Investigations of Soybean 11S Protein and Myosin Interaction by Solubility, Turbidity and Titration Studies

Solubility tests, turbidity tests, and titration experiments were employed to study the possible protein-protein interactions between purified soybean 11S protein and skeletal muscle myosin and the involvement of protein subunits in the interactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used as an analytical tool for identification of the protein species. These tests indicate that these proteins interacted at temperatures between 85°C and 100°C. Solubility and titration experiments showed that acidic subunits of soybean US protein had little or no interaction with myosin heavy chain subunits. In contrast, soybean 11S basic subunits interacted with myosin heavy chains. The SDS-PAGE method indicated that eight commercial soy protein isolates had a similar protein species composition, but certain proteins in some isolates had lost their availability for water extraction. This may account for different functional properties exhibited by differen soy protein isolates.     

Effect of transglutaminase-induced cross-linking on gelation of myofibrillar/soy protein mixtures

Microbial transglutaminase (MTGase)-catalyzed interaction and gelation of mixed myofibrillar (MPI)/soy (SPI) protein isolates were investigated at varying ionic strengths and MPI:SPI ratios, with or without SPI being preheated (80?°C). MTGase treatments in deionized water converted myosin heavy chain and actin into lower molecular-weight polypeptides, which gradually diminished as the ionic strength increased up to 0.6 M NaCl. A reduced intensity in the electrophoretic bands of soy proteins (7S and 11S except the basic subunits) was observed in all treatments, suggesting cross-linking with MPI. The enzyme treatment slightly increased the thermal transition (denaturation) temperatures of MPI/SPI but greatly enhanced (P<0.05) the elasticity of the mixed protein gels when compared with untreated samples, independent of incubation time.     

Foaming Characteristics of Commercial Soy Protein Isolate as Influenced by Heat-Induced Aggregation

The foaming properties of commercial soy protein isolate subjected to different temperatures (20–90°C) were assessed. The results revealed that the solubility and surface hydrophobicity of a 5% (w/v) commercial soy protein isolate suspension increased with increasing temperature, which increased foaming capacity and reduced foaming stability. Commercial soy protein isolate supernatant (i.e., soluble fraction) had higher foaming capacity at low temperatures (20–50°C). A high content of commercial soy protein isolate soluble fraction increased foaming capacity but decreased foaming stability. The SDS-PAGE patterns and molecular weight distribution of commercial soy protein isolate revealed that there were soluble, large molecular weight aggregates (>400 kDa) formed mainly from A and B-11S polypeptides of commercial soy protein isolate via disulfide bonds. Additionally, some aggregates also dissociated into small polypeptides and subunits after heat treatment. Commercial soy protein isolate precipitate (i.e., insoluble fraction) had a high content of proline and cysteine, which probably contributed to the foaming stability of commercial soy protein isolate.     

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.     

The relationship between breaking force and hydrophobic interactions or disulfide bonds involved in heat-induced soy protein gels as affected by heating time and temperature

Hydrophobic interactions and disulfide bonds involved in heat-induced soy protein gels were characterised by determining the dissolution kinetics of gels. Reducing SDS-PAGE results revealed that all proteins in gel network could be dissolved simultaneously by 1% (w/v) SDS solution, while a majority of glycinin (11S) A polypeptide and a moderate amount of 11S-B polypeptides, 7S-α′, α, γ, and β subunits were found in 2% (w/v) DTT dissolving samples. Stronger interaction force between proteins in gel network would result in lower dissolution constant rate. The breaking force of soy gels increased from 543 to 2171 g_force with increasing heating temperature from 85 to 100 °C, and denaturation of 11S globulins played an important role in the development of gel network. As increasing heating time from 30 to 120 min, the breaking force of gels increased from 1687 to 2175 g_force, then decreased to 1253 g_force when the time was prolonged to 240 min. Negative correlations were observed between breaking force and dissolution constant rate k_SDS or Δk, which suggested that the strengthening of both hydrophobic interactions and disulfide bonds.     

Forces Involved in Soy Protein Gelation: Effects of Various Reagents on the Formation, Hardness and Solubility of Heat-Induced Gels Made from 7S, 11S, and Soy Isolate

The effects of various reagents on the formation, hardness and solubility of heat-induced gels of soybean 7S, 11S globulins and isolate were studied. Gels were formed in 30 mM Tris HCl buffer (pH 8.0) with or without reagents by heating at 80°C for 30 min. The results indicated that electrostatic interactions and disulfide bonds are involved in the formation of 11S globulin gels; mostly hydrogen bonding in 7S globulin gels and hydrogen bonding and hydrophobic interactions in soy isolate gels. Analyses of the proteins solubilized from the gels indicated that the basic subunits of 11S globulin interact with 7S globulin in soy isolate gels. The contribution of certain acidic subunits to network formation in US soy isolate gels is limited     

Purification and identification of interacting components in a wheat starch–soy protein system

The objective of this research was to quantify the solubility, hydrophobicity and interaction characteristics of wheat–starch proteins (puroindoline, gliadin and glutenin) and protein-containing soy fractions (soy flour isolate [SFI], SFI 7S and 11S fractions, hexane-extracted textured soy flour [TVP] isolate, TVP 7S and 11S fractions, expelled, extruded soy flour [TSP] isolate, TSP 7S and11S fractions). Functional characteristics were assessed in aqueous sucrose solutions at pH 5.5 and 7.5 after heating to 25, 50, and 100 °C. Textured soy protein fractions were more soluble and had higher surface hydrophobicity profiles than their untextured counterparts. Sucrose addition decreased hydrophobicity in the textured proteins but increased it in untextured proteins. Characteristics of the isolate, as a whole, appear to be dictated by those of its 11S moiety. Dissociation constants (K_d values) for soy protein and starch-derived puroindoline were determined and indicated an extremely short association in all cases. The 11S fractions formed a complex with puroindoline in solution; however 7S fractions did not.     

Protein-Protein Interactions Between Soybean Beta-conglycinin (B1-B6) and Myosin

Protein-protein interactions between soybean beta-conglycinin (B₁- B₆) and myosin were studied by turbidity, solubility and SDS-poly- acrylamide gel electrophoresis (SDS-PAGE) analysis. Turbidity and solubility studies showed that, under the experimental conditions used, these proteins interacted at temperatures between 60° and 100°C, while SDS-PAGE analysis indicated that the interaction also occurred at 50°C. The interasction was such that no detectable complexing between these two proteins was observed. The presence of beta-conglycinin resulted in diminished aggregations of myosin heavy chains between 50° and 100°C.     

Interactions and gel strength of mixed myofibrillar with soy protein, 7S globulin and enzyme-hydrolyzed soy proteins

The mixed protein gels were prepared adding soy protein isolate (SPI), 7S globulin, enzyme-hydrolyzed soy proteins, 10- to 100-kDa ultrafiltration fraction and 0.5- to 10-kDa ultrafiltration fraction to myofibril protein isolate (MPI) gels, and five chemical interactions namely nonspecific associations, ionic bonds, hydrogen bonds, hydrophobic interactions and disulfide bonds in these gels were investigated by means of determining gel solubility within 20–75 °C. Furthermore, correlations between gel strength and different chemical interactions were evaluated statistically by Pearson’s correlation test. The gels with 0.5- to 10-kDa fraction presented the biggest gel strength below 60 °C, and the gels with SPI had better gel strength above 65 °C. At different endpoint temperatures, nonspecific associations decreased in order of MPI mixed with 0.5- to 10-kDa fraction, 10- to 100-kDa fraction, enzyme-hydrolyzed soy proteins, 7S globulin and SPI. Gels with ultrafiltration fractions had higher ionic bonds. Hydrogen bonds fluctuated in small scale below 55 °C and reduced at higher temperature. Hydrophobic interactions increased to maximum before decreasing slowly as the temperature went on. In short, both hydrophobic interactions and ionic bonds had significantly positive correlation with gel strength for mixed gels with enzyme-hydrolyzed soy proteins, whereas for the other four mixed gels, it was hydrophobic interactions and nonspecific associations.     

Effect of corn oil and amylose on the thermal properties of native soy protein and commercial soy protein isolate

Differential scanning colorimetry (DSC) was used to estimate thermal property differences between a commercial soy protein isolate (SPI) and milled defatted soy protein flour (MDF). The measurements were determined in the presence of 15, 20, 25, and 30% corn oil and 2, 4, and 6% amylose. SDS-PAGE showed that the SPI material contains aggregates as a result of the isolation procedures and processing. Upon DSC, this protein isolate showed a 7S protein transition peak at 77 °C and an 11S peak at 170 °C, while the MDF sample had a 7S peak at 69 °C and 11S peak at 177 °C. The MDF sample showed ΔH values 4 times greater than that of the SPI sample. These values reflect the effect of the isolation process on the protein. In the presence of corn oil, the MDF sample showed three transition peaks while the SPI sample displayed only two. The MDF sample demonstrated more interaction with oil than did the SPI sample. The change in the ΔH was reflective of this interaction. The addition of amylose to the SPI sample resulted in the appearance of a third peak. Amylose had a mixed effect on the two proteins; peaks of the same protein reacted differently to amylose level. Increasing the amylose level had the most influence on the third peak of the MDF sample. Amylose influence on the two proteins was attributed to a reduction of the amount of free oil in the system.     

Differential Scanning Calorimetry Analysis of the Effects of Heat and Pressure on Protein Denaturation in Soy Flour Mixed with Various Types of Plasticizers

The effects of heat and pressure on protein denaturation in soy flour were explored by an experimental design that used pressure (atmospheric to 600 MPa), temperature (room to 90 °C), time (1 to 60 min), and type of aqueous plasticizer (NaCl, sucrose, betaine, and lactobionic acid (LBA)) as factors. When 50% (w/w) soy flour‐water paste was high hydrostatic pressure (HHP)‐treated for 20 min at 25 °C, the treatment at 200 MPa showed a small effect on denaturation of only the 7S soy globulin, but the treatment at 600 MPa showed a significant effect on denaturation of both the 7S and 11S soy globulins. The treatment at 60 °C showed a less‐pronounced effect on denaturation of the 11S globulin, even at 600 MPa, but that at 90 °C showed a similar extent of denaturation of the 11S globulin at 600 MPa to that at 25 °C. Chaotropic 2N NaCl, 50% sucrose‐, 50% betaine‐, or 50% LBA‐water solutions showed protective effects on protein denaturation during HHP treatment at 25 °C. Although LBA enhanced the extent of thermostability of soy protein less than did 2N NaCl, LBA exhibited better stabilization against pressure. The results from DSC analysis demonstrated that thermostable soy proteins were not always barostable.     

预热处理对SPI水解过程多肽稳定性的影响

研究了预热处理对大豆分离蛋白(SPI)水解多肽溶液稳定性的影响。采用浊度法监测聚集过程,结果表明预热处理后的SPI蛋白在水解过程的聚集速率明显升高,但过度的热处理不利于酶促聚集速率进一步提高。采用差示扫描量热法分析其热力学性质,结果显示11S经90℃热处理解聚并和解聚的7S通过疏水相互作用形成聚集物,聚集物的形成一方面导致水解液浊度的增大,另一方面减少了酶反应位点,不利于酶的作用。     

Thermally Stimulated Luminescence in Powdered Soy Proteins

Heating powder isolated soy proteins (ISPs) in a N₂ environment produced thermally stimulated luminescence (TSL), in 2 major temperature regions, 50 to 250°C (region R1) and 250 to 350°C (region R2). In soy protein 7S fraction, strong TSL was detected in both regions with glow peak maximum (T_m) at 150 ± 15°C and at 300 ± 10°C. Two additional satellite or shoulder peaks were detected from the ISP and 7S protein fraction within region R1 at T_m = 90°C and T_m = 210°C. The soy protein 11S fraction produced a broad, poorly defined TSL peak in the low‐temperature region. Electron paramagnetic resonance spectroscopy data from the control ISP sample, deuterium sulfide‐treated ISP, ISP stored in either N₂ or O₂, and defatted soy flour, indicated that the trapped radicals present in ISP is associated with the production of the primary TSL peak at 150 ± 15°C. Activation energies required to release the trapped charges (for luminescence to occur) are approximately 0.70, 0.78, 1.50, and 1.8 eV for TSL at T_m = 100, 150, 200, and 300°C, respectively. The reaction mechanism that leads to the release of the trapped charges for TSL to occur followed a mixed order kinetic, between 1.5 and 1.8. The frequency factor varied between 10⁷/s and 10¹⁷/s.     

Texture and rehydration properties of texturised soy protein: analysis based on soybean 7S and 11S proteins

Soy protein, one of the most commonly used raw materials for texturised vegetable protein, has an important influence on texturised soy protein (TSP) with its 7S and 11S fractions. In this study, soy 7S and 11S proteins were extracted from soybean isolate and added back to the raw material to prepare TSP and analyse the effect of both on the physical properties of TSP. The results showed that the addition of 5% soy 7s or 11s protein increased the water-holding capacity (up to 9.04%) and rehydration rate (up to 25.71%) of TSP. Compared with adding soy 11s protein, adding soy 7s protein has a faster rehydration rate at a lower temperature (30 and 45 °C). After extrusion, the content of free sulphhydryl groups, total sulphhydryl groups, and disulphide bonds was significantly reduced (P < 0.05). The extrusion treatment caused degradation of the protein chains, and the proteins mainly formed insoluble polymers. Electrophoretic analysis revealed that the sodium dodecyl-sulphate (SDS) reducing the extractable rate of the precipitate after SDS non-reduction extraction of the TSP added with 5% soy 7S and 11S proteins were lower than that of the control. The proportion of different soybean protein components in TSP could change its texture, water-holding, and rehydration characteristics of it, which provides a new method for the characteristics design of TSP.     

Interactions between Whey Protein Isolate and Soy Protein Fractions at Oil–Water Interfaces: Effects of Heat and Concentration of Protein in the Aqueous Phase

ABSTRACT:  The 2 main storage proteins of soy—glycinin (11S) and β-conglycinin (7S)—exhibit unique behaviors during processing, such as gelling, emulsifying, or foaming. The objective of this work was to observe the interactions between soy protein isolates enriched in 7S or 11S and whey protein isolate (WPI) in oil–water emulsion systems. Soy oil emulsion droplets were stabilized by either soy proteins (7S or 11S rich fractions) or whey proteins, and then whey proteins or soy proteins were added to the aqueous phase. Although the emulsifying behavior of these proteins has been studied separately, the effect of the presence of mixed protein systems at interfaces on the bulk properties of the emulsions has yet to be characterized. The particle size distribution and viscosity of the emulsions were measured before and after heating at 80 and 90 °C for 10 min. In addition, SDS-PAGE electrophoresis was carried out to determine if protein adsorption or exchanges at the interface occurred after heating. When WPI was added to soy protein emulsions, gelling occurred with heat treatment at WPI concentrations >2.5%. In addition, whey proteins were found adsorbed at the oil–water interface together with 7S or 11S proteins. When 7S or 11S fractions were added to WPI-stabilized emulsions, no gelation occurred at concentrations up to 2.5% soy protein. In this case also, 7S or 11S formed complexes at the interface with whey proteins during heating.     

Deamidation and Functional Properties of Food Proteins by the Treatment with Immobilized Chymotrypsin at Alkaline pH

Ovalbumin, lysozyme, 7S globulin, 11S globulin, and gluten were treated with immobilized chymotrypsin on controlled-pore glass at pH 10 at 20°C to improve their functional properties. Optimum pH of deamidation of ovalbumin by immobilized chymotrypsin was 10, where proteolysis was very limited. Deamidation percentages of ovalbumin, lysozyme, 7S globulin, 11S globulin, and gluten were 10.0, 8.4, 6.0, 5.0, and 8.0, respectively. SDS polyacrylamide gel electrophoretic patterns of ovalbumin and lysozyme showed no difference between untreated and treated proteins, while those of soy proteins and gluten showd that larger molecular weight fractions were dissociated into smaller molecular size fractions. Solubility of gluten was greatly improved at all pHs, 2-12. Both emulsifying and foaming properties of proteins were improved by treatment with immobilized chymotrypsin.     

Improvement of functional properties of acid-precipitated soy protein by the attachment of dextran through Maillard reaction

The functional acid-precipitated soy protein (SAPP)–dextran conjugate was prepared by dry-heated storage at 60 °C under 79% relative humidity (RH) for 5 days through Maillard reaction between the ε-amino of lysine in soy proteins and the reducing-end carbonyl residue in the dextran. The covalent attachment of dextran to SAPP was confirmed by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography. Functional properties of soy protein depend on the structural and aggregation characteristics of their major components (storage globulins 7S and 11S). The conjugate seemed to be predominantly formed by 7S, and the acidic subunits of 11S in soy protein. The emulsifying properties of the SAPP–dextran conjugate were about four times higher than those of SAPP. The solubility of the protein was not enhanced as a result of preheating, but rather it was not decreased when the conjugated protein was heated at 90 °C for 20 min due to the presence of the polysaccharide. The excellent emulsifying properties of SAPP–dextran conjugate were maintained even at pH 3.0 and were further improved at pH 10.0. The object of Maillard reaction is to guarantee the suitable reaction degree, and the resulting soluble conjugate can have excellent emulsifying properties.     

Elucidation of protein aggregation in frozen cod and haddock by transmission electron microscopy/immunocytochemistry,light microscopy and atomic force microscopy

Polyclonal antibodies raised to both native cod myosin and actin as well as to aggregated proteins obtained from frozen cod stored for 11 months at ?10 °C were used to investigate disposition of muscle proteins in frozen cod and haddock fillets by transmission electron microscopy. Specimens from cod and haddock fillets, stored at ?10 °C, treated with anti‐aggregate antibody as the primary antibody, showed significantly more gold particles, especially around the protein aggregates and muscle fibres compared with fish stored at ?30 °C. Samples that were treated with anti‐myosin or anti‐actin antibody showed opposite results. Similar binding properties were observed in ELISA experiments involving the reaction of actin and myosin to both native and aggregate antibodies; thus immunological tests can be used for monitoring aggregate and texture changes in frozen stored fish. In addition, atomic force microscopy images obtained from cod muscle also indicated structural changes in frozen cod muscle proteins. The mica surface was covered with a continuous layer of muscle proteins comprising mainly small globular particles and a few large particles for the control cod sample stored at ?30 °C for 11 months. In contrast, cod fillets stored at ?10 °C showed a thin layer of proteins with small holes and an increased number of large particles denoting aggregates. Formation of ice crystals between the muscle fibres of frozen cod and haddock muscle was monitored without thawing by light microscopy at ?20 °C. The micrographs showed a greater proportion of large ice crystals and extensive protein fibre changes in fillets stored at ?10 °C compared with the control at ?30 °C. Copyright © 2004 Society of Chemical Industry     

Isoflavone Aglycone Content and the Thermal,Functional, and Structural Properties of Soy Protein Isolates Prepared from Hydrothermally Treated Soybeans

Soybeans were hydrothermally treated at 2 different temperatures (40 °C and 60 °C) and for 4 different hydration times (4, 8, 12, and 16 h) to (i) increase the isoflavone aglycone content in a soy protein isolate and (ii) evaluate the changes in thermal, functional, and structural properties of a soy protein isolate as a function of hydrothermal treatment conditions. Our study is the first to evaluate aglycone content, extraction yield, β‐glucosidase activity, differential scanning calorimetry, protein digestibility, scanning electron microscopy, water absorption capacity (WAC), foaming capacity (FC), and foaming stability of soy protein isolates prepared from hydrothermally treated soybeans. For aglycone enhancement and the extraction yield maintenance of soy protein isolates, the condition of 40 °C for 12 h was the best soybean hydrothermal treatment. The structural rearrangement of proteins that occurred with the hydrothermal treatment most likely promoted the capacity of proteins to bind to aglycone. Moreover, the structure shape and size of soy protein isolates verified by scanning electron microscopy appears to be related to the formation of hydrophobic surfaces and hydrophobic zones at 40 °C and 60 °C, respectively, affecting the protein digestibility, WAC, and FC of soy protein isolates.     

Influence of enzyme active and inactive soy flours on cassava and corn starch properties

The aim of this work was to study the influence of enzyme active and inactive soy flours on the properties of cassava and corn starches. Four starch/soy flour composites were evaluated: cassava/active soy flour (Cas/AS), cassava/inactive soy flour (Cas/IS), corn/active soy flour (Corn/AS) and corn/inactive soy flour (Corn/IS). Starch gelatinization occurred at 58.67°C for Cas and at 64.19°C for corn; gelatinization occurred at higher temperatures when soy flours were present, while ΔH diminished. The presence of AS reduced 80% the retrogradation enthalpy of Cas and 40% that of corn. Cas presented lower pasting temperature than corn starch (67.8 and 76.8°C, respectively) and higher peak viscosity (427.9 and 232.8 BU, respectively). The pasting properties of both starches were drastically reduced by soy flours, and this effect was more noticeable in Cas; AS had higher effect than IS. X‐ray diffraction pattern of retrograded samples showed that both starches recrystallisation (mainly that of Cas) was reduced when AS was added. Tan δ values decreased with AS addition to corn, but they increased when added to Cas. The images obtained using confocal laser scanning microscopy (CLSM) showed that IS was distributed as large aggregates, whereas AS distribution was more homogeneous, especially when incorporated to Cas. These results show that cassava starch interacts specifically with active soy flour (AS, mainly in native state). The delaying effect of AS on cassava starch retrogradation was clearly shown. This finding could be useful in obtaining gluten‐free breads of high quality and low retrogradation rate.