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Apr 6, 2015 - Heat-induced soy protein gels were prepared by heating protein solutions at 12%, 15% ,or 18% for 0.5, 1.0, or 2.0 h. The release of ...
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Release Behavior of Non-Network Proteins and Its Relationship to the Structure of Heat-Induced Soy Protein Gels Chao Wu, Yufei Hua,* Yeming Chen, Xiangzhen Kong, and Caimeng Zhang State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, People’s Republic of China ABSTRACT: Heat-induced soy protein gels were prepared by heating protein solutions at 12%, 15% ,or 18% for 0.5, 1.0, or 2.0 h. The release of non-network proteins from gel slices was conducted in 10 mM pH 7.0 sodium phosphate buffer. SDS-PAGE and diagonal electrophoresis demonstrated that the released proteins consisted of undenatured AB subunits and denatured proteins including monomers of A polypeptides, disulfide bond linked dimers, trimers, and polymers of A polypeptides, and an unidentified 15 kDa protein. SEC-HPLC analysis of non-network proteins revealed three major protein peaks, with molecular weights of approximately 253.9, 44.8, and 9.7 kDa. The experimental data showed that the time-dependent release of the three fractions from soy protein gels fit Fick’s second law. An increasing protein concentration or heating time resulted in a decrease in diffusion coefficients of non-network proteins. A power law expression was used to describe the relationship between nonnetwork protein diffusion coefficient and molecular weight, for which the exponent (α) shifted to higher value with an increase in protein concentration or heating time, indicating that a more compact gel structure was formed. KEYWORDS: soy protein gel, non-network proteins, release behavior, diffusion coefficients, network, protein composition

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by acidification or enzymes. Gireeshkumar et al.23,24 measured the mobility of fluorescent-labeled dextran with different molecular weights in β-lactoglobulin solutions and gels at different concentrations and different ionic strengths by fluorescence recovery after photobleaching (FRAP), and they observed a strong decrease in diffusion coefficients with an increasing tracer size and protein concentration in more homogeneous gels that were formed at lower salt concentrations. On the basis of the above studies, it can be concluded that microstructural changes in protein gels under different conditions can be characterized by tracing the mobility of the particles in the gel. Like many other globular proteins, soy protein can form a gel upon heating. Commercial soy protein isolates consist of two major components: 7S β-conglycinin and 11S glycinin globulins. The 7S component is a trimeric glycoprotein that is composed of three subunits, α (67 kDa), α′ (71 kDa), and β (50 kDa), which are associated via hydrophobic interactions, while the 11S component is composed of six subunits, each of which is disulfide linked via acidic (A, 37−42 kDa) and basic (B, 17−20 kDa) dimers. During thermal treatment, both globulins dissociate into subunits and the dissociated β subunits of 7S preferentially interact with the basic polypeptides of the 11S globulin, which may be responsible for the formation of the three-dimensional network in gels.2,3 Many studies2,3,5,25 have demonstrated that the contribution of subunits to the gel network differs and that proteins which are not involved in network formation can be isolated from the gels by centrifugation,2,26,27 extraction,2,8 or diffusion processes.26 In

INTRODUCTION Soy proteins are widely used ingredients in many food products. The ability to form a gel upon heating is one of the most important attributes of commercial soy protein products. Gel microstructures are strongly affected by the heating time, temperature, ions, protein concentration, and pH. Many studies of soy protein gels have been conducted to reveal the polypeptides/subunits that participate in network formation,1−3 thermodynamic properties,1,4,5 macrostructure,6−10 changes in protein conformation,11−13 and structure−physicochemical function relationships. However, very little is currently known about the relationship between the structural changes in the soy protein gel and the release kinetics of components that are not involved in gelation. Protein gels can be used as vehicles to deliver large-molecular-weight solutes, such as peptides and proteins.14,15 Understanding the release of proteins from gels is a subject of considerable interest and has received increased attention in recent years. The mobility of particles within protein gels clearly depends on the gel structure. A large number of studies have already reported the self-diffusion of particles in gels. Philippe et al.16 studied the diffusion of different sizes of dextran in heatdenatured β-lactoglobulin solution, aggregates, and gels by pulsed field gradient NMR (PFG-NMR) and observed a decrease in the diffusion coefficients with an increase in protein concentration or a decrease in NaCl concentration. Colsenet et al. studied the diffusion of polyethylene glycols (PEG) in casein17,18 or whey protein19,20 suspensions and gels by PFGNMR, and they found a decrease in the diffusion coefficients with an increasing protein concentration, denaturation time, or decreasing ionic strength; this decrease was more pronounced for larger PEG polymers. Feunteun et al.21,22 also studied the relationship between the self-diffusion of PEG and the casein gel structure but with different coagulation processes induced © XXXX American Chemical Society

Received: January 9, 2015 Revised: April 1, 2015 Accepted: April 5, 2015

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DOI: 10.1021/acs.jafc.5b00132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry the present study, non-network proteins, as with dextran16,23,24 or PEG,17−22 were used as “probes”. The diffusion coefficients of non-network proteins with different molecular weights in heat-induced soy protein gels, in terms of the effect of the protein concentration and heating time, were investigated. Fick’s diffusion law was used to analyze the release kinetics of the non-network proteins. The relationship between the release behavior of non-network proteins and the structure of the heatinduced soy protein gel in response to the protein concentration and heating time are discussed. The quantitative characterization of the diffusion behavior provides insight into the structure and properties of heat-induced soy protein gels.

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Determination of the Protein Concentration. The protein concentration of the samples (filtered through a cellulose acetate membrane with a pore size of 0.22 μm; Sartorius Co., Ltd., Gottingen, Germany) at different time points was determined using the bicinchoninic acid (BCA) method,29 with bovine serum albumin as a standard. The BCA reagent was prepared by mixing 50 volumes of reagent A (containing 1% BCA, 2% Na2CO3, 0.16% Na2C4H4O6, 0.4% NaOH, 0.95% NaHCO3) with 1 volume of reagent B (4% CuSO4) immediately before use. Then, 100 μL of the sample and 2 mL of BCA reagent were pipetted into a test tube and mixed thoroughly. The mixture was allowed to stand for 30 min at 37 °C and then returned to room temperature, and the absorbance was monitored at 562 nm, using 100 μL of water as a control. All of the measurements were performed in triplicate. Contents of Released Non-Network Proteins. The content (Wrp, g/100 g of total protein) of released proteins was calculated as

MATERIALS AND METHODS

Materials. Low-temperature soy meals were kindly provided by Shandong Gushen Industrial & Commercial Co., Ltd. (Dongying, Shandong, People’s Republic of China). The flakes had a protein content of 55.0% (N × 6.25, dry base) and a nitrogen solubility index of 87%. Defatted soy flakes were extracted with 85% aqueous alcohol at room temperature for 1 h with a ratio of 1/5 (w/v) flakes to solvent. The slurry was vacuum-filtered, and the filter cake was mixed with 95% (v/v) ethanol at room temperature for 1 h with a ratio of 1/2 (w/v). After it was dried for 2 days at room temperature, the meal was dispersed in distilled water (1/10, w/v) and adjusted to pH 7.0 by adding a small amount of 2 M NaOH. After it was stirred for 1 h at room temperature, the suspension was centrifuged (15800g, 30 min, 4 °C) to recover the supernatants. Soy protein was precipitated by adjusting the pH to 4.5 with 2 N HCl and centrifuged at 10000g for 30 min at 4 °C. After it was washed with distilled water, the protein precipitate was resuspended in distilled water and solubilized by adjusting the pH to 7.0. The protein dispersions were stirred overnight at 4 °C to enhance protein dissolution. The protein solution was lyophilized and stored at −18 °C. Proximate analysis showed that the dried powder had protein and ash contents of 91.65 ± 0.8% (N × 6.25) and 4.26 ± 0.04%, respectively, on a dry basis, and the 7S/11S ratio of native isolate was 0.79 ± 0.02 (determined by reducing-SDSPAGE).28 All other reagents were of analytical grade. Preparation of the Protein Gels. Rehydration of the soy protein powder was performed by thorough magnetic stirring at room temperature with 0.1 M NaCl and 10 mM sodium phosphate buffer (pH 7.0) at the desired concentration (12%, 15%, and 18%, w/v) (soy gels with a protein concentration higher than 12% are easy to handle during the release process). In each case the pH was adjusted to 7.0 with 1 N HCl or 1 N NaOH. Sodium azide was added (0.02% w/v) to each solution to prevent bacterial development. The soy protein suspensions were centrifuged at 40000g for 30 min to remove gas and then carefully placed in SEBC bottles (special glass bottles with a tight screw-threaded blue cover and with high temperature, acid, and alkali resistance, Biocolor Co., Ltd., Shanghai, People’s Republic of China). Gelation was then conducted by heating the soy protein dispersions in SEBC bottles at 95 °C, after the temperature of the solution reached the target value (95 °C), which occurred after approximately 3 min in a preliminary experiment. The solution was maintained in the water bath for exactly 30 min or for different time periods (30 min, 1 and 2 h). After heating, the bottles were immediately cooled in ice water for 5 min and stored at 4 °C overnight to ensure complete gelation before further use. Release of Non-Network Proteins from Gels. The gels (approximate 20 g) generated under different conditions were cut into 1.5 mm thick slices with a razor and transferred to 10 mM sodium phosphate buffer (pH 7.0) (10 times excess w/v) in 500 mL SEBC bottles. NaN3 (0.02% w/v) was added to each solution to inhibit bacteria growth. The SEBC bottles were gently rotated in a water bath at 25 °C. The non-network proteins in the gels were allowed to diffuse out of the gel slices for 56 h, which was sufficiently long to reach equilibrium on the basis of preliminary experiments. During diffusion, 0.5 mL samples were periodically collected and replaced with fresh buffer.

Wrp =

CnonVbuffer × 100 1000C pMgel

where Cnon, Vbuffer, Cp, and Mgel are the concentration (mg mL−1) of non-network proteins in the buffer at equilibrium (48 h), the volume (mL) of buffer used, the protein concentration (g/g) in the gel, and the weight (g) of the gel in the buffer, respectively. Contents of Released Undenatured 11S Proteins and Denatured Proteins. The contents of released proteins (Wrp, g/ 100 g of total protein) were divided into two parts. One part is the content of released undenatured 11S proteins. (The 7S component is completely denatured at 90 °C after 30 min according to Sorgentini et al.,28 and therefore, the undenatured component in the released protein is native 11S-AB. Ruan et al.30 found that the disruption of the interpolypeptide disulfide bond between A and B occurred even at 70−80 °C lower than the denaturation temperature (93.21 °C) of 11S. Therefore, the intact AB band in nonreducing SDS-PAGE can be identified as undenatured 11S.) The other part is the content of denatured proteins released from the protein gel. The content of undenatured 11S proteins (Wundenatured, g/100 g of total protein) released from the gel was calculated from the equation

Wundenatured = WrprAB

(1)

where Wrp and rAB are the content of released proteins (Wrp, g/100 g of total protein) and the ratio of the content of undenatured 11S proteins to that of released network proteins, obtained by comparing the AB band intensity with the total intensity of all of the bands in the same nonreducing SDS-PAGE lane. The content of denatured proteins (Wdenatured, g/100 g of total protein) in the released network proteins was calculated as

Wdenatured = Wrp(1 − rAB)

(2)

where 1 − rAB represents the ratio of released denatured proteins to the content of released network proteins. Network Protein Content per 100 g of Gel. The network protein content (Wnet, g/100 g of gel) was calculated using the equation ⎛ Wrp ⎞ Wnet = C p⎜1 − ⎟ 100 ⎠ ⎝

(3)

where Cp and Wrp are the protein concentration (g/g) in the gel and the content of released proteins (g/100 g of total protein). High-Performance Size Exclusion Chromatography (SECHPLC). The molecular weight distribution of the samples was determined according to the method reported by Wu et al.31 with some modifications. The samples were filtered through a cellulose acetate membrane with a pore size of 0.22 μm (Sartorius Co., Ltd., Gottingen, Germany). A Hitachi liquid chromatography system equipped with a Shodex protein KW-804 column (Shodex Separation and HPLC Group, Tokyo, Japan) was used. The elution was performed with 50 mM phosphate buffer (pH 7.0, including 0.3 M NaCl) at a flow rate of 1.0 mL/min. Each sample (20 μL) was injected B

DOI: 10.1021/acs.jafc.5b00132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. (a) Changes of protein concentration in the buffer after the gel slices (15% protein concentration) were placed in. (b) Relative nonnetwork protein mass release as a function of the square root of the diffusion time.

Figure 2. (a) SEC profiles of the samples released at different diffusion times from protein gels with a 15% protein concentration. (b) SDS-PAGE of eluates collected from the SEC-HPLC experiment. Reducing SDS-PAGE lanes 1−6: 1, marker (kDa); 2, gel; 3, diffusion sample; 4, peak 1; 5, peak 2; 6, peak 3. into the column, and the effluent was monitored at 214 nm. The column was calibrated using standard proteins (Bio-Rad) between 1350 Da and 670 kDa (vitamin B12, 1350 Da; horse myoglobulin, 17 kDa; chicken ovalbumin, 44 kDa; bovine gamma globulin, 158 kDa; thyroglobulin, 670 kDa). All of the samples were measured in triplicate, and an Agilent Chemistry Station was used to analyze the peak area. Diffusion Coefficients of Non-Network Proteins in Gels and in Water. Non-network proteins with different molecular weights released from soy gel slices were characterized by calculating the apparent diffusion coefficient (D), which was estimated on the basis of the release kinetics curve, fitted using an analytical solution of Fick’s second law:32

⎛ Dt ⎞1/2 Mt = 4⎜ 2 ⎟ ⎝ πδ ⎠ M∞

D0 =

where T, k, and η are the kelvin constant (293.5 K), Boltzmann constant (1.38 × 10−23 J K−1) and the viscosity of pure water (1.00 × 10−3 Pa s at 25 °C). The Rh values of peaks 1−3 used for calculations are 5.85 nm (Stokes radius of native 11S globulins),33 2.80 nm (ovalbumin, 45 kDa), and 2.01 nm (α-lactalbumin, 14 kDa), respectively. SDS-PAGE. SDS-PAGE was performed to examine the protein compositions according to the method proposed by Laemmli.34 Electrophoresis was performed with a continuous and dissociating buffer system: 0.375 M Tris-HC1 (pH 8.8) and 0.1% SDS for the separating gel and 0.025 M Tris-HC1 (pH 8.3), 0.192 M glycine, and 0.1% SDS for the run buffer. A 12.5% resolving gel overlaid with a 3% stacking gel was used. Aliquots of each sample were mixed with 1× sample dissolving buffer (4% SDS, 20% glycerol, 0.125 M Tris-HCl buffer, pH 6.8, 0.01% bromophenol blue) and divided into two parts: one part with 0.02% 2-mercaptoethanol (w/w) heated to 5 min at 100 °C and the other part without 2-mercaptoethanol. These two parts were centrifuged at 15000g for 10 min at room temperature. The two parts were loaded separately into SDS-PAGE gels and subjected to electrophoresis. The gel was then stained with Coomassie brilliant blue R250 and destained with deionized water that was changed two to three times. Finally, the gels were scanned using a computing densitometer (Molecular Imager Chemi Doc XRS+, Bio-Rad, USA). To investigate the protein compositions of the peak area of the SECHPLC curve, the eluates of the corresponding area were collected 50 times and then concentrated using an Amicon Ultra 15 mL centrifugal filter (Millipore Corporation, Germany) with a membrane NMWL (nominal molecular weight limit) of 3 kDa. The concentrated eluates were analyzed by SDS-PAGE. The intensities of the protein bands were analyzed using Image Lab software (Bio-Rad, USA). The content

(4)

where Mt and M∞ are the amounts of protein released from the gel at time t and the protein in the buffer in the equilibrium state (48 h), respectively, while D and δ are the diffusion coefficient and the gel thickness, respectively. Mt/M∞ can also be expressed as St/S∞ (given that the volume of the diffusion buffer is almost constant), in which St and S∞ are the SEC-HPLC peak areas at time t and at the time of equilibrium, respectively. Therefore, the diffusion coefficient of nonnetwork proteins can be calculated as

D=

k 2δ 2π 16

kT 6πηR h

(5)

where k is the slope of the relative solute release (St/S∞) versus the square root of the time curve. The diffusion coefficients in water (D0) were calculated using the Stokes−Einstein equation, for different fractions: C

DOI: 10.1021/acs.jafc.5b00132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry of each component was determined as a percentage of the component in the sample by comparing the individual band intensities with the total intensity of all of the bands in the same lane. Statistical Analysis. Experiment data were subjected to analysis of variance (ANOVA) using SAS 9.1. A least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. All of the treatments were performed in triplicate. Data are expressed as the mean ± SD (n = 3).

of soy protein components to the network structure of gels. Subunits that are weakly involved in the network should be relatively free and released from the gels by ultracentrifugation. Puppo et al.9 reported that the 11S fractions remained in the native state after heating and would exist as soluble peptides in gel matrix interstices. Diffusion Coefficients of Non-Network Proteins with Different Molecular Weights. Figure 3 shows the relative

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RESULTS AND DISCUSSION Release of Non-Network Proteins from 15% Soy Protein Gels. Changes in the Released Non-Network Protein Concentration. The protein concentrations after diffusion time of 0.5, 1, 2, 4, 6, 9, 12, 23, 27, 32, 40, 48, and 56 h were determined, and the results are displayed in Figure 1a. The concentration of released proteins increased steadily over the first 12 h, but there was essentially no change during the final 8 h. Therefore, the diffusion of non-network proteins reached an equilibrium state at approximately 48 h. The diffusion of non-network proteins as a function of the diffusion time was fitted to Fick’s second law by plotting the relative mass release of non-network proteins (Mt/M48) against the square root of the diffusion time, as shown in Figure 1b. A linear relationship was observed during the first 12 h, indicating that the initial phase of the diffusion of non-network proteins could be described using the Fickian diffusion law. Molecular Weight Distribution and Protein Composition of the Released Non-Network Proteins. Released proteins from the gel were sampled at different diffusion times, and the size distributions of the proteins were analyzed by SEC-HPLC (Figure 2a). Four peaks were observed, among which the last peak was attributed to solvent absorption. Peaks 1−3 were identified as proteins, with molecular weights of 253.9 ± 22.5, 44.8 ± 5.1, and 9.7 ± 1.6 kDa, respectively, as determined using a standard calibration curve. It is interesting to note that the areas with different protein peaks did not change synchronously during the diffusion period. The protein compositions of peaks 1−3 on the basis fo SDSPAGE are shown in Figure 2b. The protein gel per se (lane 2) contained all of the major components of soy proteins, including the 7S α′, α, γ, and β subunits as well as the 11S A, B, A3, and A5 polypeptides,35 in addition to a faint band with a size of 15 kDa protein (named 15K protein). Lane 3 shows the composition of the released proteins in the buffer. Only a very small amount of 7S α′, α, and β subunits and a moderate amount of B polypeptides were detected, in comparison to the large amount of A and 15K band. Peak 1 (lane 4) contained almost all of the protein species found in the original diffusion sample (lane 3) in addition to a faint 15K band. A nonreducing SDS-PAGE analysis (data not shown) revealed that the A and B polypeptides in the diffused sample and peak 1 actually existed in the form of AB subunits. Peak 2 (lane 5) contained several unidentified proteins with molecular weights of 23 and 20 kDa as well as small amounts of B polypeptides. On the basis of the SDS-PAGE analysis, the 15K and small amount of A5 polypeptides were eluted in peak 3 (lane 6). The SDS-PAGE analysis indicated that the proteins released from the 15% gel were composed of AB subunits of 11S and other small proteins. Because there was a very low content of 7S subunits in the diffusion buffer, it can be inferred that 7S contributed to the gel network, while a fraction of undenatured 11S and some small proteins were not involved in the formation of the gel network. Utsumi et al.2,3 also discovered a difference in the contribution

Figure 3. Cumulative release (%) of the proteins corresponding to peaks 1−3 (Figure 2a, SEC profiles) as a function of the square root of the diffusion time.

peak areas (St/S∞) of peaks 1−3, respectively, as a function of the square root of the diffusion time (t1/2). The cumulative release appeared to be proportional to the square root of the diffusion time up to approximately 70% protein release. This finding indicates that the release of peaks 1−3 was all controlled by Fickian diffusion processes in the initial stage (during the first 12 h). The diffusion coefficients (D) were calculated from the slope of the fitted lines according to eq 5. As shown in Table 1, the diffusion coefficients (D) increased from 2.90 × 10−12 to 1.64 × 10−11 m2 s−1 as the protein molecular weight decreased from 253.9 ± 22.5 to 9.7 ± 1.6 kDa. Colsenet et al.20 studied the diffusion of PEG in whey protein gels with a 75% protein denaturation ratio. They reported that the diffusion coefficient increased from 3.0 × 10−12 to 6.0 × 10−11 m2 s−1 when the molecular weight of PEG decreased from 82.25 to 1.08 kDa. Moreover, the diffusion coefficients of PEG with molecular weights of 4, 12, 20, and 36 kDa were 8.9 × 10−11, 3.8 × 10−11, 2.0 × 10−11, and 1.3 × 10−11 m2 s−1, respectively, in calcium alginate hydrogels with an alginate concentration of 16.5 g L−1.36 Table 1 also shows the diffusion coefficients in water (D0). The results show that the gel matrix decreased the diffusion of non-network proteins by approximately 1 order of magnitude. This finding is in agreement with those reported by Schillemans et al.,37 who demonstrated that the diffusion coefficients of myoglobin from chemically cross-linked dextran hydrogels decreased from 1.06 × 10−10 m2 s−1 in pure water to (0.9−1.3) × 10−11 m2 s−1 in hydrogels. Moreover, Table 1 shows the theoretical values of D/D0 using the predictive expression suggested by Davis38 on the basis of studies of diffusion in polyacrylamide gels: D = exp( −(0.05 + 10−6M )c p) D0 (5) where M and cp are the molecular weight of the solute and the polymer gel mass fraction (expressed as a percent). According D

DOI: 10.1021/acs.jafc.5b00132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Diffusion Coefficients of Non-Network Proteins in 15% Gel and Water peak

D

D0

D/D0

D/D0 by Davis’ model

1 2 3

(2.90 ± 0.16) × 10−12a (6.43 ± 0.17) × 10−12b (1.64 ± 0.46) × 10−11c

(35.78 ± 3.1) × 10−12d (76.74 ± 5.3) × 10−12e (10.69 ± 0.6) × 10−11f

0.074 0.085 0.153

0.009 0.256 0.408

Figure 4. (a) Contents of undenatured 11S proteins (Wundenatured, g/100 g of total protein) and denatured proteins (Wdenatured, g/100 g of total protein) released from the gels at different protein concentrations and the network protein content (Wnet, g/100 g of gel). The three parameters are plotted as different protein concentrations. (b) SDS-PAGE of non-network proteins released from soy gels heating for 0.5 h at different protein concentrations.

Table 2. Diffusion Coefficients of Non-Network Proteins as a Function of the Soy Protein Concentration in the Gels D (m2 s−1) protein concentration (% w/v)

peak 1

peak 2

peak 3

12 15 18

(3.43 ± 0.14) × 10−12a (2.90 ± 0.16) × 10−12b (2.11 ± 0.12) × 10−12c

(6.95 ± 0.23) × 10−12d (6.43 ± 0.17) × 10−12d (6.01 ± 0.19) × 10−12e

(16.80 ± 0.46) × 10−12f (16.43 ± 0.47) × 10−12f (16.39 ± 0.36) × 10−12f

and finally to 18%, the corresponding released undenatured 11S proteins were 3.71, 5.17, and 7.18 g, respectively, per 100 g of gel proteins, which can also be explained by the increase in AB subunits (AB + A5B3) bands from 32.1 ± 0.4% to 39.7 ± 0.7% and 48.9 ± 0.5% in the released samples from gels with protein concentrations of 12%, 15%, and 18%, respectively, by nonreducing SDS-PAGE (Figure 4b). Concomitantly, the amounts of released denatured proteins were 7.85, 7.86, and 7.49 g, respectively, per 100 g of gel proteins. Thus, the increase in the total released non-network proteins (11.56, 13.03, and 14.67 g/100 g of total protein for protein concentrations of 12%, 15%, and 18%, respectively) was contributed mainly by the increase in the undenatured AB subunits. Several studies have reported that the degree of denaturation decreased with the increase in protein concentration. Zhang et al.39 and Mills et al.40 found that the onset temperature and peak temperature (in DSC curves) increased with an increasing content of crude soy protein. Mori et al.41 also reported that the heat-induced association−dissociation of 11S globulin differs depending on the protein concentration, as evidenced by the finding that 11SAB subunits did not dissociate into A and B polypeptides at a protein concentration of 5% to the extent they did at a protein concentration of 0.5%. At the same time, other studies have demonstrated the formation of higher molecular weight aggregates1,40,41 and a decreased gelation temperature26 when soy proteins are heated at higher concentrations. Thus, it is reasonable to postulate in our studies that, although the degree

to eq 5, D/D0 depended on the molecular weight of the solute and the polymer concentration of the gel. Although the calculated D/D0 using Davis’ model also increased with a decrease in molecular weight, the predictions of eq 5 are very different from the observed values, suggesting that the diffusion behavior of solutes in soy protein gels was substantially different from that in polyacrylamide gels. In fact, polyacrylamide gels behaved like a screen with homogeneous mesh size: peaks 2 and 3 with molecular weights of less than 44.8 kDa showed a much higher diffusion rate than that of peak 1 with a molecular weight of 253.9 kDa. On the other hand, soy protein gels were more heterogeneous in nature. Therefore, no clearcut distinction between diffusion coefficients for high- and lowMW proteins was observed. Effect of Protein Concentration on the Release of Non-Network Proteins. Heat-induced soy gels were made at 95 °C and 30 min using three protein concentrations: 12%, 15%, and 18% (w/v). With an increased protein concentration, more proteins were involved in the formation gel network as shown in Figure 4a. There were 10.61, 13.05, and 15.36 g of network proteins per 100 g of gel for the 12%, 15%, and 18% protein concentrations, respectively. However, the amount of released proteins, which was calculated on the basis of net protein mass rather than the gel mass, also increased. Figure 4a shows released undenatured 11S proteins (Wundenatured) and denatured proteins (Wdenatured) calculated from eqs 1 and 2. When the protein concentration increased from 12% to 15% E

DOI: 10.1021/acs.jafc.5b00132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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study reported by Colsenet,19 the diffusion of PEG in whey protein gels was determined by NMR. They found that α shifted from 0.59 to 0.84 as the protein concentration increased from 0.1 to 0.4 g/g. These authors20 suggested that the diffusion of the probe was affected by the changes in the gel microstructure and that the increase in α occurred in accordance with a decrease in the interparticle distances in the gels. The power law exponent α would theoretically tend toward 1 for nonentangled polymer chains when the network mesh size of the gel decreases and approaches the solute radius.44 For α equal to 2, eq 6 represents the reptation model first introduced by de Gennes.42 The reptation model can be used to describe the dynamics of long polymer chain diffusion in a cross-linked gel, and according to this model, when the mesh size of the gel is less than the solute radius, the macromolecular solute would be forced to adopt a wormlike displacement behavior. The exponent determined in the present study was close to the theoretical value of 3/5 for a swollen coil in a good solvent.42 Therefore, theoretically and practically, the pore size of the soy protein gel network is larger than the radius of non-network proteins. In addition, α gradually shifted toward higher values when the protein concentration or gel network density increased. Effect of Heating Time on the Release of NonNetwork Proteins. The effect of heating time on the gel properties was investigated at 95 °C at a protein concentration of 18% (w/v) for 0.5, 1.0, and 2.0 h. Figure 6a shows that the amounts of undenatured 11S proteins released from the gels were 7.17, 3.92, and 1.54 g/100 g of total protein for heating times of 0.5, 1.0, and 2.0 h, respectively. Furthermore, the corresponding release of denatured proteins was of 7.49, 9.07, and 9.23 g/100 g of total protein, respectively. Moreover, the network protein content (Wnet, g/100 g of gel) increased from 15.36 to 15.63 and 16.09 g/100 g of gel as the heating time increased. As shown using nonreducing SDS-PAGE (Figure 6b), the intensity of the AB subunits decreased with an increase in heating time. Prolonging the heating time resulted in small increases in the contents of A polypeptides (17.1 ± 0.3%, 16.7 ± 0.5%, and 19.4 ± 0.6% for 0.5, 1.0, and 2.0 h, respectively; data not shown) and dimers of A polypeptides (2.3 ± 0.2%, 3.1 ± 0.3%, and 3.3 ± 0.2% for 0.5, 1.0, and 2.0 h, respectively; data not shown) but a slight decrease in trimers of A polypeptides from 3.2% to 0.5%. However, a remarkable increase in protein polymers was observed (7.8 ± 0.5% to 30.6 ± 0.7% for heating times from 0.5 to 2.0 h), in which A polypeptides were the main components on the basis of nonreducing/reducing diagonal electrophoresis (data not shown). Therefore, the increase in the amount of released denatured proteins (Wdenatured in Figure 6a) with an increase in heating time was probably contributed by the increase in polymers that were formed by the disassociated A polypeptides. The increase in network protein content (Wnet in Figure 6a) can be explained by the increase in the amount of dissociated B polypeptides as a consequence of the preferential interaction between the β subunit of 7S and the B polypeptides of 11S, which has been reported to contribute to the gel network.2,3 Marleen et al.45 and Philippe et al.16 also found that, upon prolonged heating of whey proteins or β-lactoglobulin, more proteins were incorporated into the gel network. Diffusion coefficients (D) for different MW fractions from gels with different heating times are illustrated in Table 3. A higher molecular weight clearly resulted in greater sensitivity to

of denaturation of 11S decreased, more proteins were involved in the gel network at higher protein concentrations. The diffusion coefficients of non-network proteins with different molecular weights in the soy gels at different protein concentration are shown in Table 2. It is clear that the diffusion coefficients of the protein fractions with higher molecular weights were substantially more affected by the gel protein concentration. Gireeshkumar23,24 found that the diffusion coefficients of dextran decreased strongly with the increase in the protein concentration of β-lactoglobulin gels and that the larger molecular size of dextran resulted in a greater decrease in the diffusion coefficient. For smaller molecular tracers (PEG) with MW values of 1 and 8.5 kDa, respectively, Colsenet18 found that the structural changes of casein gels induced by different protein concentrations had essentially no effect on their diffusion in the gel, and the same effect was observed for the changes in the diffusion coefficients of peak 3. For globular proteins, Rh is related to the cubic root of the molecular weight: Rh =

3

3M w 4πNaρ

where Mw is the molecular weight, NA is the Avogadro constant, and ρ is the density of a protein. From the Stokes−Einstein equation D=

kT 6πηR h

the following power law dependence can be obtained:

D = AM w −α

(6)

where A is a pre-exponential factor and α is the characteristic exponent. Equation 6 is often used to describe the self-diffusion of polymer chains in different systems with α ranging from 0.55 for dilute systems42 to 2 in concentrated systems.43 The diffusion coefficients determined for different protein fractions were plotted against MW on the double-logarithmic coordinate as shown in Figure 5. The exponent α yielded from the plot increased from 0.58 to 0.69 when the protein concentration increased from 12% to 18%. Favre et al.36 determined the diffusion rate of PEG with a MW value from 4 to 35 kDa in calcium alginate hydrogels, and they reported that the exponent increased from 0.768 to 1.017 as the alginate concentration increased from 8.7 to 28.5 g mL−1. On the other hand, in the

Figure 5. Power law representation of non-network protein diffusion coefficients in gels with different protein concentrations as a function of the non-network protein molecular mass. F

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Figure 6. (a) Contents of undenatured 11S proteins (Wundenatured, g/100 g of total protein) and denatured proteins (Wdenatured, g/100 g of total protein) released from the gels with a 18% protein concentration for heating for 0.5, 1.0, and 2.0 h, and the content of network protein content (Wnet, g/100 g of gel). The three parameters are plotted as the heating time at a protein concentration of 18%. (b) SDS-PAGE of non-network proteins released from 18% soy gels at different heating times (h).

Table 3. Diffusion Coefficients of Non-Network Proteins in 18% Protein Gels as a Function of Heating Time D (m2 s−1) heating time (h)

peak 1

peak 2

peak 3

0.5 1.0 2.0

(2.11 ± 0.12) × 10−12a (2.06 ± 0.14) × 10−12a (1.71 ± 0.20) × 10−12b

(6.01 ± 0.23) × 10−12c (5.75 ± 0.17) × 10−12cd (5.30 ± 0.19) × 10−12d

(16.39 ± 0.46) × 10−12e (16.36 ± 0.55) × 10−12e (16.20 ± 0.26) × 10−12e

changes in diffusion with heating time. Good fits for the diffusion coefficients shown in Table 3 with eq 6 could also be obtained, yielding α values of 0.67, 0.69, and 0.75 for heating times of 0.5, 1.0, and 2.0 h, respectively. This finding suggested that a more compact gel network was obtained with a longer heating time. Lucey et al.46 came to the same conclusion on the basis of a comparison of permeability measurements, demonstrating that the gels made from reconstituted skim milk heated at 80, 85, and 95 °C for 30 min had permeability coefficients lower than those that were heated for 15 min. Marleen et al.45 also found that prolonged heating caused a decrease in pore size by studying the effect of the duration of heating on the properties of whey protein gels. Jacoba et al.1 demonstrated that, after the onset of soy protein gelation, an increase in G′ was observed upon further heating. They suggested that G′ increased because more protein became incorporated into the network, leading to further accumulation of the network structure. Alternately, G′ might have increased as a consequence of rearrangements in the network. A type of rearrangement that might occur is fusion of the protein aggregates in the strands, which results in an increase in protein−protein interactions per cross-section and thus in denser and stiffer strands. In summary, this study demonstrated that the diffusion behavior of non-network proteins can be used to probe the structural characteristics of heat-induced soy protein gels. The major parts of the released non-network proteins were occupied by undenatured AB subunits, monomers, dimers, trimers, and polymers of A polypeptides, as well as the unidentified 15 kDa protein, while the amounts of 7S subunits and B polypeptides were very small among the released proteins. By SEC-HPLC, the released proteins could be classified into three fractions according to their size, with

characteristic molecular weights of 253.9, 44.8, and 9.7 kDa. The release behavior of non-network proteins was fitted to Fick’s second law. The increase in the gelling concentration resulted in a rise in non-network protein content in the gel, which was mainly contributed by undenatured AB subunits. A prolonged heating time reduced the amount of released nonnetwork proteins and caused the composition to shift to more denatured proteins. In all of the cases, the diffusion coefficients of non-network proteins decreased with an increasing gel network density. An increase in the molecular weight of the protein fractions increased the sensitivity of the non-network protein diffusion coefficients to the structural changes in soy gels induced by different conditions. More proteins appeared to be involved in the gel network at elevated protein gelling concentrations to form a denser matrix, whereas the structural changes in the gels during prolonged heating might have been caused by rearrangements in the network structure and probably to some extent by further incorporation of protein into the network. Due to the elucidation of the relationship between the release behavior and structure of soy protein gels, further investigations are underway to reveal whether diffusion experiments can be affected by rheological properties and different proteins (7S and 11S).

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AUTHOR INFORMATION

Corresponding Author

*Y.H.: tel, 0510-85917812; fax, 0510-85329091; e-mail, yfhua@ jiangnan.edu.cn. Funding

The work was financially supported received from the National Natural Science Foundation of China (No. 21276107), the National Great Project of Scientific and Technical Supporting G

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Programs funded by the Ministry of Science & Technology of China during the 12th five-year plan (No. 2012BAD34B04-1), and the 863 Program (Hi-tech research and development program of China, No. 2013AA102204-3). Notes

The authors declare no competing financial interest.

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