Article pubs.acs.org/JAFC
Fabrication and Characterization of Stable Soy β‑Conglycinin− Dextran Core−Shell Nanogels Prepared via a Self-Assembly Approach at the Isoelectric Point Ji-Lu Feng, Jun-Ru Qi,* Shou-Wei Yin, Jin-Mei Wang, Jian Guo, Jing-Yi Weng, Qian-Ru Liu, and Xiao-Quan Yang College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China ABSTRACT: The preparation of soy β-conglycinin−dextran nanogels (∼90 nm) went through two stages, which are safe, facile, and green. First, amphiphilic graft copolymers were formed by dextran covalently attaching to β-conglycinin via Maillard dryheating reaction. Second, the synthesized conjugates were heated above the denaturation temperature at the isoelectric point (pH4.8) so as to assemble nanogels. The effects of pH, concentration, heating temperature, and time on the fabrication of nanogels were examined. The morphology study displayed that the nanogels exhibited spherical shape with core−shell structures, which was reconfirmed by zeta-potential investigation. Both circular dichroism spectra and surface hydrophobicity analyses indicated that the conformations of β-conglycinin in the core of nanogels were changed, and the latter experiment further revealed that the hydrophobic groups of β-conglycinin were exposed to the surface of protein. The nanogels were stable against various conditions and might be useful to deliver hydrophobic bioactive compounds. KEYWORDS: soy β-conglycinin, dextran, Maillard reaction, self-assembly, nanogels
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INTRODUCTION Self-assembly is an approach that the patterns of basic unit spontaneously transform from disordered to ordered.1,2 As a category of bottom-up technique, self-assembly is an innovative method to prepare novel particles with attractive characteristics.3 Schmitt et al. reported that linear structure aggregates were formed by heating 1 wt % β-lactoglobulin at 85 °C for 15 min at pH 5.8, whereas colloidally stable microgels with spherical shapes were formed from pH 4.6 to 5.8.4 These aggregates exhibited different structures at various pH values, which may hinder their application in food processing. However, spherical aggregates fabricated by various amphiphilic polymers via self-assembly may overcome such limitation. An emphasis has been placed on synthesizing these stable spherical particles;5−8 in brief, amphiphilic block copolymers which were formed by covalent linking previously were then assembled to form core−corona particles. The Maillard reaction, first detected by Louis-Camille Maillard in 1912 and widely utilized in the food industry, is a nonenzymatic browning reaction between the ε-amino groups (in proteins, peptides, or amino acids) and the reactive carbonyl group (in reducing sugars).9 The Maillard reaction requires no chemical regents, which is safe and economical. Glycation of protein by the means of the Maillard reaction has been extensively studied and has been proved to significantly improve the functional properties of proteins, such as heat stability,10 surface properties,11 and emulsifying properties.12 Notwithstanding, there is still a lack of further research on utilizing Maillard products for intensive processing. Maillard reaction products (glycated proteins) are amphiphilic copolymers, which possess inspiring potential to construct ordered micro- or nanoscale particles by self-assembly under appropriate conditions. Wu and co-workers used © 2015 American Chemical Society
Maillard products and their hydrolysates to form spherical nanoparticles by the Maillard reaction, hydrolysis, and desolvation method,13 but the glutaraldehyde−ethanol solution they used to cross-link was poisonous, which may hinder the application of nanoparticles in a delivery system. Li and coauthors reported that lysozyme−dextran core−shell nanogels with a hydrodynamic diameter around 200 nm could be produced by two steps: Maillard reaction and heat gelation process.14 However, there is an increasing need to minimize the size of colloidal particles to nanoscale (0−100 nm), for the reason that smaller-size nanogels are able to deliver functional materials with longer circulation time in vivo; that is to say, the effects of controlled release and cellular uptake were improved.15,16 Furthermore, little research has been made on constructing plant protein based nanogels through the Maillard reaction and self-assembly. The low-cost soy protein is extensively used in the food industry due to its functional properties, which are more valuable in many instances compared with those of animal proteins.17 As a main component of soy protein, soy βconglycinin has various advantages such as lowering serum triacylgycerol, cholesterol, and lipid levels.18 Dextran is a family of 1→6-α-D-glucans with a high percentage of 1→4-α-branch linkage, and therefore it is flexible and water-soluble. Consequently, soy β-conglycinin and dextran were chosen as the sources to fabricate nanogels. The aim of this research was to evaluate the possibility of fabricating biocompatible soy β-conglycinin−dextran nanogels Received: Revised: Accepted: Published: 6075
April 9, 2015 May 12, 2015 June 11, 2015 June 15, 2015 DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
Article
Journal of Agricultural and Food Chemistry via the Maillard reaction followed by a self-assembly method; that is, the Maillard dry-heat conjugates were heated in a water bath at the isoelectric point of β-conglycinin, where it showed the lowest polydispersity index value. The size distribution, turbidity, morphology, ζ-potential, and conformation of nanogels were investigated to characterize the nanogels. The mechanism of nanogel formation was systematically analyzed, and the stability of nanogels was also observed.
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DG = [(A 0 − A1)/A 0] × 100% where A0 and A1 were the absorbance of solutions before and after glycation, respectively. Dynamic Light Scattering Measurements. The size distributions of the samples were conducted on a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcestershire, UK) with a fixed scattering angle of 173° equipped with a 4 mW He−Ne laser (633 nm wavelength). The sample was placed in a 1 cm × 1 cm cuvette (PCS8501) and analyzed three times at 25 °C. The apparent average hydrodynamic diameter (Dh) and polydispersity index (PDI) were obtained. ζ-Potential Measurements. The ζ-potentials of samples were also examined on the Zetasizer Nano-ZS instrument. ζ-Potentials were calculated by using Dispersion Technology software according to the Smoluchowski approximation in an automatic mode.23 Each sample was analyzed in triplicate. Turbidity Measurements. Native β-conglycinin, β-conglycinin/ dextran mixtures, and β-conglycinin−dextran conjugate powders were dissolved in distilled water to achieve the final protein concentration of 1 mg/mL. The pH values of the solutions were acidified to 4.8 using 0.1 mol/L HCl. The solutions were then placed in a water bath at 95 °C for different times (0−60 min).The turbidity of each sample was assessed by measuring the transmittance of sample solution at a wavelength of 540 nm on a UV2300 ultraviolet−visible spectrophotometer (Tianmei Ltd.).24 Surface Hydrophobicity (H0) Measurements. Surface hydrophobicity was obtained by using the ANS as fluorescence probe following the method described by Haskard and Li-Chan25 with some modifications. Briefly, samples were dissolved in buffer solutions (0.01 mol/L, pH 7.0) to reach desired protein concentrations (0.02−0.2 mg/mL). Aliquots of ANS stock solution (8 × 10−3 mol/L, 20 μL) were mixed with each sample solution (4 mL). The mixtures were then shaken fully on a vortex mixer and stored in the dark for 3 min before measurements. Fluorescence intensity (FI) was analyzed on an F7000 fluorescence spectrophotometer (Hitachi Co., Tokyo, Japan) with wavelengths of 390 nm (excitation) and 470 nm (emission) at 25 °C. The FI value at each concentration minus the FI value of the blank (without added ANS) was calculated as the final FI value. The initial slope of the final FI value versus protein concentration (mg/mL) was obtained by a linear regression analysis and used as an index of H0. Each sample was conducted three times. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded on an MOS-450 spectropolarimeter (BioLogic Science Instrument, Grenoble, France). The far-UV CD spectrum probed the secondary structures. Each sample was placed in a 2 mm quartz cuvette with the protein concentration of 0.1 mg/mL and scanned from 180 to 260 nm. The near-UV CD spectrum probed the tertiary structures. Each sample was placed in a 10 mm quartz cuvette with a protein concentration of 2 mg/mL and scanned over a wavelength range from 260 to 340 nm. For each measurement, the spectrum was an average of eight scans. All determinations were carried out in triplicate. Atomic Force Microscopy (AFM). AFM images were captured in tapping mode on a Multimode 8 scanning probe microscope (Bruker Inc., Billerica, MA, USA) equipped with a silicon cantilever (FESPA, Bruker Corp, Santa Barbara, CA, USA) of 125 μm length. Aliquots (2 μL) of sample previously diluted to 5 μg/mL with deionized water were deposited on a freshly cleaved mica substrate and air-dried for overnight before imaging. Scanning Electron Microscopy (SEM). SEM image was acquired on a Nova NanoSEM 430 (FEI NanoPorts, Eindhoven, The Netherlands) at 10 kV accelerating voltage. The preparation of specimen for SEM was the same as AFM. The specimen was coated with gold followed by imaging. Transmission Electron Microscopy (TEM). TEM image was recorded on a JEM-2100F electron microscopy at an accelerating voltage of 80 kV. Aliquots (10 μL) of dispersions (diluted to 3 μg/μL with deionized water, previously) were dropped on the carbon membrane support copper grid. Excessive dispersions were removed by filter paper and dried for 5 min. Ten microliters of 1% (w/v)
MATERIALS AND METHODS
Materials. Soy β-conglycinin was separated from defatted soybean flour (obtained from Shandong Gaotang Lanshan Group Corp., Shandong, China) on the basis of the method of Nagano et al.19 The protein content of β-conglycinin was 84.03% (Kjeldahl, N × 5.71). Dextran (molecular weight 48−90 kDa) and 8-anilino-1-naphthalenesulfonic acid (ANS) were supplied by Sigma (St. Louis, MO, USA). All reagents were of analytical grade unless specified. All solutions were prepared by using deionized water. Synthesis of Soy β-Conglycinin−Dextran Conjugates. Soy βconglycinin and dextran powder mixtures with weight ratios of 1:1 were dissolved in distilled water and lyophilized. The lyophilized powder was dry-heated at 60 °C with a relative humidity of 79% in a desiccators containing saturated KBr in the bottom for 4 days. The resultant samples were dissolved in distilled water and centrifuged (10000g, 15 min, 25 °C). The supernatant was then freeze-dried, and the products were soy β-conglycinin−dextran conjugates. Fabrication of Soy β-Conglycinin−Dextran Nanogels. The Maillard conjugates were dispersed in double-distilled water to reach a soy β-conglycinin concentration of 1 mg/mL. The pH of the completely solubilized conjugate solution was acidified to 4.8 by stepwise addition of 0.1 mol/L HCl. The solution was then placed in a water bath without stirring at a temperature of 95 °C for 50 min. The resultant soy β-conglycinin−dextran nanogels solution was either kept at 4 °C or freeze-dried. To evaluate the effect of pH on the size distributions of nanogels, the conjugates were acidified to pH 3.8, 4.3, 4.8, 5.3, or 5.8 and subsequently incubated in water baths at 80 °C for 30 min. Other steps were as described above. To study the effect of protein concentration, conjugate solutions at concentrations ranging from 0.5 to 10 mg/mL were used and heated at 80 °C for 30 min. Other steps were as described above. To investigate the effect of the heating temperature, the conjugate solutions were heated at 75, 80, 85, 90, or 95 °C for 30 min. Other steps were as described above. To observe the effect of heating time on size distributions of nanogels, the Maillard products were heated in water baths at 95 °C for 5−60 min. Other steps were as described above. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed according to the methodology of Laemmli20 with a 12% acrylamide separating gel and a 5% stacking gel. The samples were dissolved in 0.125 mol/L Tris-HCl buffer that contained 1% (w/v) sodium dodecyl sulfate, 2% (v/v) 2mercaptoethanol, 5% (v/v) glycerol, and 0.025% (w/v) bromophenol blue. The sample solutions (5 mg/mL, 10 μL) were heated for 5 min in boiling water before electrophoresis. After electrophoresis, the gels were stained for protein for 2.5 h and for carbohydrate for 16 h with Coomassie brilliant blue R-250 and periodate−Schiff solution, respectively.21 The gel stained for protein was then destained with 10% (v/v) methanol and 10% (v/v) acetic acid. Degree of Glycation (DG). The DG of soy β-conglycinin−dextran conjugates was estimated using trinitrobenzenesulfonic acid (TNBS) reaction according to Adler-Nissen with some modifications.22 A sample solution (1 mg/mL, 0.5 mL) was mixed with NaHCO3 (4 wt %, 0.5 mL), sodium dodecyl sulfate (0.1 wt %, 0.5 mL), and TNBS (0.1 wt %, 0.5 mL). The solution was then incubated at 40 °C for 2 h, followed by the addition of 0.25 mL of 1 N HCl to quench the reaction. The absorbance of the solution was measured by using a UV2300 ultraviolet−visible spectrophotometer (Tianmei Ltd., Shanghai, China) at a wavelength of 340 nm. DG was determined as 6076
DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
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lactoglobulin. The pI of β-conglycinin is 4.8.30 Therefore, in this study, the effect of pH on the formation of nanogels was studied by heating (80 °C) the soy β-conglycinin−dextran conjugates (1 mg/mL) at the pH range of 3.3−5.8. As shown in Figure 2, when the pH was shifted toward the pI, the mean
phosphotungstic acid was placed on the copper grid and stained for 5 min before removing it with filter paper, and the grid was dried overnight at ambient temperature. Data Analysis. All measurements were performed in triplicate if not specified. Significance of difference was analyzed using analysis of variance (ANOVA) and Tukey’s test at 5% level of probability (SPSS 17.0 for Windows statistical software).
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RESULTS AND DISCUSSION Characteristics of Soy β-Conglycinin−Dextran Conjugates Formed by Maillard Dry-Heating Reaction. The process of Maillard conjugation involves three stages.26 In the initial stage, an amino group was condensed with a carbonyl group to synthesize a Schiff base, which then cyclizes to an Nsubstituted glycosylamine. It subsequently goes through Amadori rearrangement to produce a more stable Amadori rearrangement product (ARP, 1-amino-1-deoxy-2-ketose).27 The ARP undergoes a complex series of reactions to form multiple colored compounds in the following stages.28 The reaction is often limited to the initial stage to prevent the occurrence of highly colored and poorly characterized products. The formation of Maillard-type protein−polysaccharide complexes was confirmed by SDS-PAGE and TNBS test. Figure 1A exhibits the protein staining SDS-PAGE pattern; the
Figure 2. Dh and PDI of nanogels prepared by heating the βconglycinin−dextran conjugates (1 mg/mL) in a water bath at 80 °C for 30 min at different pH values.
hydrodynamic diameter (Dh) increased, whereas the polydispersity index (PDI) decreased, implying that the nanoparticles were more uniform at pI. There was a dominant interaction, electrostatic repulsion, and hydrophobic attraction during the heat-induced gelating process. When the pH decreased from 4.3 to 3.3 or increased from 5.3 to 5.8, βconglycinin carried more charges, resulting in stronger longrange electrostatic repulsion and weaker short-range hydrophobic aggregation. Therefore, the β-conglycinin molecules were separated from each other, which may weaken the gelation ability of protein and make it unfavorable to the formation of homogeneous nanogels. On the other hand, at the isoelectric point where β-conglycinin carried zero net charge and nanogels displayed the lowest PDI value, the electrostatic repulsion can be ignored and hydrophobic attraction was strong enough to construct well-defined nanonetwork hydrogels. Figure 3 exhibits the influence of β-conglycinin concentration on the DLS result of nanogels, which were prepared by heating (80 °C) the conjugates (0.5−10 mg/mL) at pH 4.8. The increment of β-conglycinin gradually increased the Dh and PDI values of nanogels (from 79.26 to 123.65 nm and from 0.189 to 0.276, separately). A higher concentration of β-conglycinin brings molecules closer together. As a consequence, the mean particle size increased as the concentration increased. Furthermore, when the protein concentration was increased to ≥8 mg/mL, the turbidity of the solution markedly increased and the mobility of the solution sharply diminished. In the following study we adopted 1 mg/mL as the optimal protein concentration to form nanogels. A heat treatment of proteins above their denaturation temperature is a prerequisite for unfolding the hydrophobic groups and forming gels.31 Because the denaturation temperature of β-conglycinin is 60−75 °C,32 a heating temperature range of 75−95 °C was used to study the formation of nanogels. As shown in Figure 4, the Dh and PDI of nanogels did not change significantly when the heating temperature
Figure 1. SDS-PAGE patterns for β-conglycinin/dextran samples: (A) protein stain; (B) carbohydrate stain. Lanes: 1, protein marker; 2, native β-conglycinin; 3, β-conglycinin−dextran conjugates. Arrows indicate the boundary between the stacking and separating gels.
characteristic bands of β-conglycinin (subunits of α′, α, and β) were detected in lane 2, which is consistent with Zhang.29 However, when it comes to lane 3, a shift of bands toward the top of separating gels was visualized after the Maillard reaction, which is clear evidence that compounds with high molecular weight were developed. The migration of dextran can be elided because it carries no charge, the effect of noncovalent interactions on SDS-PAGE may be ignored because they were disrupted in the reducing condition, and an intense bank accumulated on the top of separating gels in carbohydrate staining pattern (Figure 1B, lane 3), all of which strongly verified the covalent attachment of dextran to β-conglycinin. The degree of glycation was evaluated by quantifying the loss of available amino groups of β-conglycinin after Maillard reaction using the TNBS method. The DG of the Maillard dryheating products prepared by us was around 16.7%. Characteristics of Soy β-Conglycinin−Dextran Nanogels Fabricated by Self-Assembly Approach. DLS is a quantitative and effective tool to investigate the particle size and size distribution of nanogels. As mentioned above, Schmitt and co-workers reported spherical microgels exhibiting colloidal stability were formed in a narrow pH range near the isoelectric point (pI) of β6077
DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
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Figure 3. DLS results of nanogels prepared by heating the βconglycinin−dextran conjugates with different concentrations at 80 °C for 30 min.
Figure 6. Turbidity of β-conglycinin, β-conglycinin/dextran mixture, and β-conglycinin−dextran conjugates incubated in a water bath at 95 °C for 0−60 min. (Inset) Photographs of β-conglycinin (A), βconglycinin/dextran mixture (B), and β-conglycinin−dextran conjugates (C) heated in a water bath at 95 °C for 60 min.
investigated. As shown in Figure 5, when the heating time increased, the particle size did not grow markedly and PDI dropped slightly. It is notable that the PDI value fell below 0.2 after 15 min of heating, indicating that homogeneous dispersed nanogels were formed in a broad heating time range of 15−60 min. We used a 50 min heat treatment to the conjugates in the following study. As shown in Figure 6, depletion flocculation appeared after a heat treatment to β-conglycinin, and a similar phenomenon was found by Chen and co-workers.33 Precipitation was also observed in the presence of free dextran, indicating that the individual dextran molecules cannot prevent the β-conglycinin molecules from aggregating. However, when the conjugates were heated, no coagula were found and the solution was translucent. Therefore, the dextran grafted to βconglycinin not only can suppress the protein coagulation but also can improve the solubility of protein. Taken together, heating 1 mg/mL soy β-conglycinin− dextran conjugates at pH 4.8 in a water bath at 95 °C for 50 min was chosen to prepare nanogels in the following study. Morphology, Formation Mechanism, and Structure of Soy β-Conglycinin−Dextran Nanogels. Morphology and Formation Mechanism. Atomic force microscopy has been proved to be helpful for detecting 3D information on nanoscale particles.34 Scanning electron microscopy is available to obtain 2D information by examining the surfaces of samples,35 whereas transmission electron microscopy is valuable for investigating the structures of specimens,36 both of which produce 2D images. DLS and AFM were carried out to determine the dimensions and morphology of β-conglycinin−dextran conjugates and nanogels as well as to probe the effect of self-assembly on the aggregation of protein. DLS data show that after self-assembly, the Dh of particles increased from 73.21 to 83.33 nm, whereas the polydispersity index reduced from 0.184 to 0.096, which indicates that the particles in nanogel solution were more homogeneous. The AFM images exhibit that the conjugates (Figure 7A) were irregular aggregations and the nanogels (Figure 7B) were approximately spherical in shape. In comparison, there were far fewer aggregates and more uniform globules in the nanogels than conjugates. The DLS and AFM analyses imply that self-assembly of the amphiphilic conjugates
Figure 4. Dh and PDI of nanogels prepared by heating 1 mg/mL βconglycinin−dextran conjugates at different temperatures for 30 min.
improved. Because the lowest PDI value appeared at 95 °C, we chose a heating temperature of 95 °C for nanogel synthesis. The size distribution of nanogels (Figure 5) and the turbidity of native β-conglycinin and β-conglycinin/dextran mixture as well as nanogels (Figure 6) as a function of heating time were
Figure 5. Dh and PDI of nanogels prepared by heating 1 mg/mL βconglycinin−dextran conjugates at 95 °C for different times. 6078
DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
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Figure 7. AFM images and size distribution of β-conglycinin−dextran conjugates (A) and nanogels (B); SEM image (C) and TEM image (D) of βconglycinin−dextran nanogels.
can form spherical particles and deplete self-aggregates of βconglycinin. The SEM image presented in Figure 7C illustrates that the well-dispersed nanogels were of globular shape with narrow particle distribution. The TEM image (Figure 7D) verifies that the nanogels were characterized by spherical shapes with core− shell structure having a dark shell and a light core. The phenomenon found in the TEM image may be rationalized by the following mechanism: a heat treatment to conjugates could cause the β-conglycinin to unfold, promote macroscopic aggregates, and form macro gels, but the dextran covalently
attached to the β-conglycinin through Maillard reaction restrained the β-conglycinin from aggregating due to the steric hindrance of dextran; it, therefore, served as the barrier for the inner β-conglycinin molecules, which cross-linked with each other by attractive forces (e.g., hydrophobic, disulfide bonds).37 Hence, the flexible and hydrophilic dextran chains constituted the dark shell region, whereas the hydrophobic cross-linked βconglycinins constituted the light core region (Figure 7D). ζ-Potential. The ζ-potential of β-conglycinin, β-conglycinin/ dextran mixture, and β-conglycinin−dextran nanogels was monitored at various pH values (Figure 8). The samples 6079
DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
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Figure 8. Zeta potential of native β-conglycinin, β-conglycinin/dextran mixture, and β-conglycinin−dextran nanogels at different pH values.
were positively or negatively charged at the pH above or below pI, and they carried zero net charge near the pI. It is worth noting that the ζ-potential of nanogels was pronouncedly lower in magnitude compared with native β-conglycinin as well as mixture, and the difference was even larger when the pH was farther from the pI. Harada and Kataoka synthesized core− shell-like polyion complex micelles developed from lysozyme and poly(ethylene glycol)−poly(aspartic acid) block copolymer (PEG−P (ASP)).38 The ζ-potential of these associates was about zero because the PEG corona could sterically stabilize the micelles. Riley and co-workers reported that the ζ-potential of nanoparticles prepared from poly(lactic acid) (PLA)−PEG copolymers was negligible when compared with PLA nanoparticles, and they attributed this phenomenon to the PEG segment that shielded the carboxyl acid end groups of the PLA chains.39 Likewise, the ζ-potential profile of nanogels in Figure 8 further confirmed the aforementioned finding that βconglycinin cores were covered with nonionic dextran blocks. The coated dextran shielded the inner protein and, therefore, it weakened the electrostatic repulsion of protein in different cores. However, the nanogel solution still carried several net charges at the pH farther from the pI. This phenomenon may be attributed to the presence of native β-conglycinin in the solution, which neither conjugated with dextran nor fabricated nanogels. Circular Dichroism Spectroscopy. CD spectroscopy has been proved as a reliable method to reveal the conformational structure of protein.40 The far-UV spectrum (in the region of 190−250 nm) estimates the secondary conformation of protein, whereas the near-UV fingerprint (in the region of 260−340 nm) exhibits the tertiary structure of protein. The far-UV curves of native β-conglycinin, β-conglycinin/ dextran mixture, β-conglycinin−dextran conjugates, and nanogels were presented in Figure 9A. The spectrum of native βconglycinin showed two negative dichroic peaks around 208 and 217 nm, a positive bank in the vicinity of 195 nm, and a zero-crossing near 200 nm, suggesting that the secondary composition of native β-conglycinin was mainly β-sheet.41 There was no significant difference in secondary structure among the native protein, mixture, and conjugates. However, as to nanogels, the mean residue ellipticity at 208 and 217 nm largely decreased, which indicated that the secondary conformation of nanogels changed and the β-sheet content increased. The near-UV spectra are shown in Figure 9B. The native βconglycinin exhibited a positive peak at 270 nm, and the
Figure 9. Far-UV CD spectra (A) and near-UV CD spectra (B) of native β-conglycinin, β-conglycinin/dextran mixture, and β-conglycinin−dextran conjugates and nanogels.
dichroic curve of mixture was similar to that of β-conglycinin. However, a decrease in the magnitude of the bank was observed after glycation, demonstrating that aromatic side chains were exposed to some extent during Maillard reaction.42 It is worth noting that the positive peak of nanogels disappeared and the intensity of the spectrum was relatively low, clearly indicating that the tertiary structure of protein was totally destroyed after further heating of the Maillard dry-heat conjugates. Surface Hydrophobicity. H0 was determined by fluorescence probe methods using the ANS probe, which can access the hydrophobic groups on the surface of proteins and bind with the aromatic amino acid residues. H0 has been used to reflect the 3D structure and conformational changes of protein.25 Figure 10 exhibits the changes of H0 at pH 7.0 with various specimens. The surface hydrophobicity of the mixture decreased slightly compared with the native β-conglycinin, suggesting that the free dextran had little effect on the H0 of protein. Moreover, there was no substantial difference (P < 0.05) in H0 between the conjugates and native β-conglycinin. This phenomenon might be attributed to the conjecture that H0 is determined not only on the degree of partial denaturation of β-conglycinin during the Maillard dry-heat process, which is prone to increase it, but also on the degree of glycation, which is prone to decrease it. However, further heat treatment of conjugates caused a pronounced enhancement in H0 of the nanogels. It is well-known that heat treatment will lead to the 6080
DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
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the tertiary structure of nanogels was changed, which is consistent with the previous CD study. Because the core of nanogels was composed of cross-linked β-conglycinins, the hydrophobic groups of which were exposed to the surface of protein, it is likely that several well-defined hydrophobic compartments were developed inside the core of nanogels, which might be available to deliver hydrophobic compounds due to the hydrophobic attraction between βconglycinins and compounds. Stability of Soy β-Conglycinin−Dextran Nanogels. The nanogels prepared at pH 4.8 were then incubated at various pH values. Figure 11A illustrates that the particle size and size distribution of nanogels did not change significantly from pH 2 to 12, suggesting that the nanogels were resistant to pH change. When the pH was farther from the pI, the β-conglycinins in the core were prone to separate from each other, but the crosslinking structure of β-conglycinins, which were formed in the heating process, made the nanogels remain stable in solution. On the other hand, when the pH was at/around the pI, the βconglycinins in different cores tended to aggregate due to the lack of electrostatic repulsion; however, the glucan shells acted as shields to prevent the proteins from aggregating owing to the hydrophilicity and steric hindrance of dextrans. The results were in accordance with the aforementioned self-assembly mechanism of nanogels.
Figure 10. Surface hydrophobicity (H0) of native β-conglycinin, βconglycinin/dextran mixture, β-conglycinin−dextran conjugates, and nanogels. Different letters (a, b) above columns indicate significant differences (P < 0.05).
thermal denaturation of protein and subsequent exposure of nonpolar amino acids. Consequently, the surface hydrophobicity of β-conglycinin in nanogels was higher than that of the native protein and conjugates. The result indicates that
Figure 11. (A) Dh and PDI of nanogels under different pH conditions; (B) size distribution of freshly prepared nanogels and after storage for 1, 2, 3, or 4 months; (C) size distribution of nanogels as prepared and diluted to 0.1 mg/mL; (D) size distribution of nanogels as prepared and freeze-dried followed by rehydration. 6081
DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
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No aggregates were found in the nanogel solution and, together with the particle size and polydispersity values (Figure 11B), were maintained after 4 months of storage at 4 °C without antiseptics, indicating that the nanogels were initially in thermodynamic equilibrium.38 The narrow Gaussian fit of nanogels (Figure 11C), which were diluted to 0.1 mg/mL (10fold), reconfirms that the cross-linking structures of nanogels are fairly stable. Furthermore, the lyophilized nanogels were water-dispersible, and there is no considerable change in the particle size of nanogels that were freeze-dried followed by rehydrating versus the original ones (Figure 11D). All of these outstanding properties are favorable to the practical application of nanogels in the food industry. The synthesis of nanogels was illustrated in Figure 12. Amphiphilic soy β-conglycinin−dextran conjugates were
We appreciate the Chinese National Natural Science Fund (serial no. 31370036, 31301432), the Research Fund for Pearl River S&T Nova Program of Guangzhou (201506010063), and the project supported by Guangdong Natural Science Foundation (S2013010012097) for financial support. Notes
The authors declare no competing financial interest.
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Figure 12. Illustration of the synthesis of β-conglycinin−dextran nanogels.
formed by dextran covalently attaching to protein via the Maillard reaction. The amphiphilic graft copolymers subsequently self-assembled to fabricate core−shell nanogels. The grafted dextran afforded a shell to the inner cross-linked β-conglycinins and in turn improved the stability of nanogels. The formerly buried hydrophobic groups were exposed to the surface of β-conglycinin and constructed several hydrophobic compartments in the core, which may offer great potential to deliver hydrophobic compounds due to the hydrophobic interaction between the nanogels and the compounds. In conclusion, soy β-conglycinin−dextran nanogels were successfully produced via a self-assembly method at the isoelectric point of β-conglycinin. The nanogels were spherical with core−shell structures, as evidenced by AFM, SEM, and TEM as well as ζ-potential studies, and the hydrodynamic diameter was around 90 nm. Surface hydrophobicity and CD analyses clearly indicated that the structures of β-conglycinins were destroyed and the hydrophobic groups were exposed to the surface of protein to form hydrophobic compartments in the cores. The nanogels were fairly stable against long-term storage, pH change, lyophilization, or dilution. These valuable properties and the low toxicity of nanogels may provide a promising way to deliver hydrophobic compounds, and further research will emphasize the loading and unloading bioactive substance.
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DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
Article
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DOI: 10.1021/acs.jafc.5b01778 J. Agric. Food Chem. 2015, 63, 6075−6083
