Stable and pH-Sensitive Protein Nanogels Made by Self-Assembly of

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Stable and pH-Sensitive Protein Nanogels Made by Self-Assembly of Heat Denatured Soy Protein Nannan Chen,† Lianzhu Lin,† Weizheng Sun,† and Mouming Zhao*,†,‡ †

College of Light Industry and Food Sciences, and ‡State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China S Supporting Information *

ABSTRACT: In this study, we examined the possibility of preparing stable soy protein nanogels by simply heating homogeneous soy protein dispersion. The protein nanogels formed were characterized by z-average hydrodynamic diameter, polydispersity index, turbidity, ζ-potential, morphology, and their stability to pH and ionic strength change. Soy protein dispersion (1% w/v) was homogeneous around pH 5.9 where it had the lowest polydispersity index (∼0.1). Stable and spherical nanogels were formed by heating soy protein dispersion at pH 5.9 under 95 °C. They sustained constantly low polydispersity index (∼0.1) in the investigated pH range of 6.06−7.0 and 2.6−3.0. The nanogels were pH-sensitive and would swell with pH change. They were stable at 0−200 mM NaCl concentration. Denaturation of soy glycinin was the prerequisite for the formation of stable nanogels. Soy protein nanogels had a core−shell structure with basic polypeptides and β subunits interacting together as the hydrophobic core; and acid polypeptides, α′, and α subunits locating outside the core as hydrophilic shell. The inner structure of soy protein nanogels was mainly stabilized by disulfide bonds cross-linked network and hydrophobic interaction. Soy protein nanogels made in this study would be useful as functional ingredients in biotechnological, pharmaceutical, and food industries. KEYWORDS: soy protein, heating, nanogels, stable, pH-sensitive, swell

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INTRODUCTION Designed colloidal particulate systems are finding increasing utilization within the biotechnological, pharmaceutical, and food industry for application as encapsulation and delivery systems, or to modulate the physicochemical and sensory properties of foods.1,2 Roughly spherical aggregates have been variously called nano- or microparticles. The borderline between nano and micro is situated at a diameter of 100 nm, but this criterion is not always strictly upheld.3 The expression nanogels or microgels conveys that the particles are constituted of cross-linked network and swell considerably in a good solvent depending on the cross-linking level.4,5 The turbidity of dispersions is often observed to decrease when the particle swells.4 There has been considerable interest in the formation of biopolymer nanoparticles from proteins and/or polysaccharides as they are more biocompatible and food safe. Alternatively, protein nanoparticles can be formed by controlled thermal denaturation of protein at certain pH. Previously, protein nanogels have been prepared from ovalbumin and ovotransferrin and from oppositely charged ovalbumin and lysozyme by heating the mixture at pH 7.0 and 10.3, respectively.6,7 Recently, it was discovered that stable microgels can be formed by heating β-lactoglobulin in a narrow pH range around 5.8.8−11 Stable suspensions of roughly spherical whey microgels have also been produced in a similar way.12,13 The protein nanoparticles produced by heat denaturation of lactoferrin were resistant to pH change and salt addition.14 Narrowly dispersed nanoparticles were formed by the self-assembly of β-casein and lysozyme under heating.15 There is strong incentive for using low-cost vegetable sources of protein in the world economy. Among them, soy protein © 2014 American Chemical Society

provides all of the essential amino acids needed to fulfill human nutritional requirements. Its protein value is essentially equivalent to that of food proteins of animal origin. Since the 1960s, soy protein products have also been used as nutritional and functional food ingredients in every food category available to the consumer.16 Glycinin and β-conglycinin are the two major components of soy protein. Glycinin is composed of acidic and basic polypeptides, which is linked by a single disulfide bridge, except for the acidic polypeptide A4.17,18 Three different β-conglycinin subunits are known as α′, α, and β, which are associated via hydrophobic interactions.19,20 Above the isoelectric point, the onset denaturation temperature of glycinin is around 80−90 °C, and β-conglycinin starts to denature around 60−75 °C.21 As compared to the extensive study of animal origin protein nanoparticles, those on fabricating nanoparticles using soy protein are relatively few, and most of the studies are about the functionality of the nanoparticles. Soy protein nanoparticles were formed by sequential treatments of heating at 95 °C for 15 min at pH 7.0 and then electrostatic screening with NaCl addition.22 These soy nanoparticles were found to have a good potential to act as Pickering-type stabilizers. In another study, calcium was added to induce a nanoscale network formation after the primary alkaline/heat treatment and the following pH neutralization of soy protein solution.23 In vitro study suggested that these soy protein nanoparticles were nontoxic and mainly Received: Revised: Accepted: Published: 9553

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preheating time needed to reach the selected final temperature (it would take a maximum of 6 min to reach 95 °C in this experiment). pH Stability of Soy Protein Nanogels. The effects of pH on the properties of the protein nanogels prepared by heating protein dispersion (pH 5.9) at 95 °C for 30 min were measured by modifying the pH of the dispersions from 2.6 to 7.0. Samples without heat were used as control. For samples withdrawn from different temperatures and times, pH was adjusted to 7.0. Characteristics of particles in different pH were further analyzed. Salt Stability of Soy Protein Nanogels. The effects of salt addition on the stability of the protein nanogels prepared by heating protein dispersion (pH 5.9) at 95 °C for 30 min was measured by adjusting the NaCl concentration of the dispersion from 0−200 mM. Samples without heat were used as control. For the samples withdrawn from different temperatures and times, NaCl concentration of dispersions was adjusted to 150 mM. Sedimentation Stability. The stability of the samples to sedimentation after pH and NaCl concentration adjustment was recorded by taking photographs using a digital camera after they had been stored at 4 °C for 24 h. Particle Size, Polydispersity, and Surface Charge Measurements. Protein dispersion was diluted to 0.1% (w/v) for particle size, polydispersity, and surface charge measurements using a commercial dynamic light scattering and microelectrophoresis device (Nano-ZS, Malvern Instruments, Worcestershire, UK). The particle size was reported as the z-average hydrodynamic diameter (Dh), polydispersity was reported as polydispersity index (PDI), while the particle surface charge was reported as the ζ-potential. The Dh, PDI, and ζ-potential were analyzed by Dispersion Technology Software (DTS) version 4.20 supplied by the manufacturer (Malvern Instruments Ltd.). PDI were calculated by the cumulant analysis method of the autocorrelation function. The autocorrelation function (G(t)) was calculated from the fluctuation of the scattered intensity with time: In(G(t)) = a + bt + ct2. t is the time (s), and a, b, and c are terms of polynomial fit calculated. The polydispersity index was calculated from the ratio 2c/b2. Turbidity Measurement. The turbidity of soy protein dispersions was determined by measuring the absorbance of the dispersions without dilution at λ 500 nm using a UV−vis spectrophotometer (Genesys 10s, Thermo Fisher, America). Microscopic Observations. Transmission electron microscopy (TEM) was used with the negative staining method. Observations were made with JEM-2100F transmission electron microscope operating at 200 kV (JEOL, Japan). A drop of sample was deposited onto a Formvar-carbon-coated copper grid, and excess of product was removed after 5 min using filter paper. The grid was dried at room temperature for 5 min. Phosphotungstic acid (1%) was then added for 5 min, with excess being removed as before. Samples (pH 5.9) with or without heat and the corresponding samples with pH adjusted to pH 2.6 and 7.0 were observed. Electrophoresis. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a discontinuous buffered system, using 12% separating gel and 5% stacking gel according to our previous method with minor modification.24 Soy protein dispersion (pH 5.9) after heating at 95 °C for 30 min was adjusted to pH 6.4, 7.0, and 8.0. Then samples with and without pH adjustment were ultracentrifuged at 156 000g for 30 min, which was supposed to separate the protein particles and soluble proteins.25 The supernatant and the sample without ultracentrifugation (pH 5.9 after heat) (250 μL) were then mixed with 250 μL of 0.06 M Tris−HCl buffer (pH 6.8), containing 2% (w/v) SDS, 25% (v/v) glycerol, 0.1% (w/v) bromophenol blue, and 5% (v/v) 2-mercaptoethanol (2-ME). Molecular weight marker made from rabbit phosphorglase B (97.4 kDa), bovine serum albumin (66.2 kDa), rabbit actin (43 kDa), bovine carbonic anhydrase (31 kDa), human growth hormone (22 kDa), and hen egg white lysozyme (14.4 kDa) was used as reference. Particle Size of Nanogels in Various Solvents. To unravel the interactive forces involved in the formation and maintenance of the nanogels structure, the z-average hydrodynamic diameter of the soy protein nanogels in the presence of 8.0 M urea, 1% SDS, and 30 mM DTT, alone or in combinations, was also determined according to the

distributed in cytoplasm when uptaken into Caco-2 cells. The above studies suggest that soy protein nanoparticles have some unique qualities. However, much more studies are needed to find a convenient way to produce stable soy protein nanoparticles, to elucidate the mechanism of particles formation, and to illustrate their inner structure. In this investigation, we explored the possibility to produce stable soy protein nanogels by heating soy protein dispersion at the critical pH point where it had the lowest polydispersity index. The z-average hydrodynamic diameter, polydispersity index, ζ-potential, turbidity, and morphology were evaluated to characterize the nanogels. Their resistance to pH and ionic strength change was also investigated. Influence of heat load (temperature and time) on the stability of soy protein particles would be discussed. A possible mechanism of the nanogels formation was proposed. Soy protein nanogels produced in this study would be useful functional ingredients to modify the optical or rheological properties of food products, or to encapsulate and deliver bioactive ingredients.

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MATERIALS AND METHODS

Materials. Defatted soy flakes were purchased from Yuwang Group (Shangdong, China). Soy protein (94.2% protein (Kjeldahl, N × 6.25), 4.5% moisture, and 1.3% ash) was prepared from defatted soy flakes by the method described in this Article. BCA (bicinchoninic acid) protein assay kit and dithiothreitol (DTT) were purchased from Dingguo (Beijing, China). 1-Anilinonaphthalene-8-sulfonic acid (ANS) was purchased from Sigma-Aldrich (St. Louis, MO). Iodoacetamide was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All other chemicals were of analytical reagent grade and obtained in China. Soy Protein Preparation. Defatted soy flakes powder was mixed with 15-fold (in weight) deionized water, and the mixture (pH 6.8) was adjusted to pH 7.5 with 2.0 M NaOH. After being stirred for 2 h, the resulting suspension was centrifuged at 8000g for 20 min at 4 °C to remove the insoluble material. The pH of the supernatant then was adjusted to pH 4.5 with 2.0 M HCl, and the precipitate was collected by centrifugation at 8000g for 10 min at 4 °C. The precipitate was then redissolved with 5-fold (in weight) deionized water, and the pH was adjusted to 7.0 with 2.0 M NaOH. The neutral soy protein solution was dialyzed against deionized water at 4 °C for 48 h and then freezedried and stored at 4 °C until use. Soy Protein Dispersion Preparation. The soy protein dispersion was prepared by dispersing soy protein powder in deionized water (1.1%, w/v) and stirring at 25 °C for 2 h. The soy protein dispersion was left for 12 h at 4 °C to allow complete hydration. Afterward, the dispersion was centrifuged at 10 000g for 15 min to remove insoluble matter. The protein concentration was analyzed using BCA assay. The protein concentration then was adjusted to 1% (w/v). Creation of Soy Protein Nanogels. The pH of soy protein dispersion was modified to 2.6−7.0 by stepwise addition of 2.0 M HCl. Samples were withdrawn at different pH values for size and polydispersity index analysis. For producing nanogels, soy protein dispersion was adjusted to the pH (5.9) where the dispersion had the lowest polydispersity index and poured into 100 mL glass vials (with a screw-top and solid cap) and heated in a water bath without stirring at 95 °C for 30 min. After the heat treatment, samples were immediately cooled in ice water to 4 °C. Correspondingly, soy protein dispersion (pH 5.9) without heat was used as control. For the study of the effects of holding temperature on the stability of particles, soy protein dispersion (pH 5.9) was heated at 50−95 °C for 30 min (room temperature was 25 °C). For the study of the effects of holding time on stability of particles, soy protein dispersion (pH 5.9) was heated at 95 °C for 0−60 min. Samples were withdrawn at different temperatures and times for further analysis. It should be noted that the reported heating time zero did not take into account the 9554

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method described above. To unravel if the disulfide bonds were accessible, nanogels were first dispersed in 30 mM DTT; second, it was added with iodoacetamide to remove the DTT;26 and finally SDS was added (final concentration 1%). The corresponding particle size of this nanogel dispersion was then measured. All solvents were prepared with 20 mM pH 8.0 Tris-HCl buffer. Sulfhydryl (SH) Group Content. SH group content before and after heat treatment of soy protein dispersion at pH 5.9 was measured using Ellman’s reagent.27 Surface Hydrophobicity Measurement. Surface hydrophobicity before and after heat treatment of soy protein dispersion was evaluated using ANS as the fluorescent probe according to the method of Alizadeh-Pasdar and Li-Chan.28 Protein dispersion was diluted with 10 mM pH 7.0 sodium phosphate buffer. Fluorescent intensity was measured via a F7000 fluorescence spectrophotometer (Hitachi Co., Japan). Intrinsic Fluorescence of Tryptophan. Intrinsic fluorescence of the tryptophan before and after heat treatment of soy protein dispersion was measured on a F7000 fluorescence spectrophotometer. Protein concentration was diluted to 0.002% with 10 mM sodium phosphate buffer (pH 7.0). Excitation and emission slits were set at 5 nm. The excitation wavelength was 295 nm to excite tryptophan. The emission spectra were recorded from 300 to 400 nm with a scan speed of 240 nm/min. Each spectrum was the average of two scans and corrected for a protein-free sample. Statistical Analysis. Statistical calculations were performed using the statistical package SPSS 11.5 (SPSS Inc., Chicago, IL) for one-way ANOVA. Least-squares difference was used for comparison of mean values among treatments and to identify significant differences (p < 0.05) among treatments.

translucent, but the dispersion was stable and homogeneous, without precipitation. However, protein particles formed at pH 5.8−6.0 were unstable with pH change, taking the particles formed at pH 5.9 as an example. Similar particle size and PDI distribution were observed when pH increased from 5.9 to 7.0, indicating a relatively reversible process. The appearance of the dispersion gradually became transparent (Figure 3A). The soy protein dispersion was unstable in the pH range of 3.5−5.5 as secondary aggregation of protein particles happened and the particles precipitated (Figure 3A). Under pH 3.5, protein precipitates were dissociated and the solution became transparent again (Figure 3A), but PDI was larger than 0.25 (data are no shown), indicating the relative poly dispersiveness of the protein dispersion. Characteristics of Protein Nanogels Made by Heat Treatment. To make protein particles stable, thermal treatment was performed on soy protein dispersion of pH 5.9 under 95 °C for 30 min (the effects of changing the heating protocol are discussed later in this Article). This pH was supposed to make the largest production of stable particles within a narrow range of particle size as it had the lowest PDI. After heat treatment, the pH of the protein dispersion increased from 5.9 to 6.06, which might be the result of self-assembly of protein particle. The increase of pH after thermal treatment was also observed by Donato et al. when they heated dematerialized β-lactoglobulin solution at 70 and 85 °C under mild acidic pH conditions (pH 5.7−5.9).8 The behavior of the heated soy protein particles was totally different from that without heat. In the pH range of 6.06−7.0, the particle size increased gradually with the increasing pH (Figure 2A). Surprisingly, the turbidity decreased with the increased particle size, while the PDI value was still very low and remained relatively constantly at about 0.1. This result excluded the possibility that the size changes were caused by the aggregation of the protein particle. Instead, the nanoparticles swelled with the increased pH, containing large amounts of water. It should be pointed out that swelling was the most important characteristic of nanogels.4,5 Therefore, nanoparticles formed after heating in our study were deemed as nanogels. The appearance of the dispersion was still translucent in pH 7.0 (Figure 3B), indicating the irreversible change of protein particles after heating. In pH 5.9 and 5.8, the particle size, turbidity, and the PDI value increased, which was due to the aggregation of the protein particle. Precipitation of nanogels happened when the protein dispersion pH was in the range of 3.7−5.7 (Figure 3B), but these aggregates were redispersible when the dispersion pH was out of this range. In the pH 2.6− 3.2, with the decreased pH, particle size at first decreased and then increased gradually (Figure 2B). The visual appearance of the dispersion was still translucent (Figure 3B). The PDI value decreased to 0.1 at pH 3.0 and remained constantly around 0.1 thereafter. Interestingly, in pH 2.6−3.0, the turbidity decreased with increased particle size. Therefore, nanogels were stable and would also swell in this pH range. The ζ-potential was higher than that before heating when the pH increased from 6.06 to 7.0 (Figure 2C), indicating the redistribution of particle inner structure. The internal and external charge distribution of the aggregated proteins was optimized to promote the structuring of nanogels, resulting in more negatively charged residues exposing. The higher surface charge would contribute to the stability of the protein nanogels as the electrostatic repulsion between them was enhanced. In the pH range 2.6−3.2, the ζpotential was lower than that without heating. For the salt stability, precipitation occurred, when the ionic strength

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RESULTS AND DISCUSSION Characteristics of Protein Particles before Heat Treatment. The initial pH of soy protein dispersion was 7.0. As shown in Figure 1, when the pH decreased, there was no

Figure 1. Effects of pH on the z-average hydrodynamic diameter and polydispersity index of soy protein dispersion before thermal treatment. pH was adjusted from 7.0 to 5.7 (■); from 5.9 to 7.0 (□).

significant increase of particle size above pH 6.2. With the further decreased pH, the surface charge on the protein particle was neutralized, reducing the electrostatic repulsion between particles, which would induce protein aggregation. Therefore, at pH 6.2, particle size significantly increased, indicating the large particle formation. The particle size then increased gradually with a sudden increase at pH 5.7. Correspondingly, the PDI decreased gradually from 0.52, with a sudden decrease at pH 6.2, reaching the lowest at pH 6.0, 5.9, and 5.8, with the value below 0.1. After that, PDI began to increase. Therefore, in the pH range of 5.8−6.0, aggregation would lead to a relatively monodisperse protein particle distribution. The particle size was relatively large and the corresponding dispersion was 9555

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Figure 3. Influences of pH and NaCl concentration on the visual appearance of soy protein dispersion (pH 5.9) without (A,C) or with heating (B,D) at 95 °C for 30 min. Influence of holding temperature (for 30 min) on the salt stability (150 mM NaCl) of soy protein dispersion (E). Influence of holding time (under 95 °C) on the salt stability (150 mM NaCl) of soy protein dispersion (F).

2.6 and exhibited a narrow range of apparent lengths. Besides, the apparent lengths of protein particles were larger than that at pH 5.9, which might be the swelling of protein nanogels. These results were in accordance with particle size distribution characteristics at various pH values as described before. The observed particle size by TEM was different from the Dh, which might be due to the air-drying process and phosphotungstic acid addition. All of the above observations led us to conclude that spherical soy protein nanogels with pH and salt stability as well as swelling characteristics were formed by heating soy protein dispersion (pH 5.9) under 95 °C for 30 min. Their stability was closely related to thermal treatment. Therefore, we further did some experiments on the influence of holding temperature and time on the stability of soy protein particles. Effect of Holding Temperature on the Protein Particle Stability. As shown in Figure 5A, before the pH was adjusted to 7.0, there was a very little increase of particle size at 50 °C. Thereafter, particle size decreased gradually from 65 °C, reached the lowest at 80 and 85 °C, and subsequently increased at 90 °C. The turbidity observed almost a similar trend (Figure 5B). Around pH 6.0, the onset denaturation temperature in deionized water is about 85 °C of soy glycinin and 65 °C for βconglicinin.21 Therefore, the decrease of particle size was supposed to relate to the denaturation of soy β-conglicinin. The increase of particle size after 85 °C was supposed to relate to the denaturation of glicinin. PDI value was relatively constant around 0.1 (Figure 5C). The ζ-potential increased significantly after 60 °C (Figure 5D). Thus, higher temperature contributed to the exposing of negative charge residues. After the pH of the protein dispersion was adjusted to 7.0, both particle size and turbidity increased with the holding temperature. Before 70 °C, particle size was smaller and PDI value was significantly higher than that before pH adjustment, proving the dissociation of

Figure 2. Effects of pH on the z-average hydrodynamic diameter (■), polydispersity index (▲), and turbidity (□) of soy protein dispersion after thermal treatment (A, pH 5.8−7.0; B, pH 2.6−3.2) and ζpotential of particles (C) before (●) and after thermal treatment (○).

increased to 50 mM NaCl for protein dispersion (pH 5.9) without heat treatment (Figure 3C). After heat treatment, however, even the NaCl concentration increased to 200 mM, and no precipitation occurred (Figure 3D). When protein concentration increased from 1% to 5%, particle size and PDI would increase, but they still had pH and salt stability as well as swelling characteristics (see the Supporting Information). To shed more light on the soy protein nanogels, the morphology of the soy protein particles was observed by TEM performed on selected samples. At pH 5.9 without heat, protein particles were mainly characterized by a spherical shape and exhibited a narrow range of apparent lengths (Figure 4B). When pH increased to 7.0 or decreased to 2.6, the number of spherical particles decreased, and some irregular particles were observed. They exhibited a wide range of apparent lengths. At pH 5.9 after heat, protein particles were still mainly characterized by a spherical shape and exhibited a narrow range of apparent lengths (Figure 4E). The spherical particles could still be found when pH increased to 7.0 or decreased to 9556

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Figure 4. TEM negative-staining micrographs of soy protein dispersions (pH 5.9) without heat: pH adjusted to 2.6 (A), not adjusted (pH 5.9) (B), adjusted to 7.0 (C); and the corresponding heat treated dispersion with pH adjusted to pH 2.6 (D), not adjusted (pH 5.9) (E), and adjusted to 7.0 (F). The scale bar represents 2 μm.

Figure 5. Effects of holding temperature (30 min) on the z-average hydrodynamic diameter (A), turbidity (B), polydispersity index (C), and ζpotential (D) of protein particles. Open squares (□) represent the protein dispersion at pH 5.9, while the solid squares (■) represent the protein dispersion with pH adjustment to 7.0.

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Figure 6. Effects of holding time at 95 °C on the z-average hydrodynamic diameter (A), turbidity (B), polydispersity index (C), and ζ-potential (D) of protein particles. Open squares (□) represent the protein dispersion at pH 5.9, while the solid squares (■) represent the protein dispersion with pH adjustment to 7.0.

particles. After 70 °C, protein particles gradually swelled as the particle size increased but turbidity decreased. Therefore, the denaturation of β-conglicinin might have induced the swelling of protein particles. Yet before 85 °C, the particle was unstable, as the PDI value increased after pH adjustment. The surface charges of all samples after pH adjustment were larger than those before and increased more noticeably for sample after heating above 80 °C. When the ionic strength of the protein dispersion was adjusted to 150 mM, the above 75 °C treated samples did not precipitate after 24 h (Figure 3E). The above observations indicated that the denaturing of soy glycinin was crucial for the formation of stable soy protein nanogels. Effect of Holding Time on the Protein Particle Stability. As shown in Figure 6A, before the pH was adjusted to 7.0, particle size decreased significantly at 5 min, then increased mildly within the 60 min. It has been proved by many investigations that β-conglycinin could prevent thermal aggregation of glycinin.29,30 This explained the limited increasing of particle size as the time increased. Correspondingly, turbidity decreased remarkably at 5 min, and then mildly increased as the time increased (Figure 6B). The PDI value was still constantly around 0.1 (Figure 6C). The ζ-potential increased significantly after heating for 5 min, and remained relatively constant thereafter (Figure 6D). When the pH was adjusted to 7.0, the particle size of samples without heat treatment decreased significantly, while that with heat treatment increased remarkably and remained relatively constant as the time increased. The turbidity of the control sample decreased significantly and became transparent, while that of the thermal treated sample decreased mildly. For dispersion without heat treatment, the PDI increased from 0.1 to 0.47 after pH adjustment, proving the dissociation of particles, while that of the thermal treated sample still remained at a constant value around 0.1. The ζ-potential of all samples increased after pH adjustment, while that of samples with heat treatment

increased more significantly than the control. This indicated that after heat treatment, more negatively charged residues were exposed. All of the above observations indicated that nanogels began to form after 5 min under 95 °C. Besides, the increase of particle size, surface charge, and the decrease of turbidity after 5 min were relatively constant, indicating that when soy protein nanogels formed, their swelling capacity under a certain pH was constant regardless of heating time. When ionic strength increased to 150 mM NaCl, the protein dispersion without heat treatment precipitated; however, after heat treatment even for 5 min, no precipitation occurred (Figure 3F). The above observation proved that the stable nanogels formed within a very short time at 95 °C. Yet it should be pointed out that with the further increased time to 5 h, the stability of nanogels was disturbed and precipitation occurred. Structure Characteristics of Soy Protein Nanogels. Before ultracentrifugation, reducing SDS-PAGE (Figure 7) observed all of the main subunits of soy protein (lane 5). However, after ultracentrifugation (lane 4), only small amounts of α′, α, acid polypeptides, and some lower molecular fraction were still remaining in the supernatant, indicating that all of the basic poly peptides, β subunits, and most of the α′/α subunits, acid polypeptides took part in the formation of nanogels. When pH increased (lanes 1−3), the content of α′, α, and acid polypeptides in the supernatant increased, indicating that these free α′, α, and acid polypeptides might locate on the surface of the nanogels. They might interact with the nanogels through electrostatic interaction so when the electrostatic repulsion force increased, some of them were “pealed” from the surface. This result was in accordance with Chen et al.25 Besides, α′/α subunits have an extension region that has been proved to limit the aggregation of soy protein.31 They located on the surface of the nanogels so that the size of the nanogels was relatively constant despite heating time as indicated formerly (Figure 9558

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hydrophobic residues to a more polar environment (Table 1), although it was not significantly different in value. The disulfide bonds between different subunits formed during the heating process as SH decreased significantly (Table 1). To further elucidate the definite inner force stabilizing the nanogels, nanogels were dispersed in various kinds of protein-perturbing solvents, and the particle size was measured. As shown in Figure 8, 30 mM DTT alone could not dissociate

Figure 7. Electrophoretic patterns of soy protein nanogels and supernatant protein fraction of different pH nanogels dispersion after ultracentrifugation. Lane M: marker proteins. Lanes 1, 2, and 3 represent supernatant protein fraction in pH 8.0, 7.0, and 6.4 protein dispersions by adjusting the pH of initial nanogels dispersion (pH 6.06). Lane 4 represents the supernatant protein fraction in the initial soy nanogels dispersion. Lane 5 represents the protein electrophoretic pattern of the initial nanogels dispersion. Soy protein constituents: α′, α, and β represent subunits for β-conglycinin; A and B represent the acidic and basic polypeptides for glycinin.

Figure 8. Effects of various protein-perturbing solvents on the zaverage hydrodynamic diameter of nanogels. S1, 20 mM pH 8.0 TrisHCl buffer; S2, 30 mM DTT; S3, 1% SDS; S4, 8.0 M urea; S5, urea +DTT; S6, SDS+DTT; S7, iodoacetamide was added into nanogels dispersion in S2 to remove the DTT first and then SDS was added. Different letters represent significant difference (p < 0.05).

the nanogels. In the 1% SDS and 8 M urea, the particle size increased significantly. This might be that the nanogels were unfolded by SDS and urea, which could disturb the hydrophobic interaction and hydrogen bonds, respectively. Yet they still could not dissociate the nanogels into smaller particles. However, when nanogels were dispersed in SDS and urea solution combined with DTT, the particle size decreased significantly, especially for that of SDS and DTT solution. These results indicated that the inner structure of nanogels had a disulfide bonds cross-linked “skeleton”. These disulfide bonds play a crucial role in the stability of nanogels. They were inaccessible without the destroying of hydrophobic interaction as when DTT was removed by iodoacetamide, and addition of SDS could not dissociate the particles. Therefore, hydrophobic interaction was also very important in the formation of nanogels. As compared to hydrophobic interaction and disulfide bonds, hydrogen bonds seemed to play a relatively minor role in the stabilization of the nanogels structure. This kind of structure might be related to the pH and salt stability of the nanogels. According to the above discussion, the proposed model of the soy protein nanogels formation pathway was illustrated in Figure 9. In conclusion, soy protein nanogels with a hydrophobic core and a hydrophilic shell have been made successfully by a simple

6A). When heated above 80 °C, both β-conglicinin and glicinin unfolded and dissociated into monomers (polypeptides).32 The hydrophobic residues exposed and induced the aggregation. In soy protein, basic polypeptides are highly hydrophobic and would preferentially interact with β subunits.32 On the other hand, basic polypeptides have an isoelectric point around 8.0− 8.5,33 while other subunits (α′, α, β, and acid polypeptides) have isoelectric points around 4.0−5.5.34 Therefore, in the pH range 6.06−7.0, basic polypeptides were positively charged and others were negatively charged. Combined with the increased surface charge after heating, it was reasonable to propose that the positively charged basic polypeptides together with β subunits were buried and located in the core of the nanogels, forming the hydrophobic core, while highly negatively charged α′, α, and acid polypeptides were located on the surface of the nanogels as they were relatively more hydrophilic.31,35 This kind of core−shell model has also been proposed by Ren et al. when they studied the protein particle formation mechanism in defatted soymilk.36 Association of monomers was mainly driven by hydrophobic interaction. After the hydrophobic aggregation, there were still some hydrophobic residues exposed that increased the surface hydrophobicity of nanogels (Table 1). The red shift of λmax for tryptophan residues further confirmed the exposure of Table 1. Surface Hydrophobicity (S0), λmax of Tryptophan Residues, and Sulfhydryl (SH) Content of Soy Protein (pH 5.9) before and after Heatinga samples

S0 × 103

λmax (nm)

SH (nmol/mg)

pH 5.9 heat−pH 5.9

91.13 ± 1.22 a 167.82 ± 11.4 b

337.5 ± 0.14 a 338.7 ± 0.15 a

5.19 ± 0.18 a 3.25 ± 0.07 b

a

Values in the same column followed by different letters are of significant difference (p < 0.05).

Figure 9. Diagrammatic depiction of soy protein nanogels formation. 9559

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thermal process under a certain pH value. They are resistant to pH and ionic strength change. They will swell when the electrostatic repulsion force is large enough. Further work will be carried out to explore their inner structure with other technology as well as their functionality.

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ASSOCIATED CONTENT

* Supporting Information S

Figures 1S, 2S, and 3S showing the influence of protein concentration on the particle size, PDI, and salt stability of nanogels, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel./fax: +86 20 87113914. E-mail: [email protected]. Funding

This work was supported by the National High Technology Research and Development Program of China (863 Program) (no. 2013AA102201), the National Natural Science Foundation of China (no. 31171783), and the Fundamental Research Funds for the Central Universities (2013ZB0017). Notes

The authors declare no competing financial interest.

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