Article pubs.acs.org/JAFC
Structural and Gel Textural Properties of Soy Protein Isolate When Subjected to Extreme Acid pH-Shifting and Mild Heating Processes Qian Liu,†,‡,§ Rui Geng,†,‡ Juyang Zhao,‡ Qian Chen,‡ and Baohua Kong*,‡,§ ‡
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, People’s Republic of China Synergetic Innovation Center of Food Safety and Nutrition, Harbin, Heilongjiang 150030, People’s Republic of China
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ABSTRACT: Changes in the structural and gel textural properties were investigated in soy protein isolate (SPI) that was subjected to extreme acid pH-shifting and mild heating processes. The SPI was incubated up to 5 h in pH 1.5 solutions at room temperature or in a heated water bath (50 or 60 °C) to lead to protein structural unfolding, followed by refolding at pH 7.0 for 1 h. The combination of pH-shifting and heating treatments resulted in drastic increases in the SPI gel penetration force (p < 0.05). These treatments also significantly enforced the conversion of sulphydryl groups into disulfides, increased the particle size and hydrophobicity values, reduced the protein solubility (p < 0.05), and strengthened the disulfide-mediated aggregation of SPI. The intrinsic fluorescence spectroscopy results indicated structural unravelling when protein was subjected to acidic pH-shifting in combination with heating processes. The slight loss of secondary structure was observed by circular dichroism. These results suggested that pH-shifting combined with heating treatments provide great potential for the production of functionalityimproved SPI, with the improved gelling property highly related to changes in the protein structure and hydrophobic aggregation. KEYWORDS: pH shifting, heating, soy protein isolate, protein structure, gel textural property
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INTRODUCTION Soy protein as a health-promoting and inexpensive functional composition is widely used in meat products, nutraceutical products, and beverages.1 In general, untreated soy proteins display only limited functionality, such as emulsifying, gelation, and other processing properties.2 Therefore, additional studies are needed to modify the soy protein isolate (SPI) structures to exhibit broadened functionality, such as solubility, emulsification, and gelation. The ability of SPI to form gel is usually regarded as a very important functional property of SPI, and thus, it is worth probing into the inner link between the structure and gel characteristics of soy protein.3 The thermal-induced protein gel is a complicated process that includes protein aggregation and unfolding before forming a complex network structure.4 The molecular weight and size of the protein aggregates have a relationship to the gel network and gel properties.5 As the major components in the SPI, 7S and 11S play a decisive role in the gelling property. One study found that 7S and 11S mainly affect the gel viscoelasticity and hardness and that the B subunit of 11S and β subunit of 7S undergo macromolecule aggregation by electrostatic interactions, which were considered to have an important effect on the gel properties.6 Damodaran and Kinsella7 reported that the existence of 7S definitely inhibits the dissociation of the 11S subunits. Hence, both protein aggregation and each subunit play an important role in gelation. In recent years, numerous researchers have made some effects to change the protein structures to produce certain conformations that are in favor of producing a desirable functionality.8 When exposed to extreme pH conditions, many globular proteins undergo significant conformational changes, typically referred to as the molten globule (MG) state.8 The discovery of an extreme pH-shifting-induced intermediate state © XXXX American Chemical Society
in SPI has resulted in an interest in investigating acid or alkaline treatments to improve the functionality of these protein.9−13 Jiang et al.13 proved that acidic or alkaline pH treatments could cause MG structure formation in SPI, resulting in enhanced emulsifying ability. Some studies have revealed that extreme pH treatments might dissect protein into subunits, thus leading to improved SPI functional properties,13 particularly the enhanced gelation of mixed myofibrillar and soy protein sols.14 Heating is the easiest method to enhance the functional properties of protein, producing numerous desirable structural changes in the SPI, in favor of protein gel formation.14 It is conceivable that SPI becomes more sensitive to temperature changes after exposure to extreme pH values. The heating temperature in the present study was 50 or 60 °C. The SPI is substantially but not completely denatured (unfolded) by the extreme pH treatment. However, the MG state (a metastable state) induced by pH shifting renders soy proteins more susceptible to further structural changes and hydrophobic aggregation. Hence, the low temperatures of 50 and 60 °C, which would not normally affect the structure of 7S or 11S globulins in control SPI, now become effective. When the pH shifting is combined with mild heating, a number of desirable structural and functional changes can occur. A clear knowledge of the gel textural profiles of soy proteins treated by acidic pH shifting alone or pH shifting combined with heating is crucial for the food industry. The aim of this research was to evaluate the effect of extreme acid pH-shifting combined with 50 or 60 °C heating treatments on the gelation and structural properties Received: December 15, 2014 Revised: May 3, 2015 Accepted: May 4, 2015
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DOI: 10.1021/acs.jafc.5b01331 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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emission at 484 nm and excitation at 365 nm (both with a slit width of 5 nm) on a fluorescence spectrophotometer (Hitachi F-4500, Hitachi, Ltd., Tokyo, Japan). The initial FI slope versus protein concentration plot was used to determine the protein surface hydrophobicity. Intrinsic Fluorescence Emission Spectra. The intrinsic emission fluorescence spectra of the protein solutions were obtained using a fluorometer (F-4500 model, Hitachi, Tokyo, Japan). The protein solutions (0.2 mg/mL) were prepared in 10 mM phosphate buffer (pH 7.0). The protein solutions were excited at 295 nm, and the emission spectra were recorded from 300 to 400 nm with a constant slit of 2.5 nm for both excitation and emission. Circular Dichroism (CD). Far-ultraviolet (UV) CD spectra of the protein samples were determined at 25 °C using a CD spectropolarimeter (Mos-450, Biologic, Claix, France) to measure the influence of the different treatments on the secondary structure according to the methods of Jiang et al.,13 with some modification. Each SPI sample (0.5 mg/mL) was dissolved in 10 mM phosphate buffer at pH 7.0 and scanned 5 times between 190 and 250 nm to obtain an averaged value. Phosphate buffer was used as the blank solution. The bandwidth, step resolution, and scan rate were 1.0 nm, 0.2 nm, and 100 nm/min, respectively. The residue ellipticity (θ) values were calculated as [θ] (deg cm2 dmol) = (100XM)/LC, where X is the signal (millidegrees) obtained by the CD spectrometer, M is the average molecular weight (MW) of amino acid residues (assumed to be 115), C is the protein concentration (mg/mL) of the sample, and L is the cell path length (1 cm). Electrophoresis. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) was conducted as described by Liu and Xiong.18 The stacking gel and resolving gel contained 5 and 12% acrylamide, respectively. Electrophoresis samples were prepared both without and with β-mercaptoethanol. For the SDS−PAGE samples without β-mercaptoethanol, 1 mM N-ethylmaleimide (NEM) was added to prevent the formation of disulfide artifacts. Statistical Analyses. The experiment was replicated 3 times, and for each of the replications, at least triplicate samples were analyzed. The data were analyzed using the general linear model procedure. An analysis of variance (AOV) was performed to determine the significance of the main effects. Tukey’s procedures were used to identify the significant differences (p < 0.05) between the means.
of SPI and to identify the relative structural contributions that produced the functionality improvements.
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MATERIALS AND METHODS
Materials. Defatted soy flakes were obtained from Harbin HighTech Group (Harbin, Heilongjiang, China). Ethylenediaminetetraacetic acid (EDTA), 2-nitro-5-thiobenzoate (NTB), and 1-anilino-8naphthalene-sulfonate (ANS) were obtained from Sigma Chemical Co. (St. Louis, MO). All of the reagents and chemicals were of analytical grade. Preparation of SPI. SPI was prepared according to the method of Jiang et al.13 and Chen et al.15 The samples were freeze-dried and stored in a 4 °C cooler. The protein content of the prepared SPI was 94.76% (w/w), as determined by the Kjeldahl method. pH-Shifting and Heating Process-Treated SPI. The pH-treated SPI was prepared according to Jiang and Xiong.13 Suspensions of SPI (30 mg/mL protein) was centrifuged at 12000g for 15 min at 4 °C. The supernatant was adjusted to pH 1.5 with 2 M HCl. After holding at pH 1.5 for 0, 1, 3, or 5 h under different temperature conditions, room temperature (20 ± 2 °C), 50, or 60 °C, to unfold the protein, the solutions were neutralized with 2 M NaOH to pH 7 and kept at this pH for 1 h to make partial refolding. Meanwhile, to remove any salts brought from the pH adjustments, proteins from the treatments were precipitated at pH 4.5, washed 3 times, and then resolubilized at neutral pH.14 The control sample did not treat with pH shifting and heating. The SPI samples treated in a 50 or 60 °C water bath were used for comparison. Testing of Gel Strength. The SPI (120 mg/mL) samples were suspended in deionized water, and gels were formed in 25 × 40 mm (length × diameter) glass vials in a 90 °C water bath for 30 min. Then, the gels were stored overnight at 2−4 °C. Before analysis, the gels were equilibrated at room temperature (22−24 °C) for 30 min. Gel strength of the SPI gels was analyzed with a model TA-XT2 texture analyzer (Stable Micro Systems, Ltd., Godalming, U.K.) attached to a 5 kg load cell. The gels were axially penetrated to a depth of 12 mm at a speed of 50 mm/min with a P/0.5 flat-surface cylindrical probe (12 mm in diameter). The penetration force, defined as the force required to rupture the gel, was expressed as gel strength. Protein Solubility. Protein samples (treated and control SPI) were completely dispersed (20 mg/mL) in sodium phosphate buffer (10 mM, pH 7.0). Then, the protein solutions were centrifuged at 10000g for 15 min. Protein concentrations of the supernatants and whole suspensions were determined by the Biuret method. Protein solubility was calculated according to the method of Jiang et al.13 Particle Size Distribution. Each SPI suspension (0.2 mg/mL, in 10 mM sodium phosphate buffer at pH 7.0) was transferred to a square cuvette for dynamic laser light scattering analysis (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, U.K.). The particle size was expressed as the average of at least six readings of the volume surface mean diameter (D32) and volume mean diameter (D43). Total Sulfhydryl (SH) and Exposed SH Contents. SH content was measured according to the method of Beveridge et al.16 SPI samples (15 mg) were suspended in 5.0 mL reaction buffer (0.09 M glycine, 0.086 M Tris, and 4 mM EDTA at pH 8.0) with (total SH) or without (exposed SH) 8 M urea. After the addition of 50 μL of Ellman’s reagent, the resultant suspensions were incubated for 1 h at room temperature (23 ± 1 °C) with occasional vibrating and then centrifuged for 15 min at l0000g. The absorbance of the supernatant was read at 412 nm with the reagent buffer as the blank. The protein content of samples was measured by the micro-Kjeldahl method. The total SH and exposed SH contents in micromoles per gram of protein were calculated using the extinction coefficient of NTB, which is 13 600 M−l cm−l. Surface Hydrophobicity. The surface hydrophobicity of the samples was measured using ANS as a fluorescence probe.17 Briefly, 4 mL aliquots of the SPI samples (0.005−0.5 mg/mL) were dissolved in 0.01 M phosphate buffer at pH 7.0 and mixed with 50 μL of 8 × 103 M ANS. After the reaction had proceeded for 10 min at room temperature, the fluorescence intensity (FI) was determined with an
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RESULTS AND DISCUSSION Penetration Testing. Penetration testing is useful way to determine gel properties. For samples treated with pH-shifting alone or 50 °C mild heating alone, there were no significant changes (p > 0.05) in the penetration force with the increased incubation time (Figure 1). The penetration force of the sample
Figure 1. Penetration force of SPI after acidic pH-shifting combined with heating treatments at various holding times. B
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Figure 2. Solubility of SPI after pH-shifting combined with heating treatments at various holding times.
Figure 4. Total and exposed SH contents of SPI after pH-shifting combined with heating treatments at various holding times.
Figure 5. Surface hydrophobicity of SPI after acidic pH-shifting combined with heating treatments at various holding times.
Figure 3. Particle size of SPI solutions after pH-shifting combined with heating treatments at various holding times: (a) volume surface mean diameter (D32) and (b) volume mean diameter (D43).
0.05). However, the combination of pH-shifting with 50 and 60 °C heating treatments led to drastic increases in the penetration force of SPI gels (p < 0.05), with the value increased by 44.1
with 60 °C heating alone for 5 h was 2.26 N, and it was significantly higher than that of control samples (2.13 N) (p < C
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Figure 6. Intrinsic emission fluorescence spectra of SPI after acidic pH-shifting combined with heating treatments at various holding times.
hydrogen and hydrophobic bonds, and Mleko and Foegeding22 revealed the increase in shear moduli of whey protein gels and increase in temperature during heating mainly to the presence of hydrophobic forces. Protein Solubility. Solubility can affect protein surfaceactive properties. As depicted in Figure 2, the solubility of control SPI was 94.4%, with heating the SPI at 50 and 60 °C alone causing no significant change in solubility (p > 0.05). When the sample was treated with pH 1.5 shifting, the solubility decreased sharply with increasing incubation times (p < 0.05), which dropped to 77.9% after 5 h. Yuan et al.23
and 69.0% after holding for 5 h, respectively, compared to control SPI. A protein gel can be stabilized by both non-covalent and covalent forces. The gelation or coagulation of SPI was shown to include covalent cross-links and hydrophobic interactions.19 Results from SDS−PAGE (Figure 8) and SH content measurements (Figure 4) confirmed the formation of disulfide bonds during heating. Some researchers have suggested that this disulfide bond formation helps to reinforce the gel structure.20 Furthermore, O’Kane et al.21 also showed that network formation by the legumin proteins was caused by D
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Figure 7. CD spectra of SPI after acidic pH-shifting combined with heating treatments at various holding times.
that the solubility had a obvious correlation with surface hydrophobicity of whey protein, which was affected by the aggregation state of the protein. The results were also supported by SH contents, surface hydrophobicity, and SDS− PAGE analysis, as discussed below. Particle Size Determination. The volume surface mean diameter (D32) and volume mean diameter (D43) of control or modified SPI dispersions were measured by a dynamic laser light scattering instrument (Figure 3). The D32 and D43 of control SPI were 102.3 and 188.2 μm, respectively, and heating the SPI at 50 and 60 °C alone caused no significant change in particle size (p > 0.05). As expected, pH-shifting treatment
reported that, because of the effect of acid (pH 2.0) treatment, SPI would unfold, aggregate, and expose more hydrophobic residues. Meanwhile, similar trends were observed for samples treated with pH 1.5 + 50 or 60 °C, which rendered more solubility reduction than that for the pH 1.5 shifting treatment alone for 1, 3, and 5 h, respectively (p < 0.05). This phenomenon indicated that SPI was more susceptible to denaturation under extremely low pH-shifting combined with heating conditions. Jiang et al.13 suggested that the decrease in protein solubility was mainly contributed to the formation of disulfide cross-linking and hydrophobic aggregation under the extremely low pH-shifting treatments. Liu et al.24 also noted E
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Figure 8. SDS−PAGE pattern of SPI after acidic pH-shifting combined with heating treatments at various holding times. Con: SPI solutions at pH 7.0 without pH-shifting and heating treatment. The SDS−PAGE samples were prepared (a and c) with or (b and d) without β-mercaptoethanol. MW: molecular weight marker (Da). SPI constituents: α′ (86 kDa), α (66 kDa), and β (51 kDa) for conglycinin; A, the acidic subunit (34−43 kDa), B, the basic subunit (17−26 kDa) for glycinin.
decreased (p < 0.05) by 22.6, 9.2, 10.18, 45.5, and 80.23% at 5 h for the samples treated with pH 1.5 shifting, 50 °C heating, 60 °C heating, pH 1.5 + 50 °C, and pH 1.5 + 60 °C, respectively. The acidic pH-shifting treatments alone had little effect (p > 0.05) on the total SH after treating for 5 h, which was in accordance with the results of Jiang et al.13 The heating treatment at 50 °C alone also had minimal effect on decreasing the total SH (p > 0.05), and the effect of 60 °C treatment was larger than the effect observed for 50 °C treatment (p < 0.05). However, the acidic pH-shifting or heating treatments alone had significant effects in decreasing the exposed SH (p < 0.05) of SPI for 5 h of incubation, which were different from the effects on the total SH. The combination of pH-shifting and heating treatments could significantly enforce the conversion of SH groups into disulfides (p < 0.05). The amount of detectable total SH and exposed SH in the pH 1.5 + 60 °C samples after incubation exhibited more decreases than observed in the pH 1.5 + 50 °C samples. The decreased number of SH contents can be used to explain the improved gel texture of SPI gel in Figure 1. It was noteworthy that incubations over 1 h resulted in a loss of SH contents, which were mainly due to protein structure opening in control proteins being particularly sensitive to the heating as well as the formation of S−S bonds leading to a decreased number of total and exposed SH. The disulfide covalent
significantly increased the particle size of SPI (p < 0.05), and D32 and D43 increased 1.26- and 1.42-fold after 5 h of incubation, respectively. Moreover, SPI samples treated with pH 1.5 + 50 or 60 °C exhibited a larger particle size than that with the pH 1.5 shifting treatment alone (p < 0.05). Yuan et al.23 pointed out that the mean hydrodynamic diameter of SPI obviously increased after acid (pH 2.0) treatment for 1 h. Gulseren et al.25 found that non-covalent interactions were the immediate driving force for the protein aggregation. In addition, according to Kohyama et al., 26 hydrophobic interactions between protein molecules induced by the aggregation of the protein were the predominant factors for a fine gel network structure. Hu et al.27 also suggested that exposition of hydrophobic residues might facilitate the formation of protein−protein aggregates, leading to a better gel network and water-holding capacity. Consequently, it can be concluded that complicated hydrophobic aggregation of SPI occurs under pH-shifting combined with mild heating treatments. SH Contents. The pH-shifting and heating treatments caused some changes in the SH contents that seemed to have some relation to protein unfolding. The total SH and exposed SH in the SPI samples decreased with increasing incubation times (p < 0.05; Figure 4). The total SH in control SPI samples was 4.495 μmol/g of protein, and the value significantly F
DOI: 10.1021/acs.jafc.5b01331 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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The blue shift was mainly attributed to an alter in the threedimensional positions and the shift of the Trp microenvironment toward a more hydrophobic microenvironment.39 Jiang et al.13 reported that some buried side chains could be exposed to the polar surface after acid pH-shifting treatments. Wu et al.40 noted that blue shifts of the maximum emission wavelength revealed that the previously exposed Trp residues of SPI were buried in the interior of the modified SPI molecules. As expected, the intrinsic fluorescence results are consistent with those from the ANS analyses (Figure 5), which expressed the gradual exposure of hydrophobic clusters initially buried in the extreme pH-shifting combined with heating processes. CD Spectra. To understand the impact of acidic pH and heating processes on the secondary structure of SPI, the soy protein samples were subjected to CD determination. According to Kelly and Price,41 the ellipticities at 222 and 208 nm allow for the evaluation of the α-helix content in a protein. Moreover, the blue shift of the peak wavelength and the attenuation of the 210 nm negative peaks suggest losses in the α-helical structure content. As shown in Figure 7a, the CD spectra for the pH-shifting samples were not markedly altered during the first 1 h of incubation; however, after 3−5 h of treatment, the negative peak changed and blue shifted. Jiang et al.8,13 noted that the CD spectra of the pH-shifted SPI samples incubated for shorter times exhibited only slight changes. However, after acidic pH-shifting combined with heating treatments, considerable changes occurred in the SPI (panels b and c of Figure 7). The negative peak attenuated with the holding time and the peak minimum shifted from 209 to 207 nm in the pH-shifting + 50 °C sample incubated for 5 h, which indicates partial disruption of the α helix. A similar result was obtained for samples under pH-shifting + 60 °C treatment, where the peak minimum reduced to 206 nm. To prove that this change was not exclusively caused by heating, the controls for 50 and 60 °C heating alone were measured and are shown in panels d and e of Figure 7. Clearly, no obvious changes in the CD spectra were observed, and the secondary α-helix conformation did not appear to be disrupted by mild heating treatments alone. These results indicate that a change in secondary structure is commonly observed among samples subjected to a combination of pH 1.5 shifting and mild heating processes. In addition, there was a negatively linear correlation between surface hydrophobicity and the α-helix structure content of SPI, which was supported by the results of Hou and Chang.42 SDS−PAGE. SDS−PAGE of the protein samples treated with β-mercaptoethanol or n-ethylmaleimide (NEM) was used to study the role of S−S linkages in forming protein aggregates in protein samples incubated under acidic pH and heating conditions (Figure 8). The SDS−PAGE patterns of the control SPI had the characteristic bands corresponding to the subunits of β-conglycinin (α′, α, and β) and glycinin (A and B). As shown in panels b and d of Figure 8, under non-reducing conditions (+NEM), large polymers were observed in both the pH-shifting and pH-shifting and heating samples that accumulated at the top of the gel. These high-molecular-weight (MW) bands were disulfide covalent polymers and aggregates from soy globulins. The gradual disappearance of the α′, α, and β subunits in the pH 1.5 + heating samples during incubation suggested that β-conglycinin was the precursor of those highMW polymers. Moreover, the glycinin subunits (A and B) showed unremarkable changes during incubation.
polymers have been illustrated by the SDS−PAGE results (Figure 8). Some researchers reported that acidation leads to the removal of disulfide bonds; however, with consistent heating treatments, the repeated formation of intra- and intermolecular disulfide bonds and the occurrence of oxidation may give rise to the loss of total SH and reaction SH contents.28 The reason that total SH contents have no significant changes in pH 1.5 shifting treated samples may be due to the lower pH-treated samples only displaying limited denaturation (as proven by the hydrophobicity and CD analysis; Figures 5 and 7) and disulfide bonds being relatively stable at acidic conditions.29 The decrease in the SH content incubation of pH-shifting + heating samples was maybe due to the formation of disulfide bonds by SH oxidation and SH/SS interchange reactions, which is favored by heating.30 This result revealed that the SH contents or disulfide linkages in SPI were important residue factors for the protein conformational stability. Surface Hydrophobicity. Surface hydrophobicity allows for the detection of changes in the distribution of hydrophobic groups at the surface, which are caused by changes in the molecular structure of protein upon denaturation. The other factors that may cause an increase in surface hydrophobicity include the expansion of peptide chains or the dissociation of protein subunits.31 Studies had reported that treatment conditions, including temperature, time, and protein concentration, influenced protein surface hydrophobicity.32 In our study, acidic pH-shifting treatment alone showed a slight effect on the surface hydrophobicity of soy protein, and surface hydrophobicity rose significantly (p < 0.05) only after 5 h of treatment (Figure 5). Conversely, the acidic pH-shifting combined with heating (50 °C for more than 3 h or 60 °C for more than 1 h) markedly increased the hydrophobicity values of SPI (p < 0.05), where the pH 1.5 + 60 °C treatment samples had the highest hydrophobicity. As Jiang et al.8 reported, at the extreme acidic pH conditions, most of the proteins in SPI might be deacetylated and the removal of charged COO− groups would mainly account for the increased surface hydrophobicity, which resulting in enhanced attractive interactions between individual protein molecules. Temperature increases cause proteins to unfold, exposing the SH and hydrophobic groups. For example, glycinin has secondary structure alterations and a surface hydrophobicity that increases upon heating.33,34 Heating the SPI, which consists of both glycinin and β-conglycinin, induces the development of specific interactions among the subunits35 and leads to the formation of soluble complexes between the β subunit of β-conglycinin and the basic subunit of glycinin.36 However, in Figure 3, heating the SPI at 50 and 60 °C alone caused no significant change in the hydrophobicity value (p > 0.05). Hence, acidic pH-shifting treatment might cause proteins to unravel their spatial structure and render a MG state13,37 when combined with mild heating and would further disrupt the structure, with more hydrophobic groups exposed on the surface, as the temperature and time increase. Emission Fluorescence Spectroscopy Analysis. Intrinsic fluorescence spectroscopy reflect the movement of protein side chains.38 As depicted in panels a, b, and c of Figure 6, the λmax of the samples treated with pH 1.5 shifting, pH 1.5 + 50 °C, and pH 1.5 + 60 °C, respectively, had marked shifts in the maximum emission wavelength compared to the control SPI, while the fluorescence spectra of both 50 and 60 °C heating samples had no obvious blue shift (panels d and e of Figure 6). G
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(6) German, B.; Damodaran, S.; Kinsella, J. E. Thermal dissociation and association behavior of soy proteins. J. Agric. Food Chem. 1982, 30, 807−811. (7) Damodaran, S.; Kinsella, J. E. Effect of conglycinin on the thermal aggregation of glycinin. J. Agric. Food Chem. 1982, 30, 812−817. (8) Jiang, J.; Xiong, Y. L.; Chen, J. Role of β-conglycinin and glycinin subunits in the pH-shifting-induced structural and physicochemical changes of soy protein isolate. J. Food Sci. 2011, 76, C293−302. (9) Matsudomi, N.; Sasaki, T.; Kato, A.; Kobayashi, K. Conformational changes and functional properties of acid-modified soy protein. Agric. Biol. Chem. 1985, 49, 1251−1256. (10) Wagner, J. R.; Sorgentini, D. A. Thermal and electrophoretic behavior, hydrophobicity, and some functional properties of acidtreated soy isolates. J. Agric. Food Chem. 1996, 44, 1881−1889. (11) Wagner, J. R.; Guéguen, J. Surface functional properties of native, acid-treated, and reduced soy glycinin. 1. Foaming properties. J. Agric. Food Chem. 1999, 47, 2173−2180. (12) Liang, Y.; Kristinsson, H. G. Structural and foaming properties of egg albumen subjected to different pH-treatments in the presence of calcium ions. Food Res. Int. 2007, 40, 668−678. (13) Jiang, J.; Chen, J.; Xiong, Y. L. Structural and emulsifying properties of soy protein isolate subjected to acid and alkaline pHshifting processes. J. Agric. Food Chem. 2009, 57, 7576−83. (14) Jiang, J.; Xiong, Y. L. Extreme pH treatments enhance the structure-reinforcement role of soy protein isolate and its emulsions in pork myofibrillar protein gels in the presence of microbial transglutaminase. Meat Sci. 2013, 93, 469−76. (15) Chen, N. N.; Zhao, M. M.; Sun, W. Z.; Ren, J. Y.; Cui, C. Effect of oxidation on the emulsifying properties of soy protein isolate. Food Res. Int. 2013, 52, 26−32. (16) Beveridge, T.; Toma, S. J.; Nakai, S. Determination of SH- and SS-groups in some food proteins using Ellman’s reagent. J. Food Sci. 1974, 39, 49−51. (17) Sorgentini, D. A.; Wagner, J. R. Comparative study of structural characteristics and thermal behavior of whey and isolate soybean proteins. J. Food Biochem. 1999, 23, 489−507. (18) Liu, G.; Xiong, Y. L. Electrophoretic pattern, thermal denaturation, and in vitro digestibility of oxidized myosin. J. Agric. Food Chem. 2000, 48, 624−630. (19) Tang, C. H.; Wu, H.; Yu, H. P.; Li, L.; Chen, Z.; Yang, X. Q. Coagulation and gelation of soy protein isolates induced by microbial transglutaminase. J. Food Biochem. 2006, 30, 35−55. (20) Zhou, F.; Zhao, M.; Su, G.; Cui, C.; Sun, W. Gelation of salted myofibrillar protein under malondialdehyde-induced oxidative stress. Food Hydrocolloids 2014, 40, 153−162. (21) O’Kane, F. E.; Happe, R. P.; Vereijken, J. M.; Gruppen, H.; van Boekel, M. A. J. S. Heat-induced gelation of pea legumin: Comparison with soybean glycinin. J. Agric. Food Chem. 2004, 52, 5071−5078. (22) Mleko, S.; Foegeding, E. A. pH induced aggregation and weak gel formation of whey protein polymers. J. Food Sci. 2000, 65, 139− 143. (23) Yuan, B. E.; Ren, J. Y.; Zhao, M. M.; Luo, D. H.; Gu, L. J. Effects of limited enzymatic hydrolysis with pepsin and high-pressure homogenization on the functional properties of soybean protein isolate. LWT - Food Sci. Technol. 2012, 46, 453−459. (24) Liu, C. M.; Zhong, J. Z.; Liu, W.; Tu, Z. C.; Wan, J.; Cai, X. F.; Song, X. Y. Relationship between functional properties and aggregation changes of whey protein induced by high pressure microfluidization. J. Food Sci. 2011, 76, E341−E347. (25) Gulseren, I.; Guzey, D.; Bruce, B. D.; Weiss, J. Structural and functional changes in ultrasonicated bovine serum albumin solutions. Ultrason. Sonochem. 2007, 14, 173−183. (26) Kohyama, K.; Sano, Y.; Doi, E. Rheological characteristics and gelation mechanism of tofu (soybean curd). J. Agric. Food Chem. 1995, 43, 1808−1812. (27) Hu, H.; Fan, X.; Zhou, Z.; Xu, X. Y.; Fan, G.; Wang, L. F.; Huang, X. J.; Pan, S. Y.; Zhu, L. Acid-induced gelation behavior of soybean protein isolate with high intensity ultrasonic pre-treatments. Ultrason. Sonochem. 2013, 20, 187−195.
For the reducing SDS−PAGE (panels a and c of Figure 8), the large MW aggregates and polymers were completely dissociated into the corresponding α′, α, and β subunits of βconglycinin. All protein samples expressed a similar electrophoretic pattern. These results demonstrate that disulfide crosslinking of β-conglycinin was responsible for the production of protein aggregates and polymers during the pH-shifting and heating processes, which also explains the increased protein gelation observed in Figure 1. Both inter- and intramolecular SH/SS interchanges are dynamic and are expected in the observed protein polymer formation.14 Such reactions more likely would result in less SH groups and more S−S linkages, which might lead to some favorable changes in the protein functional properties. In conclusion, our results demonstrated that the pH treatments under extremely acidic (pH 1.5) conditions combined with proper heating (50 and 60 °C) produced structurally modified SPI samples that displayed enhanced gelling ability. The pH-shifting and heating denaturation induced an increase in the particle size, surface hydrophobicity, and SH content and a decrease in the solubility, resulting in protein cross-linking. The CD and fluorescence spectroscopy analyses revealed secondary and tertiary structural changes in pH-shifting and heating samples. Overall, the improvement in the protein gelling property induced by pH-shifting combined with heating treatments was party related to the change in secondary and tertiary structures and mainly attributed to the connection and interaction between protein subunits through hydrophobic association, hydrogen bonds, and disulfide bonds. The pH-shifting combined with mild heating treatments give great potential for the production of functionality-improved SPI.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-451-55191794. Fax: +86-451-55190577. Email:
[email protected]. Author Contributions †
Qian Liu and Rui Geng contributed equally to this work.
Funding
This study was supported by the Development Program of China (863 Program) (Grant 2013AA102208-2), the Program for Young Talents of Northeast Agricultural University (Grant 14QC40), and the Foundation of Science and Technology in Heilongjiang (Grant GC13B212). Notes
The authors declare no competing financial interest.
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