g of protein, respectively). Leucine, phenylalanine + tyrosine, and histidine content of JCDM and protein fractions met the Food and Agriculture Organization/World Health Organization recommendation for children. The protein solubility (PS) profiles showed minimum values (5.3%–59.7%) at pH 5–6 and maximum at pH 2 (79.7%–81.6%) and above pH 10 (84.6%–89.8%). These findings suggest that JCDM proteins could be used in the formulation of juice or protein-based beverages. All the proteins showed the highest values for foam expansion (231%–285%) at pH 9. JCDM and the albumin fraction formed highly stable foams at pH 9, while the globulin and glutelin foams were stable at pH 3 and 2, respectively. Protein with stable foams, like those from jatropha are suitable for application in ice cream, mousse, among others. The emulsion activity index had similar behavior as foam expansion, but did not follow a specific trend. Thus, the proteins are suitable for use in salad dressing, sausages, comminuted meats, and mayonnaise. Taken together, JCDM protein and its soluble protein fractions have strong promise as alternative proteins for food structuring.     

Thermal properties and adhesion strength of modified soybean storage proteins

Soy proteins have shown great potential for adhesive and resin applications. This investigation characterized the thermal and adhesive properties of the major soy protein components conglycinin (7S) and glycinin (11S) after chemical modification. These globulins were extracted from defatted soy flour, then modified with either sodium hydroxide, sodium dodecyl sulfate (SDS), or urea. Modified 7S, 11S, and mixtures of 7S and 11S at varying ratios were evaluated for gluing strength with cherry veneer plywood and for thermal denaturation using DSC. Adhesive strength and water resistance were significantly improved for all proteins modified with sodium hydroxide. Gluing strength and water resistance were improved for SDS- and ureamodified proteins containing greater portions of 7S globulins. The opposite behavior was observed for proteins containing large amounts of 11S globulins. DSC results showed that the temperatures of denaturation (T _d ) decreased for the proteins modified with sodium hydroxide or urea, whereas the T _d values of proteins modified with SDS were similar to the unmodified proteins. These results suggested that, at the concentrations studied, sodium hydroxide or urea could denature soybean protein more effectively than SDS, resulting in lower protein thermal stability. Soybean proteins with high ratios of 11S had more ordered structures, as evidenced by the high enthalpy values of protein denaturation observed in DSC measurements.     

Protein fractionation and properties of salicornia meal

Salicornia bigelovii Torr. is an annual salt-marsh oilseed plant. Hexane-defatted salicornia meal was extracted sequentially with 0.5 M sodium chloride (2x), water, 70% ethanol, and 0.1 N sodium hydroxide (2x). Each sodium chloride extract was dialyzed against deionized water and centrifuged to separate a water-soluble fraction (albumin) from a salt-soluble fraction (globulin) before freeze-drying. Ethanol extracts and neutralized sodium hydroxide extracts (glutelin) were dialyzed against water and freeze-dried. Globulin accounted for the highest amount of protein nitrogen, followed by glutelin and albumin. SDS-PAGE of reduced albumin, globulin, and glutelin showed a number of protein bands. Nitrogen solubility of defatted salicornia meal from pH 2 to 11 indicated a minimum solubility of 22%, around pH 4.5. Nonprotein nitrogen of defatted meal was 23% of total nitrogen, higher than defatted soybean, sunflower, and rapeseed meals. Albumin had the highest proportion of lysine and sulfur amino acids per 16 g nitrogen among all the fractions analyzed.     

Water and solute transport across cellulose acetate membranes in the treatment of soybean whey by reverse osmosis

In processing full-fat soy flour to produce an acid-precipitated lipid protein concentrate, there results a by-product whey fraction which, because of its high biological oxygen demand, represents a serious disposal problem. Processing of food waste streams by reverse osmosis has received considerable attention because of its low theoretical energy requirement, since no phase change is involved. A series of statistically designed and analyzed experiments were conducted on a pilot-plant reverse osmosis unit to study the effect of the operating parameters on solute and solvent transport in cellulose acetate membranes. Sucrose and sodium chloride solutions were tested in addition to soybean whey to relate the mixed solute system in whey to that of single-solute organic and inorganic feed solutions. Water flux was shown to have an Arrhenius dependency on temperature, and some membrane compaction was observed with the more porous membrane. Concentration polarization for sucrose and sodium chloride solutions increased linearly with water flux. Solute flux for soybean whey solutions decreased with molarity and was independent of pressure, whereas solute rejection increased with temperature and pressure and was independent of molarity. Good agreement was obtained using the derived parameters A, B, and τ for soy whey in the diffusion transport model when compared to the observed experimental values.     

Temperature and cultivar effects on soybean seed oil and protein concentrations

The soybean [Glycine max (L.) Merr.] industry is interested in cultivar and climate effects on seed composition. These factors may underlie the known geographic variation in seed protein and oil concentrations. Regression analyses were used to test hypotheses of the effect of temperature and cultivar on oil and protein concentrations of soybean seed using a large data set from the U.S.A. Soybean Uniform Tests. The data set included 20 cultivars representing 10 maturity groups across 60 locations (latitude 29.4 to 47.5° N) for a total of 1863 cultivar by location by year observations. Temperature was determined for each observation as the average daily mean temperature from predicted first pod (first pod at least 5 mm long), using the SOYGRO phenology model, to observed maturity. The mean temperature ranged from 14.6 to 28.7°C among the observations. Linear, quadratic, and linear plateau regression models of oil and protein concentrations vs. temperature were evaluated. The quadratic model gave the best-adjusted R ² values for oil and protein with temperature, of 0.239 and 0.003, respectively. The analyses showed that the oil concentration increased with increasing temperature and approached a maximum at a mean temperature of 28°C. Unaccounted variation in the protein concentration may be from other factors such as photoperiod, water stress, or high temperatures during seed fill. Protein plus oil had a linear relationship with temperature (adjusted partial R ²=0.183). These data document the contribution of climate and cultivar to geographic variability of oil and protein concentrations in the United States.     

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.