Preparation of Gelatin Films Incorporated with Tea Polyphenol

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Preparation of Gelatin Films Incorporated with Tea PolyphenolNanoparticles for Enhancing Controlled Release Antioxidant Properties Fei Liu, John Antoniou, Yue Li , Jiang Yi, Wallace Yokoyama, Jianguo Ma, and Fang Zhong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00003 • Publication Date (Web): 31 Mar 2015 Downloaded from http://pubs.acs.org on April 4, 2015

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Journal of Agricultural and Food Chemistry

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Preparation of Gelatin Films Incorporated with Tea

2

Polyphenol-Nanoparticles for Enhancing Controlled Release Antioxidant

3

Properties

4

Fei Liu,† John Antoniou,† Yue Li,† Jiang Yi,† Wallace Yokoyama,‡ Jianguo Ma,† and

5

Fang Zhong*,†

6 7

†

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of Food Science and Technology, Jiangnan University, Wuxi 214122, P.R. China

9

‡

Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School

Western Regional Research Center, ARS, USDA, Albany, CA 94710, United States

10 11 12

*Corresponding author:

13

Fang Zhong

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Tel: +86-510-85197876, Fax: +86-510-85197876, E-mail: [email protected]

1

ACS Paragon Plus Environment


15 16

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ABSTRACT Gelatin

films

incorporated

with

chitosan

nanoparticles

of

various

17

free/encapsulated tea polyphenols (TP) ratios were prepared in order to investigate the

18

influence of different ratios on physico-chemical and antioxidant properties of films.

19

The TP containing nanoparticles were prepared by cross-linking chitosan

20

hydrochloride (CSH) with sulfobutylether-β-cyclodextrin sodium (SBE-β-CD) at

21

three different encapsulation efficiencies (EE, ~50%, ~80% and ~100%) of TP. The

22

stability of TP-loaded nanoparticles was maintained during film drying process from

23

the analysis of free TP content in the redissolved film solutions. Composite films

24

showed no significant difference in visual aspects while the light transmittance

25

(250-550 nm) was decreased with incorporation of TP. Nanoparticles appeared to be

26

homogeneously dispersed within the film matrix by microstructure analysis (SEM and

27

AFM). TP loaded films had ferric reducing and DPPH radical scavenging power that

28

corresponded to the EEs. Sunflower oil packaged in bags made of gelatin films

29

embedded with nanoparticles of 80% EE showed the best oxidation inhibitory effect,

30

followed by 100% EE, 50% EE and free TP over 6 weeks storage. However, when the

31

gelatin film was placed over the headspace and not in contact with the oil the free TP

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showed the best effect. The results indicate that sustained release of TP in the

33

contacting surface can ensure the protective effects which vary with free/encapsulated

34

mass ratios, thus improving antioxidant activities instead of increasing the dosage.

35 36

KEYWORDS:

Gelatin

film,

tea

polyphenols,

nanoparticles, oil, oxidation inhibition 2


cyclodextrin,

chitosan

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INTRODUCTION

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Lipid oxidation or oxidative rancidity, besides microbial growth, is the main

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cause of food spoilage particularly for food rich in polyunsaturated fatty acids.1,2 In

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order to prevent oxidation and extend shelf life, synthetic antioxidants like BHA, BHT,

41

TBHQ and PG have been widely used as food additives for decades. With the concern

42

of undesirable side effects and potential risks, the search for natural antioxidants as

43

alternatives to synthetic antioxidants is therefore of increasing attention in food

44

applications recently.

45

Direct addition of antioxidants into food may encounter the limitation that once

46

the activity of antioxidants was neutralized by the reaction with food compounds, the

47

protection ceases and the quality of the food degrades at an increased rate.1 Since the

48

lipid oxidation process are generally induced from the surface of foodstuffs, many

49

efforts have been made to incorporate antioxidants into food packaging/edible film to

50

control the oxidation of food product,3-6 with the expection that antioxidants released

51

by a controlled diffusion to the food can retard the oxidation and extend the shelf life.7

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Natural antioxidants such as resveratrol,8 curcuma ethanol extracts,9 and green tea

53

extracts or tea polyphenols (TP)6,10 have been successfully incorporated to fabricate

54

antioxidant active packaging or edible films. According to Gómez-Estaca, et al.1, one

55

of the valid solutions for slowing down the releasing process of active compounds in

56

films is the inclusion of antioxidants in the matrix of nanoparticles that increase the

57

tortuosity of the diffusion path. Furthermore, the problem that antioxidants may loss

58

activity with time thus eliminating the antioxidant activity of films remains despite 3



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that the hydrophilic antioxidants (like TP) can be incorporated directly into films.10

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Nano-encapsulation before being incorporated in the film matrix can also protect TP

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or other active substances from premature reactions with oxygen and light.

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Tea catechins, the main compounds in TP can form inclusion complexes with

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β-cyclodextrin and the complex can maintain the antioxidant capacity of tea

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catechins.11 Chitosan nanoparticles could be formulated based on the ionic gelation

65

between chitosan and sulfobutylether-β-cyclodextrin (SBE-β-CD),12 allowing an

66

efficient encapsulation of TP. Although gelatin films incorporated with TP-loaded

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chitosan-tripolyphosphate nanoparticles have been studied before by our research

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group,13 there was no direct comparison between the encapsulated TP and the free TP

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in the film. Free TP would be homogenously distributed in the film whereas the

70

encapsulated TP would be slowly released from nanoparticles to regions of film

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without antioxidants. Moreover, it has been reported that incorporation of nisin in the

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form of a nano-emulsion (half free and half encapsulated) showed the best results

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against food pathogens when compared to completely encapsulated (100%) and free

74

nisin.14 The migration of some free TP to the film surface may be required in order to

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inhibit the initial oxidation, and perhaps both free TP and encapsulated TP within the

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film matrix are necessary to guarantee long term oxidative stability. For TP chitosan

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nanoparticles-incorporated films, it is still not known what differences in antioxidant

78

properties would be presented when different free/nano-encapsulated active agent

79

ratios are applied.

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In addition to carrying and protecting bioactive compounds, nanoparticles such 4


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as blank chitosan nanoparticles can improve the barrier, thermal and mechanical

82

properties of films such as those based on hydroxypropyl methylcellulose (HPMC),15

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starch,16 tara gum17 and gelatin.18 Among them, the addition of chitosan nanoparticles

84

with smaller particle sizes resulted in stronger mechanical properties and better barrier

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properties as observed by de Moura, et al.15 in HPMC films. Therefore the inclusion

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of antioxidants in chitosan nanoparticles into polymer matrices might bring a twofold

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advantage: the performance improvement of food packaging/edible film material and

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the provision of an additional antioxidant function.19

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In this study, TP was encapsulated in the matrix of CSH and SBE-β-CD

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nanoparticles and the TP containing nanoparticles were added into gelatin films. As a

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natural antioxidant, TP is subject to oxidation. In order to determine if encapsulation

92

improved antioxidative lifetime of TP and protected it from degradation, gelatin films

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were also prepared embedded with free TP. The objective of this study was to

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investigate the antioxidant activities of gelatin films incorporated with free TP and

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TP-loaded

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encapsulation efficiencies.

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MATERIALS AND METHODS

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Materials. Gelatin (type B from bovine skin) and glycerol were purchased from

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China Medicine (Group) Shanghai Chemical Reagent Co. (Shanghai, China). CSH,

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molecular weight 100 kDa and degree of deacetylation 86%, derived from crab shells

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was obtained from Golden-Shell Biochemical Co., Ltd. (Hangzhou, China).

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SBE-β-CD (food grade), molecular weight 2.082 kDa and an average degree of

CSH-SBE-β-CD

nanoparticle

(CSN)

suspensions

5


with

different


103

substitution 6, was purchased from Kunshan Chemical Industries Co., Ltd. (Jiangsu,

104

China). TP, polyphenol content ≥98% and catechins ≥90% (determined by

105

manufacturer), was obtained from Qiangsheng Medicine Science Technology Co., Ltd.

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(Shanghai, China). 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from

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Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were analytical grade.

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Preparation of CSNs. CSNs were prepared according to a previously reported

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method,20 with minor modifications. Briefly, 24 mL of 2, 4 or 8 mg/mL SBE-β-CD in

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distilled water was added drop-wise to 72 mL of 0.5, 1 or 2 mg/mL CSH aqueous

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solutions, respectively, while under vigorous magnetic stirring leading to the

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formation of nanoparticles via the ionic gelation mechanism. The mass ratios of

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CSH/SBE-β-CD were controlled at 3:4 (w/w) in all cases. TP-loaded CSNs were

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prepared by adding an aqueous TP solution to the SBE-β-CD solution to obtain a

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concentration of 1 mg TP/mL and stirred for 24 h at room temperature to promote

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inclusion complex formation. The TP-SBE-β-CD solution was combined with the

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CSH solution. The nanoparticle suspensions were immediately analysed or used in

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film forming. All TP loaded films contained the same total amount of TP.

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Characterization of CSNs. The z-average particle size and size distribution of the

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nanoparticle suspensions were determined by dynamic light scattering (DLS,

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Zeta-PALS + BI-90Plus, Brookhaven Instrument Co., Holtsville, NY) at a fixed

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scattering angle of 90° at 25 ± 1 °C. All measurements were run in quadruplicate. The

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morphology of nanoparticles was examined by a high-performance digital imaging

124

transmission electron microscope (TEM, JEOL 2100, Hitachi High-Technologies 6


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125

Corporation, Tokyo, Japan). The TEM samples were prepared by placing one drop of

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the suspension on a copper grid and staining with 2% (w/v) phosphotungstic acid, and

127

air drying. Once dried, the sample was imaged by the microscope with an accelerating

128

voltage of 100 kV.

129

Encapsulation Efficiency of TP loaded-CSNs. The encapsulation efficiency (EE) of

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TP in CSNs was determined according to Hu, et al.21 Briefly, TP-loaded nanoparticles

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were carefully transferred into an AmiconUltra-4 centrifugal filter device (Millipore

132

Co., Billerica, MA) with a 10,000 MW cut-off Ultracel membrane. After

133

centrifugation at 4000 × g for 10 min, the concentration of TP in the ultrafiltrate was

134

determined by the Folin-Ciocalteu method. Prior to measurement, the filtrate was

135

5-fold diluted with distilled water. The total content of TP was calculated from the TP

136

concentration in the aqueous medium before CSNs formation. The EE of TP was

137

calculated using the Formula (1):

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EE (%) = ((Total Content of TP – Content of TP in Ultrafiltrate)

139

/Total Content of TP) × 100

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Preparation of Films. The gelatin films were prepared by our previously reported

141

method with some modifications.22 Briefly, 8% (w/v) gelatin solutions were prepared

142

by hydrating gelatin powders in distilled water for 1 h at room temperature and then

143

dissolved by heating at 65 °C with continuous stirring. Glycerol (1.6%) was added as

144

a plasticizer in order to make the films less brittle and easier to handle. The

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composition of the gelatin films including films with TP-loaded nanoparticles

146

formulations are shown in Table 1. The 8% gelatin-1.6% glycerol solutions (25 mL)

(1)

7



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were diluted with water (75 mL) or nanoparticle suspensions to 2% (w/v) gelatin in all

148

film-forming solutions. A homogeneous solution was achieved after stirring for 1 h.

149

Films were prepared by casting the 2% gelatin solutions (35 mL) in square Petri

150

dishes (10 cm × 10 cm) and drying at 25 °C in an oven to a constant weight

151

(approximately 36 h). The films were peeled off and conditioned at 25 °C and 53%

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relative humidity (RH) for at least 48 hours in a controlled environmental chamber

153

(Shengxing Experimental Equipment Co., Ltd. Shanghai, China) before testing.

154

Total Phenol Assay. The total phenolic content of films was estimated by the

155

Folin-Ciocalteu method according to Jouki, et al.23 Initially cut conditioned films (2.5

156

cm × 2.5 cm) were weighed and redissolved in 10 mL of distilled water at 40 °C for 1

157

minute, and centrifuged (10000 x g, 10 min) at 20 °C. Supernatant (1 mL) and

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distilled water (1 mL) were thoroughly mixed with Folin-Ciocalteu reagent (5 mL, 10%

159

v/v). After 6 min, 4 mL of 7.5% (w/v) sodium carbonate was added. The mixture was

160

stirred thoroughly and allowed to stand at room temperature for 60 min prior to

161

recording the absorbance at 765 nm in a spectrophotometer (Shimadzu UV-2450,

162

Japan). The concentration of total phenol was expressed as mg gallic acid equivalent/g

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film sample according to the following Formula (2):

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Total Phenolic Content = (C × V )/M

165

(2)

where C is the concentration of gallic acid obtained from the standard curve

166

(mg/mL), V is the volume of the supernatant (mL) and M is the weight of film (g).

167

Characterization of Films.

8


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Visual Appearance and Light Transmission. The film-forming solutions and films

169

were evaluated visually for uniform color and the presence of insoluble particles. The

170

ultraviolet and visible light transmittance properties of the films were measured

171

according to the method of Bitencourt, et al.9 Film samples of 10 x 25 mm were

172

placed in the light path of a spectrophotometer (UV-2450, Shimadzu, Tokyo, Japan).

173

Transmittance was recorded from 200 to 800 nm in triplicate.

174

Microstructure. Cross-section morphology of films was analyzed using a field

175

emission scanning electron microscope (SEM, S-4800, Hitachi, Japan). Films were

176

cryofractured by immersion in liquid nitrogen and conditioned in a desiccator at 25 °C

177

before measurement. Samples were mounted on the specimen holder using

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double-sided adhesive tapes and then sputter coated with gold under vacuum. The

179

samples were scanned with an accelerating voltage of 1 kV.

180

An atomic force microscope (AFM, Bruker Dimension Icon, Bruker Co.,

181

Germany) was used to observe surface morphology of films in tapping mode, and 50

182

µm × 50 µm images under ambient conditions were obtained. All samples were

183

analyzed in triplicate using a Bruker Nanoscope software (Version 1.40) to calculate

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the roughness values. The obtained parameters were: Ra, which is the arithmetic

185

average of the absolute values of the surface height deviations (Z) measured from the

186

mean plane and Rq, which is the root mean square average of height deviations taken

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from the mean image data plane. Ra and Rq were calculated from the following

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Formulas (3) & (4):

9



1

N ∑j=1 Zj

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189

Ra =

190

=

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Determination of Antioxidant Activities In Vitro. In vitro antioxidant properties of

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films were performed based on the procedure reported by Wu, et al.10 The supernatant

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was prepared as stated in Total Phenol Assay part and was collected for further

194

analyses including ferric reducing power and DPPH radical scavenging activity.

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Ferric Reducing Power. The determination of reducing power was measured as

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described by Li, et al.24 with slight modifications. Briefly, 1.0 mL of redissolved film

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solution was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL

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potassium ferricyanide (1%, w/v). After incubating at 50 °C for 20 min and rapidly

199

cooling, 2.5 mL of trichloroacetic acid (10%, w/v) was added to the mixture followed

200

by centrifugation at 10000 x g for 10 min. The obtained supernatant (2.5 mL) was

201

treated with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride (w/v). The

202

absorbance of the reaction mixture was record at 700 nm (Shimadzu UV-2450, Japan)

203

after 10 min reaction. Higher absorbance indicates higher reducing power.

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DPPH Radical Scavenging Activity. The method described by Yi, et al.25 was used

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to evaluate the DPPH radical scavenging activity, with slight modifications. Two mL

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of film supernatant was mixed with 2.5 mL (0.1 mM) of DPPH dissolved in ethanol.

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The mixture was kept in dark for 30 min at room temperature followed by

208

centrifugation at 8000 x g for 5 min. The absorbance was measured at 517 nm. The

209

assay was carried out in triplicate. The DPPH radical scavenging activity was

210

calculated as follows (5):

N

(3)

∑

(4)

10


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DPPH Scavenging % =100 × (1- (As - Ab )/Ac )

(5)

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where As represents the absorbance of sample after film supernatant was added,

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Ab the absorbance of film supernatant alone and Ac the absorbance of control as

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distilled water was mixed with DPPH solution instead of sample.

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POV and TBARS of Sunflower Oil in Contact with or without Touching Films.

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Two methods were used to evaluate the antioxidant properties of films in contact with

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oil or above the headspace of the oil. The antioxidant properties of the films in contact

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with the oil was determined by the method of Bao, et al.13 The films were transformed

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into 4 cm x 4 cm bags by heat sealing and 1 mL of fresh sunflower oil was added to

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the bags. The antioxidant properties of the films placed over the headspace of the oil

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was determined according to Jongjareonrak, et al.4 and Wu, et al.26 10 mL of fresh

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sunflower oil was poured into a glass cell (30 mm diameter × 50 mm height) and

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covered with the film (4 cm × 4cm) sealed with glue and parafilm. All samples were

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stored in a humidity controlled chamber (53 ± 2% RH) at 28 °C. Samples for peroxide

225

value (POV) and thiobarbituric acid reactive substances (TBARS) analysis were

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collected at intervals of 1 week over a period of 6 weeks. Samples without film

227

packaging were also assessed as control.

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POV Analysis. POV was determined by AOCS Cd 8-53 Official Method.27 The

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amount of oil sample analyzed varied and depended on its oxidation degree. The oil

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sample was dissolved in 50 mL of a mixture of acetic acid/chloroform (3:2 v/v) within

231

a 250 mL Erlenmeyer flask. Then saturated KI solution (0.5 mL) was added and the

232

flask was allowed to stand for 1 min with occasional agitation. After addition of 11



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distilled water (50 mL), the mixture was titrated against sodium thiosulfate (0.01 M)

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with starch as an indicator. A blank titration was treated the same but without

235

containing oil. POV (meq of oxygen/kg) was calculated using the following Formula

236

(6):

237

POV = 1000 × S × c/m

(6)

238

In this formula, S is the volume of sodium thiosulfate solution (blank corrected)

239

in mL; c is the normality of sodium thiosulfate solution and m is the weight of oil

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sample in grams.

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TBARS Analysis. TBARS were detected using AOCS Cd 19-90 method.28 The oil

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sample (50-200 mg) was solubilized in 10 mL of 1-butanol, mixed with 10 mL of 0.2%

243

TBA in 1-butanol, heated at a 95 °C water bath for 2 h and rapidly cooled in an ice

244

bath. The absorbance was measured at 532 nm against a corresponding blank

245

(reaction with all the reagents and treatments except the oil). The standard curve was

246

calculated from the TBARS reaction of a series of aliquots (0.1-1 mL) of 0.2 mM 1, 1,

247

3, 3-tetra-ethoxypropane (95%) prepared in 1-butanol. The results were expressed as

248

µmol malonaldehyde (MDA)/g of oil (n=3).

249

Statistical Analysis. Data were presented as mean value ± standard deviation. The

250

data were analyzed by one-way analysis of variance (ANOVA) using the SPSS 19.0

251

package (IBM, New York). Duncan’s-multiple range test was used to determine the

252

significant differences of the mean values (P

Preparation of Gelatin Films Incorporated with Tea Polyphenol

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