Improved chemical stability and cellular antioxidant activity of resveratrol in zein nanoparticle with bovine serum albumin-caffeic acid conjugate

Food Chemistry 261 (2018) 283–291

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Improved chemical stability and cellular antioxidant activity of resveratrol in zein nanoparticle with bovine serum albumin-caffeic acid conjugate Yuting Fana, Yuexiang Liua, Luyu Gaoa, Yuzhu Zhangb, Jiang Yia, a b

T

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College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China Western Regional Research Center, ARS, USDA, Albany, CA 94710, United States

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article: Resveratrol (PubChem CID: 445154) Caffeic acid (PubChem CID: 689043) DCFH-DA (PubChem CID: 104913) ABAP (PubChem CID: 1969) L-ascorbic acid (PubChem CID: 54670067)

In this study, bovine serum albumin (BSA)-caffeic acid (CA) conjugate was prepared with free radical-induced grafting method. The CA to BSA ratio of the conjugate was 115.7 mg/g. In vitro antioxidant activity assays suggested that BSA-CA conjugates had stronger antioxidant activity than BSA. Resveratrol-loaded zein encapsulated with BSA and BSA-CA conjugate core-shell nanoparticles were prepared with antisolvent method. Particle sizes were 206.3 nm, and 217.2 nm for BSA and BSA-CA, respectively. The encapsulation efficiencies (EEs) were 85.3% and 86.5% for zein-BSA and zein-BSA-CA nanoparticles, respectively. SEM results indicated that both nanoparticles were spherical with mean diameter approximately 200 nm and smooth surfaces. Both thermal and UV light stability of resveratrol was significantly improved after nanoencapsulation. BSA-CA conjugate showed remarkably greater protection than BSA against resveratrol degradation. Cellular antioxidant activity (CAA) study confirmed that resveratrol in both zein-BSA and zein-BSA-CA nanoparticles had significant higher antioxidant activities than resveratrol alone.

Keywords: Resveratrol Zein BSA Caffeic acid Nanoparticle CAA

1. Introduction Resveratrol (trans-3,4′,5-trihydroxystilbene), a naturally occurring polyphenol mainly existed in grapes and red wine, has been shown to have a lot of health-promoting effects, such as, antioxidant activity, anti-inflammatory, antiaging and antidiabetogenic property (Baur et al., 2006), and anti-amyloidogenic activity (Marambaud, Zhao, & Davies, 2005). Recent studies also showed that resveratrol may prevent diet-induced obesity, increase mitochondrial function, physical stamina, glucose tolerance in mice (Park et al., 2012), and protect against and ameliorate the symptoms of metabolic diseases. Resveratrol has attracted a lot of attention not just from food science, but also cosmetics, and pharmaceutical industries. However, resveratrol is insoluble and unstable in aqueous phases. The development of a liquid formulation to stabilize resveratrol, protect it from degradation, enhance its water dispersibility, and achieve specific location release is a great challenge. Nanoparticle-based delivery system with mesoporous silica, zein, and zein-polyphenol conjugates have been shown to improve its oral bioavailability and anti-inflammatory effects recently (Juère et al., 2017; Liu et al., 2018; Penalva et al., 2015). Due to the cost-effective and generally recognized as safe (GRAS) properties, protein-based nanoparticles are ideal delivery systems and

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have been widely used for liable oxidized nutraceutical encapsulation. β-Lactoglobulin, α-lactalbumin, soybean protein, gliadin, and zein have been used (Arroyo-Maya et al., 2012; Fan, Zhang, Yokoyama, & Yi, 2017; Joye, Davidov-Pardo, & McClements, 2015; Livney, 2010; Pujara, Jambhrunkar, Wong, McGuckin, & Popat, 2017). Among these proteins, zein is a class of proline-rich corn (Zea mays) proteins. Almost 75% amino acid residues of zein are hydrophobic, resulting in its unique aqueous-alcohol solubility, and film-forming property (Lawton, 2002). The low solubility in aqueous phase but good solubility in alcohol makes it particularly suitable for encapsulating and delivering functional bioactives that have poor aqueous dispersibility but high ethanol dispersibility (Patel & Velikov, 2014). Recently, zein has been used for nanoencapsulation of hydrophobic nutraceuticals, including curcumin, vitamin D3, essential oil, and resveratrol. Nevertheless, the use of zein nanoparticles as a nano-delivery system may be limited due to their low stability and aggregation in aqueous systems (Hu & McClements, 2014) as a result of high surface hydrophobicity and close-to-neutral-pH isoelectric point (6.2) (Shukla & Cheryan, 2001). Zein colloidal particle aggregation can be reduced by encapsulating the particles with biopolymers. Sodium caseinate and β-lactoglobulin were used as stabilizer (Chen, Zheng, McClements, & Xiao, 2014; Davidov-Pardo, Pérez-Ciordia, Marı́n-Arroyo, & McClements, 2015).

Corresponding author. E-mail address: [email protected] (J. Yi).

https://doi.org/10.1016/j.foodchem.2018.04.055 Received 24 November 2017; Received in revised form 23 March 2018; Accepted 16 April 2018 Available online 17 April 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.

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for proteins. UV–vis analysis results confirmed that no free CA or free Lascorbic acid was detectable in the dialyzed conjugate solutions. The BSA-CA conjugate solution was lyophilized (Labconco, MO) and stored at -20 ˚C for further use.

Even though the dispersity of zein nanoparticles can be ameliorated, the protection of encapsulated hydrophobic nutraceuticals from degradation is still limited, especially at harsh conditions such as high temperature and UV light. Protein-polyphenols conjugates have been used as stabilizers for delivery of nutraceuticals due to its great emulsifying activity and antioxidant activity. Four main approaches were used for fabrication of protein-polyphenol conjugates so far, such as activated ester (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl))-mediated method, enzyme-mediated strategy, alkaline treatment, and free radical induced grafting approach (Liu, Ma, Gao, & McClements, 2017). However, activated ester-mediated method has the possibility to produce harmful products. Furthermore, organic solvents are also used and cause potential risk. Enzyme-mediated strategy is strictly restricted due to the high costs of enzyme. Alkaline treatment may cause the oxidation and degradation of polyphenols. Compared to other three approaches, free radical method is a much safe, green, and facile approach and is widely used recently to obtain protein-polyphenol conjugates. Free radical-induced conjugation approach is employing an H2O2/L-ascorbic acid redox pair, as radical initiator, to functionalize proteins with phenolic acids (polyphenols) in a singlestep. Bovine serum albumin (BSA), which is also a component of whey protein showed excellent gelation activity, surface activity, and nutraceutical binding ability (Livney, 2010; Matsudomi, Rector, & Kinsella, 1991). However, no protein-polyphenol conjugate based on BSA has been reported. In this study, BSA-caffeic acid (CA) conjugate, prepared with free radical-induced grafting method, was characterized with SDS-PAGE, and far CD, and used as an emulsifier to stabilize resveratrol-loaded zein nanoparticle. The physical state and the interaction among resveratrol, zein, and BSA-CA conjugate was investigated with ATR-FTIR, and XRD. Thermal and UV light stability of free resveratrol, resveratrol in zein nanoparticles with or without BSA and BSA-CA conjugate was analyzed. Cellular antioxidant activity (CAA) of resveratrol was also evaluated with Caco-2 cell models.

2.3. Evaluation of phenolic groups by Folin–Ciocalteu reagent The CA amount in BSA-CA conjugate was monitored with a forementioned method (Hu et al., 2016). Briefly, 0.5 mL of BSA-CA conjugate was mixed with 1 mL of Folin–Ciocalteu reagent for 5 min in the dark, then 2 mL of 20% sodium carbonate (Na2CO3) was added. The reacting substances was then mixed and kept for 1 h at 25 °C. The absorbance was obtained with a UV–vis spectrometer (UV2600, Shimadzu, Japan) at 747 nm. The conjugating amounts of BSA-CA conjugates were presented as milligrams of gallic acid equivalent per gram of BSA-CA conjugate. 2.4. Characterization of BSA-CA conjugate 2.4.1. SDS-PAGE Aliquots of BSA or BSA-CA conjugates (protein content 5 mg/mL) were boiled in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol) containing 100 mM β-mercaptoethanol for 5 min and separated by electrophoresis on 4–20% gels. The electrophoresis was performed at 120 V. After running, the gel was stained using the Colloidal Coomassie Brilliant Blue G-250 Staining protocol (Fan et al., 2017). The stained gel was scanned with a Chemilmager™ 4400 (Alpha Innotech, CA, U.S.). 2.4.2. Circular dichroism (CD) CD spectra were collected at 20 °C with a J-815CD spectrometer (Jasco, Tokyo) in the wavelength ranging from 195 to 260 nm with 2.0 mm quartz cuvettes. BSA, and BSA-CA conjugates were dissolved with a 10 mM phosphate buffer (PB, pH 7.0) at a protein concentration of 0.2 mg/mL. Scanning was performed at 50 nm/min. Ten scans were averaged and the CD spectra were displayed as mean residue ellipiticity (degrees cm2/dmol).

2. Materials and methods 2.1. Materials

2.4.3. DPPH radical scavenging activity DPPH radical scavenging activity of BSA, BSA-CA conjugate, and CA was analyzed as described previously (Yi, Lam, Yokoyama, Cheng, & Zhong, 2015). The absorbance was measured at 517 nm.

Bovine serum albumin (BSA) (≥98%), resveratrol (≥98%), L-ascorbic acid, hydrogen peroxide (30%, w/w), 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), fluorescein, caffeic acid (CA), gallic acid, Trolox, and 2′,7′dichlorofluorescin diacetate (DCFH-DA) were obtained from SigmaAldrich (St. Louis, MO). All other analytical grade chemicals and reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) (containing 4.5 g/L D-glucose and GlutaMAX™), penicillin and streptomycin (100×), fetal bovine serum (FBS), TrypLE™ Select, Hanks’ balanced salt solution (HBSS), and phosphate buffer solution (PBS)(10×) were purchased from GIBCO (Grand Island, NY, U.S.). Caco-2, a human epithelial colon adenocarcinoma cell line, was purchased from the American Type Culture Collection (Manassas, VA, U.S.).

2.4.4. Ferric reducing power The reducing power of BSA, BSA-CA conjugate, and CA was measured as described previously in our published paper with slight modifications (Yi et al., 2015). The absorbance was measured at 700 nm. 2.4.5. Oxygen radicals antioxidant capacity (ORAC) The antioxidant capacity of BSA, BSA-CA conjugate, and CA was analyzed with oxygen radicals antioxidant capacity assay as described previously (Zulueta, Esteve, & Frígola, 2009). In each well, 50 μL of fluorescein (70 nM) and 50 μL (5 μg/mL) of sample, control (PB, pH 7.0), or standard (trolox, 20 μM) were mixed. The mixture was kept at 37 °C for 15 min, and then 25 μL of AAPH (221 mM) was added. After that, 96-well plate was put into a fluorescence microplate reader (Synergy HTX, BioTek, Vermont, USA). The fluorescence was detected at 535 nm every 5 min for 90 min at 37 °C with the excitation at 485 nm. The ORAC values of BSA, BSA-CA conjugate, and CA were presented as μM Trolox equivalents (μM TE).

2.2. Preparation of BSA-CA conjugate BSA-CA conjugate was fabricated with free radical-induced methods as described previously (Spizzirri et al., 2009) with slight modifications. In brief, 0.5 g BSA was fully dissolved in 50 mL ultrapure water, and 0.5 mL of 10.0 M H2O2 and 0.25 g L-ascorbic acid were added gradually. The mixture was then stirred at 25 °C under atmospheric air for 2 h. After that, 0.25 g CA was added to the mixture and continued to stir for 24 h. The free unreacted CA in reaction solution was then removed by dialysis against ultrapure water for two days with about 10 times water changes at 4 °C. The dialysis bags had a 3 kDa molecular weight cutoffs

ORAC (μMTE) =

Ct × (AUCs−AUCb) × k (AUCt −AUCb)

(1)

where Ct is the concentration (μM) of Trolox (20 μM), k is the sample dilution factor, and AUC is the area below the fluorescence decay curve of the sample, blank, and Trolox, respectively. 284

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AUC = 1+

∑ i=1

fi/5 f0

resolution. A background spectrum was obtained for each sample. The powder sample was placed on the center of the crystal surface. Two hundreds and fifty-six scans were taken for the background and the sample.

(2)

where f0 is the initial fluorescence and fi/5 is the fluorescence at time i/ 5.

2.6.5. XRD Crystal forms of pure resveratrol, zein, BSA-CA conjugate, and resveratrol-loaded zein nanoparticle with BSA-CA conjugate were analyzed by XRD (Philips X’pert, Netherland). Samples were added to the flat XRD glass slide using a die to make a rectangular shape before being placed on the operating floor for detection. The operation conditions were 40 kV and 40 mA with Cu as the X-ray source (λ = 1.54). Samples were scanned from 3° to 60° at a scanning rate of 4° per min using a continuous scan over the 2θ range. Data was processed with X’pert data software (Philips, Netherland).

2.5. Preparation of resveratrol loaded zein nanoparticle with BSA and BSACA conjugate Resveratrol (10 mg/mL) was dissolved in pure ethanol as stock solution. Zein (20 mg/mL) was dissolved in 70% aqueous ethanol solution. Resveratrol stock solution was added dropwise to zein solution at a ratio of 1:5 (v/v). To coat the resveratrol-loaded zein nanoparticles with BSA and BSA-CA conjugates, an aliquot (3.0 mL) of zein-resveratrol ethanol solution was added into BSA or BSA-CA conjugate solution dropwise. The resulting mixture was stirred using a magnetic stirrer at 800 rpm for 60 min. The final ratio of zein to BSA or BSA-CA conjugate was 10:1 (w/w). Finally, the ethanol was removed using a rotating evaporator (Rotavapor R110, Büchi Crop., Switzerland). All experiments were performed in triplicate. The obtained samples were then lyophilized and kept at −20 °C for further use.

2.7. Stability 2.7.1. Chemical stability Resveratrol (control), resveratrol-loaded zein, zein-BSA, and zeinBSA-CA nanoparticles redispersed in PB (pH 7.0, 10 mM) were stored at 25 and 50 °C in incubators in the dark for 30 days immediately after preparation, and equal aliquots were taken at certain intervals for resveratrol concentration determination. Resveratrol dissolved in DMSO, and then dispersed in PB was used as control. High temperature (50 °C) was used to accelerate the process of resveratrol degradation. Resveratrol stock solution was prepared by dissolving in DMSO at a concentration of 200 μg/mL. A calibration curve was obtained to quantify resveratrol in control or nanoparticles ranging from 0.1 to 10.0 μg/mL. Nanoparticles without resveratrol were used as blanks and the absorbance was used to calibrate the absorbance from proteins. Absorbance of resveratrol was analyzed with a UV–vis spectrometer (UV2600, Shimadzu, Japan) at 305 nm. The path length was 1 cm.

2.6. Characterization of nanoparticle 2.6.1. Particle size, zeta-potential, and polydispersity index Particle size (Dz), polydispersity index (PDI), and zeta-potential of all resveratrol-loaded nanoparticles were measured by dynamic light scattering (Zetasizer Nano ZSE, Malvern Instruments, Worcestershire, UK) (Yi et al., 2016). Nanoparticles were diluted 100-fold with ultrapure water, and adjusted to neutral pH with NaOH. All samples were measured in triplicate at 25 °C. 2.6.2. Encapsulation efficiency (EE) and loading amount (LA) EE and LA of resveratrol in zein nanoparticles with BSA or BSA-CA conjugate were analyzed with a recently reported method with minor modification. Free resveratrol was evaluated by analyzing the amount of resveratrol transferred into the ultrafiltrate receiver after being centrifuged at 2000g for 1 h with an Amicon Ultra-3 K centrifugal filter device (Millipore Corp., Billerica, MA, USA). The amount of resveratrol in the ultrafiltrate was determined by UV–vis spectrophotometer as described in Section 2.7.1. Resveratrol loaded in zein nanoparticles remained in filter unit. EE and LA were calculated with the following formula:

EE (% )= 100 ×

LA (% )= 100 ×

2.7.2. UV light stability The stability of resveratrol to light-induced degradation was assessed using a UV light lamp and cabinet. An aliquot of 5 mL of each sample was poured into a Petri dish (60 mm diameter) and exposed to UV light at 365 nm for 120 min. The remaining resveratrol was measured with a UV–vis spectrometer (UV2600, Shimadzu, Japan) at 305 nm at various time intervals. Nanoparticles without resveratrol were used as blanks. 2.8. Bioaccessibility and digestion stability

total amount of resveratrol−free resveratrol total amount of resveratrol

(3)

loaded amount of resveratrol total amount of nanoparticles

(4)

Briefly, 7.5 mL sample was mixed with 7.5 mL simulated gastric fluid (2.0 mg/mL porcine pepsin, 0.1 M HCl, and 0.10 M NaCl), and pH was adjusted to 2.0 by dropwise addition of 1 M HCl. The sample was incubated in a water bath under stirring continuously at 250 rpm in an incubator at 37 °C for 1 h (Thermo Scientific, NH, U.S.) After gastric phase, the sample was then adjusted to pH 7.0 using NaOH or HCl solution. then 15 mL simulated intestinal fluids (bile salts 4.0 mg/mL; pancreatin 1.0 mg/mL; PBS 0.1 M, and pH 7.0) was added, then stirring continuously at 250 rpm and 37 °C in an incubator (Thermo Scientific, NH, U.S.) for 2 h to simulate the temperature and movement of the intestine. The bioaccessibility of resveratrol was determined by the amount in micelles after digestion divided by total amount before digestion. An aliquot of the digesta was centrifuged at 8000 rpm with a benchtop centrifuge (Thermo Scientific, NH, U.S.) for 30 min at 10 °C, and the supernatant was filtered using a 0.45 μm filter. After that, the sample was diluted in DMSO and resveratrol was measured as described in Section 2.7.1.

2.6.3. SEM The particle size, shape, and morphology of resveratrol-loaded zein nanoparticles encapsulated with BSA or BSA-CA conjugate was observed with SEM (Hitachi S-4700, CA, U.S.). Freeze-dried nanoparticles powders were added and adhered onto conductive carbon tape. Gold particles were deposited onto the powders with a sputter coater under vacuum to avoid the charging effect prior to observation. An electron accelerating voltage of 10 kV was used. 2.6.4. ATR-FTIR Characterizations of pure resveratrol, zein, BSA-CA conjugate, and resveratrol-loaded zein nanoparticle with BSA-CA conjugate were determined by ATR-FTIR. For ATR-FTIR spectrometry, infrared spectra were obtained at room temperature with an ATR-FTIR spectrophotometer (Nicolet iS10, Thermo-Scientific, WI, USA) in the wavenumber range of 4000–650 cm−1. All spectra were collected at 4 cm−1

2.9. CAA Human colon carcinoma cell monolayers (Caco-2 cells) were 285

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weakened. Similar results were also found for BLG when conjugated with chlorogenic acid, suggesting CA may successfully bound to BSA (Fan et al., 2017). Secondary structure analysis also showed unordered secondary structure of BSA was increased after conjugated with CA with the decrease of order structure (α-helix, and β-sheet structure) (not shown). The results indicated that the interior hydrophobic amino acids were exposed after conjugated with CA.

maintained in DMEM containing 10% FBS, 1× nonessential amino acid, and 1× penicillin and streptomycin at 37 ˚C in an incubator (Sanyo, Osaka, Japan) with 95% humidity and 5% CO2. To determine the CAA of free resveratrol, resveratrol-loaded zein nanoparticle with BSA and BSA-CA conjugate, cell-based antioxidant assays were performed based on a method reported in the literature (Wolfe & Liu, 2007). After 48 h incubation, the media was removed and Caco-2 cells were rinsed with PBS. Triplicate wells were treated with 100 µL of free resveratrol, resveratrol-loaded zein-BSA nanoparticles, and resveratrolloaded zein-BSA-CA nanoparticles in treatment media at various concentrations for 4 h at 37 °C. Free resveratrol was prepared by dissolving in dimethyl sulfoxide (DMSO) and dispersing in PBS. Caco-2 cells were rinsed with PBS, and 100 µL of 25 µM DCFH-DA in treatment medium was added to each well and incubated for 1 h. After that, Caco-2 cells were rinsed with PBS three times, 100 µL 600 µM ABAP was added to the cells, and the plate was immediately placed into a fluorescence microplate reader (Synergy HTX, BioTek, Vermont, USA). Emission fluorescence intensity at 535 nm (with excitation at 485 nm) was recorded every 5 min for 1 h at 37 °C. Control wells were treated with DCFH-DA and ABAP and blank wells were treated with DCFH-DA without ABAP. The sample background wells were treated with resveratrol-loaded nanoparticle and DCFH-DA without ABAP. To calculate the CAA value of samples, the area under the curve of fluorescence versus time was integrated with the following formula,

(∫ SA− ∫ BA)/(∫ CA− ∫ BA) × 100

CAA value = 100−

3.2. Antioxidant activity of BSA-CA conjugate DPPH scavenging activity, reducing power, and oxygen-radical antioxidant capacity (ORAC) assays were used to evaluate the antioxidant activities of BSA, BSA-CA conjugate, and CA. As depicted in Fig. 2S, the antioxidant activity of all three samples was dose-dependent. BSA showed little DPPH scavenging activity and the highest value was only 9.0% at 1.0 mg/mL. CA showed the highest DPPH scavenging activity. The DPPH scavenging activity of BSA-CA conjugate was remarkably higher than that of BSA at all concentration, suggesting BSA’s chemical antioxidant activities were sharply improved after conjugation with CA. Notably, no significant differences were found between BSA-CA conjugate and CA when the concentration was no less than 0.4 mg/mL. The DPPH scavenging activity values were 89.7% and 91.9% for BSA-CA conjugate and CA at 0.4 mg/mL, respectively (Fig. 2S). Reducing power was analyzed through the calculation of the inhibition degrees of the conversion of Fe3+ to Fe2+ by antioxidants. Identical to DPPH scavenging activity, the reducing power of BSA was lowest at all concentration among BSA, BSA-CA conjugate, and CA. At 1.0 mg/mL. The reducing power of BSA was only 0.010, while the value soared to 0.662 after CA conjugation, indicating the reducing power was remarkably improved 66.2 times. The results obviously suggested that the reducing power of BSA were greatly increased after grafting with CA. CA had the highest reducing power. ORAC assay was cost-effective and time-efficient, and has been widely used as a standard tool to measure the antioxidant activity of nutraceuticals. Similarly, BSA-CA showed remarkably higher oxygen radical scavenging ability than BSA. The ORAC values were 546.4, 4073.9, and 4823.5 for BSA, BSA-CA, and CA, respectively. The results suggested that BSA-CA conjugates had approximately 7.5 times higher ORAC value than BSA. CA also had the highest ORAC assay. The great antioxidant activity of polyphenols has been mainly attributed to the higher number of hydroxyl group substituents. The hydroxyl groups of CA bound to BSA possibly resulted in the improvement of antioxidant activities of BSA–CA conjugates. Furthermore, the exposure of hydrophobic amino acid after conjugation with CA was possibly another reason for it, as depicted by far UV CD. The antioxidant activities of other proteins like ovalbumin, gelatin, α-lactalbumin, and β-lactoglobulin have also been appreciably enhanced by conjugation with catechin, gallic acid, chlorogenic acid, or EGCG (Fan et al., 2017; Feng, Cai, Wang, Li, & Liu, 2018; Spizzirri et al., 2009; Yi, Fan, Zhang, & Zhao, 2016). A synergistic increase in the antioxidant activity of BSA and polyphenols in inhibiting lipid oxidation was also reported (BonoliCarbognin, Cerretani, Bendini, Almajano, & Gordon, 2008). The information obtained above clearly indicated that BSA-CA conjugate can be used as an effective emulsifier for the stabilization, protection, and delivery of functional compounds.

(3)

where ∫ SA, ∫ BA, and ∫ CA are the integrated area of the signal from free resveratrol (dissolved in DMSO), resveratrol-loaded zein-BSA nanoparticles, and resveratrol-loaded zein-BSA-CA nanoparticles, the blank curve or sample background curve, and i the control curve, respectively. The medium effective dose (EC50) was analyzed for free resveratrol (dissolved in DMSO), resveratrol loaded zein-BSA nanoparticles, and resveratrol loaded zein-BSA-CA nanoparticles from the curve of log (Fa/Fu) against log concentration of resveratrol (μg/mL), and EC50 was determined from the plot of log (Fa/Fu) = log 1 = 0, where Fa = CAA, Fu = 100−CAA. 2.10. Statistical analysis All experiments were performed in triplicate and the data were expressed as mean ± STD. The results were subjected to the analysis of variance (ANOVA) with the SPSS 17.0 package (IBM, New York). The significance was defined at the 95% confidence level. 3. Results and discussion 3.1. Characterization of BSA-CA conjugate The reaction ratio between protein and polyphenols has already been optimized previously and 1:0.5 (w/w) between BSA and CA was chosen for BSA-CA conjugate preparation. As depicted in Fig. 1A, a clear band, corresponding to native BSA with molecular weight of 66 kDa, was observed in lane 2. After CA conjugation, the molecular weight of BSA increased slightly, indicating CA was successfully conjugated onto BSA with the free radical method (lane 3). A far-UV CD spectrometer was used to analyze the secondary structure compositions of BSA conjugated with CA, as shown in Fig. 1B. BSA is a globular protein with a high α-helix content, confirmed by two strong negative bands at approximately 208 and 222 nm, respectively. This was consistent with the X-ray crystallographic results which showed that the BSA structure is predominantly α-helical with the remaining polypeptide residing in turns and extended or flexible regions between subdomains, and no β-sheets. After conjugation, hypsochromic shifts were observed for the negative band at both 208 and 222 nm. And intensity of the negative bands between 208 and 222 nm was

3.3. Characterization of resveratrol-loaded zein nanoparticle The mean particle diameter of resveratrol-loaded nanoparticles without BSA coating was 897.5 nm (Table 1). Some colloidal nanoparticles of zein appeared to be flocculated or aggregated and large zein particles were visible. EE was 51.6%, and LA was 4.9%. The results indicated that zein particles aggregation formed possibly through hydrophobic interactions. In the presence of BSA or BSA-CA conjugate, the mean particle diameters of zein particles in the colloidal solutions were 206.3 nm, and 286

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Fig. 1. SDS-PAGE results (A) under reducing conditions of BSA and BSA-CA conjugates. Lanes: 1, protein markers; 2, native BSA; 3, BSA-CA conjugates; Far-UV CD spectra (B) of BSA and BSA-CA conjugates in the wavelength range of 195–260 nm.

characteristic absorption peaks were obtained for both proteins. Two prominent bands, corresponding to amide I (1600–1650 cm−1) and amide II (1500–1530 cm−1) were found. An absorption band of OeH stretching were found at 3203 cm−1 from resveratrol’s FTIR spectrum. Furthermore, FTIR spectrum of resveratrol showed characteristics peaks at 1601 cm−1, 1580 cm−1, 1512 cm−1, 1453 cm−1, 1367 cm−1, and 964 cm−1, probably due to aromatic eC]Ce stretching, olefinic eCeCe stretching, aromatic ring eC]Ce stretching, aromatic ring eC]Ce stretching, eCeOe stretching vibration, and trans olefinic eC]Ce stretching, respectively (Pujara et al., 2017). Most of the characteristic peaks of resveratrol disappeared when it was loaded in zein and zein-BSA-CA nanoparticles (Fig. 3A), and this may indicate the formation of nanoparticles that reduced the bending and stretching of the bonds in resveratrol due to the incorporation of resveratrol within the nanoparticles. Resveratrol (control) was highly crystallized, whereas encapsulated resveratrol in nanoparticle is amorphous, evidenced by XRD results below. The absence of new peaks in the resveratrol-loaded zein-BSA-CA nanoparticle indicated that the proteins and resveratrol are physically attached to each other and no covalent reaction exhibited. The incorporation of resveratrol into zein and zeinBSA-CA nanoparticles resulted in shifts in the peaks associated with the aliphatic and aromatic moieties, suggesting that interactions between resveratrol and proteins occurred by hydrogen bonds, hydrophobic interactions, or electrostatic interactions. Similar results were also reported by Davidov-Pardo et al. (Davidov-Pardo, Joye, & McClements, 2015).

217.2 nm, respectively (Table 1). Particle size distribution graphs showed that both the particle sizes of resveratrol-loaded zein-BSA and zein-BSA-CA nanoparticles were relatively small, and were unimodal and narrow (Fig. 2). Zeta-potential values were −33.8 mV, and −37.6 mV for zein nanoparticles coated with BSA and BSA-CA conjugate, respectively, whereas zeta-potential value was −5.7 mV for zein particle in the absence of a stabilizer. The results may suggest that zein nanoparticles was successfully encapsulated with BSA and BSA-CA conjugate. Presumably, the β-lactoglobulin molecules adsorbed on the hydrophobic parts, located on outside surfaces of the zein particles, and therefore prevented precipitation. Previous studies have also confirmed that edible protein emulsifiers, like sodium caseinate and β-lactoglobulin, can prevent the flocculation or aggregation of zein particles. Hence, the emulsifier plays an important role in the formation and stabilization of zein colloidal nanoparticles (Chen et al., 2014; Patel, Hu, Tiwari, & Velikov, 2010). The EEs of resveratrol were 85.3% and 86.5% for zein nanoparticles coated with BSA and BSA-CA conjugate, respectively. And the LAs of resveratrol were 7.2% and 7.3% for zein nanoparticles coated with BSA and BSA-CA conjugate, respectively. SEM results showed that both resveratrol-loaded zein nanoparticles coated with BSA and BSA-CA conjugates were spherically shaped and homogeneously dispersed (Fig. 2B and C). The mean particles diameters obtain in SEM experiments (approximately 200 nm in diameters) were consistent with the results by dynamic light scattering.

3.3.1. ATR-FTIR ATR-FTIR spectroscopy is an excellent method for chemical analysis, through analyzing the wavelength and intensity of the absorption of FTIR radiation by a sample (Yang, Yang, Kong, Dong, & Yu, 2015). The ATR-FTIR spectra (4000–650 cm−1) of resveratrol, zein, BSA-CA conjugate, and resveratrol-loaded zein-BSA-CA nanoparticle were shown in Fig. 3 to analyze the intermolecular interaction between resveratrol and proteins. The zein and BSA-CA spectra (Fig. 3) were very similar and the

3.3.2. XRD In this study, XRD, a rapid and widely used analytical technique primarily used for phase identification of a crystalline material was performed to analyze the amorphous nature of resveratrol encapsulated in BSA-CA. As depicted in Fig. 3B, pure resveratrol was highly crystallized and exhibited sharp diffraction peaks in the 2θ range of 5–50°. Similar results were also reported by Pujara et al. (2017). The amorphous nature of zein and BSA-CA is confirmed from the broad peak

Table 1 Characteristics (mean particle diameter, zeta-potential, PDI, encapsulation efficiency (EE, %), and loading amount (LA, %)) of resveratrol-loaded zein nanoparticles encapsulated with BSA and BSA-CA conjugate (n = 3).

a

Characteristics

Particle diameter (nm)

Zeta-potential (mV)

PDI

EE (%)

LA (%)

Zein Zein-BSA Zein-BSA-CA

897.5 ± 64.3 206.3 ± 5.8 217.2 ± 4.6

−5.7 ± 0.7 −33.8 ± 1.0 −37.6 ± 1.3

0.536 ± 0.073 0.215 ± 0.020 0.202 ± 0.023

51.6 ± 2.7 85.3 ± 2.7 86.5 ± 2.8

4.9 ± 0.3 7.2 ± 0.2 7.3 ± 0.3

Significant differences within a column are denoted with letters (P < 0.05). 287

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Fig. 2. Particle size distributions (A) and SEM graphs of resveratrol-loaded zein nanoparticles with BSA (B), and BSA-CA conjugate (C), respectively.

further confirmed that resveratrol-loaded zein-BSA-CA nanoparticle showed the highest chemical stability compared to resveratrol alone in aqueous solution, zein nanoparticle and zein-BSA nanoparticle. This was mainly due to the high content of amino acids with antioxidant activity including Trp, Phe, and Tyr occurring in proteins (zein and BSA) (Joye, Davidov-Pardo, & McClements, 2015), having partly free radicals scavenging and prooxidative metal chelating ability (Elias, Kellerby, & Decker, 2008). Furthermore, the protein layers inhibited the interaction between pro-oxidants (like metal ions, and free radicals) and encapsulated resveratrol. The thicker the layers of proteins around resveratrol, the higher the protection of resveratrol from degradation. Additionally, the disruption of tertiary structure after CA conjugation may cause the exposure of amino acid residues with antioxidant activity, leading to the increase of protection against resveratrol degradation. Lastly, the antioxidant activities (DPPH scavenging activity, reducing power, and ORAC) of BSA have confirmed to be improved appreciably after conjugation with CA (Fig. 2). The high antioxidant activity of CA was mainly due to the higher number of hydroxyl group substituents, resulting in higher hydrogen atom donation and the termination of free radicals (Sato et al., 2011; Seyoum, Asres, & El-Fiky, 2006). The hydroxyl groups of CA added to BSA probably resulted in improved antioxidant activities of BSA−CA conjugate.

between 10° and 25°. However, no obvious diffraction peaks belonging to resveratrol were found in the resveratrol-loaded zein nanoparticle and zein-BSA-CA nanoparticles, suggesting the complete nano-encapsulation of resveratrol in zein-BSA-CA nanoparticles and its amorphous nature within zein-BSA-CA nanoparticles.

3.4. Chemical stability 3.4.1. Thermal stability Chemical stability of liable nutraceuticals is vital as a consequence of its high relevance with the corresponding biological activity. Alltrans-resveratrol is far more biologically active than its isomers. Almost no biological activity occurred for the degradation products of resveratrol. Resveratrol is easily isomerized, degraded, and oxidized, especially at alkaline pH or under UV light (Zupančič, Lavrič, & Kristl, 2015). Storage assays showed that resveratrol is degraded within a few weeks in aqueous systems, mainly attributed to spontaneous oxidation. At 25 °C, the chemical stability is relatively high (Fig. 4). By way of example, the retention rate of resveratrol alone (control) was 81.2% after 30 d storage. After loading in zein particles, the retention rate was slight increased to 87.6%. Compared to resveratrol (control), higher chemical stability was observed in zein particle. Zein particle may protect resveratrol from interaction of pro-oxidants in aqueous phase. The retention rate for zein-BSA nanoparticle was 91.5%. Resveratrol in zein-BSA-CA nanoparticle was stable and highest retention (96.3%) was found, and less than 4% of resveratrol was lost. The results clearly indicated BSA-CA conjugate significantly improved the chemical stability than zein and zein-BSA. At neutral pH, higher temperatures further accelerated the resveratrol degradation. At 50 °C (Fig. 4B), the retention rate of resveratrol (control) remarkably decreased to 51.3%, indicating heat treatment appreciably promoted its degradation. Retention rate of resveratrol in zein-BSA-CA nanoparticle (87.6%) is significantly higher than that in zein-BSA nanoparticle. The accelerated stability experiments at 50 °C

3.4.2. UV light stability UV light can accelerate the degradation of resveratrol (Trela & Waterhouse, 1996). Approximately 65% resveratrol was degraded when stored under UV light for only 120 min, whereas the retention rate was 85.5% after 2 d storage at 50 °C, indicating that UV light may be a factor more important than thermal treatment for resveratrol loss (Fig. 4C). The retention rate increased to 45.3% when loaded in zein particle. The retention rates were 53.8% and 62.7% for zein-BSA and zein-BSA-CA nanoparticle, respectively, after 120 min treatment. The zein-BSA nanoparticle showed greater protections for resveratrol from degradation than zein particle. Aromatic groups and the double bonds 288

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Fig. 3. ATR-FTIR spectra (A) and X-ray diffraction graphs (B) of resveratrol (black curve), zein (blue), BSA-CA conjugate (red), and resveratrol-loaded zeinBSA-CA conjugate nanoparticles (greenish blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of BSA could absorb UV light to protect easily-oxidized nutraceuticals from degradation. Approximately 10% higher retention rate of resveratrol in zein-BSA-CA nanoparticle than in zein-BSA were observed. This was mainly attributed to the strong UV absorption ability of CA which showed strong protection against resveratrol from UV light-induced degradation. CA has also proved to provide a satisfactory photoprotection to the skin against UV-mediated oxidative damage (Saija et al., 2000), and the protection of liposomes from UV radiation-induced peroxidation and reaction with nitrogen oxides originated from sodium nitroprusside was also efficient (Saija et al., 1999). 3.5. Bioaccessibility and digestion stability Fig. 4. Chemical stability of resveratrol in zein nanoparticles with BSA or BSACA conjugate at 25 °C (A) and 50 °C (B), respectively during 30 d storage. UV stability (C) of resveratrol in zein nanoparticles with BSA or BSA-CA conjugates during 120 min storage at room temperature. Values were expressed as mean ± STD, n = 3. Values at the same storage time with different letters (a−d) are significantly different (p < 0.05).

A dynamic in vitro gastrointestinal model was used to monitor the effects of BSA or BSA-CA conjugate coating on resveratrol bioaccessibility loaded in zein nanoparticle. As depicted in Fig. 4S, the bioaccessibility of free resveratrol (control) was 43.6% due to its insolubility in water. After loaded in zein particles, the bioaccessibility of resveratrol increased to 62.7% (p < 0.05) because of the increased aqueous solubility. BSA coating further increased resveratrol bioaccessibility to 73.4%. This was mainly attributed to the decrease of mean particle size after BSA coating which increased the interaction between digestive enzyme and protein nanoparticle and the transfer of resveratrol from

nanoparticle to micelles increased. The bioaccessibility of resveratrol was not affected by CA conjugation when compared to the one encapsulated with BSA (p > 0.05). After gastrointestinal digestions, the retention of resveratrol was 289

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Covalently bound CA showed no adverse impacts on the cellular uptake of resveratrol-loaded zein-BSA-CA nanoparticle. 4. Conclusion Resveratrol-loaded zein nanoparticles was encapsulated with BSACA conjugate, synthesized by free radical-induced grafting method. Encapsulated resveratrol in both nanoparticles had better stability than free resveratrol (control) and resveratrol-loaded zein particle. Compared to zein-BSA nanoparticle, resveratrol in zein-BSA-CA nanoparticles had higher stability under both thermal and UV light treatment, which was mainly attributed to the higher antioxidant activities (DPPH scavenging ability, reducing power, and ORAC). Both zein-BSA nanoparticle and zein-BSA-CA nanoparticle had remarkably higher CAA than resveratrol (control), whereas no significant differences were found between zein-BSA nanoparticle and zein BSA-CA nanoparticle. The present study showed that zein-BSA-CA nanoparticles delivery system has great potential for application in food industry as nutrition supplements and functional beverage for enhancing the chemical stability and biological activity of liable nutraceuticals.

Fig. 5. Comparison of the CAA values and determination of the EC50 of free resveratrol, resveratrol-loaded zein nanoparticle with BSA and BSA-CA conjugate, respectively. Different numbers mean significant difference.

Acknowledgement measured and the results confirmed that it remained chemically stable, without remarkable loss for all samples (Fig. 4S), suggesting that the delivery system may slow or prevent the degradation or metabolism and increase the bioaccessibility of resveratrol after digestion.

This research was financially supported by the National Natural Science Foundation of China (No. 31601512) and Young Scholars' Scientific Research Startup Funding from Shenzhen University (No.2016010).

3.6. CAA Conflict of interest To assess the biological activity of nutraceuticals, Caco-2 cells based CAA model was used to evaluate the in vitro antioxidant activity of resveratrol (control), resveratrol in zein-BSA nanoparticle and zeinBSA-CA nanoparticle. Added DCFH-DA that cannot be absorbed by Caco-2 cells until being hydrolyzed by esterases occurring in cells. DCFH can be oxidized to fluorescent DCF by pro-oxidants (free radicals). The increase of fluorescence from DCF induced by peroxyl radicals (ABAP) was restrained by absorbed resveratrol (Wolfe & Liu, 2007). The CAA of free resveratrol, resveratrol-loaded zein-BSA, and zeinBSA-CA nanoparticles in Caco-2 cells was shown in Fig. 5. CAA values of resveratrol in all three samples increased in a dose-dependent manner. After loading in zein-BSA nanoparticle and zein-BSA-CA nanoparticle, the CAA value of resveratrol increased remarkably (p < 0.05) compared with that of free resveratrol at the same concentration (Fig. 5). Medium effective dose (EC50) was calculated from the point of Log(Fa/Fu) = 0. EC50 values of resveratrol-loaded zein-BSA and zein-BSA-CA nanoparticles were 20.8 µg/mL and 19.3 µg/mL, whereas the value was 41.7 µg/mL for resveratrol (control). Both zeinBSA and zein-BSA-CA nanoparticles exhibited higher antioxidant ability than free resveratrol, indicating nanoparticle-based delivery system could improve the uptake and bioavailability of encapsulated antioxidant ingredients into Caco-2 cells. Improved chemical stability may be an important reason. Recent studies demonstrated that soybean protein nanoparticles can protect resveratrol from the possible autooxidization of resveratrol in an mildly alkaline aqueous phase, similar to the pH in CAA experiment (Pujara et al., 2017). Furthermore, nanoparticles have high surface area for peroxyl quenching due to smaller particle size. Resveratrol molecules dissolved in DMSO may form resveratrol aggregates after dispersed in PB for its low water-solubility. The absorption of resveratrol aggregates was probably inhibited by its large size. Lastly, XRD results also confirmed that resveratrol-loaded zein-BSA-CA nanoparticles was amorphous. The bioavailability of the amorphous resveratrol was higher than crystalline resveratrol since the melting point and the induction of intermolecular attractive forces were usually lower for noncrystalline ingredients. No significant differences were observed between zein-BSA and zein-BSA-CA nanoparticles.

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