Novel Soy β-Conglycinin Core–Shell Nanoparticles As Outstanding

May 22, 2019 - (22−26) As the pH is adjusted from 7.0 to 2.0(24,25) or 11.0–12.5,(22,23) the structure of apoferritin or ferritin is gradually dis...
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Cite This: J. Agric. Food Chem. 2019, 67, 6292−6301

Novel Soy β‑Conglycinin Core−Shell Nanoparticles As Outstanding Ecofriendly Nanocarriers for Curcumin Ling-Ling Liu,† Peng-Zhan Liu,† Xiu-Ting Li,‡ Ning Zhang,§ and Chuan-He Tang*,†,‡ †

Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, P. R. China Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Fangshan 100031, P. R. China § Department of Food Science and Engineering, Jinan University, Guangzhou 510632, P. R. China Downloaded via UNIV OF SOUTHERN INDIANA on July 25, 2019 at 08:33:04 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: The development of high-performance nanocarriers for nutraceuticals or drugs has become one of the topical research subjects in the functional food fields. In this work, we for the first time propose a novel and ecofriendly process to obtain a kind of nanostructured soy β-conglycinin (β-CG; a major soy storage globulin) as outstanding nanocarriers for poorly soluble bioactives (e.g., curcumin), by a urea-assisted disassembly and reassembly strategy. At urea concentrations > 4 M, the structure of β-CG gradually dissociated into its separate subunits (α, α′, and β) and even denatured (depending on the type of subunits); after dialysis to remove urea, the dissociated subunits would reassemble into a kind of core−shell nanostructured particles, in which aggregated β-subunits acted as the core while the shell layer was mainly composed of α- and α′-subunits. The core−shell nanoparticles were favorably formed at protein concentrations of 1.0−2.0 wt %. Curcumin crystals were directly introduced into the β-CG solution at high urea concentrations (e.g., 8 M) and would preferentially interact with the denatured β-subunits. As a consequence, almost all of the curcumin molecules were encapsulated in the core part of the reassembled core− shell nanoparticles. The loading amount of curcumin in these nanoparticles could reach 18 g of curcumin per 100 g of protein, which far exceeds those reported previously. The encapsulated curcumin exhibited a high water solubility, extraordinary thermal stability, and improved bioaccessibility, as well as a sustained release behavior. The findings provide a novel strategy to fabricate a kind of high-encapsulation-performance, organic solvent-free, and biocompatible nanocarrier for hydrophobic nutraceuticals and drugs. KEYWORDS: soy β-conglycinin, nanocarrier, curcumin, disassembly, reassembly, core−shell nanostructure

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INTRODUCTION The development of nanocarriers for drugs and nutraceuticals is one of the topical research interests in the pharmaceutical and food fields.1−3 Nanocarriers can be used to enhance water solubility, stability, and even bioaccessibility (or bioavailability) of poorly soluble bioactives. In many cases, nanocarriers can also be designed to act as targeted-delivery or controlledrelease systems. Polymers from natural or synthetic sources are currently applied as the starting materials to fabricate nanocarriers. Protein-based nanocarriers have been wellrecognized to be a kind of effective delivery system for drugs or nutraceuticals,4−7 thanks to their nontoxicity, biodegradation, and biocompatibility, as well as being “generally recognized as safe” (GRAS). Many proteins, e.g., caseins, whey proteins, and soy proteins, are actually natural nanocarriers for hydrophobic bioactives themselves.8−14 In this regard, most of the bioactives are directly bound on the surface of the proteins to form a kind of nanocomplex through hydrophobic interactions. Although the usage of proteins as natural nanocarriers for bioactives is more simple, facile, and practical than other nanosized delivery systems, its limitationa are also obvious, e.g., limited bioactive loading amount, accelerated instability of colloidal systems, and even high susceptibility to chemical degradation (during further processing, e.g., drying). In contrast, the emerging assembly/selfassembly technology of proteins provides an alternative © 2019 American Chemical Society

strategy to develop high-encapsulation-performance nanocarriers for bioactives. The unique nanostructures of many proteins, e.g., casein micelles or oligomeric globulins, make them act as potential nanocarriers for encapsulation, protection, and delivery of drugs or nutraceuticals. The potential of casein micelles (CM) to perform as nanocarriers has been widely investigated during the past decade.15−21 CM can be assembled from sodium caseinate or β-casein. Semo et al.18 for the first time reported that reassembled CM from sodium caseinate exhibited similar characteristics to naturally occurring CM. The reassembled CM has been successfully used as nanocarriers for hydrophobic nutraceuticals or drugs, including vitamin D2,18 β-carotene,17 ω-3 polyunsaturated fatty acids,20 curcumin,15,16 and mitoxantrone (a hydrophobic anticancer agent).19 In most of the cases, hydrophobic bioactives are usually dispersed in an organic solvent and introduced prior to the self-assembly or reassembly of CM. Recently, Pan et al.21 proposed an organicfree self-assembly process to fabricate casein nanoparticles as nanocarriers for curcumin, using the pH-shifting technique, based on the consideration that curcumin can be charged and dispersed at alkali pH values. Received: Revised: Accepted: Published: 6292

October 25, 2018 April 14, 2019 May 21, 2019 May 22, 2019 DOI: 10.1021/acs.jafc.8b05822 J. Agric. Food Chem. 2019, 67, 6292−6301

Article

Journal of Agricultural and Food Chemistry

the LA in these reassembled nanoparticles was so high, almost all of the encapsulated curcumin molecules were present in an amorphous state. The nanoencapsulation greatly led to enhanced stability and bioaccessibility of curcumin. More importantly, no organic solvent is required to solubilize curcumin or other hydrophobic bioactives, before the nanoparticle formation, because they can be solubilized directly in 6−8 M urea. The findings of this work indicate that oligomeric globulins from plant sources can be developed into a kind of outstanding nanocarrier with a unique core−shell nanostructure for hydrophobic bioactives, by an ecofriendly diassembly/ reassembly process with high-encapsulation performance.

Ferritin or apoferritin, usually characterized by a spherical architecture of 24 similar subunits, is another well-recognized storage protein with an interesting potential to perform as a natural nanocarrier for nutraceuticals or drugs.22−26 As the pH is adjusted from 7.0 to 2.024,25 or 11.0−12.5,22,23 the structure of apoferritin or ferritin is gradually dissociated, and when the pH is adjusted back to 7.0, the corresponding dissociated subunits will be reassembled into initially spherical molecules. This disassembly and reassembly has successfully encapsulated anthocyanin, β-carotene, and hydrophobic drugs in reassembled ferritin or apoferritin. Recently, Yang et al.27 reported that the application of 20 mM urea could facilitate the polyphenols (e.g., epigallocatechin gallate, EGCG) to be permeated in the cage of an apoferritin, and when the urea was removed by dialysis, ferritin−polyphenol coassemblies were formed. In contrast, no works have been available addressing the fabrication of nanocarriers originating from other nanostructured oligomeric gloublins by self-assembly or reassembly. Soy glycinin (SG) and β-conglycinin (β-CG) are the two major storage globulins, usually present in the 11S and 7S forms, respectively. These two globulins, with a good surface hydrophobicity, have been confirmed to act as effective nanocarriers for hydrophobic or even hydrophilic nutraceuticals, e.g., vitamin D and B12, folic acid, and curcumin, by means of formation of nanocomplexes.12,13,28,29 In the nanocomplexes of β-CG and curcumin, the loading amount (LA) of curcumin could reach 3 g of curcumin/100 g of protein.12 Although the maximal potential of these isolated globulins to load curcumin or other bioactives has not been reported, it is expected that an appropriate structural unfolding of the protein would greatly increase their binding capacity for bioactives. For example, in the soy protein isolate (SPI) case, the maximal LA could reach 14.4 g of curcumin/100 g of protein, if the SPI was ultrasonic-treated prior to the nanocomplexation.11 Herein, the objective of our work was to report a facile fabrication process of a kind of unique core−shell nanoparticle from urea-dissociated β-CG, as (nano)carriers for curcumin (as a representative hydrophobic bioactive), by means of a disassembly and reassembly strategy. This novel process is more facile, organic solvent-free, and efficient as compared with those previously reported. These reassembled core−shell nanoparticles were formed based on the preferential ureainduced unfolding and aggregation of different subunits (α, α′, and β) of β-CG, with their structure and properties delicately modulated by varying the urea concentration ([U], in the range 1−8 M). Previous works had studied the urea-induced dissociation/reconstitution of subunits of β-CG and found that 6 M urea can completely disassociate β-CG molecules into separate subunits, and the dissociated subunits can be reconstituted to form a similar 7S-structure (to their initial structure) when urea is removed.30,31 On the basis of these pioneering works, the urea-induced disassembly and reassembly of β-CG was revisited using the laser scattering and spectroscopic techniques, as affected by variation in [U] in the range 0−8 M as well as protein concentration (c, 0.1−6.0 wt %). It was found that when the [U] was >4 M, a kind of novel core−shell nanoparticle could be fabricated from ureadissociated β-CG, which exhibited an outstanding capacity to load curcumin. The maximal LA of these reassembled nanoparticles could reach ∼18.0 g of curcumin/100 g of protein, which far exceeds those reported previously. Although

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MATERIALS AND METHODS

Soy β-CG (lyophilized) was isolated according to the process of Nagano et al.,32 from defatted soy flakes (Shandong Yuwang Industrial and Commerical Co. Ltd., China). The protein content of this β-CG sample was 88.30 ± 1.54% (determined by Dumas method, with a nitrogen conversion factor of 6.25). Curcumin (∼98% purity, from Curcuma longa), 1-anilinonaphthalene-8-sulfonate (ANS−) reagent, porcine bile extract (B8631), pancreatin (from porcine pancreas, P1750, 4 × USP), and pepsin (from porcine gastric mucosal, P7000, 975 units/mg of protein) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.). Unless stated, all other chemicals were of analytical grade. Urea-Induced Disassembly and Reassembly of β-CG. Two sets of experiments were performed to study the urea-induced disassembly and reassembly of β-CG at pH 7.0. The first set of experiments was carried out on a β-CG solution at a given protein concentration (c) of 2.0 wt % but varying concentrations of urea ([U], 0−8 M). The solid urea was directly added into the β-CG solution to reach the required [U] (2−8 M) in the final mixtures, under the stirring conditions. After the completion of urea addition, the β-CG solutions at [U] = 0−8 M were further incubated at 4 °C for >10 h for the complete interaction between urea and the protein. Then, a dialysis against water was applied to remove urea with a membrane (with a molecular weight cutoff of 8 kDa), for 72 h (3 times to change the water), to initiate the reassembly of β-CG from urea-dissociated β-CG. Another set of experiments was performed at [U] = 8 M but varying c values of 0.1−6.0 wt %, in order to study the influence of c on the structure of reassembled β-CG. In this case, the reassembled βCG samples were obtained with the same process as described earlier. Characterization of Untreated or Dissociated/Reassembled β-CG. SDS-PAGE and Native-PAGE. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE were conducted according to He et al.33 The protein solutions (c = 2 wt %) were diluted with the sample buffer containing 25% (v/v) glycerol, 5.0 wt % β-mercaptoethanol, 2.0 wt % SDS, and 0.1 wt % bromophenol blue, to a final c of ∼0.2 wt %. For SDS-PAGE experiments, all the diluted samples were vortexed (10 s), heated (100 °C, 3 min), and then centrifugated (10 000g, 5 min). Next, aliquots (10 μL) of these samples were loaded onto the 5 wt % acrylamide stacking gel and 12 wt % separating gel containing 0.1 wt % SDS. For native-PAGE, the experiments were carried out using a discontinuous buffer system on a 7.5% separating gel and 2.5% stacking gel. No SDS or β-mercaptoethanol was applied in the Tris-HCl buffer, and the diluted samples (unheated) were vortexed and centrifugated under the same conditions (as mentioned earlier). Particle Size Distribution and z-Average Diameter (Dz). The particle size distribution (PSD) profiles of particles in the β-CG solutions (at c = 2.0 wt %) at [U] = 0−8 M, or further after the removal of urea by dialysis (see earlier), were measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS instrument (Malvern Instruments). The DLS was determined at a fixed angle of 173° at 25 °C. The Dz of the particles was calculated based on the assumption that all protein particles are in the spherical form, according to the Stokes−Einstein equation. 6293

DOI: 10.1021/acs.jafc.8b05822 J. Agric. Food Chem. 2019, 67, 6292−6301

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Figure 1. (A) SDS-PAGE or native-PAGE patterns of untreated or reassembled β-CG samples. The numbers 1−4 represent the reassembled β-CG samples from urea-dissociated β-CG at [U] = 2, 4, 6, and 8 M, respectively, followed by a dialysis. The α, α′, and β correspond to the three major subunits of β-CG. (B) Representative size distribution profiles of particles of β-CG at [U] = 0−8 M, as well as of the corresponding reassembled βCG samples. The arrow indicates the formation of large aggregate nanoparticles in the reassembled β-CG sample. (C) z-average diameter (Dz) of urea-dissociated and reassembled β-CG samples, as a function of [U] (0−8 M). The data are the means ± SD (n = 3). All the experiments were performed at a constant c of 2.0 wt %. Particle Morphology. The particle morphology of untreated β-CG, or reassembled β-CG without or with curcumin loaded (at c = 2.0 wt %), was assessed bytransmission electron microscopy (TEM) tecnique, with an XFlash 5030T instrument (Bruker Instruments Co. Ltd., Karlsruhe, Germany). One drop of samples (c = 20 μg/mL) on 230-mesh copper grids were negatively stained with 1% (w/v) phosphotungstic acid. After drying for 12 h at 25 °C, the TEM observations were performed at 80 kV voltage. ζ-Potential and Surface Hydrophobicity (Ho). The ζ-potential of particles was determined using the Nano-ZS instrument (Malvern Instruments Ltd.), in combination with a multipurpose autotitrator (model MPT-2, Malvern Instruments Ltd.) at 25 °C. The H0 was determined using ANS− (at c = 0.1 wt %) as the fluorescence probe with a F-7000 fluorescence spectrophotometer (Hitachi Co., Japan). The fluorescence was excited at 390 nm and emitted at 470 nm. A linear equation of the maximal fluorescence intensity (vertical axis) against c (horizontal axis) could be obtained, and the slope meant H0. All the determinations were repeated in three replicates on individually prepared samples, and the means were applied for the significant analyses. Intrinsic Fluorescence and Near-UV CD Spectroscopy. The intrinsic Trp fluorescence spectra of protein samples, at approximately c = 0.01 wt %, were determined with the same fluorescence spectrophotometer above at 25 °C. The fluorescence was excited at 280 nm, with emission in the range of 300−440 nm recorded. The slit width was set at at a constant 5 nm, and the scanning speed was fixed at 240 nm/min. The near-UV CD spectra of untreated or reassembled β-CG samples, in the range 250−340 nm, were obtained using a Chirascan spectrometer (Applied Photophysics Ltd., U.K.) at 25 °C. The spectra were an average of three scans with a bandwidth of 1 nm and a scanning rate of 60 nm/min. The applied c was precisely kept at 1

mg/mL. Each spectrum was normalized with that of a protein-free buffer. Thermal Denaturation. The thermal denaturation of proteins was evaluated using a TA Q200-DSC thermal analyzer (TA Instruments, New Castle, DE, U.S.A.). Each sample (∼2.0 mg) was mixed with 10 μL of phosphate buffer (0.01 M; pH 7.0) in aluminum liquid pans. After equilibrating for 6 h at 25 °C, the hermetically sealed pans were heated from a temperature of 50 to 90 °C at a heating rate of 5 °C/ min. In each DSC experiment, an empty pan was applied as the reference. The protein denaturation characteristics, including denaturation temperature (Td) and enthalpy change of the endotherm (ΔH), were obtained. Encapsulation of Curcumin in Reassembled β-CG Nanoparticles. All the encapsulation experiments of curcumin in reassembed β-CG nanoparticles were performed at constant c = 2.0 wt % and [U] = 8 M. First, β-CG solutions (2.0 wt %) containing 8 M urea were obtained as described earlier and incubated at 4 °C for >10 h. Next, curcumin crystals were gradually added to the β-CG urea solution at [U] = 8 M, to achieve a required concentration of 1.0−6.0 mg/mL, under stirring conditions for at least 2 h. After incubation in an amber bottle at 4 °C for 12 h, the mixtures were dialyzed against water to remove urea in the system (as earlier). The resultant suspensions containing curcumin-loaded samples were further filtered through a 0.45 μm hydrophilic filter to ensure that any insoluble material was removed. Last, the pH of the final suspensions was adjusted to 7.0. For the control, curcumin crystal was directly added to a β-CG solution (c = 2.0 wt %) in the absence of urea, to a c of 1.0 mg/mL (relative to the solution). Determination of Encapsulation Efficiency and Loading Amount. The encapsulated curcumin in the reassembled β-CG nanoparticles was extracted with ethyl acetate as follows. Ethyl acetate (3 mL) was mixed with 0.2 mL of the suspensions (containing curcumin-loaded β-CG nanoparticles; c = 2 wt %). After vortexing for 1 min, the resultant mixtures were left for layering. After that, the 6294

DOI: 10.1021/acs.jafc.8b05822 J. Agric. Food Chem. 2019, 67, 6292−6301

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individual subunits (α, α′, and β) will keep a separated state in the system. The PSD of β-CG (at c = 2.0 wt %) at [U] = 0−8 M was determined using DLS, as displayed in Figure 1B (top panel), while their Dz data are summarized in Figure 1C. The PSD profile of untreated β-CG was monomodal with the peak centered at ∼13.9 μm, and its Dz was ∼27 nm (Figure 1B and C). Considering that the dimension of 7S-form globulins is approximately 12.5 nm × 12.5 nm × 3.75−4.0 nm,35 the observation thus confirmed that, under the current conditions, β-CG would be mainly present in a 9S form with two identical cyclic 7S ensembles facing each other.30 Upon increasing [U] from 0 to 6 M, it can be well-observed that the main PSD peak at ∼13.9 nm progressively deceased in magnitude, and concurrently, a new PSD peak at ∼6.7−5.0 nm appeared with the peak position progressively decreasing from 6.7 to 5.0 nm (Figure 1B, top). Accordingly, the Dz of β-CG progressively decreased from 27 to ∼10 nm, as the [U] increased from 0 to 4−6 M (Figure 1C). Previous works indicated that 6 M urea completely dissociates β-CG into its separate subunits.31,34 Thus, the observations clearly demonstrated that increasing the [U] from 0 to 6 M led to a gradual transformation of 9S- or 7S-form β-CG molecules into separate subunits. When the [U] was further increased from 6 to 8 M, the PSD peak (at ∼5 nm) of dissociated β-CG on the contrary slightly shifted to a larger size, and the Dz noticeably but insignificantly increased (Figure 1B and C). Thanks to the higher ratio of hydrophobic amino acids, the β-subunit exhibits a much higher tendency to denature and aggregate than the αand α′-subunits, at [U] = 6 M (Thanh and Shibasaki30), or upon heating at 90 °C.34 Thus, the increase in Dz at [U] = 8 M relative to that at [U] = 6 M might be largely ascribed to the urea-induced structural unfolding and aggregation of the dissociated subunits, especially that of the dissociated βsubunit. Reassembly of β-CG from Its Urea-Dissociated Subunits. The reassembly of β-CG (at c = 2.0 wt %) from ureadissociated subunits at [U] = 2−8 M was carried out by the removal of urea through dialysis. The PSD profiles of different reassembled β-CG samples obtained at [U] = 2−8 M, as well as their Dz values, are included in Figure 1B and C. No noticeable changes in the PSD profiles and the Dz were obtained between untreated (control) and reassembled ([U] = 2−4 M) β-CG samples (Figure 1B and C), indicating that, at these [U] values, the urea-induced disassembly and reassembly of β-CG were highly reversible. When the [U] was increased up to 6 M, the PSD profile of the reassembled β-CG sample became bimodal, with a new shoulder peak at ∼28 nm appearing; the shoulder PSD peak became more distinct when the [U] was further increased to 8 M (Figure 1B). At [U] = 8 M, the relative volume of the shoulder peak in the reassembled β-CG sample was similar to that of the initial PSD peak (centered at 15.8 nm; Figure 1C). Surprisingly, the Dz of the reassembled β-CG samples remarkably and progressively increased from 25.8 to 43.6 nm, upon increasing [U] from 4 to 8 M (Figure 1C), indicating formation of larger sizes of nanoparticles at higher [U] values. This is well in agreement with the morphological observations of the untreated β-CG and fabricated β-CG nanoparticles (obtained at [U] = 8 M) using TEM (Figure 2A and B), where the size of the fabricated nanoparticles basically ranged from 20 to 100 nm, while for the untreated β-CG, most of particles were in the range 10−30 nm. The size of the nanoparticles in the fabricated β-CG nanoparticles (at [U] = 6−8 M) was large so that they could

organic phases (the top layer) were obtained. The curcumin concentration in the organic phases was determined spectrophotometically at 420 nm using a UV754N UV−vis spectrophotometer (Precision & Scientific Instrument, Shanghai, China). The percentage of encapsulation efficiency (EE%) of encapsulated curcumin was calculated as the relative ratio of the amount of encapsulated curcumin to the total amount. The loading amount (LA) of curcumin (μg per 100 mg) was calculated from the relative ratio of amount of encapsulated curcumin to total protein. X-ray Diffractometry. The crystallization of curcumin, free or in the β-CG nanoparticle (obtained at c = 2.0 wt %, [U] = 8 M, and a curcumin concentration in the protein−urea solution of 1.0 mg/mL), was evaluated using a Bruker AXS D8 Advance diffractometer (Karlsruhe, Germany) at 25 °C, with a continuous scanning mode. The X-ray diffraction profiles of untreated β-CG, or its physical mixture with curcumin (at a similar curcumin-to-protein ratio to that in the reassembled sample), were also determined as the controls for comparison. The X-ray diffractometry (XRD) experiments were performed using Ni as the X-ray radiation (λ = 0.15418 nm), with 2θ scanning from 4° to 50° at a speed of 0.02/s. The results were analyzed using Jade 5.0 software. Heat Stability Test. The heat stability of free or encapsulated curcumin was evaluated by monitoring its degradation kinetics at 80 °C. All the fresh curcumin solutions or dispersions (at pH 7.0) in amber bottles were heated in a water bath at 80 °C for an incubation period up to 3 h. The free curcumin dispersion (control) was prepared by mixing curcumin absolute ethyl alcohol solution with water at a volume ratio of 1:25. The curcumin remaining after heating for different periods was determined as above. In Vitro Simulated Digestion Experiments. The stability and bioaccessibility of free or encapsulated curcumin were evaluated using an in vitro gastric (60 min) and intestinal (120 min) digestion model according to Chen et al.10 Free or encapsulated curcumin solution or dispersion (20 mL) was mixed with 20 mL of 0.1 M HCl (pH 1.5) in a shaker (at 37 °C, with a rate of 95 rpm) for 10 min. After the pH of the mixtures was readjusted to 1.5 with 2.0 M HCl, pepsin powder (8 mg) was added under a shaking condition to initiate the pepsin digestion. At the end of gastric digestion (60 min), the resultant digests were adjusted to pH 7.0 with 4.0 M NaOH, and bile extract powder (400 mg) and pancreatin powder (20 mg) were sequentially added to initiate the intestinal digestion (60−120 min). The curcumin remaining in the supernatants (obtained after centrifugation at 10 000g for 30 min) of digests at different incubation periods was determined as earlier. The stability of free or encapsulated curcumin during the digestion was evaluated by monitoring the degradation kinetics of curcumin as earlier. The bioaccessibility of free or encapsulated curcumin was defined as the percentage of remaining curcumin in the supernatants (as earlier) at the end of the whole digestion (180 min). Statistical Analysis. All the data were statiscally analyzed using an analysis of variance (ANOVA), with the means compared with a least significant difference (Tukey HSD) at a confidence interval of 95%.

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RESULTS AND DISCUSSION Urea-Induced Disassembly and Reassembly of β-CG. Structural Dissociation and Unfolding. Figure 1A shows the SDS-PAGE and native-PAGE profiles of the current β-CG sample. The SDS-PAGE profile of this β-CG mainly exhibiting α, α′, and β subunits is basically the same as that observed by Nagano et al.32 The native-PAGE profile of this β-CG is basically the same as previously observed by He et al.33 β-CG is usually present in a 9S form at pH 7.0 and ionic strengths 4 M. The reversible disaasembly and reassembly of β-CG at [U] = 2−4 M nearly did not change its ζ-potential and H0, while in the irreversible case at [U] = 6−8 M, the reassembly resulted in a progressive decrease in ξ-potential, as well as a considerable increase in H0 (Table 1). The observations Table 1. Surface Characteristics (ξ-Potential and Surface Hydrophobicity, Ho) of Different Reassembled β-CG Samples, Obtained from Urea-Dissociated β-CG at Different Urea Concentrations ([U], 0−8 M)a urea concentrations 0 2 4 6 8

M (control) M M M M

ξ-potential (mV)

H0

−27.00 ± 2.0 a −24.61 ± 3.0 a −21.79 ± 3.4 a −12.93 ± 2.1 b −8.80 ± 1.2 b

3498 ± 829 a 3572 ± 820 a 3871 ± 873 ab 5942 ± 568 bc 6191 ± 754 c

Values are means ± SD (n = 3). Values in the same group with different letters (a, b) are significantly different (p < 0.05).

a

further confirmed that the application of high concentrations (6−8 M) of urea changed the structural characteristics of βCG, e.g., decreased charge density and increased hydrophobic clusters exposed to the aqueous environment. To unravel the molecular mechanism for the [U]-dependent disassembly and reassembly of β-CG, the urea-induced conformational changes of β-CG, as well as the differences between untreated and reassembled β-CG samples, were characterized using intrinsic Trp fluorescence, near-UV CD spectroscopy, and DSC technique, as displayed in Figure 3. For oligomeric globulins, the conformational changes may occur at quaternary and/or tertiary levels. However, in many cases, only the conformational changes at the tertiary level of a protein can be monitored by detecting intrinsic fluorescence characteristics of hydrophobic amino acids, e.g., Trp, Phe, or Tyr. The 6296

DOI: 10.1021/acs.jafc.8b05822 J. Agric. Food Chem. 2019, 67, 6292−6301

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Figure 3. (A) Typical intrinsic fluorescence spectra of urea-dissociated and reassembled β-CG at [U] = 0−8 M. The excitation wavelength was 280 nm. (B, C) Typical near-UV CD spectra (B) and DSC profiles (C) of reassembled β-CG, obtained from urea-dissociated β-CG at [U] = 0−8 M. The DSC experiments were performed on freeze-dried reassembled β-CG samples, with a heating program from 50 to 90 °C at a heating rate of 5 °C/min.

Figure 4. Schematic illustration for (A) the urea-induced disassembly of β-CG, as well as the reassembly of dissociated subunits to form core−shell β-CG nanoparticles, at different [U] values, and (B) the formation of curcumin-loaded reassembled β-CG nanoparticles from urea (8 M)dissociated β-CG.

treatment does not change the structure of β-subunit, limiting the intersubunit preferential hydrophobic interaction of βsubunit. As a consequence, all the dissociated subunits tend to reassemble in a random way, forming a comparable quaternary structure to their initial one when reassembled. In contrast, at [U] = 6−8 M, the structure of β-subunit would fully unfold,

and initially buried hydrophobic clusters within the subunit were gradually exposed. In this case, the preferential hydrophobic interaction between unfolded β-subunits considerably increases, and as a result, a kind of reassembled β-CG sample with a core−shell nanostructure will be formed. The ureainduced disassembly of β-CG, as well as the reassembly of 6297

DOI: 10.1021/acs.jafc.8b05822 J. Agric. Food Chem. 2019, 67, 6292−6301

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Journal of Agricultural and Food Chemistry dissociated subunits to form core−shell β-CG nanoparticles, is illustrated in Figure 4A. Influence of Protein Concentration. Besides the [U], the urea-induced dissociation of β-CG, as well as its reassembly from the dissociated subunits, is also affected by the c. Figure 5

stable tertiary structure via a structural rearrangement, and as a result, these subunits could keep separate from each other when reassembled. When the c was high (e.g., 4−6 wt %), the strong intermolecular (and intersubunit) hydrophobic interactions would greatly limit the occurrence of the preferential hydrophobic interactions of dissociated β-subunits. Due to this consideration, all the nanoencapsulation experiments in the following section were performed on the reassembled β-CG nanoparticles obtained at c = 2.0 wt % and [U] = 8 M. Nanoencapsulation of Curcumin in Reassembled βCG Nanoparticles. There are two advantages for the reassembled β-CG nanoparticles to act as nanocarriers for poorly soluble bioactives, e.g., curcumin. One is that curcumin crystals can be directly introduced in 8 M urea solution containing β-CG. Our preliminary experiments indicated that the maximal dispersion capacity of curcumin in 8 M urea can reach 6.1 mg/mL (data not shown), which is ∼554 540 times that of free curcumin in water at 25 °C.40 The other is that, rather than binding on the surface of protein molecules, e.g., in the case of forming soy protein nanocomplexes with curcumin,9−11 curcumin is incorporated in the core part of the core−shell nanoparticles (Figure 4B), because at 8 M urea curcumin with a highly hydrophobic nature is expected to readily interact with the urea-denatured β-subunit of β-CG through hydrophobic interactions. The nanoencapsulation of curcumin at concentrations of 1− 6 mg/mL (in the 8 M urea suspensions) in the β-CG nanoparticles was obtained at c = 2.0 wt %, in terms of EE% and LA. Figure 6A shows that all the curcumin-loaded suspensions containing the reassembled β-CG nanoparticles were yellow in appearance, and the turbidity progressively increased with increasing the initial curcumin concentration ([Cur]) in the range 1.0−6.0 mg/mL. No precipitation occurred in the suspensions even after a storage of 7 days (data not shown). The observations clearly indicated that curcumin was effectively encapsulated in the reassembled βCG nanoparticles, and the solubility in water was remarkably improved. At [Cur] = 1.0 mg/mL, the EE% and LA were about 78.8% and 30.8 μg/mg protein, respectively (Figure 6B). The LA could further progressively increase up to ∼180 μg/mg protein, when the [Cur] was increased to 6.0 mg/mL, although in this case, the EE% decreased to ∼61% (Figure 6B). The maximal LA (∼180 μg/mg protein) is remarkably higher than the maximal LA reported for curcumin encapsulated in the

Figure 5. z-average diameter (Dz) of urea (at 8 M)-dissociated and reassembled β-CG nanoparticles, as a function of c (0.1−6.0 wt %). Each datum is the mean ± SD (n = 3).

shows the influence of protein concentration (c) (0.1−6.0 wt %) on the Dz of urea (at [U] = 8 M)-dissociated and reassembled β-CG samples. In this case, the Dz progressively declined from 16.4 to 11.6 nm with the c increasing from 0.1 to 2.0 wt %; after that, the Dz contrarily increased, as the c increased from 2 to 6 wt % (Figure 5). The progressive decrease in Dz in the low c range (2 wt %) would be associated with the enhanced intermolecular hydrophobic interactions between urea-denatured β-CG molecules at higher c values.39 When the c was increased from 0.1 to 2.0 wt %, the Dz of the reassembled β-CG samples considerably increased from about 20.0 to 43.7 nm, but it dramatically declined when the c was >2.0 wt % (Figure 5). The observations suggest that the reassembly of β-CG from its dissociated subunits into a kind of core−shell nanoparticle was more favorably formed at appropriate c values, e.g., 1−2 wt %. Under very diluted conditions, the urea-dissociated subunits tended to form a new

Figure 6. (A) Visual observations of curcumin, encapsulated in untreated β-CG (control) or reassembled β-CG nanoparticles at c = 2.0 wt %. The [Cur] (from left to right) was 1.0, 2.0, 4.0, and 6.0 mg/mL, respectively. (B) Encapsulation efficiency (EE%) and loading amount (LA) of curcumin in the reassembled β-CG nanoparticles, as affected by the [Cur] (1−6 mg/mL). Each datum is the mean ± SD (n = 3). 6298

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peaks corresponding to free curcumin were observed (Figure 7), indicating that the nanoencapsulation completely inhibited the crystallization of curcumin. The inhibited crystallization of curcumin by the encapsulation could be largely due to its transformation from a crystal to an amorphous state. A similar inhibition of curcumin crystallization has been previously observed for curcumin-loaded soy protein isolate (SPI) nanoparticles, prepared by a desolvation and evaporation process.43,44 Improved Stability and Bioaccessibility of Curcumin. Curcumin molecules in an aqueous environment are very prone to chemical degradation, even at ambient temperature.46,47 In this work, the influence of the nanocapsulation (in the reassembled β-CG nanoparticles) on the stability of curcumin was evaluated by a heating at 80 °C or during an in vitro simulated digestion at room temperature (Figure 8). At 80 °C, free curcumin in water (at [Cur] = 0.2 mg/mL) fast degraded, as the time proceeded, and approximately 57% and 72% of curcumin were degraded when the suspension containing free curcumin was heated for 0.5 and 3.0 h, respectively (Figure 8A). In contrast, almost 90% of encapsulated curcumin survived, after the whole heating period of 3 h (Figure 8A). The observations clearly indicated that the nanoencapsulation remarkably improved the heat stability of curcumin in an aqueous environment. A similar improvement by the nanocapsulation was observed in the in vitro simulated stomach and intestinal digestion at room temperature, although in this case, curcumin (free or encapsulated) was more susceptible to degradation (Figure 8B). The improvement of curcumin stability in the current work was remarkably better than that of curcumin encapsulated in the nanocomplexes with SPI.10,11 In the latter cases, almost 70% and 60% of encapsulated curcumin were degraded after heating for 3 h at 95 and 85 °C, respectively, while in the current work, it was ∼10% degraded after 3 h of heating at 80 °C. The differences in stability of encapsulated curcumin might be associated with the differences in location in the fabricated nanoparticles. In the cases of SPI nanocomplexes, curcumin molecules were mainly bound on the surface of the protein nanoparticles, via hydrophobic interactions. In the current work, almost all of the curcumin molecules were incorporated within the interior of the β-CG nanoparticles, which would not be accessible to aqueous solution. In addition, the bioaccessibility of free or encapsulated curcumin was also assessed using the whole in vitro digestion (3 h) model, with the results included in Figure 8B (inset). Free curcumin had a bioaccessibility of 21.5%, which is close to that reported in our previous work (∼20%).10 When encapsulated in the reassembled β-CG nanoparticles, the bioaccessibility of curcumin was increased up to ∼40.6% (Figure 8B, inset), indicating the improvement of bioaccessibility. Although the improvement of bioaccessibility of curcumin by the nanoencapsulation in the current work was remarkably poorer than that observed for the nanocomplexation with SPI (60−90%; 10), it should be noteworthy that most of thr encapsulated curcumin molecules still remained in the reassembled β-CG nanoparticles. The observations suggested that the nanoencapsulation in the reassembled nanoparticles imparted the encapsulated curcumin a sustained release behavior during the in vitro digestion. Also, it can be argued that the reassembled β-CG nanoparticles could perform as nanocarriers for potentially colon-targeted delivery of curcumin.

nanocomplexes with heated and sonicated soy protein isolate (144.5 μg/mg protein),11 as well as that (19 μg/mg of casein) of curcumin remaining in the supernatant of casein nanocapsules.21 The maximal water solublility of curcumin at c = 2.0 wt % (∼3.68 mg/mL) was 334 500 times that (11 μg/L) of free curcumin in water.40 The improvement in curcumin solubility is remarkably better than that (131 000-fold) reported for micelles of a synthesized diblock copolymer.41 The influence of the incorporation of curcumin (at [Cur] = 1 mg/mL) on the particle morphology of the reassembled βCG nanoparticles (formed at [U] = 8 M) was investigated by TEM, as included in Figure 2. The TEM observations indicated that, when the curcumin was loaded, the size of the reassembled β-CG nanoparticles remarkably increased, and most of the nanoparticles became more homogeneous in size distribution and smooth in surface (Figure 2B and C). Interestingly, no crystals of curcumin were observed on the surface of the reassembled β-CG nanoparticles or in the system (Figure 2C), suggesting that, although the LA was high, all the curcumin molecules were encapsulated within the interior of the nanoparticles. If we look at typical curcumin-loaded reassembled β-CG nanoparticle in more detail, it can be wellobserved that the nanoparticle had a weakly stained hydrophilic shell layer and a heavily stained core (with curcumin) (Figure 2D). A similar core−shell structure with a hydrophilic shell layer has been previously observed for β-CG-dextran nanogels using the TEM.42 The TEM observations indirectly support the proposed formation mechanism for curcuminloaded reassembled β-CG nanoparticles. Inhibited Crystallization of Curcumin. The crystallinity of curcumin in the reassembled β-CG nanoparticles, formed at c = 2 wt %, [U] = 8 M, and [Cur] = 1.0 mg/mL, was assessed by XRD (Figure 7). Free curcumin exhibited three major XRD

Figure 7. Representative XRD spectra of curcumin-loaded reassembled β-CG nanoparticles, or the physical mixture of curcumin and β-CG (with the same composition as in the reassembled sample). The curcumin-loaded reassembled β-CG nanoparticle powder was obtained from urea (8 M)-dissociated β-CG (at c = 2 wt %) in the presence of 1.0 mg/mL of curcumin, followed by dialysis and freezedrying. The XRD spectra of free curcumin and untreated β-CG are also included.

peaks at [2θ] of around 8, 17, and 25°, respectively (Figure 7), which is comparable to that reported by Teng et al.43,44 and Jia et al.,45 implying a high degree of crystallinity. The XRD pattern of the simple physical mixture with β-CG was almost the same as that of free curcumin, but when the curcumin was loaded in the reassembled β-CG nanoparticles, no major XRD 6299

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Figure 8. Degradation kinetics of curcumin, free or encapsulated in the reassembled β-CG nanoparticle in water, at 80 °C (A) or during in vitro simulated digestion process (B). Free curcumin suspension was prepared by diluting the curcumin ethanol solution (5 mg/mL; 100% ethanol) with 39 mL of deionized water to 0.2 mg/mL. The bioaccessibility of free or encapsulated curcumin after the whole in vitro digestion process is displayed within (B) (inset). Each datum is the mean ± SD (n = 3). (4) Chen, L.; Remondetto, G. E.; Subirade, M. Food protein-based materials as nutraceutical delivery systems. Trends Food Sci. Technol. 2006, 17, 272−283. (5) Elzoghby, A. O.; Abo El-Fotoh, W. S.; Elgindy, N. A. Caseinbased formulations as promising controlled release drug delivery systems. J. Controlled Release 2011, 153, 206−216. (6) Elzoghby, A. O.; Samy, W. M.; Elgindy, N. A. Protein-based nanocarriers as promising drug and gene delivery systems. J. Controlled Release 2012, 161, 38−49. (7) Tarhini, M.; Greige-Gerges, H.; Elaissari, A. Protein-based nanoparticles: From preparation to encapsulation of active molecules. Int. J. Pharm. 2017, 522, 172−197. (8) Livney, Y. D. Milk proteins as vehicles for bioactives. Curr. Opin. Colloid Interface Sci. 2010, 15, 73−83. (9) Tapal, A.; Tiku, P. K. Complexation of curcumin with soy protein isolate and its implications on solubility and stability of curcumin. Food Chem. 2012, 130, 960−965. (10) Chen, F. P.; Li, B. S.; Tang, C. H. Nanocomplexation of curcumin and soy protein isolate: Influence on curcumin stability and/or bioaccessibility and in vitro protein digestibility. J. Agric. Food Chem. 2015, 63, 3559−3569. (11) Chen, F. P.; Li, B. S.; Tang, C. H. Nanocomplexation of soy protein isolate with curcumin: Influence of ultrasonic treatment. Food Res. Int. 2015, 75, 157−165. (12) David, S.; Zagury, Y.; Livney, Y. D. Soy β-conglycinin-curcumin nanocomplexes for enrichment of clear beverages. Food Biophys. 2015, 10, 195−206. (13) Levinson, Y.; Israeli-Lev, G.; Livney, Y. D. Soybean βconglycinin nanoparticles for delivery of hydrophobic nutraceuticals. Food Biophys. 2014, 9, 332−340. (14) Pujara, N.; Jambhrunkar, S.; Wong, K. Y.; McGuckin, M.; Popat, A. Enhanced colloidal stability, solubility and rapid dissolution of resveratrol by nanocomplexation with soy protein isolate. J. Colloid Interface Sci. 2017, 488, 303−308. (15) Esmaili, M.; Ghaffari, S. M.; Moosavi-Movahedi, Z.; Atri, M. S.; Sharifizadeh, A.; Farhadi, M.; Yousefi, R.; Chobert, J. M.; Haertlé, T.; Moosavi-Movahedi, A. A. Beta casein-micelle as a nano vehicle for solubility enhancement of curcumin: food industry application. LWT 2011, 44, 2166−2172. (16) Pan, K.; Luo, Y.; Gan, Y.; Baek, S. J.; Zhong, Q. pH-driven encapsulation of curcumin in self-assembled casein nanoparticles for enhanced dispersibility and bioactivity. Soft Matter 2014, 10, 6820− 6830. (17) Sáiz-Abajo, M. J.; González-Ferrero, C.; Moreno-Ruiz, A.; Romo-Hualde, A.; González-Navarro, C. J. Thermal protection of βcarotene in re-assembled casein micelles during different processing technologies applied in food industry. Food Chem. 2013, 138, 1581− 1587.

In conclusion, novel core−shell nanostructured particles with aggregated β-subunits as the core and hydrophilic α- and α′-subunits as the shell layer have been facilely fabricated from β-CG, with an urea-induced dissembly and reassembly strategy. The unique core−shell nanoparticles were favorably formed at [U] = 6−8 M and c = 1−2 wt %. This kind of reassembled β-CG nanoparticle was confirmed to act as an outstanding nanocarrier for curcumin. There were many advantages for this nanoencapsulation, e.g., no organic solvent used, extremely high encapsulation efficiency, and extraordinary stability for encapsulated curcumin. The maximal LA of curcumin (18 wt %) reported in this work far exceeds those reported previously. The encapsulated curcumin in the fabricated nanoparticles exhibited an extraordinary thermal stability and a good sustained-release behavior. The findings of this work provide an important strategy using a facile and ecofriendly process to fabricate a kind of high-encapsulationperformance nanocarrier for poorly soluble nutraceuticals or drugs.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Peng-Zhan Liu: 0000-0003-2703-1993 Chuan-He Tang: 0000-0002-8769-2040 Funding

This work was part of the projects supported by The National Key Research and Development Program of China (2017YFD0400200), the NNSF of China (serial nos. 21872057 and 31471695), and GDHVPS (2017). Notes

The authors declare no competing financial interest.

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