Article pubs.acs.org/JAFC
Pickering Emulsion Gels Prepared by Hydrogen-Bonded Zein/Tannic Acid Complex Colloidal Particles Yuan Zou,† Jian Guo,† Shou-Wei Yin,† Jin-Mei Wang,† and Xiao-Quan Yang*,†,‡ †
Food Protein Research and Development Center, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China ‡ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China ABSTRACT: Food-grade colloidal particles and complexes, which are formed via modulation of the noncovalent interactions between macromolecules and natural small molecules, can be developed as novel functional ingredients in a safe and sustainable way. For this study was prepared a novel zein/tannic acid (TA) complex colloidal particle (ZTP) based on the hydrogen-bonding interaction between zein and TA in aqueous ethanol solution by using a simple antisolvent approach. Pickering emulsion gels with high oil volume fraction (φoil > 50%) were successfully fabricated via one-step homogenization. Circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements, which were used to characterize the structure of zein/TA complexes in ethanol solution, clearly showed that TA binding generated a conformational change of zein without altering their supramolecular structure at pH 5.0 and intermediate TA concentrations. Consequently, the resultant ZTP had tuned near neutral wettability (θow ∼ 86°) and enhanced interfacial reactivity, but without significantly decreased surface charge. These allowed the ZTP to stabilize the oil droplets and further triggered cross-linking to form a continuous network among and around the oil droplets and protein particles, leading to the formation of stable Pickering emulsion gels. Layer-by-layer (LbL) interfacial architecture on the oil−water surface of the droplets was observed, which implied a possibility to fabricate hierarchical interface microstructure via modulation of the noncovalent interaction between hydrophobic protein and natural polyphenol. KEYWORDS: zein, tannic acid, colloidal particle, hydrogen bond, Pickering emulsion gel
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INTRODUCTION Food-grade colloidal structures such as colloidal particles, complexes, and microcapsules can be generated by utilization of molecular interactions between food macromolecules and small molecular weight compounds or mutual interactions of two macromolecules. They have shown good potential for applications in the food, beverage, and dietary supplement industries. These novel structures used for the formulation and delivery of bioactive molecules1,2 or enhancement in the functionality of food-grade biopolymers3,4 have been realized as a new generation of food functional ingredients. Colloidal complexation can be induced between two or three food compounds that possess high reactivity and show tendency to form noncovalent interaction. Noncovalent interactions, such as hydrophobic, cation−π, electrostatic, and hydrogen bonding, can be exploited to obtain microstructures with important properties. These interactions among food compounds generally pose a problem, which may result in physical instability, such as aggregation and precipitation. However, colloidal complexation of the reactive compounds, based on the balance between different noncovalent interactions by modulating the pH, ionic strength, temperature, and concentration ratio, can be considered as a feasible solution to both technical and nutritional functionality.2,5 Moreover, these food-grade colloidal particles and complexes were made from natural materials and formed via modulation of the noncovalent interactions, avoiding the use of harsh chemical cross-linkers. Thus, they hold promise for producing functional foods in a safe and sustainable way. We had studied the colloidal © XXXX American Chemical Society
complexation of soy protein with a biosurfactant stevioside (STE) and their synergistic interfacial properties. It was found that these weakly interacting mixtures exhibited greater foaming capacity3 and considerable emulsification stability.6 Patel et al.4 studied the colloidal complexes of methylcellulose (MCE) and tannic acid (TA), and the complexes showed excellent surface activity and were further utilized for stabilization of foams and emulsions. As a kind of natural polyphenol and food-grade material with a long history of industrial application, tannic acid (TA) is widely used in clarifying the undesirable haze in beers and fruit juices, and it also serves as a structurant and formulation aid.7 TA belongs to a group of hydrolyzable tannins and contains digalloyl ester groups connected to a glucose core. It has diverse biological properties including antioxidant, antibacterial, and antiviral properties.8,9 Because of the presence of abundant hydroxyls (around 25 hydroxyl groups), TA can participate in noncovalent interactions including ionic pairing and hydrogen bonding. Its amphiphatic nature further contributes to hydrophobic, p−π, and π−π interactions. TA can interact with a wide range of small molecules (such as alkaloids5) and macromolecules including polysaccharides,4 proteins,10 and synthetic polymers.11 Kharlampieva and co-workers had successfully prepared layer-by-layer (LbL) assemblies and Received: May 12, 2015 Revised: July 28, 2015 Accepted: July 30, 2015
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DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
ation of zein and TA was due to noncovalent interaction (mainly hydrogen bonding), which was more specific than the hydrophobic interaction in zein−SS complexes. Because of the multiple biological properties of TA, including antioxidant and antibacterial properties, these emulsions might inherently possess excellent chemical stability and some health benefits, and they could be developed as novel functional food ingredients.
multilayer microcapsules based on TA and synthetic polymers (poly-N-vinylpyrrolidone) via hydrogen-bonding interaction, for drug encapsulation, controlled delivery, and long-term storage. TA−protein interactions are essentially physical: hydrophobic and hydrogen bond mediated.7 The tannin affinities for human salivary proline-rich proteins (PRP) were found to be related to astringency,12 and tannin−gliadin interactions for haze formation13 had been investigated extensively. For PRP, including gliadin13 and kafirin,14 hydrogen bonds between the hydroxyl groups of tannins and the carbonyl groups of proteins are believed to be involved in the complexation of the proteins and tannins. PRP can also interact with pentagalloyl glucose of TA via hydrophobic interactions with the pyrrolidine ring of proline. Below the tannin critical micelle concentration (CMC), the protein polypeptides surround the tannins; above the tannin CMC, tannins act as bridges to cross-link the polypeptides for forming aggregates or agglomerates of proteins.15 Stabilization of the oil−water interface with colloidal particles, which leads to the formation of Pickering emulsions, is known to display long-term stability against coalescence, and the potentials for texture modification, calorie and fat reduction, and bioactive compound delivery have emerged.16 As compared to conventional emulsifiers, the main benefits of Pickering emulsions are high physical/chemical stabilities. The particlestabilized interface can be modified after emulsion formation to bring additional functionality; this modification of interfacial properties can be in response to a trigger, and Pickering emulsions can serve as templates for preparing materials that could not be obtained from conventional emulsions.16 Studies on protein-based particles used as Pickering emulsion stabilizers are still scarce, although they show clearly a growing research field. Zein, a major protein of corn, is capable of forming selfassembled nanoparticles to stabilize Pickering emulsion due to its amphiphilic character. Therefore, zein-based particles possess promising potential to serve as food-grade Pickering emulsion stabilizers in the food industry. de Folter and coworkers have employed unmodified zein colloidal particles to stabilize Pickering emulsion, but it was very unstable to creaming and the aqueous phase was turbid.17 Fortunately, we have reported the stabilization of oil−water interfaces with zein complex colloidal particles.18,19 Our findings showed that the complexation of zein particles and the surfactant sodium stearate (SS) was due to nonspecific hydrophobic interaction, which resulted in enhanced adsorption and accumulation of zein particles at the oil−water interface. The resultant Pickering emulsions exhibited superior stability against both coalescence and creaming.18 More recently, we fabricated dual-functional zein/chitosan complex particles (ZCP), which were loaded with antioxidant curcumin, and further prepared antioxidant Pickering emulsions via manipulation of the interface framework. The combination of steric hindrance from ZCP-based interfacial architecture and interfacial cargo of curcumin gave rise to emulsions with favorable oxidative stability.19 However, modifiers such as SS and chitosan are either synthetic or have a poor organoleptic acceptation, which limit their application in food formulations. Moreover, emulsions stabilized by zein−SS complexes presented oil droplets in the top phase after a few days of storage. In this work, TA, a natural polyphenol, was used as an efficient tool to control the self-assembly behavior of zein, so as to fabricate stable and edible zein/TA colloidal particles (ZTP) and consequently prepare stable Pickering emulsions. Interestingly, the results indicated that the complex-
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MATERIALS AND METHODS
Materials. Zein (purity is around 88−96%), tannic acid, and fluorescent dyes (Nile Red and Nile Blue A) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). α-Zein is the major component of zein, which has two subtypes, Z19 and Z22, confirmed by SDS-PAGE. Corn oil was obtained in a local supermarket (Guangzhou, China). Other chemicals were of analytical grade. Colloidal Particle Synthesis. The zein colloidal particles were prepared via an antisolvent precipitation technique,19 with modification. In brief, an aliquot of 2.5 g of zein powder was dispersed in 100 mL of a 70:30 ethanol/water (v/v) solution and stirred using a magnetic stirrer for 2 h, then stored overnight at 4 °C to allow complete hydration. Zein stock solutions with different TA concentrations (expressed as zein/TA ratios, 1:0−1:0.5 w/w) at different pH values (3.0, 5.0, 7.0; the pH was adjusted with 2 mol/L HCl and 2 mol/L NaOH) were heated in a water bath at 55 °C for 50 min with slow stirring. The preheated zein−TA solutions (10 mL) were quicklyt poured into 25 mL of Millipore water (18.2 MΩ·cm) under continued stirring (1000 rpm). After 15 min of stirring, the ethanol in zein−TA solutions were further removed, and preconcentration of colloidal particle was performed by using an RV 10 digital rotary evaporator (IKA-Works Inc., Germany). Finally, the zein concentration in the solutions (8.3 mL) was 3% (w/v). The obtained solutions were centrifuged at 4000g for 10 min to remove insoluble aggregates, and then they were stored at 4 °C for further use. For field emission scanning electron microscope (FE-SEM) observation and contact angle measurement, the resultant ZTP colloidal particles were lyophilized for 24 h (Christ Delta 1-24 LSC, Martin Christ, Germany) and then ground into homogeneous powders. Particle Size and Zeta (ζ)-Potential Measurements. The ZTP colloidal particles were diluted to 1 mg/mL with Millipore water, and the pH was adjusted to 3.5; then the particle size, ζ-potential, and polydispersity index (PDI) were measured using a Nano ZS Zetasizer instrument (Malvern Instruments, Worcestershire, UK). All measurements were carried out at 25 °C, and the results are reported as averages of three readings. Morphology Observation. The morphology of the freeze-dried ZTP powders was observed by FE-SEM. The powder samples were loosely glued onto a conductive adhesive mounted on a stainless steel stage, subsequently coated with a thin conductive gold and platinum layer using a sputter coater (Hummer XP, Anatech, Union City, CA, USA). They were observed with a Zeiss Merlin FE-SEM (Zeiss, Oberkochen, Germany). Contact Angle Measurement. The three-phase contact angle (θow) of colloidal particles was measured using an OCA 20 AMP (Dataphysics Instruments GmbH, Germany), which was described in our previous research.18 The ZTP powders were prepared as pellets of 13 mm in diameter and 2 mm in thickness, and the pellets were placed into an optical glass cuvette containing purified corn oil. Next, a drop of pH 3.5 Milli-Q water (5 μL) was deposited on the surface of the pellets using a high-precision injector. After 4 min for equilibration, the drop image was photographed using a high-speed video camera, and the profile of the droplet was numerically solved and fitted to the Laplace−Young equation. Contact angles were measured on each of four pellets per sample, and three measurements were performed for each pallet. Circular Dichroism (CD) Spectroscopy. A Chirascan spectropolarimeter (Applied Photophysics Ltd., UK) was used for monitoring the configuration of zein in 70% (v/v) aqueous ethanol after heating with TA. The samples were diluted to 0.1 mg/mL by 70% (v/v) B
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Size distribution and morphology of the zein/TA complex colloidal particle (ZTP) prepared at (A) various pH values (3.0, 5.0, and 7.0) with a zein/TA ratio of 1:0.2 and (B) various zein/TA ratios (from 1:0 to 1:0.5 w/w) at pH 5.0. FE-SEM images showed the ZTP prepared at pH 5.0 (C) and 7.0 (D) with the same zein/TA ratio of 1:0.2. if the observed asymmetric particles are approximated by a rectangular prism with edges of a, b, and c (b and c ≪ a), knowledge of Rg and Rc gives edge a with the largest length from the equation a2 = 12(Rg2 − Rc2).21 Fourier Transform Infrared Spectroscopy (FTIR). The FTIR spectra of ZTP powder were recorded at 400−4000 cm−1 using a Bruker VERTEX 70 spectrometer (Germany) equipped with a narrow-band mercury cadmium telluride detector with a resolution of 2 cm−1. Preparation of Pickering Emulsion. The Pickering emulsions were prepared by resultant zein/TA colloidal particles with different concentrations (0.25−1.5 wt %). All emulsions were prepared by homogenizing the mixture of 50 v % corn oil and 50 v % corresponding concentration particle suspensions. In brief, corn oil (5 mL) was added to the particle suspensions (5 mL) in a glass vial, and the resultant mixtures were sheared using an Ultra-Turrax T10 homogenizer (IKA-Works Inc., Germany) at 23000 rpm for 2 min at room temperature to yield Pickering emulsions. Droplet Size Distribution of Emulsion. The oil droplet size distribution of emulsions was determined using a Malvern MasterSizer 3000 (Malvern Instruments Ltd., Worcestershire, UK). The samples were diluted with deionized water. The refractive indices of water and corn oil were taken as 1.33 and 1.52, respectively. Droplet size measurements were reported as the volume-average diameter, d4,3 = Σnidi4Σnidi3, where ni is the number of particles with diameter di. All of the experiments were performed in three replicates. Confocal Laser Scanning Microscopy (CLSM). The microstructure of emulsions was observed with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems Inc., Heidelberg, Germany). The emulsions were dyed with a mixed fluorescent dye solution consisting of 1 mg/mL Nile Red and 1 mg/mL Nile Blue A. The stained emulsions were placed on concave slides and covered with
aqueous ethanol solution with different pH values (3.0, 5.0, 7.0), which was also used as the reference buffer. The CD spectroscopy was recorded from 190 to 260 nm in a 1 mm path length quartz cuvette, expressed as mean residue ellipticity in deg cm2 dmol−1. From the CD spectra, fractional contents of the secondary structure (α-helix, β-sheet, β-turn, and random coil) of the zein−TA complex in 70% (v/v) aqueous ethanol were calculated according to the CONTIN/LL program in CD Pro software. Small-Angle X-ray Scattering (SAXS). SAXS experiments were performed using a SAXSess camera (Anton-Paar, Graz, Austria) to collect information about the structure parameters and the shape of zein/TA complexes in 70% (v/v) aqueous ethanol solutions. A PW3830 X-ray generator with a long fine-focus sealed glass X-ray tube (PAN Alytical Almelo, The Netherlands) was operated at 40 kV and 50 mA. The samples with a zein concentration of 1.0 wt % were filled into a capillary of 1 mm diameter and 0.01 mm wall thickness. The capillary was placed in a TCS 120 temperature-controlled sample holder unit (Anton Paar) along the line-shaped X-ray beam in the evacuated camera housing. A shot exposure period of 900 s was used to acquire the scattering data. The sample-to-detector distance was 261.2 mm, and the temperature was kept at 25.0 °C. Data analyses were done by Guinier methods. The overall radius of gyration (Rg) calculated from the initial slope of a Guinier plot as ln(I(q)) against q2, where I (q) is the scattering intensity and q = (4π/λ) sin (θ/2) is the modulus of the scattering vector with θ the scattering angle and λ the wavelength of the X-ray beam. The limit of Guinier plots is usually defined as qRg < 1. The I(0) was evaluated at very small angles, and the intensity is represented as I(q) = I(0) exp(−1/3(qRg)2). If one of the particle dimensions is much larger than other two dimensions, the radius of gyration of the cross-section (Rc) is obtained in a larger q range than that used in the Rg analyses and calculated from the slope of the cross-section plots as ln(I(q)q) against q2 (qRc < 1). Moreover, C
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry coverslips. They were observed with a 100× oil immersion lens and an argon krypton laser having an excitation line of 488 nm and a helium neon laser (He/Ne) with excitation at 633 nm. Rheology of Emulsion Gels. The dynamic oscillatory measurement of Pickering emulsion gels was performed at 25 °C with the frequency ranging from 1 to 10 rad/s (all of the measurements were conducted within the linear viscoelastic region), using a Haake RS600 rheometer (Thermo Electron Co., Waltham, MA, USA) between two parallel plates (d = 35 mm) with a gap of 1.0 mm. The elastic (G′) and loss (G″) moduli as a function of the frequency were recorded. All of the experiments were performed in three replicates. Statistics. Statistical analyses were performed using an analysis of variance (ANOVA) procedure of the SPSS 21.0 statistical analysis program, and the differences between means of the trials were detected by a least significant difference (LSD) test (P ≤ 0.05).
exhibited colloidal stable, nanosized (∼99 nm), and nearly monodisperse size distribution (PDI ≈ 0.11). FE-SEM images (Figure 1C,D) further confirmed that the particles formed at pH 5.0 were relatively small and well dispersed, whereas ZTP particles formed at pH 7.0 were aggregated clusters. The ZTP prepared at pH 5.0 with zein/TA ratios of 1:0.2 actually possessed much higher ζ-potential (about +51 mV), which contributed to the strong electrostatic repulsion among particles. However, when the zein/TA ratio was increased from 1:0.3 to 1:0.5, the ζ-potential of ZTP declined to +35 mV and the particle dispersions became unstable (Figure 1B; Table 1), indicating that the excess TA resulted in the aggregation and complex precipitation of ZTP. Moreover, the ζ-potentials of TA colloidal particles (prepared via an antisolvent precipitation technique stated under Materials and Methods; TA concentration in ethanol aqueous of 5 mg/mL) prepared at pH 3.0, 5.0, and 7.0 and finally adjusted to a pH of 3.5 were +9.32, −2.67, and −4.26 mV (data not shown), respectively. Zein is positively charged below its isoelectric point (pI 6.2).23 After the antisolvent and evaporation procedure, the pH of the ZTP dispersion decreased from pH 5.0 to 3.48 (data not shown). The protonation of zein at such a pH resulted in a strong positive surface charge on colloidal particles (about +51 mV); thus, this long-range electrostatic repulsion was able to stabilize zein in water, which was its poor solvent.17 However, the final pH of ZTP formed at pH 3.0 was much lower (about 2.85; data not shown), and these particles had lower surface charge (+33 mV), leading to colloidal instability and precipitation. Recently, Kharlampieva and co-workers11,24 studied the TA−poly(N-vinylpyrrolidone) (TA−PVPON) multilayer microcapsules formed at pH 5.0 and 7.4, respectively. They found that TA was in its neutral and partially protonated form at pH 5.0, and this facilitated strong hydrogen-bonding interactions between the hydroxyl groups on TA and the carbonyl groups on the pyrrolidone rings in PVPON. This complexation further suppressed the ionization of TA, resulting in the improvement of their drug encapsulation and long-term storage.11,24 In a similar way, hydrogen-bonding interaction between protonated TA and zein might dominate in the ZTP formed at pH 5.0. At pH 3.0 and 7.0, TA was in partial ionization of hydroxyl groups, leading to the formation of electrostatic complexes with low surface charge and unstable colloidal dispersions. The interfacial wettability of the particles plays a crucial role for the preparation of emulsions stabilized by particles or Pickering emulsions. The near-neutral wettability (θow ∼ 90°) can facilitate efficient packing of particles at the oil−water interface and create a steric barrier against the coalescence of oil droplets. The wettability of the zein particles had been successfully modified by ionic surfactant, sodium stearate, via hydrophobic interaction18 or by a positively charged polysaccharide, chitosan, via weak electrostatic interaction,19 as we reported previously. In this study we tried to tune the wettability of zein particles via modulating the hydrogenbonding interaction between TA and zein. Figure 2 shows typical images of water droplets deposited to zein films immersed in corn oil. The wettability of zein particles expressed as oil-in-water three-phase contact angles (θow) was calculated,19 which is shown in the insets of Figure 2. The θow of bare zein particles formed at pH 5.0 was around 107.13°, which was consistent with our previous result (θow ∼ 112.7°).19 These particles actually did not favor interfacial particle adsorption and formation of oil-in-water Pickering emulsion. The ZTP
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RESULTS AND DISCUSSION Preparation of Zein−TA Complex Colloidal Particles (ZTP). Compared with the traditional antisolvent approach that was used to fabricate zein colloidal particles,18,19 we adopted a modified method to prepare ZTP. In brief, both zein and TA were dissolved in 70% (v/v) aqueous ethanol solution and heated to enhance their interaction, rather than directly diluting the aqueous ethanol solution of zein with TA aqueous solution. Heating can unravel some α-helical chain structures of zein by breaking hydrogen bonds, followed by realignment and reorganization into a β-sheet conformation.21 It can be hypothesized that hydrogen bonds of α-helical structures and β-sheets may be broken during incubation, leading to the formation of random coils, which may provide complexation sites for TA to interact. The complexation sites are probably the carbonyl groups of the polypeptide, which hydrogen bond with the hydroxyl groups of the TA.22 Due to the protonation, neutralization, and ionization of TA in solutions under varied pH, TA exhibited the capability to facilitate hydrogen-bonded interaction with macromolecules through pH-dependent regulation.11,17 Therefore, the colloidal stability of ZTP was explored at pH 3.0, 5.0, and 7.0, respectively. Figure 1 shows the morphology and size distribution of ZTP formed at different pH values and TA concentrations (expressed as zein/ TA ratio). Table 1 shows the mean size, polydispersity (PDI), and ζ-potential values of ZTP as a function of pH and zein/TA ratio. Unlike the ZTP prepared at pH 3.0 and 7.0, which showed severe aggregation, greater particle size (∼200 nm, determined by FE-SEM), and multiple-modal size distribution (PDI ≈ 0.74, pH 3.0), the ZTP prepared at pH 5.0 with intermediate TA concentration (zein/TA wt/wt = 1:0.2) Table 1. Mean Size, PDI, and ζ-Potential of ZTP Prepared under Varied pH and Zein/TA Ratiosa sample
PDI
mean ζ-potential (mV)
0.74 ± 0.05a 0.11 ± 0.00b 0.14 ± 0.00b
33.35 ± 1.65b 51.50 ± 0.50a 33.05 ± 0.15b
mean size (nm)
pH (zein/TA (w/w) = 1:0.2) 3.0 249.45 ± 12.95a 5.0 99.12 ± 0.37c 7.0 148.75 ± 5.65b zein/TA (w/w) (pH 5.0) 1:0.0 96.27 ± 0.87c 1:0.1 109.00 ± 1.20b 1:0.2 99.12 ± 0.37bc 1:0.3 103.80 ± 1.00bc 1:0.5 202.95 ± 7.05a
0.09 0.15 0.11 0.16 0.57
± ± ± ± ±
0.01c 0.03b 0.00bc 0.01b 0.01a
57.10 56.90 51.50 49.35 35.55
± ± ± ± ±
3.10a 1.50a 0.50ab 1.55b 0.45c
a
Mean values in table followed by different lower case letters are significantly different at P ≤ 0.05 by Duncan’s multiple-range tests. D
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Oil-in-water three-phase contact angles (θow) of zein particles with different pH values and TA concentrations: (A) ZTP prepared at different pH values with the same zein/TA ratio of 1:0.2 (w/w); (B) ZTP prepared at pH 5.0 with different zein/TA ratios. θow values (insets) were determined for corn oil phases.
Figure 3. CD spectra of zein in 70% (v/v) aqueous ethanol with different pH values (A) and TA concentrations: (A) with different pH at the same zein/TA ratio of 1:0.2 (w/w); (B) with different zein/TA ratios at pH 5.0. (Insets) Fractional contents of the secondary structure in zein after heat treatment.
oblong structure with an axial ratio of 6:1−12:1, which was evidenced by SAXS experiments.25,27 The extended configuration of zein in protic solvents could provide increased accessibility to the reactive sites of protein for TA, thus modifying their functionality. The secondary structure of zein in 70% (v/v) aqueous ethanol was monitored in the farultraviolet (UV) region. The CD profiles of zein with different pH values and TA concentrations are shown in Figure 3. The fractional contents of secondary structure (α-helix, β-sheet, βturn, and random coil) of zein were calculated, and they are shown in the insets of Figure 3. First, because the CD spectrum of TA was almost the same as that of the buffer (70:30 v/v ethanol/water solutions), TA did not have its own CD pattern to interfere with zein (data not shown). The CD spectra of zein exhibited the characteristics of α-helix with two strong negative ellipticity values at around 208 and 222 nm. CD ellipticity at 222 nm has been widely used to estimate α-helix content in proteins.27 Compared to the complexes at pH 5.0, a slight
formed at pH 3.0 and 7.0 as well as at pH 5.0 with excess TA concentrations (zein/TA ratios of 1:0.3−1:0.5) had much lower θow (70−77°) and surface charge (+30 mV) concurrently. These implied that the excess TA and ionization of TA facilitated the formation of electrostatic complexes. In contrast, the ZTP formed at pH 5.0 with a zein/TA ratio of 1:0.2 had near-neutral wettability (θow ∼ 86°) without decreasing their surface charge significantly, suggesting that this ZTP could be utilized to yield stable Pickering emulsion. These interactions might be dominated by hydrogen bonding between the carbonyl moieties of zein and the hydroxyl groups of the neutral TA at acidic pH and caused an increase in the hydroxyl groups at the surface of zein particles, thereby lessening their hydrophobic characteristics. Structure Characterization of Zein/TA Complexes and Particles. The structure and conformation of α-zein in various solutions had been extensively studied.25−27 The consensus is that α-zein in aqueous ethanol solution exists as an extended E
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. FTIR spectra of zein/TA complex colloidal particle (prepared at pH 5.0 with a zein/TA ratio of 1:0.2) (A), zein/TA physical mixture (mixed the zein and TA powder with a zein/TA ratio of 1:0.2) (B), TA (C), and zein particle (D).
Table 2. Structural Parameters of Zein−TA Complex in 70% (v/v) Aqueous Ethanol Obtained by SAXSa pH (zein/TA = 1:0.2 w/w) Rg (nm) a (nm) I(0)
zein/TA (w/w, pH 5.0)
3.0
5.0
7.0
1:0.0
1:0.1
1:0.2
1:0.3
1:0.5
2.50 8.66 0.45
3.25 11.25 0.67
5.63 19.50 1.01
2.90 10.03 0.52
2.24 7.76 0.38
3.25 11.25 0.67
2.35 8.15 0.42
2.17 7.61 0.45
a
Rg, overall radius of gyration of zein; a, longest edge in an elongated rectangular prism, which approximates the asymmetric particles of zein; I(0), extrapolation of intensity to an angle of zero.
increase of α-helix content and decrease of β-sheet for zein/TA complexes at pH 3.0 and 7.0 were observed, but the difference was not significant (Figure 3A). Cabra et al.21 found that the secondary structure of zein in aqueous ethanol solutions strongly depended on the pH on the basis of CD measurements. It was also shown that the α-helix and β-sheet contents at pH 5.0 were around 33 and 12%, which were similar to our results. Huang and co-workers27 compared the dissolution behavior of α-zein in aqueous ethanol with that in acetic acid solutions by SAXS, which indicated that the protonation of acetic acid imposed on zein. The positive surface charges of zein and reduction of hydrophobic interaction with the presence of the acetate solvation layer, which was able to provide long-range electrostatic repulsion, enhanced the solution stability of zein. As a weak acid, TA completely protonates and ionizes at pH 3.0 and 7.0, respectively.11 Therefore, the increase of α-helix content at pH 3.0 and 7.0 might contribute to the formation of electrostatic complexes between zein and protonated or ionized TA. Figure 3B clearly shows evidence for a conformational change of zein in aqueous ethanol solution that was induced by TA binding. It can be seen that binding of TA causes an obvious transformation from α-helix to β-sheet configuration of zein at pH 5.0, and the greatest shift generated on the zein−TA complexes with intermediate TA concentration (a zein/TA ratio of 1:0.2). The protein interaction with polyphenol driven by hydrophobic attraction and enhanced with hydrogenbonding network had been well confirmed.28 The interaction between milk β-lactoglobulin (β-LG)29 and tea tannins (epigallocatechin gallate, EGCG) was studied in detail,27
which indicated that the β-LG conformation was altered in the presence of EGCG with an increase in β-sheet and α-helix, which helped to enhance the structural stabilization of protein. The conformational change of zein due to the TA binding is dependent on the TA concentration. When the zein/TA ratio was increased to 1:0.3−1:0.5, the fractional contents of the secondary structure in zein were close to that of pure zein (Figure 4B), implying that the binding model of zein and TA would be changed. Recent findings indicated that for native unfolded protein, such as β-casein30 and PRP,15 interactions with tannin were dependent on the colloidal state and the concentration of tannins. Small tannins below their CMC generate a conformational change of the protein surrounding or “wrapping” the tannins. However, larger tannins with high concentration led to aggregation and complex precipitation by a bridging effect. Zein, which is a proline-rich protein, adopts an extended and flexible conformation in aqueous ethanol solution,21,27 and its binding with TA might comply with the similar model. SAXS experiments suggested that α-zein in aqueous ethanol solution existed as an extended oblong structure with an axial ratio of 6:1.27 In this study, SAXS was used to monitor the possible changes of the contour structure of zein in aqueous ethanol with TA binding. On the basis of Guinier plots for the oblong structure of zein, the structural parameters, including overall gyration radius (Rg), the longest edge in an elongated particle (a), and the extrapolation of intensity to an angle of zero I(0) of zein, were calculated and are shown in Table 2. It was noteworthy that there was a little difference in Rg and I(0) values of zein/TA complexes when compared with those of F
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry pure zein. Rg values of zein in all ethanol solutions at pH 5.0 with different zein/TA ratios are between 2.17 and 3.25 nm, which were equal to or even less than that of pure zein (2.90 nm), indicating that the TA binding at pH 5.0 did not disturb the contour structure of zein in aqueous ethanol, although small changes were recorded in its secondary structure (Figure 3). A similar result was observed for β-casein.30 Small tannins could increase the number of proteins per micelle without changing their size, leading to compaction of micelles. In the case of pH 7.0, the Rg and a values of zein/TA complexes increased significantly. They were nearly twice greater than those of the ZTP prepared at pH 3.0 and 5.0, indicating the formation of complex aggregates. This might explain why the ZTP formed at pH 7.0 was larger than that formed at pH 5.0 (Figure 1; Table 1). FTIR spectra of zein colloidal particles, which were used to characterize the noncovalent interaction of zein and TA, are shown in Figure 4. The absorption peak at 3308 cm−1 is assigned to the NH stretching vibration of the amide I from zein, whereas the absorptions at 1657 and 1540 cm−1 are attributed to CO of amide I and CNH of amide II of zein, respectively (Figure 4D).31 Wide bands in the range of 3600−3100 cm−1 are attributed to OH stretching of the phenolic or hydroxyl group of TA (Figure 4C).32,33 Both CO and NH bonds were involved in the hydrogen bonding between different elements contributing to the secondary structure of zein. For ZTP (Figure 4A), the absorption at 1657 cm−1 related to the CO bond (amide I) of zein overlapped the TA band at 1614 cm−1 (aromatic CO bonds). Therefore, only a single band at 1656 cm−1 was observed, and the new component at 3356 cm−1 appeared. Collectively, the results revealed the hydrogen-bonding interaction in nature for the zein/TA complex colloidal particles. For the zein/TA physical mixture (mixture of the zein and TA powder with a zein/TA ratio of 1:0.2), the absorbance peaks at 1386, 1311, 1237, and 1170 cm−1 were almost similar to those of the zein particle and the peaks at 1448 and 869 cm−1 were similar to those of TA, but all peaks above were significantly different from those of the ZTP. Moreover, the range below 1500 cm−1 is significant for deformation, bending, and ring vibrations, which can be very specific for a substance or for different types of substitution; thus, this range is frequently referred to as the “fingerprint region” of a spectrum.34 Pickering Emulsions and Gels Prepared by ZTP. As shown in Figure 5, Pickering emulsions prepared by using bare zein colloidal particle and ZTP (prepared at pH 5.0) as stabilizers were compared in terms of storage stability. The bare zein colloidal particle was actually not a good emulsifier for Pickering emulsions, which showed severe aggregation and oil release on the top phase of fresh emulsions. In contrast, the emulsions stabilized by ZTP showed no sign of oil separation even after 30 days of storage. Actually, these emulsions immediately transformed from the liquid state to the solid state and exhibited arrested dynamics. This further contributed to the long-term stabilization action (Figure 5). Interestingly, it could be seen that the emulsion stabilized by ZTP with relatively low particle volume fractions (0.25−1.0 wt %/v) showed creaming and serum release, but significant increase of the serum in the bottom phase was not observed in the prolonged 30 days of storage, which had been observed in typical Pickering emulsion stabilized by zein/chitosan particles (ZCP).23 Nevertheless, emulsions prepared with high particle fractions (1.0−1.5 wt %/v) exhibited homogeneous gels
Figure 5. Visual observation of fresh emulsion gels prepared by ZTP with varied zein/TA ratios by different particle concentrations (0.25− 1.5%, w/v) for fresh (0 days) and 30 days of storage. ZTP was prepared at pH 5.0.
without creaming and phase separation after 30 days of storage. These homogeneous emulsion gels are remarkable given the formation of protein continuous networks. CLSM was used to characterize the microstructure of emulsions prepared with ZTP by different particle fractions, and the obtained images are shown in Figure 6. Droplets were rendered visible by the fluorescent protein particles (labeled in red) that covered the oil droplets (labeled in green). For the emulsions stabilized solely by zein particles, few protein molecules were absorbed around the oil droplets. In contrast, the interfacial protein network around the oil droplets was
Figure 6. CLSM images of the emulsions stabilized by pure zein colloidal particles (1.5% w/v) and ZTP with different particles contents (0.5, 1.0, and 1.25% w/v). Protein was stained red and the oil phase, green. ZTP was prepared at pH 5.0 with a zein/TA ratio of 1:0.2. G
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 7. Droplet size distribution profiles (A) and rheological property (B) of Pickering emulsion gels prepared by ZTP with different particle fractions: storage moduli, G′ (solid symbols) and loss moduli, G″ (open symbols). ZTP was prepared at pH 5.0 with a zein/TA ratio of 1:0.2.
There are two kinds of protein-based emulsion gels. One is emulsion-filled protein gel, in which oil droplets act as filler particles incorporated in the continuous protein network. The other is protein-stabilized emulsion gels or particulate gels in which protein molecules on the surface connect together and form a network. Both of these two types have totally different rheological behaviors.37 We therefore investigated the rheological properties of the Pickering emulsion gels, which are presented in Figure 7B using frequency sweep tests. In all cases, the storage modulus (G′) slightly but progressively increased with the increase of particle fractions, indicating their noncovalent “physical” cross-links in nature.37 The increased intermolecular noncovalent cross-links among ZTP particles strengthened the gel network. The G′ of emulsion gels with low particle fraction (0.25−0.75% w/v) showed less frequency dependence, which was the hallmark of particulate gels,38 whereas the G′ of the emulsion gels with high particle fraction (1.0−1.5% w/v) showed more frequency dependence. Hence, it can be concluded that the shift of gelling model from particulate gels to protein-based emulsion gels occurs with the increase of the colloidal particle loading, which involves crosslinking of the continuous phase. The enhanced reactivity of ZTP was due to the complexation of zein and TA, and it would be a key to explain the formation of Pickering emulsion gels. At high particle fractions, excess ZTP might trigger cross-links via hydrogen-bonded interaction and form a continuous network among and around the well-dispersed oil droplets (as active filler particles), leading to the formation of strong emulsion gels.
found in the emulsions stabilized by ZTP, confirming the formation of an oil-in-water (O/W) Pickering emulsion. Furthermore, higher magnification confocal images clearly showed that the multilayer interfacial architecture was formed on the oil−water surface. These microstructures were typically hierarchical, which probably arose from the predominantly LbL assembly behavior of zein particles and TA.24,35 We previously found that the ZCP could facilitate the formation of particlebased interfacial network architecture, and the ultrastable Pickering emulsions finally formed. However, to the best of our knowledge, this was the first observation of this ordered morphological signature on the oil−water interface, which implied a possibility to fabricate hierarchical interface microstructure via modulation of the noncovalent interaction between hydrophobic protein and natural polyphenol. The droplet size distributions and the mean droplet diameter (d43) for emulsions stabilized by ZTP were measured to assess the emulsifying ability and physical stability, which are shown in Figure 7A. The oil droplets in fresh emulsions prepared with ZTP had approximately uniform size with a very narrow peak in their distribution, which remained unchanged even after 30 days of storage, confirming their long-term storage stability against coalescence (data not shown). In addition, the mean droplet diameter (d43) showed the particle fraction-dependent behavior. It decreased with increase of the ZTP fraction. This behavior was further supported by the CLSM observation (Figure 6). We believe these results can be attributed to the competition for surface space of oil droplets with more surfaceactive TA micelles. As a hydrolyzed tannin, TA is actually an amphiphilic polyphenol, which can self-assemble to form micelles when their concentration is above the CMC (about 10 mM for hydrolyzed tannins).15,36 Accordingly, TA might form micelles with high ZTP fractions, resulting in interfacial particle displacement, and, consequently, decrease the size of oil droplets. Our previous findings showed that the amphiphilic stevioside (STE) micelles, competitively adsorbed on the oil− water interface with soy protein isolate (SPI), formed a mixed interfacial layer, resulting in a decrease in particle size and evident enhancement in the physical stability of SPI-based emulsions.6 Moreover, the emulsion stabilized with zein alone showed oil release on the top phase, which was not suitable to evaluate the diameter, so we did not show its diameter.
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DISCUSSION The novel colloidal structure, which is based on food-grade materials and formed via modulation of noncovalent interactions, has emerged with potential as a functional ingredient for the formulation and delivery of bioactive molecules or enhancement in the functionality of food biopolymers.1,2 In this study the novel zein/TA complex colloidal particle (ZTP) based on the antisolvent approach and the hydrogen-bonding interaction between zein and TA was successfully generated. These complex colloidal particles with tuned wettability and enhanced interfacial reactivity can be utilized for the preparation of stable Pickering emulsions gels by one-step homogenization. To better understand the underlying mechH
DOI: 10.1021/acs.jafc.5b03113 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 8. Schematic mechanism for the formation of the Pickering emulsions gels prepared with zein/TA complex colloidal particles.
Funding
anism of ZTP-stabilized Pickering emulsion gels, we propose a schematic illustration to explain the formation pathway of Pickering emulsion gels, shown in Figure 8. The protonation and ionization of TA are both critical to understanding the colloidal behavior with protein,11,24 which can be modulated efficiently via changing the pH and ionic strength of the solution. Furthermore, the colloidal state of TA affects the nature of their interaction with proteins,15,39 especially for proline-rich proteins such as zein. Zein adopted an extended configuration in aqueous ethanol solution,25,27 which exhibited a different tannin binding model compared with compacted protein in aqueous solution. TA in the neutral and partially protonated form at pH 5.0 with intermediate TA concentration facilitated strong hydrogen-bonding interactions between the hydroxyl groups on TA and the carbonyl moieties on the pyrrolidone rings of the proline-rich domain in zein. This specific interaction generated a conformational change of zein without altering their supramolecular structure (Figure 3; Table 2). The TA binding consequently affected the selfassembly behavior of zein during the antisolvent process, leading to the formation of zein colloidal particles that were coated or “wrapped” by TA (Figure 8). This complexation further suppressed the ionization of TA,11 and it caused an increase in the hydroxyl group on the surface of zein particles at acidic pH (∼3.48), thereby lessening their hydrophobic characteristics without decreasing surface charge (+51 mV) significantly (Table 1). The hydrogen-bonded zein/TA complex colloidal particle, evidenced by FTIR, with nearneutral wettability (Figure 2) and enhanced reactivity (due to the neutral TA coating), was capable of facilitating the multilayer interfacial architecture on the oil−water surface of the oil droplets (Figure 6), resulting in the formation of Pickering emulsion. In addition, the hydrogen-bonding interaction further triggered cross-links between the colloidal particles to form a continuous network among and around the oil droplets, leading to the formation of stable Pickering emulsion gels. In conclusion, stable colloidal particles with near-neutral wettability and high interfacial reactivity, which is fabricated by modulating the noncovalent interactions between small molecules (TA) and macromolecules (zein), can be used as building blocks with reaction activity to form novel edible Pickering emulsion gels with orderly interfacial architecture. This study implies a possibility to fabricate hierarchical interface microstructure via modulation of the noncovalent interaction between hydrophobic protein and natural polyphenol.
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This research was supported by grants from the Special Fund for Agro-scientific Research in the Public Interest (Grant 201303071), the Chinese National Natural Science Foundation (No. 31371744, 21406077), and the Project of National Key Technology Research and Development Program for the National High Technology Research and Development Program of China (863 Program: 2013AA102208). Notes
The authors declare no competing financial interest.
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