Food Chemistry 231 (2017) 19–24

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Food Chemistry

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Encapsulation of epigallocatechin gallate in zein/chitosan nanoparticlesfor controlled applications in food systems

http://dx.doi.org/10.1016/j.foodchem.2017.02.1060308-8146/� 2017 Published by Elsevier Ltd.

⇑ Corresponding authors.E-mail addresses: yibinzhou@ahau.edu.cn (Y. Zhou), xcwan@ahau.edu.cn (X.

Wan).1 Both authors contributed equally to this work.

Jin Liang a,1, Hua Yan a,1, Xiulan Wang a, Yibin Zhou a,⇑, Xueling Gao a, Pradeep Puligundla b, Xiaochun Wan a,⇑a State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, ChinabDepartment of Food Science & Biotechnology, Gachon University, Seongnam 13120, South Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 October 2016Received in revised form 6 January 2017Accepted 21 February 2017Available online 22 February 2017

Keywords:EGCGZeinChitosan nanoparticlesControlled releaseFatty food simulant

The objective of this study was to prepare chitosan nanoparticles (CS NPs) coated with zein as a promis-ing encapsulation and delivery system for epigallocatechin gallate (EGCG). The factors influencing thenanoparticle fabrication, including zein concentration, zein/chitosan weight ratio and EGCG encapsula-tion percentage, were systematically investigated. The physicochemical and structural analysis showedthat the electrostatic interactions and hydrogen bonds were the major forces responsible for nanoparti-cles formation. The transmission electron microscopy study revealed the spherical nature with smoothsurface of obtained nanoparticles. The release profile of EGCG showed a burst effect, followed by slowrelease. EGCG release was relatively higher from zein/chitosan nanoparticles (zein/CS NPs) with higherDPPH scavenging activity, than that of NPs without zein coating in 95% ethanol fatty simulant. Theseresults indicated that controlled-release of EGCG from zein/CS NPs and its corresponding antioxidantactivities in 95% ethanol fatty simulant may provide long-term protection against oxidation for fattyfoods.

� 2017 Published by Elsevier Ltd.

1. Introduction

Tea is one of the most popular beverages in the world whichcontains polyphenols. It has relatively strong medicinal and thera-peutic values and shows anti-ageing, anti-oxidant, weight loss andantidepressant activities (Tang et al., 2013). EGCG has been identi-fied as a major tea polyphenol (Khan & Mukhtar, 2007; Kuzuhara,Suganuma, & Fujiki, 2008). Due to the presence of multiple aro-matic phenol rings in its structure, EGCG has higher antioxidantactivity than non-phenolic or monophenolic antioxidants. How-ever, in the presence of relatively high temperature, oxygen con-centrations and pH, EGCG is oxidized easily (Shpigelman, Cohen,& Livney, 2012). Therefore, several approaches, including encapsu-lation, have emerged for its effective protection againstdegradation.

Chitosan has many distinctive biological properties such asnon-toxicity, biodegradability and antimicrobial activity. It hasbeen widely used for biomaterial applications (Siripatrawan &Harte, 2010). However, chitosan has a drawback; under neutraland basic environments, chitosan molecules lose the charge and

precipitation occurs near their pKa of 6.5 (Liang et al., 2015). There-fore, in our previous study (Liang et al., 2010, 2011), two differentwater-soluble chitosan derivatives, carboxymethyl chitosan (CMC)and chitosan hydrochloride (CHC) were chosen for nanoparticlefabrication, as they can form self-assembled CS NPs in an aqueoussolution due to their chemical structures containing car-boxymethyl and amino groups.

Zein protein is mainly used as edible coating material in thefood and drug industry (Hurtado-Lopez & Murdan, 2006). It canrelease a drug at a controlled level to meet the dosage require-ments, and is suitable for release at the site of absorption, whichis the goal of modern pharmaceutical research. It can also aid inthe enhancement of bioavailability of the biologically active sub-stance and nutrients (Zhong & Jin, 2009). Zein is insoluble in waterand therefore can be used for encapsulating purposes. It can assistin controlled release of bioactive core material from carriers innon-aqueous environment, to meet different functional require-ments such as antioxidant and antibacterial activities, and prolon-gation of shelf life (Wu, Wen, Yang, Xu, & Yin, 2011). Furthermore,zein together with chitosan could be prepared into nanoparticlesand easily formulated into edible films for food packaging(Cheng, Wang, & Weng, 2015).

Therefore, it is of great interest to study whether the encapsu-lation of EGCG in nanoparticles fabricated using chitosan and zeinshell materials could provide a controlled-release property and

Table 1Different formulations of nano-complex developed in this study.

Samples Zein (mg) Volume of CHC-CMC (ml) EGCG (mg)

A1 72 42 5A2 144 42 5A3 216 42 5A4 288 42 5B1 216 30 5B2 216 42 5B3 216 54 5C1 216 42 2C2 216 42 5C3 216 42 8

20 J. Liang et al. / Food Chemistry 231 (2017) 19–24

enhance its stability in fatty food simulants. In the current study,EGCG was encapsulated into CS NPs with and without zein coating.The comparison of the two delivery systems was studied, in termsof their physicochemical properties, namely particle size, zetapotential and encapsulation efficiency. The molecular interactionswere investigated by Fourier transform infrared spectroscopy(FTIR) and X-ray diffraction (XRD). The controlled release propertyof encapsulated EGCG in fatty food simulant was also studied.

2. Materials and methods

2.1. Materials

Zein was purchased from Beijing Solarbio Science & Technology,Co. Ltd. (Beijing, China). CHC with 85% deacetylation and CMC with83% deacetylation were supplied by Jinan Haidebei Marine Bio-engineering Co., Ltd. (China). EGCG (98.0% purity) was purchasedfrom Wu Hu Tianyuan Science & Technology Development Co.,Ltd. The other chemicals used were of analytical grade. All the solu-tions used in the experiments were prepared using ultrapurewater, which was obtained from a Millipore (Millipore, Milford,MA, USA) Milli-Q water purification system.

2.2. Preparation of EGCG-encapsulated zein/CS NPs

Different amounts of zein were dissolved in 20 ml aqueousethanol solutions (75% v/v). Initially, CMC was dissolved in differ-ent volumes of pure water and then the resultant solution addeddropwise to different amount of EGCG dissolved in 30 ml pure dis-tilled water containing CHC under magnetic stirring at 600 rpm for30 min (Liang et al., 2010). This was followed by adding the zeinsolution dropwise with magnetic stirring for another 30 min(Luo, Zhang, Cheng, & Wang, 2010). The formulations prepared inthis study were shown in Table 1. The freshly prepared nanoparti-cles in solutions were subjected to particle size, zeta potential andencapsulation efficiency measurements. The samples for releaseprofile and anti-oxidation measurements were freeze-dried imme-diately after ethanol removal under vacuum rotary evaporation,and stored at �18 �C for further assay. Meanwhile, EGCG-encapsulated CS NPs without zein and zein/SC NPs without EGCGwere prepared as control.

2.3. Particle size and zeta potential

Dynamic laser scattering (DLS) and zeta potential measure-ments were performed using a commercial laser light scatteringinstrument (DelsaMax PRO, Beckman Coulter Instruments) at25 �C and with a 90� scattering angle.

2.4. Morphology observation

Morphological structures of CS NPs prepared by using CHC andCMC materials and EGCG-encapsulated zein/CS NPs were deter-mined by Transmission Electron Microscopy (TEM) (HT-7700 Hita-chi, Japan). The samples were prepared by dropping solutions ontocopper grids coated with the support film. The samples stayed inthe copper network for 2–3 min. The samples were then immersedin phosphotungstic acid stain and stained for 2–3 min. After natu-ral drying, the samples were placed under TEM for observation.

2.5. Fourier transform infrared spectroscopy and X-ray diffraction

The chemical structures of zein, EGCG, CS NPs, EGCG-encapsulated CS NPs with or without coating zein were deter-mined using a FTIR spectrophotometer (Nicolette is 50, Thermo

Scientific, USA) with an attenuated total reflection (ATR) cell. Allsamples were prepared as KBr pellets and were scanned againsta blank KBr pellet background. The spectra were acquired in therange of 550–4000 cm�1 wave numbers, with a 4 cm�1 resolution.

X-ray diffraction (XRD) analysis of zein, EGCG, CS NPs, EGCG-encapsulated CS NPs and EGCG-encapsulated zein/CS NPs wereperformed using X-ray diffraction (Rigaku TTR Ⅲ) withbackground-free sample holders. The data were collected over anangular range from 5� to 50� 2-theta in continuous mode using astep size of 0.02� 2-theta and step time of 5 s.

2.6. Encapsulation efficiency

The encapsulation efficiency of EGCG-encapsulated zein/CS NPswas defined as the drug content that is entrapped into polymericmatrix and calculated as follows:

Encapsulation efficiency ¼ ðA� BÞ=A� 100

A = total amount of EGCG in added solution; B = total amount ofEGCG in supernatant after ultrafiltration.

The total EGCG amount was the added EGCG to polymericmatrix solution. The free EGCG was obtained by calculating theEGCG content that was ultracentrifuged at 15,000 rpm for 30 minand 25 �C in a refrigerated centrifuge (Allegra 64R Centrifuge Beck-man Coulter) (Liang et al., 2010). The free EGCG content in the fil-trate was measured using high performance liquidchromatography (HPLC) (Hong et al., 2014). The 1260 series HPLCsystem from Agilent Technologies, Inc., equipped with a 1260 ser-ies quaternary pump, a diode array detector, and a 1260 series vac-uum degasser and a Hypersil BDS C-18 column (Elite Technologies,Inc.) was used. The column heater was set at 30 �C. The standard/extracted sample (10 ml) was injected into the HPLC system. Themobile phase consisted of 0.4% acid water (A) and acetonitrile(B). The gradient was as follows: 10–25% B from 0 to 20 min; lineargradient to 10% B from 20 to 22 min; and then kept at 10% B for10 min. Total run time was 32 min, and the flow rate was 1.0 ml/min. Samples were detected at 280 nm and quantified accordingto the calibration curve of EGCG. All measurements were per-formed in triplicate.

2.7. Release profile of EGCG from zein/CS NPs

The EGCG-encapsulated zein/CS NPs samples were freeze-driedand then introduced into food simulants to investigate the releaseprofile of EGCG from zein/CS NPs. Two kinds of food simulants with95% and 50% ethanol were used to perform the release studies. Ingeneral, the 95% ethanol can be regarded as a simulant for fats,oil and fatty foods due to similar hydrophobicity. The 50% ethanolcan be regarded as a simulant for oil in water emulsions and alco-holic beverages (Iniguez-Franco et al., 2012). The release of EGCGfrom zein/CS NPs was determined by adding freeze-dried samplesand 30 ml of food simulant into 50 ml centrifuge tubes with screw

J. Liang et al. / Food Chemistry 231 (2017) 19–24 21

caps. Release tests were conducted in the dark at 25 �C for 95% and50% ethanol simulant, respectively. The food simulant (0.5 ml) wascollected and the content of EGCG was determined at predeter-mined times (0, 1, 2, 4, 6, 8, 10, 24, 48, 72, 96, 144, 168, 192,216, 240 h). All measurements were conducted in triplicate(Noronha, de Carvalho, Lino, & Barreto, 2014).

2.8. DPPH radical scavenging activity

A previously described method was used to evaluate the DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, withslight modifications (Siripatrawan & Harte, 2010). Food simulant(100 ll) was mixed with 1 ml (0.1 mM) of DPPH, dissolved in etha-nol. The mixture was kept in the dark for 30 min at room temper-ature. The absorbance was measured at 517 nm using a UV–visiblespectrophotometer DU-730 (Beckman Coulter, USA). The assay wascarried out in triplicate. The DPPH radical scavenging activity wasdetermined according to:

DPPH scavenger ð%Þ ¼ ð1� ½A sample=A control�Þ � 100

where ‘‘A sample” was the sample absorbance after mixed DPPHsolution, ‘‘A control” was the pure water absorbance of mixed DPPHsolution.

3. Results and discussion

3.1. Particle size and zeta potential

The effects of different formulations on particle size and poly-dispersity index (PDI) of EGCG-encapsulated zein/CS NPs are sum-marized in Table 2. As the zein concentration increased from 72 to216 mg, the average particle size of nanoparticles decreased from240.6 to 197.6 nm (Samples A1, A2 and A3). However, particle sizeincreased to 205.5 nm upon further increase of the zein concentra-tion to 288 mg (Sample A4). In the absence of zein, average particlesize of EGCG-encapsulated CS NPs was 215.4 nm (data not shown).After addition of zein, particle size was increased to 240.6 nm. Onzein addition, the interaction between hydrogen bonds wasenhanced, and thus the particle size increased. With furtherincreases in EGCG content, EGCG might migrate to the internalparts of the nanoparticles, resulting in a decrease of hydrodynamicdiameter. However, when the zein content increased to 288 mg,the particle size of the complex began to increase due to the forma-tion of larger self-assembled nanospheres. This interesting phe-nomenon has also been reported by Luo et al (Luo, Zhang,Whent, Yu, & Wang, 2011). And, in the presence of zein, due tothe hydrophobic interaction of zein, the structure of EGCG-encapsulated zein/CS NPs was denser (Li, Yin, Yang, Tang, & Wei,2012). The reduced particle size might be a result of the electro-static interactions between zein/CS NPs and EGCGmolecules. How-ever, when the concentration of zein was increased to 288 mg,

Table 2Particle size, surface charge and encapsulation efficiency of different samples.

Samples Partical size (nm) Zetapotential (m

A1 240.6 ± 19.5 31.2 ± 0.9A2 225.4 ± 6.7 33.3 ± 0.6A3 197.6 ± 7.7 34.7 ± 1.1A4 205.5 ± 13.5 33.3 ± 0.8B1 224.5 ± 20.0 31.6 ± 2.1B2 198.5 ± 5.3 34.9 ± 1.5B3 155.5 ± 11.5 27.5 ± 3.1C1 191.6 ± 3.5 32.0 ± 0.9C2 196.6 ± 2.3 35.1 ± 1.0C3 158.2 ± 5.2 21.2 ± 3.1

particle size of EGCG-encapsulated zein/CS NPs began to increasedue to the formation of larger self-assembled nanospheres. As vol-umes of dissolved pure water increased from 30 to 54 ml, particlesize of EGCG-encapsulated zein/CS NPs decreased from 224.5 to155.5 nm (Samples B1, B2 and B3). With the increase of volumeof the solution, the concentration of the solution and particle sizeof EGCG-encapsulated zein/CS NPs were decreased. As amountsof EGCG increased from 2 to 5 mg, particle size ofEGCG-encapsulated zein/CS NPs increased from 191.6 to196.6 nm (Samples C1, C2). However, particle size decreased to158.2 nm as EGCG amount increased to 8 mg (Sample C3). All sam-ples had a small PDI less than 0.3. This indicated that the particlesize of EGCG-encapsulated zein/CS NPs was not as homogenousas other formulations.

As soon as CHC, CMC, EGCG and zein were mixed together in thesolution, the nanoparticles were formed spontaneously with a sig-nificant positive surface charge, which was obtained by the zetapotential measurement (21–35 mV). The nanoparticles exhibiteda small PDI, in the range 0.1 to 0.3. The zeta potential of theEGCG-encapsulated CS NPs was +31 mV (data not shown). Afterbeing coated by zein, the zeta potential of the nanoparticlesbecame highly positive at +35 mV. As zein concentration increasesfrom 72 to 216 mg, the zeta potential of EGCG-encapsulated zein/CS NPs also increased from +31.2 to +34.7 mV (Samples A1, A2 andA3). However, zeta potential was decreased to +33.3 mV, as zeinconcentration increases to 288 mg (Sample A4). As volumes of dis-solving medium increased from 30 to 42 ml, the zeta potential ofEGCG-encapsulated zein/CS NPs increased from +31.6 to 34.9 mV(Samples B1, B2). With the further increase of volume to 54 ml,the zeta potential decreased to +27.5 (Sample B3). As amounts ofEGCG increase from 2 to 5 mg, zeta potential of EGCG-encapsulated zein/CS NPs increased from +32.01 to +35.1 mV(Samples C1, C2). However, zeta potential decreased to +21.2 mVas EGCG amount increases to 8 mg (Sample C3).

3.2. Encapsulation efficiency

Encapsulation efficiency is defined by the percentage of EGCGcarrying on zein/CS NPs. The encapsulation efficiencies of the dif-ferent formulations are shown in Table 2. In the study, the encap-sulation efficiency ranged from 61% to 80%. As zein concentrationincreases from 72 to 288 mg, the encapsulation efficiencyincreased from 65.0 to 80.7% (Samples A1, A2, A3 and A4). As vol-umes of dissolving aqueous medium increase from 30 to 42 ml, theencapsulation efficiency increased from 61.4 to 80.2% (Samples B1,B2). With further increase of the solution volume to 54 ml, theencapsulation efficiency decreased to 65.4% (Sample B3). Asamounts of EGCG increase from 2 to 5 mg, the encapsulation effi-ciency increased from 80 to 78.5% (Samples C1, C2). However,the encapsulation efficiency decreased to 70% as EGCG amountincrease to 8 mg (Sample C3).

V) PDI Encapsulation efficiency

0.23 ± 0.01 65.0% ± 5.30.23 ± 0.01 67.0% ± 8.50.23 ± 0.01 79.1% ± 6.40.22 ± 0.01 80.7% ± 3.50.23 ± 0.11 61.4% ± 11.60.23 ± 0.01 80.2% ± 2.30.19 ± 0.04 65.4% ± 9.00.22 ± 0.01 80.0% ± 10.00.23 ± 0.01 78.5% ± 3.10.09 ± 0.03 70.0% ± 3.0

4000 3500 3000 2500 2000 1500 1000 500

(a)

(b)

(c)

(d)

(e)

3357

3377

3424

34273416

1150

1654

1617

1654

1534

1410

1535

1323

1448

1241

1449

1411

1320 1245

Wavenumbers (cm-1)

Fig. 2. FTIR spectra of single ingredients and their complex samples.

22 J. Liang et al. / Food Chemistry 231 (2017) 19–24

3.3. Morphological observation

The morphological observations of CS NPs and EGCG-encapsulated CS NPs with or without coating zein were performedby TEM after natural drying, and the respective micrographs wereshown in Fig. 1. The size of the CS NPs and EGCG-encapsulated CSNPs exhibited nanospheres with smooth surface (Fig. 1A and B).However, the diameter was not homogenous, varying from 50 to300 nm. As shown in Fig. 1C, the particle size of EGCG-encapsulated zein/CS NPs that were formed as spherical nanopar-ticles with smooth surface and more homogeneous diameter wasaround 100–200 nm (Fig. 1D), compared with two other samples.It could be speculated that the uniform particle size may due tothe outer layer of the zein protein. This observation was consistentwith a previous study (Hong et al., 2014).

3.4. FTIR and XRD characterization

FTIR was applied to characterize the intermolecular interactionsof nanoparticles. The representative spectra of EGCG, zein, CS NPsand EGCG-encapsulated CS NPs coating with or without zein areshown in Fig. 2. The peaks of EGCG at 3357 cm�1 showed theprominent OAH stretching, at 1690–1500 cm�1 and 1220–1000 cm�1 involved C@C stretching and C@O stretching, respec-tively. In the original spectra of zein, the bands of hydrogen bondswere at 3377 cm�1. However, compared with CS NPs, infraredspectra of EGCG-encapsulated CS NPs exhibited a new peak at1150 cm�1, and the characteristic peaks of EGCG were not

Fig. 1. TEM of single ingredients and their complex samples (A: CS NPs; B: EGCG-encapsuof EGCG/CS-zein NPs).

observed. This phenomenon may be due to a peak of a broad bandarising at the range 1700–1550 cm�1 overlapping peaks. It alsoindicates the presence of hydroxyl molecules and amino interac-tion between EGCG and chitosan molecule (Luo et al., 2011). Com-pared with zein and EGCG-encapsulated CS NPs, EGCG-encapsulated zein/CS NPs has some new peaks at 1320 and1245 cm�1 in the infrared spectrograph. This indicates an interac-tion between zein and chitosan molecule. The hydrogen bondpeaks of EGCG-encapsulated CS NPs and EGCG-encapsulated

lated CS NPs; C: zein/CS NPs; D: EGCG-encapsulated zein/CS NPs; E: Size distribution

J. Liang et al. / Food Chemistry 231 (2017) 19–24 23

zein/CS NPs are also in different locations. The peaks of zein andEGCG-encapsulated zein/CS NPs at 1534 and 1535 cm-1 alsoappeared, corresponding typically to carboxyl groups. In addition,zein contains highly hydrophobic molecules, so hydrophobic inter-actions may also contribute to formation of nanoparticles (Lianget al., 2015). These results show that EGCG could be encapsulatedinto zein/CS NPs.

The XRD patterns of EGCG, zein, CS NPs and EGCG-encapsulatedCS NPs coating with or without zein are shown in Fig. 3. The majorcharacteristic peaks of EGCG appeared at 8.41, 11.93, 15.45, 16.88,

Fig. 3. XRD of single ingredients and their complex samples.

Fig. 4. The release rate of EGCG from nanoparticles into fatty food simulants containing 5activity with 50% ethanol (C) or 95% ethanol condition (D).

20.68, 21.37, 24.48, 25.75, 28.57 and 36.89 degree, indicative oftheir highly crystalline nature (Luo et al., 2013). In contrast,EGCG-encapsulated CS NPs showed one flatter peak rather thansharp peaks. Zein, and EGCG-encapsulated zein/CS NPs alsoshowed two flatter peaks. The EGCG specific peaks were notobserved suggesting that EGCG in the nanoparticles did not existin a crystalline form, thereby providing additional evidence ofencapsulation (Uskokovic & Desai, 2014).

3.5. Release profile and antioxidant activity of EGCG from zein/CS NPs

Fig. 4(A and B) display the release profiles of EGCG from CS NPsand zein/CS NPs in food simulants containing 50% or 95% ethanol.The 95% ethanol is regarded by the U.S. Food and Drug Administra-tion (U.S. FDA) as simulant for fats, oil and fatty foods due to itssimilar hydrophobicity. The EGCG amount was determined in anHPLC by aliquots at 30 �C, during 10 days. Results showed thatthe release rate of the EGCG increased from CS NPs coated withzein compared to uncoated ones in 95% ethanol. The release pro-files of the two types nanoparticles in 50% or 95% ethanol exhibitedan initial burst effect which occurred within one day, followed by asustained release over 10 days, with a typical Fick’s curve (Noronhaet al., 2014).

Recent study on catechin release from films demonstrated asimilar pattern of EGCG diffusion at 25 �C with 95% ethanol as sim-ulant. Using 50% ethanol condition, up to 70% release of encapsu-lated EGCG was noted in 24 h from the EGCG-encapsulated CSNPs and EGCG-encapsulated zein/CS NPs. During the following10 days, the amount of EGCG measured was from 60% to 70% inthe EGCG-encapsulated CS NPs and EGCG-encapsulated zein/CSNPs, respectively. The amount of EGCG in the CS NPs was similar

0% ethanol (A) or 95% ethanol condition (B) and its corresponding DPPH scavenging

24 J. Liang et al. / Food Chemistry 231 (2017) 19–24

to that in zein/CS NPs. In 95% ethanol condition, the released levelof EGCG from the EGCG-encapsulated CS NPs was up to 22%. In thecase of zein/CS NPs, EGCG was released up to 32%. During the fol-lowing 10 days, the release amount of EGCG in the CS NPs main-tained at 20–25%, and at 30–35% in the zein/CS NPs. From theseresults, it can be concluded that, in 50% ethanol solution, the ratesof release of EGCG from the CS NPs and zein/CS NPs are the same.However, in 95% ethanol solution, a 32% release of EGCG from thezein/CS NPs indicate that the wall material may show a controlledrelease effect more than the CS NPs. Therefore, the CS NPs coatedwith zein may be used to release bioactives like EGCG in fat andoil foods.

Fig. 4(C and D) shows the antioxidant activities of EGCG-encapsulated CS NPs with and without zein coating. The resultsshowed that DPPH scavenging activity of EGCG-encapsulatedzein/CS NPs was significant. The EGCG-encapsulated CS NPs with-out zein coating showed reasonable radical scavenging activity in50% ethanol solution, but in 95% ethanol solution, they showed rel-atively low scavenging activity. The EGCG-encapsulated zein/CSNPs in 50% ethanol solution have exhibited similar antioxidantactivities to that of the EGCG-encapsulated CS NPs. In contrast,the radical scavenging rate of the EGCG-encapsulated zein/CSNPs in 95% ethanol solution showed a more than 4 fold increasein comparison to the EGCG-encapsulated CS NPs samples. As CSNPs have zein coating, high antioxidant activities can be antici-pated in 95% ethanol solution compared to that of the uncoatedones. The results suggested that zein/CS NPs loaded with EGCGimproved the antioxidant activity of EGCG in 95% ethanol solution.

4. Conclusion

EGCG-encapsulated zein/CS NPs were successfully preparedunder mild conditions. Physicochemical analyses suggested thatelectrostatic interactions and hydrogen bonds are the main forcesin the nano-complex. Upon coating EGCG-encapsulated CS NPswith zein, particle size was dramatically reduced and zeta poten-tial was increased and became highly positive, depending on for-mulation type. Zein coating greatly improved the controlledrelease property of EGCG and the corresponding antioxidant activ-ity in both 50% and 95% ethanol conditions, indicating that EGCG-encapsulated zein/CS NPs could be used as a novel source for EGCGsupplementation in foods, therapeutic treatment or new film coat-ing material. Further studies are required before using this nano-complex as an active material for edible films.

Acknowledgement

This work was supported by National Modern Agriculture Tech-nology System (CARS-23), Anhui Major Demonstration Project forLeading Talent Team on Tea Chemistry and Health, the NationalNatural Science Foundation of China (31301448), National foreignhigh-end experts project (GDW20153400195) and Anhui ProvinceNatural Science Foundation (Grant No. 1508085SMC217 and No.1708085MC86).

References

Cheng, S. Y., Wang, B. J., & Weng, Y. M. (2015). Antioxidant and antimicrobial ediblezein/chitosan composite films fabricated by incorporation of phenoliccompounds and dicarboxylic acids. Lwt-Food Science and Technology, 63(1),115–121.

Hong, Z. Y., Xu, Y. Q., Yin, J. F., Jin, J. C., Jiang, Y. W., & Du, Q. Z. (2014). Improving theeffectiveness of (�)-epigallocatechin gallate (EGCG) against rabbitatherosclerosis by EGCG-loaded nanoparticles prepared from chitosan andpolyaspartic acid. Journal of Agricultural and Food Chemistry, 62(52),12603–12609.

Hurtado-Lopez, P., & Murdan, S. (2006). Zein microspheres as drug/antigen carriers:A study of their degradation and erosion, in the presence and absence ofenzymes. Journal of Microencapsulation, 23(3), 303–314.

Iniguez-Franco, F., Soto-Valdez, H., Peralta, E., Ayala-Zavala, J. F., Auras, R., & Gamez-Meza, N. (2012). Antioxidant activity and diffusion of catechin and epicatechinfrom antioxidant active films made of poly(L-lactic acid). Journal of Agriculturaland Food Chemistry, 60(26), 6515–6523.

Khan, N., & Mukhtar, H. (2007). Tea polyphenols for health promotion. Life Sciences,81(7), 519–533.

Kuzuhara, T., Suganuma, M., & Fujiki, H. (2008). Green tea catechin as a chemicalchaperone in cancer prevention. Cancer Letters, 261(1), 12–20.

Li, K. K., Yin, S. W., Yang, X. Q., Tang, C. H., & Wei, Z. H. (2012). Fabrication andcharacterization of novel antimicrobial films derived from thymol-loaded zein-sodium caseinate (SC) nanoparticles. Journal of Agricultural and Food Chemistry,60(46), 11592–11600.

Liang, J., Li, F., Fang, Y., Yang, W., An, X., Zhao, L., et al. (2010). Response surfacemethodology in the optimization of tea polyphenols-loaded chitosannanoclusters formulations. European Food Research and Technology, 231(6),917–924.

Liang, J., Li, F., Fang, Y., Yang, W., An, X., Zhao, L., et al. (2011). Synthesis,characterization and cytotoxicity studies of chitosan-coated tea polyphenolsnanoparticles. Colloids and Surfaces B-Biointerfaces, 82(2), 297–301.

Liang, H. S., Zhou, B., He, L., An, Y. P., Lin, L. F., Li, Y., et al. (2015). Fabrication of zein/quaternized chitosan nanoparticles for the encapsulation and protection ofcurcumin. RSC Advances, 5(18), 13891–13900.

Luo, Y., Wang, T. T., Teng, Z., Chen, P., Sun, J., & Wang, Q. (2013). Encapsulation ofindole-3-carbinol and 3,30-diindolylmethane in zein/carboxymethyl chitosannanoparticles with controlled release property and improved stability. FoodChemistry, 139(1–4), 224–230.

Luo, Y. C., Zhang, B. C., Cheng, W. H., & Wang, Q. (2010). Preparation,characterization and evaluation of selenite-loaded chitosan/TPP nanoparticleswith or without zein coating. Carbohydrate Polymers, 82(3), 942–951.

Luo, Y. C., Zhang, B. C., Whent, M., Yu, L. L., & Wang, Q. (2011). Preparation andcharacterization of zein/chitosan complex for encapsulation of alpha-tocopherol, and its in vitro controlled release study. Colloids and Surfaces B-Biointerfaces, 85(2), 145–152.

Noronha, C. M., de Carvalho, S. M., Lino, R. C., & Barreto, P. L. M. (2014).Characterization of antioxidant methylcellulose film incorporated with alpha-tocopherol nanocapsules. Food Chemistry, 159, 529–535.

Shpigelman, A., Cohen, Y., & Livney, Y. D. (2012). Thermally-induced beta-lactoglobulin-EGCG nanovehicles: Loading, stability, sensory and digestive-release study. Food Hydrocolloids, 29(1), 57–67.

Siripatrawan, U., & Harte, B. R. (2010). Physical properties and antioxidant activityof an active film from chitosan incorporated with green tea extract. FoodHydrocolloids, 24(8), 770–775.

Tang, D. W., Yu, S. H., Ho, Y. C., Huang, B. Q., Tsai, G. J., Hsieh, H. Y., et al. (2013).Characterization of tea catechins-loaded nanoparticles prepared from chitosanand an edible polypeptide. Food Hydrocolloids, 30(1), 33–41.

Uskokovic, V., & Desai, T. A. (2014). In vitro analysis of nanoparticulatehydroxyapatite/chitosan composites as potential drug delivery platforms forthe sustained release of antibiotics in the treatment of osteomyelitis. Journal ofPharmaceutical Sciences, 103(2), 567–579.

Wu, L. Y., Wen, Q. B., Yang, X. Q., Xu, M. S., & Yin, S. W. (2011). Wettability, surfacemicrostructure and mechanical properties of films based on phosphorusoxychloride-treated zein. Journal of the Science of Food and Agriculture, 91(7),1222–1229.

Zhong, Q. X., & Jin, M. F. (2009). Nanoscalar structures of spray-dried zeinmicrocapsules and in vitro release kinetics of the encapsulated lysozyme asaffected by formulations. Journal of Agricultural and Food Chemistry, 57(9),3886–3894.