Ph-shifting Induced Structural Changes and Functionality Enhancements of Soy Proteins in Molten Globule State
|Course||Of Food Science|
|Keywords||soy proteins pH-shifting treatment molten globule functional properties protein film|
The structure-function relationship is a main determinant factor for the usefulness and suitability of a protein ingredient in food applications. The molten globule (MG) structure, an intermediate state retaining much of the secondary structure but having a partially unfolded tertiary structure, is a folding pattern observed in many function-improved protein ingredients. Recent efforts in developing such protein ingredients have concentrated on pH-shifting treatments of meat proteins and some enzymes to improve protein functionality, which is, exposing proteins to an extreme pH condition to unfold the proteins followed by readjustment of the pH to neutrality to refold the proteins. The overall objectives of this research were to investigate the influence of pH-shifting treatments on the structure and functionality of soy proteins, and to explore the potential application of soy proteins treated under optimal pH-shifting conditions established in the preparation of well-textured muscle protein gel products. The ultimate goal was to create a theoretical basis on which to develop novel processing technologies for the expansion of the utility of soy proteins as value-added functional food ingredients.Soy protein isolate (SPI) and itsβ-conglycinin (7S) and glycinin (11S) fractions were subjected to structural unfolding by holding (0, 0.5, 1, 2, and 4 h) in acidic (pH 1.5-3.5) or alkaline (pH 10-12) pH solutions, followed by refolding (1 h) at pH 7. The pH-shifting treatments resulted in a substantial rise (P < 0.05) in protein surface hydrophobicity and intrinsic tryptophan fluorescence intensity, significant dissociation of disulfide-linked basic and acidic 11S subunits, and the exposure of tyrosine. The pH-shifting, notably at pH 12, produced soluble, disulfide-linked polymers from 11S and reduced (P < 0.05) its enthalpy (ΔH) but not the temperature of denaturation (Tm). The response of 7S globulins to pH-shifting treatments was generally less than that of 11S globulins. After pH-shifting, soy proteins adopted a molten globule-like conformation that largely maintained the original secondary structure and an overall compactness but lost some tertiary structure.Structurally-modified SPI and its 7S and 11S fractions as described above were tested for emulsifying properties with soybean oil. The pH-shifting treatments at both acidic and alkaline pH led to markedly improved (P < 0.05) emulsifying activity (EAI) as well as emulsion stability (ESI) for SPI. The pH-shifting also significantly increased (P < 0.05) EAI and ESI of 11S and, to a lesser extent, 7S. The pH-shiftinh treated 11S also produced more uniform and smaller oil droplets than 7S.The influence of pH-shifting (at pH 1.5 or 12 for 1 h) on the solubility of SPI, 7S, and 11S in the molten globule state was investigated in 0, 0.1, and 0.6 M NaCl solutions at pH 2-8. The pH12-shifting resulted in up to 2.5-fold increases in SPI solubility in the pH 6-7 range, especially at 0 M NaCl. The pH1.5-shifting had generally lower effect on protein solubility. The 11S exhibited a solubility pattern similar to that of SPI, but the solubility of 7S was not affected by pH-shifting except at 0.6 M NaCl. All proteins had reduced sensitivity to thermal aggregation after structurally alteration by pH-shifting.The effect of pH-shifting (at pH 1.5 or 12 for 1 h) on film-forming properties of SPI at different temperatures, and the physicochemical and microstructural characteristics of resulting films were studied. Glycerin-plasticized films were prepared at 30% relative humidity from pH-treated SPI solutions unheated or preheated at 50, 60, 70, or 80°C for 30 min. The pH 12-treated SPI spontaneously formed a transparent, slightly yellowish film at 20°C, which had the greatest elongation (E), while pH 1.5-treated and untreated SPIs required preheating at 50 and 70°C, respectively, to form films. Covalent cross-linking induced by heating contributed to reduced film protein leachability and water vapor permeability (WVP) but increased film tensile strength (TS). Films formed from both pH1.5- and pH12-treated SPIs were superior to the film formed from untreated SPI in E (2-fold larger, P < 0.05) despite slightly reduced TS and WVP. Disulfide bonds formed between previously dissociated A and B subunits of 11S were shown to be a major force in pH12- and pH1.5-shifting treated SPI films. The pH12-shifting treated SPI film had a smooth surface but a stranded interior texture as viewed under light and scanning electron microscopies.pH-shifting treated SPIs (at pH 1.5 or 12 for 1 h), which exhibited increased solubility and surface activity, were incorporated (0.25-0.75% protein) into myofibrillar protein isolates (MPI, 3%) sols (in 0.6 M NaCl at pH 6.25) with or without 0.1% microbial transglutaminase (TG). Static penetration tests and dynamic rheological measurement of the MPI sols during gel formation upon linear heating (20–80°C, 1°C/min) showed significant enhancements of MPI gelling ability by the presence of pH-shifting treated SPIs, notably pH1.5-shifting treated SPI, as well as preheated SPI (90°C, 3 min). A 7-h incubation with TG accentuated gel-strengthening effect of these SPIs. The B subunit in 11S was proved to be the main component that participated in the composite gels. However, the addition of native SPI impaired MPI gelation. The MPI gelling properties were also greatly improved (P < 0.05) by the addition of 10% soybean oil emulsions stabilized by pH-shifting treated SPI and not by emulsions prepared with native or preheated SPI. The major forces in the pH-shifting treated SPI and MPI composite gel system were hydrophobic interactions and disulfide bonds with the cross-linking of previously dissociated A and B subunits of 11S.The results from this research suggested that 11S globulins of SPI were more responsive to pH-shifting treatments than 7S globulins, and were largely responsible for the structural changes of SPI. The SPI in the molten globule state exhibited a stronge amphiphilicity and markedly improved emulsifying and film-forming properties, and was capable of promoting the gelation of myofibrillar protein and enhancing the rheological and structural characteristics of the final gels. These original findings can be valuable to new processing technology developments aimed at broadening the commercial application of soy proteins.