Kinetics and Antioxidant Capacity of Proanthocyanidins Encapsulated

Article pubs.acs.org/JAFC

Kinetics and Antioxidant Capacity of Proanthocyanidins Encapsulated in Zein Electrospun Fibers by Cyclic Voltammetry Hualin Wang,*,†,§ Lilan Hao,† Baicheng Niu,† Suwei Jiang,† Junfeng Cheng,† and Shaotong Jiang‡,§ †

School of Chemistry and Chemical Engineering and ‡School of Biotechnology and Food Engineering, Hefei University of Technology, 230009 Hefei, Anhui, PR China § Anhui Institute of Agro-Products Intensive Processing Technology, 230009 Hefei, Anhui, PR China S Supporting Information *

ABSTRACT: The proanthocyanidins encapsulated in zein (zein-PA) fibers was via electrospinning technique. The kinetics and antioxidant capacity of PA from zein fibers was investigated by cyclic voltammetry. Circular dichroism was used to investigate the secondary structure change of zein and its influence on the shape of fibers. The addition of PA caused a significant increase in viscosity and made fibers wider. These hydrogen bonds between zein and PA molecules would favor the α-helix change and decrease the β-folds of zein in electrospinning solutions, leading to a round-shaped tendency of fibers and enhancing the thermal properties slightly. Zein-PA fibers showed high encapsulation efficiency close to 100%, and the encapsulated PA retained its antioxidant capacity in fibers. Zein-PA fibers showed a good controlled release toward PA, and the predominant release of PA from fibers was Fickian diffusion, which could be well described by first-order model and Hixson−Crowell model. KEYWORDS: kinetics, antioxidant capacity, zein-PA fibers, electrospinning, cyclic voltammetry

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INTRODUCTION Proanthocyanidins (PA), a group of biologically active polyphenolic flavonoids, widely present in cereals, legume seeds, fruits, and juices.1 Because of its polyphenolic structure, PA has displayed a broad spectrum of biological, pharmacological, and chemoprotective properties against oxidative stress and free radicals.2−4 However, PA can be easily oxidized upon exposure to oxygen during processing or storage, so only a small proportion of PA is bioavailable after oral administration. 5−7 Encapsulation is a common approach for protecting substances of interest from harsh environments.8 Therefore, an effective encapsulating and controlled-release technology is urgent toward PA. Electrospinning is a simple and low-cost technique for fabricating the fibers with ultrasmall diameters.9 The fibers have been widely used in controlled drug release, owing to their high porosity, large surface area to volume ratio, and flexibility in encapsulation.10−13 Recently, electrospun fibers have gradually attracted more interest in active food packaging, which were versatile to achieve by incorporating antimicrobial agents into the nanofibrious matrixes, such as gallic acid in zein,14,15 retinyl acetate in poly(vinyl alcohol) containing β-cyclodextrin,16 solid triclosan/cyclodextrin inclusion complexes in poly(lacticacid),17 eugenol/cyclodextrin inclusion complexes in poly(vinyl alcohol),18 cinnamon essential oil in poly(vinyl alcohol)/βcyclodextrin,19 and glucose oxidase in PVA/CS/tea extract.20 Besides, electrospinning has been also proven to be a promising method for stabilization of antioxidants including β-carotene,21 fish oil,22 tea polyphenols,23 and curcumin.24 In the present work, PA was encapsulated in zein fibers via electrospinning technique. Zein, a prolamine in corn maize, is a nontoxic, biocompatible, and biodegradable polymer. Owing to its good oxygen barrier and high thermal resistance,25,26 zein films and zein micro-/ © XXXX American Chemical Society

nanoparticles can be used for encapsulation of aromas and flavors,27 essential oils,28 controlled release of active additives,29 and as an active food packaging material.30,31 Furthermore, the zein has good fiber-forming ability. It is convenient to fabricate electrospun fibers from the solution containing zein.14,21,22,32,33 Herein, our attention was focused on the change of the secondary structure of zein by the addition of PA in electrospinning solutions and its influence on the shape of electrospun fibers by circular dichroism (CD). Electrochemical measurements may provide us some important physicochemical parameters (redox potential, number of electrons, electron-transfer rate constant, etc.) to reveal antioxidant properties.34 Cyclic voltammetry (CV) has become a widely used tool to study the antioxidant capacities from anodic peak current intensity and peak oxidation potential.35−37 In addition, the contents of antioxidants could be easily worked out on the basis of the values of peak potential and current. Therefore, CV is an effective and convenient method not only in evaluating the antioxidant capacities but also in determining the contents of antioxidants. CV has been successfully used to determine the content of polyphenols in wines by Mannino et al.,38 Kilmartin et al.,39 and Rebelo et al.40 Encouraged by these achievements, CV was first developed to determine the contents of PA released from zein fibers. The present work is to investigate the kinetics and antioxidant capacity of PA encapsulated in zein (zein-PA) fibers by CV. To this aim, the zein-PA fibers were fabricated via electrospinning techniques; the effects of PA contents on the morphology fibers, thermal property, and encapsulation Received: February 1, 2016 Revised: March 25, 2016 Accepted: April 1, 2016

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DOI: 10.1021/acs.jafc.6b00540 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Cyclic voltammograms of gallic acid standards in the electrolyte solution (12% v/v ethanol, 0.033 M L-tartaric acid, 0.1 M NaCl, pH 3.6, 25 °C).

efficiency of zein-PA fibers as well as the interaction between zein and PA were investigated.

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solutions. Background resulting from the ethanol aqueous solutions was subtracted from the CD signal by mechanical adjustment in 5 nm steps. Secondary structures were determined by SELCON3, CONTIN, and CDSSTR programs, using a protein database that indicates α-helix, β-sheet, β-turns, and unordered structures proportions.42 Fourier Transformed Infrared Spectroscopy. FTIR spectra were used to investigate the interaction between zein and PA in the fibers and measured on a Nicolet 6700 spectrometer (Thermo Nicolet, USA) with KBr pellets. X-ray Diffraction. X-ray diffraction (XRD) analysis was performed on a D/MAX2500 V diffractometer (Rigaku, Japan) using Cu Kα radiation (λ = 0.15418 nm) to determine the crystal phase of samples. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) was conducted on a DSC Q2000 (TA Instruments, New Castle, USA). Samples by weight of about 7.0 mg were weighed in aluminum pans and hermetically sealed. The thermal analyses were performed within a temperature range of 40−250 °C at a scanning speed of 10 °C/min under nitrogen gas flow at a flow of 50 mL/min. Encapsulation Efficiency. The sample with dry weight of 0.01 g was washed with 20 mL of Milli-Q water to remove the surface PA and then dissolved in 50 mL of 80% ethanol aqueous solution. The amount of PA remaining the solution was measured using the Folin-Ciocalteu method with some modifications.40,43 The encapsulation efficiency (EE) of PA in fibers was calculated as follows:

MATERIALS AND METHODS

Materials. Zein from maize (≥97%, CAS number 9010-66-6) was obtained from Sigma-Aldrich (St Louis, MO, USA). Proanthocyanidins (PA, from grape seeds, specification: 95% OPC, appearance: brown-red powder) was provided by Shanghai Jiaoyuan Biochemical Co., Ltd. (Shanghai, China). Gallic acid, Folin-Ciocalteu’s reagent, sodium hydroxide, L-tartaric acid, sodium chloride, and ethanol were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and absolute ethanol was used as solvent. Sodium carbonate was procured from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). All the chemicals and reagents were of analytical grade. All solutions were prepared with Milli-Q water from a purification system (Millipore, Bedford, MA, USA). Preparation of Fibers. Zein solutions (28%, w/w) were prepared by dissolving zein powder in 80% ethanol aqueous solutions (ethanol/ deionized water = 4/1, w/w) assisted with magnetic stirring at 25 °C for 1 h. The required PA was added into the above solution to obtain an electrospinning solution (PA contents: 2.5, 3.0, 3.5, 4.0, and 4.5%, all based on the weight of zein); correspondingly, the PA encapsulated in zein fibers were named zein-PA 2.5, zein-PA 3.0, zein-PA 3.5, zeinPA 4.0, and zein-PA 4.5 fibers, respectively. The PA encapsulated in zein (zein-PA) fibers were fabricated on an electrospinning device consisting of a syringe, a needle (0.41 mm internal diameter), a copper sheet, a ground electrode, and a highvoltage power supply (DW-P403-1ACCC, Tianjin Dongwen, China). On the basis of experimental data, electrospinning processing was carried out at room temperature with relative humidity (RH) at 50%. The supplied voltage was kept at 15 kV, and tip-to-collector distance (TCD) was kept at 12 cm. The as-spun fibers were dried overnight in a vacuum oven at 25 °C. Scanning Electron Microscopy. The morphologies of samples were observed with scanning electron microscopy (SEM, SU8020, Hitachi, Japan) by applying an accelerating voltage of 20 kV on specimen sputter-coated with gold. SEM images were analyzed by Image Tool software, and a total of 50 counts were used to calculate the average diameter of fibers. Conductivity and Viscosity. Conductivity and viscosity of electrospinning solutions were measured at 25 °C to evaluate their influences on the morphology of fibers. Conductivity was tested by an electric conductivity meter DDL-801 (Shanghai, China). Viscosity was measured by a NDJ-5S rotational viscometer (Shanghai, China). Circular Dichroism. To investigate the influence of PA in electrospinning solutions and the electrospinning process on the secondary structure of zein, circular dichroism (CD) was performed in a 1.0 mm quartz cell at 25 °C using a JASCO J1500 spectropolarimeter (JASCO, Tokyo, Japan) with wavelength range of 190−260 nm.41 The 0.2 mg mL−1 samples were prepared in 80% ethanol aqueous

EE (%) =

Actual PA concentration × 100 Theoretical PA concentration

(1)

A calibration curve of gallic acid (ranging from 0.01 to 0.05 mg/ mL) was prepared, and the results determined from regression equation of the calibration curve (y = 4.7640x + 0.0619, R2 = 0.99) were expressed as mg of gallic acid equivalents per mg of the PA in sample. In this method, 1 mL of testing solution was mixed with 5 mL of 10-fold-diluted Folin-Ciocalteu phenol reagent. After 5 min, 4 mL of sodium carbonate solution (7.5%) was added to the mixture, shaken thoroughly, and diluted to 25 mL by adding Milli-Q water. The mixture was vortexed for 15 s and allowed to stand for 30 min at room temperature for color development. Absorbance was measured at 765 nm using a UV-754 PC spectrophotometer (Shanghai Jinghua, China). Cyclic Voltammetry. The zein-PA fibers antioxidant capacity was evaluated by CV, and antioxidant activity of PA was evaluated by the anodic peak at 0.45−0.49 V on cyclic voltammograms. CV data was recorded using a potentiostat (CHI 660D, Chenhua, Shanghai). The voltammetric experiments were carried out in an electrochemical cell of three electrodes. The working electrode was a 3 mm glassy carbon disk electrode (MF-2012) was cleaned by polishing with 0.05 μm alumina powder (CF-1050) for 1 min between runs, washed with Milli-Q water, and then sonicated for 2−3 min. A BAS Ag/AgCl reference electrode (+207 mV vs SHE) was used in conjunction with a platinum wire counter electrode. The cyclic voltammograms were B


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Journal of Agricultural and Food Chemistry recorded on freshly polished glassy carbon electrode by scanning the potential from −1.0 to 1.0 V at a scanning rate of 100 mV/s. The gallic acid standard calibration curve (Figure 1B) was obtained in 50 mL of electrolyte solutions (12% v/v ethanol, 0.033 M L-tartaric acid, 0.1 M NaCl, adjusted to pH 3.6 by NaOH) by plotting the total charge of the anodic oxidation peaks versus the concentration of gallic acid (0.7−4.6 mM) according to Figure 1A. On the basis of Figure 1B, a regression equation (Figure 1B, in set: y = 0.03x + 0.004, R2 = 0.996) was obtained, and the results were expressed as mM gallic acid equivalents per mM of the PA in sample. A weight of 0.5 g of fibers was added into 44 mL of Milli-Q water and placed on a shaker platform (120 rpm) at preset temperatures for 3 h to ensure the release equilibrium and then the fibers removed. A volume of 50 mL of testing electrolyte solution (12% v/v ethanol, 0.033 M L-tartaric acid, 0.1 M NaCl, pH 3.6) was obtained by adding needed ethanol, L-tartaric acid, NaCl, and NaOH. The testing solutions were measured by CV at 25 °C. Release Profile and Mechanism. The release profiles of PA from zein-PA fibers was carried out in 44 mL of Milli-Q water at 25 °C and placed on a shaker platform (120 rpm) according to the method reported by Neo et al. with some modifications.14 At different intervals, the fibers was removed from the solution, and then the needed ethanol, L-tartaric acid, NaCl, and NaOH were added into the system to obtain 50 mL of testing electrolyte solution (12% v/v ethanol, 0.033 M L-tartaric acid, 0.1 M NaCl, pH 3.6). The testing solutions were measured by CV at 25 °C, and the amount of the released PA was calculated by the regression equation (Figure 1B, in set). The accumulative release percentage of PA from the zein-PA fibers was calculated using the following equation: M Accumulative release (%) = t × 100% M0

Figure 2. SEM images of fibers (a) zein, (b) zein-PA 2.5, (c) zein-PA 3.0, (d) zein-PA 3.5, (e) zein-PA 4.0, and (f) zein-PA 4.5.

change into α-helix (“Influence of PA on the Secondary Structure of Zein” section). In addition, the zein-PA fibers showed an increase in average diameter with the increase of PA contents (Figure 2). The viscosity and electrical conductivity of electrospinning solutions and the average diameters (Davg) of the fibers were summarized in Table 1. The data showed that Table 1. Viscosity and Electrical Conductivity of Zein-PA Electrospinning Solutions and Size of Zein-PA Fibersa PA content (%) 0 2.5 3.0 3.5 4.0 4.5

(2)

where Mt (mg) is the released PA at an arbitrary time t and M0 (mg) is the total encapsulated in the fibers. The Ritger and Peppas equation44 was used to describe the release profile of PA in zein-PA fibers as follows:

M t /M∞ = kt n

viscosity (mPa s) 305 340 392 414 458 472

± ± ± ± ± ±

3.3 4.2 5.2 4.5 4.2 6.6

conductivity (mS cm−1) a a a a b b

119.2 110.3 106.9 101.5 99.6 96.7

± ± ± ± ± ±

2.4 3.5 3.4 2.0 2.5 2.4

a a a a a a

Davg (nm) 636 662 696 719 742 776

± ± ± ± ± ±

6.5 8.4 7.5 6.8 8.2 8.5

a a a a b b

a

Data with the same letter in the same column indicate that they are not statistically different (p > 0.05). The data (mean ± SD) are results from three independent experiments.

(3)

where Mt (mg) is the released PA at an arbitrary time t, M∞ (mg) is the released PA at equilibrium, k is the release rate constant, and n indicates the release exponent suggesting the nature of the release mechanism.14 For further analysis of the release profiles, four more kinetic release models were adopted to fit the experimental data including zero-order model (Mt/M∞ = kt), first-order model [1−Mt/M∞ = e−kt], Higuchi model (Mt/M∞ = kt1/2), and Hixson−Crowell model [(1−Mt/M∞)1/3 = −kt].14,45,46 Statistical Analysis. All experiments were performed in triplicate and repeated three times. Statistical analysis was analyzed using the unpaired Student’s t test (n = 3), and the values were expressed as the means ± standard deviation (SD). The threshold for statistical significance was set at p < 0.05.

the addition of PA caused no obvious change in electrical conductivity of electrospinning solutions but had a significant increase in viscosity, which was associated with the formation of intermolecular hydrogen bonds between PA and zein molecules. In contrast, the addition of PA would also increase the intrinsic viscosity of the polymeric solution owing to the structure of polymeric nature and a relatively high molecular weight of PA. Herein, the size of fibers was prominently dependent on the viscosity of electrospinning solutions. As a result, the higher viscosity resulted in the difficult splitting of droplets, and the fibers became wider.50−53 Especially, as PA content reached 4.0% and above, the fibers became uneven and unsmooth on surface (Figure 2e,f). As a result, a PA content of 3.5% was determined to be the optimum value to fabricate continuous and uniform round fibers (Davg = 719 ± 6.8 nm). Influence of PA on the Secondary Structure of Zein. Circular dichroism (CD) is a common method for studying conformational changes in protein secondary structure. Herein, CD was used to explore the influence of PA in electrospinning solutions and the electrospinning process on the secondary structure of zein. Figure 3 shows the CD spectra of PA-zein electrospinning solutions with different PA contents. Each curve showed two negative broad peaks at about 209−213 and 222−224 nm and a positive one around 195 nm, indicating a typical of protein with α + β structure, in which the α-helix band intensity was stronger than the β-sheet structure.42 The

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RESULTS AND DICUSSION Morphology. Figure 2 illustrates SEM images of zein-PA fibers with different PA contents. As it could be seen from Figure 2, the morphology and size of fibers were strongly dependent on PA contents. The zein fibers showed a ribbonlike morphology (Figure 2a) and had an average diameter of 636 ± 6.5 nm. The formation of ribbonlike fibers should be associated closely with unexpanded β-folds of zein.47−49 It was noteworthy that the fibers tended to be round-shaped by the addition of PA. Because all the zein and zein-PA electrospun fibers were fabricated using 80% ethanol, this phenomenon was likely to be caused by the hydrogen bonds between PA and zein molecule, which would help expand and unfold the β-folds of zein to C


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decreased again, whereas the β-sheet, β-turns, and unordered showed an inverse tendency. A probable reason was that the hydrogen bonds between PA and zein molecule would favor the change of α-helix from the β-sheet, β-turn, and unordered structure. Nevertheless, when PA content reached 4.0% and above, the higher viscosity caused by intermolecular hydrogen bonds PA and zein molecules would inhibit the α-helix change. It could be concluded that the addition of PA in low contents would decrease the β-folds of zein in electrospinning solutions and helped the fibers tend to be round-shaped (Figure 2a−d). In addition, the electrospinning process also caused a slight decrease in β-sheet for each given fibers as compared to the corresponding electrospinning solutions (Figure S8 and Table S1), but the decrease did not change the declining tendency of β-folds of zein with the addition of PA in low contents. Structure Investigation. Figure 4 illustrates the FTIR spectra of zein, PA, and representative fibers (zein-PA 3.5). In the spectrum of zein, the characteristic peaks for zein were present at 3301 cm−1 (N−H stretching); 3072, 2962, 2930, and 2871 cm −1 (C−H stretching of aliphatic groups); and 1458 cm−1(−CH2 bending). Additionally, the characteristic peaks of peptide backbone were observed at 1648 cm−1 (amide I, CO stretching), 1533 cm−1 (amide II, N−H bending), and 1240 cm−1 (amide III, C−N stretching).54 In the spectrum of PA, a peak at 3405 cm−1 corresponded to stretching modes of the different −OH groups; peaks at 1610 and 1520 cm−1 were due to the −CC−O deformation of heterocyclic ring in the dominant planar trans form and skeletal stretching mode of the o-disubstituted aromatic ring, respectively;55,56 a peak at 1442 cm was attributed to the −CH deformation and aromatic ring vibration;57 peaks at 1358 and 1286 cm−1 were associated with the −OH bending and ester C−O stretching, respectively; peaks at 1206, 1140, 1109, and 1070 cm−1 were due to C−C stretching and various phenol and ether C−O stretching;58 and the absorbance at 827 and 777 cm−1 were ascribed to C−H aromatic stretching and out-of-plane bending.55,56,59 As compared to zein and PA, the characteristic absorbance of zein and PA were presented in the spectra of zein-PA. Meanwhile, the −OH stretching at 3405 cm−1 of PA shifted lower to 3298 cm−1, and CO stretching at1648 cm−1 shifted lower to 1622 cm−1. It could be speculated that there existed intermolecular hydrogen bonds (O···H−O) between zein and PA in zein-PA matrix (Figure 4B). Figure 5A shows the XRD patterns of PA powder, zein, and zein-PA fibers. The PA diffraction pattern exhibited a diffuse background pattern with two diffraction halos, indicating that

Figure 3. CD spectra of PA-zein solutions with different PA contents at 25 °C.

influence of PA on the protein structure was noted from the obtained spectra. The addition of PA caused a shift in the negative maximum from 209 to 213 nm. Meanwhile, two negative peaks were decreased, and the positive peak was increased. This provided evidence of successive changes in the secondary structure of zein. On the basis of Figure 3, the zein secondary structure contents (α-helix, β-sheet, β-turns, and unordered) were calculated using SELCON3, CONTIN, and CDSSTR programs. The averaged results of the three methods were summarized in Table 2. With increasing PA contents, αTable 2. Secondary Structure Proportions of Zein in Electrospinning Solutions Obtained from Deconvoluted CD Spectraa PA content (%) 0 2.5 3.0 3.5 4.0 4.5

α-helix (%) 55.6 74.2 80.3 82.0 66.7 60.1

± ± ± ± ± ±

2.8 3.7 4.0 4.1 3.3 3.0

ac b b b c c

β-sheet (%) 6.7 4.7 3.8 3.5 5.5 5.9

± ± ± ± ± ±

0.3 0.2 0.2 0.1 0.2 0.3

a b b b c a

β-turns (%) 13.8 9.8 7.9 7.6 11.3 12.7

± ± ± ± ± ±

0.7 0.5 0.4 0.3 0.6 0.6

a b b b c a

unordered 22.9 12.5 8.6 8.1 16.5 21.8

± ± ± ± ± ±

1.1 0.6 0.4 0.4 0.8 1.1

a b b b c a

a

Secondary structures were determined by SELCON3, CONTIN, and CDSSTR programs. Data with the same letter in the same column indicate that they are not statistically different (p > 0.05). The data (mean ± SD) are results from three independent experiments.

helix structure of zein increased from 55.6 ± 2.8% (native zein) to its maximum value at 80.3 ± 4.0% (PA 3.5%) and then

Figure 4. (A) FTIR spectra of PA, zein and zein-PA 3.5 fibers. (B) Proposed structure of zein-PA. D


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Figure 5. (A) XRD patterns of PA, zein, zein-PA fibers. (B) DSC curves of zein and zein-PA fibers.

97.89 ± 1.4%; zein-PA 3.5: 99.01 ± 1.6%; and zein-PA 4.5: 98.02 ± 2.1%). Similarly, Neo et al.25 reported gallic acid loaded zein electrospun fiber mats with an EE value of almost 100%; Moomand et al.63 obtained fish-oil-loaded isopropanolbased zein electrospun fibers with an EE value beyond 96%. These results indicated that electrospinning was an efficient tool to obtain submicrometer-structured materials containing the targeted concentration of functional component by onestep. Antioxidant Capacity of PA in Fibers. In the present work, the antioxidant capacity and release of PA in zein-PA fibers were investigated by CV in an electrolyte solution. Figure 6 showed the cyclic voltammograms of zein-PA fibers in

the PA was amorphous. Zein fibers showed two broad peaks at 2θ = 8.87 and 19.40°, corresponding to two d-spacings at 9.9 and 4.6 Å, which were attributed to interhelix packing and zein α-helix backbone, respectively.60,61 The XRD patterns showed that the change tendency of the peak areas was in accord with that of CD data. It was noteworthy that the two diffraction peaks at 2θ = 8.87 and 19.40° of zein were broadened in the pattern of zein-PA fibers as compared to zein fibers, and this change was enhanced with the increase of PA. It was deduced that the intermolecular hydrogen bonds between zein and PA disturbed the spacing of the original interhelix packing of zein chains and original α-helix structure of zein. Similar phenomenon was observed in zein fibers containing cyclodextrins by Kayaci et al.61 Thermal Property. DSC curves of zein and zein-PA fibers are showed in Figure 5B. Each curve displayed a broad endothermic peak, which was due to the evaporation of bound water or volatile component from the molecules and often termed as dehydration temperature (TD).25,62 The glass transition temperature (Tg) was associated with the chain mobility of zein. According to DSC curves, the values of TD and Tg were summarized in Table 3. Because PA structured more Table 3. TD, Tg, and EE of PA-Zein Fibersa PA content (%) 0 2.5 3.5 4.5

TD (°C) 78.98 80.07 83.37 86.09

± ± ± ±

1.2 1.5 1.8 2.0

Tg (°C) a a a b

156.72 160.90 162.91 165.08

± ± ± ±

2.0 1.8 2.4 2.5

EE (%) a a a b

97.89 ± 1.4 a 99.01 ± 1.6 a 98.02 ± 2.1 a

a

Figure 6. Cyclic voltammograms of zein-PA fibers at different release temperatures.

−OH groups in molecular chain, the incorporation of PA would be unfavorable to the evaporation of bound water or volatile component from the molecules and caused an increase in TD; furthermore, the intermolecular hydrogen bonds between zein and PA decreased the chain mobility of zein. As a result, Tg of zein-PA fibers increased slightly with increasing PA contents. Encapsulation Efficiency of Fibers. EE is an important parameter in determining whether the encapsulation method is feasible for a given delivery application. The EE of PA in zeinPA fibers was calculated using eq 1 and listed in Table 3. As could be seen from Table 3, all the tested zein-PA fibers showed extremely high EE values close to 100% (zein-PA 2.5:

electrolyte solution at different temperatures. As could be seen from Figure 6, all zein-PA fibers revealed an anodic peak at 0.45−0.49 V, which might be associated with the reversible oxidation of −OH group at positions 3 and 4 on the β-ring, in a two-electron reaction to an o-quinone.40 The anodic peak confirmed that the encapsulated PA retained its antioxidant capacity in fibers. Besides, the intensity of oxidation peak in CV results was associated with the release temperature. As expected, high temperature favors the release of PA from nanofibrous matrix; correspondingly, intensity of oxidation peak was enhanced. However, as temperature reached 50 °C, the intensity of oxidation peak was almost disappearing owing to the oxidation of PA at high temperature before testing. Therefore, the release profile of PA from zein-PA fibers was carried out in Milli-Q water at 25 °C.

Data with the same letter in the same column indicate that they are not statistically different (p > 0.05). The data (mean ± SD) are results from three independent experiments.

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Figure 7. (A) Cyclic voltammograms of the representative fibers (zein-PA 3.5) at different release times. (B) Release profiles of PA from fibers: Mt/ M0 versus time t.

Fickian diffusion and n ≥ 0.89 indicates super Case II diffusion.44,45 As it could be seen from Table 4, the zein-PA fibers showed n values below 0.45. Therefore, the predominant release of PA from zein-PA fibers was Fickian diffusion, which indicated that the release of PA was partially attributed to the diffusion or permeation through the swollen zein fibrous matrix and partly through the water filled pores and channels in the matrix structure.64 Moreover, the values of n decreased with the increase of PA (zein-PA 2.5: 0.4400 ± 0.01; zein-PA 3.5: 0.3900 ± 0.01; and zein-PA 4.5: 0.9276 ± 0.04), suggesting that lower PA content favored the Fickian diffusion. Similar decrease in the values of n was found in gallic acid loaded zein fiber mats with the increase of gallic acid contents presented by Neo et al.14 Among values of R2 for each model (Table 4), the release kinetics for PA from zein-PA fibers showed the best correlation with the first-order model with R2 ranging from 0.9887 ± 0.03 to 0.9992 ± 0.02. This information indicated that the PA transport in fibers might be the rate-limiting step. In addition, the R2 from Hixson−Crowell model still showed higher values ranging from 0.9737 ± 0.02 to 0.9922 ± 0.01, suggesting that the PA release from zein-PA fibers could be also well-described by Hixson−Crowell model. These results indicated that PA release from fibers may be more consistent with a diffusion mechanism than with a matrix erosion mechanism.65 Electrospinning was an efficient tool for one-step encapsulation of a functional component. The morphology and size of fibers were strongly dependent on PA contents. The addition of PA caused a significant increase in viscosity and made the fibers become wider. The hydrogen bonds between zein and PA molecules would favor the change of α-helix from the β-sheet, β-turns, and unordered structure of zein, correspondingly; decreased the β-folds of zein in electrospinning solutions and helped fibers tend to be round-shaped. Moreover, these hydrogen bonds caused a slightly increase in Tg and TD. Zein-PA fibers showed significantly high values of EE close to 100%, and the encapsulated PA retained its antioxidant capacity in fibers. The zein-PA fibers showed a good controlled release toward PA, and the predominant release of PA from zein-PA fibers was Fickian diffusion, which could be well described by first-order model and Hixson−Crowell model. The results show that the zein fibers may have potential as an ideal encapsulation and controlled-release matrix for these materials that are easily oxidized or decomposed under atmosphere or light.

Release Profile of PA from Fibers. To assay the release behavior and acquiring basic information for release kinetics, the accumulative release percentages of PA from zein-PA 2.5, zein-PA 3.5, and zein-PA 4.5 fibers were measured by CV (Figure 7A, the representative fibers zein-PA 3.5; the other two were shown in the Figure S1). On the basis of the CV results and the regression equation (Figure 1B, inset), the accumulative release percentages were calculated by eq 2 and plotted versus time in Figure 7B. Each curve showed a slight initial burst release within the first 10 min and then reached a plateau after a gradual increase in accumulative release percentage, suggesting a good controlled release behavior of zein-PA fibers. The initial burst release was mainly associated with the dissolution of PA on or near the surface of fibers. Afterward, the entrapped PA molecules would take a longer time to be released from inner core of the fiber matrix and resulted in the gradual increase in accumulative release percentage. Additionally, the release of PA from fibers was mainly controlled by a diffusion driving force derived from PA contents; as a result, the accumulative release increased with the increase of PA contents at each given time. Release Kinetics. To assess the mechanism of PA release from fibers, release fractions (Mt/M∞) as a function of time t (Figure S2) were analyzed by Peppas equation, zero-order, firstorder, Higuchi, and Hixson−Crowell models (Figures S3−S7). Correspondingly, the correlation coefficients (R2) for each model and release exponent (n) were summarized in Table 4. Ritger and Peppas proposed the criteria for release kinetics from swellable systems, which indicated n = 0.45 as Fickian diffusion, whereas 0.45 < n < 0.89 indicates an anomalous nonTable 4. Correlation Coefficients (R2) According to the Different Models and the Release Exponent (n, Peppas) Used to Describe the Release of PA from Zein-PA Fibersa model Peppas n zero-order first-order Higuchi model Hixson−Crowell

zein-PA 2.5 0.9254 0.4400 0.8020 0.9992 0.9560 0.9913

± ± ± ± ± ±

0.03 0.01 0.03 0.02 0.01 0.02

zein-PA 3.5 0.9020 0.3900 0.7461 0.9977 0.9343 0.9922

± ± ± ± ± ±

0.02 0.01 0.03 0.01 0.02 0.01

zein-PA 4.5 0.9276 0.3300 0.6545 0.9887 0.9067 0.9737

± ± ± ± ± ±

0.04 0.02 0.04 0.03 0.02 0.02

a

Values of n and R2 are from Figure S3 and Figures S3−S7, respectively (in the Supporting Information). The data (mean ± SD) are results from three independent experiments. F


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b00540. Cyclic voltammograms for zein-PA 2.5 and zein-PA 4.5 fibers, release kinetics and modeling from five models, and secondary structure of zein in PA-zein fiber solutions. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 551 62901450. Fax: +86 551 62901450. E-mail: [email protected]. Funding

The research was supported by National Natural Science Foundation of China (31371859 and 31171788). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Wu for conducting circular dichroism analyses of samples.

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REFERENCES

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Kinetics and Antioxidant Capacity of Proanthocyanidins Encapsulated

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