Behavior of Zein in Aqueous Ethanol under Atmospheric Pressure

Jul 31, 2017 - Behavior of Zein in Aqueous Ethanol under Atmospheric Pressure Cold Plasma Treatment. Shuang Dong, Jian-ming Wang , Li-min Cheng, ...
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Behaviour of zein in aqueous ethanol under atmospheric pressure cold plasma (ACP) treatment shuang dong, Jian-ming Wang, Li-min Cheng, Yan-li Lu, Shu-hong Li, and Ye Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02205 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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Journal of Agricultural and Food Chemistry

Behaviour of zein in aqueous ethanol under atmospheric pressure cold plasma (ACP) treatment Article Type: Research Paper

KEYWORDS: Atmospheric Cold Plasma; zein solutions; physicochemical properties; secondary structure

Corresponding Author: Jian-ming Wang E-mail addresses: [email protected] Key Laboratory of Food Nutrition and Safety, Ministry of Education; College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China Tel: +86-22-60912398 Fax: +86-22-60912398

Co-corresponding Author: Ye Chen E-mail addresses: [email protected] Key Laboratory of Food Nutrition and Safety, Ministry of Education; College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China Tel: +86-22-60912402 Fax: +86-22-60912402

First Author: Shuang Dong Order of Authors: Shuang Dong, Jian-ming Wang*, Li-min Cheng, Yan-li Lu, Shu-hong Li, Ye Chen*

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Behaviour of zein in aqueous ethanol under atmospheric

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pressure cold plasma (ACP) treatment

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Shuang Dong, Jian-ming Wang*, Li-min Cheng, Yan-li Lu, Shu-hong Li, Ye Chen*

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(Key Laboratory of Food Nutrition and Safety ,Ministry of Education , College of Food Engineering

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and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China)

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ABSTRACT: The effects of atmospheric cold plasma (ACP) on zein in aqueous

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ethanol (80%, v/v) were investigated including particle size distribution, molecular

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structure, and content of free sulfhydryl (free-SH) group and disulfide bond, etc. The

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film-forming properties of zein films were also characterized. After ACP treatment,

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the particle size of zein aggregates showed a remarkable decrease and uniform

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particle distribution. There was a downward trend both in pH value and viscosity with

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the increasing ACP treatment intensity. Moreover, the increase of disulfide bonds

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concentration was suggested to be correlated to the compact structure strengthened by

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crosslinking between zein molecules. It was proved from SEM micrographs that

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plasma could significantly decrease the aggregation degree of zein micelles. There

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was a slight decrease of the peak intensity in UV and fluorescence spectra compared

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with native zein, indicating the bulk structure of zein solution had not been disrupted.

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The reinforced flexibility and tensile strength of zein films had been observed after

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treatment on film-forming solution. This study provided an experimental basis for the

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investigation on behavior of plasma-treated protein in solution.

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KEYWORDS: Atmospheric Cold Plasma; zein solutions; physicochemical properties;

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secondary structure 2

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INTRODUCTION

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As a promising novel food ingredient, zein is the major storage protein from corn

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which is generally regarded as safe “GRAS” status. More than half of the amino acid

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residues in zein are hydrophobic, which make it one of the few proteins that can be

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solubilized in aqueous ethanol (60 ~ 90%, v/v) solutions but not in pure water.1,2 Zein

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forms spherical colloidal nanoparticles in aqueous ethanol which are normally applied

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for controlled and targeted delivery of bioactive components in food, pharmaceutical

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and biotechnological industries.3,4 Additionally, strong intermolecular disulfide bonds

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together with hydrophobic interactions between zein molecules constitute the

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molecular basis of film formation.5,6 However, some inherent defects of natural zein

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limit its application in modern industry, such as weak solubility in water and poor

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mechanical properties of films.7 With the growing demand on products that are

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biodegradable and renewable raw materials, developing methods for better utilization

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of zein becomes meaningful and valuable. Efforts have been made in recent decades

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to modify the functional properties and structure of zein.7-9 In summary, most of these

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studies mainly focused on chemical and biological methods which indeed improved

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the properties of zein effectively, while little attention had been paid to the edibility

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for food safety in consideration of some toxic compounds applied in processes.

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Moderate modification of zein with an alternative technology is in request for current

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food and material industries.

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As a nonhazardous and future processing method, atmospheric cold plasma (ACP)

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is an efficient technology with widespread application in cleaning, sterilization and 3

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surface modification. Cold plasma at atmospheric pressure can be obtained by

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exposing a gas/mixture of gases to an electric field, which in turn accelerates the

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charged particles, leading to collisions with the heavy species (e.g. ions and neutrals).

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Such non-thermal technology has gained great popularity because it avoids the

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undesirable effects generated when heat treatments are applied to food matrices.10

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Moreover, ACP was proved to be an effective modification method without damaging

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bulk properties due to the treatment only affected a few nanometers (10 nm or less)

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below the surface of materials.11-13 In last decades, plasma modification technology

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has been mainly applied on surface treatment for polymer material.14,15 However,

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limited studies were conducted on liquid materials, among which were mostly on

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starch solution and wastewater.16-18 To the best of our knowledge, little information is

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available to identify the effects of plasma treatment on protein in solution. Plasma is

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consisted of positive and negative ions, free radicals, and excited or non-excited

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molecules etc. and these particles possess energies comparable to or exceeding the

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non-covalent bond such as intermolecular forces energies (a few eV) of the protein

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system.19,20 Therefore plasma might be expected to be a potential modification

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technology for zein solution as ACP could induce an alternation in intermolecular

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forces within proteins.

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It was reported that plasma modification could induce the depolymerization in

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polymeric material.21 Therefore, it was supposed that plasma could reduce the degree

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of aggregation and obtain smaller micelles by the breakage of secondary bonds. With

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this regard, we aim to study the effects of ACP treatment on zein solution in aqueous 4

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ethanol, and characterize physicochemical properties of zein solution and films,

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seeking the potential modification mechanism induced by ACP treatment.

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MATERIALS AND METHODS

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Materials

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Zein (regular grade) was purchased from Wako Pure Chemical Industries, Ltd.

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(Osaka, Japan). Alcohol (HPLC grade) was purchased from Sigma-Aldrich (Shanghai,

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China). Other chemicals were reagent grade.

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ACP treatment of zein in aqueous ethanol

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Zein (3%, w/v) was dissolved in 80% (v/v) aqueous ethanol. As our preliminary

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experiments proved, zein showed optimum solubility in 80% ethanol solution which

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was usually used for film preparation as an excellent solvent. Afterwards, zein

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solutions were incubated at 60°C for 10 min for sufficient dissolution and cooled

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down to ambient condition (25 ± 0.1°C). In order to investigate the effect of treatment

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voltage and time on physicochemical properties and structural of zein, zein solution of

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10 mL was placed in the quartz reactor and subjected to plasma treatment under

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different conditions as follows. Voltage group was conducted at fixed treatment

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duration (3 min), in which the voltage was adjusted to 0, 50, 55, 60, 65 and 70 v,

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respectively. Duration group was conducted at fixed voltage (60 v), in which the

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duration was set to 0, 1, 2, 3, 4 and 5min, respectively. All performances were

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conducted with a current of 1.0 ± 0.2 A.

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The schematic of plasma equipment is shown in Figure 1. ACP was conducted 5

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by using a DBD-50 Plasma Reactor (Suman Co., Ltd, China). The two electrodes

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(outer diameter = 50 mm) are covered with dielectric layers (made of quartz) to assure

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the uniform microdischarges. In our treatment, the heat impact on zein has been

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excluded even under the maximum voltage 70 v. After treatment, all samples were

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stored under refrigerated conditions (4°C) for 12 h until further measurements.

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Preparation of zein film

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Zein (10%, w/v) was dispersed in 80% (v/v) ethanol solution and continuously

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stirred at 60°C for 10 min until fully dissolved (10 min). After ACP treatment under

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different conditions, 10 mL film forming solution was casted into a polypropylene

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mould (150 mm × 250 mm) dried at an ambient condition (25 ± 0.1°C) until the

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solvent was completely evaporated. Before further test, zein films were stored in

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desiccator (relative humidity 50%) under 25 ± 0.1°C for equilibration.

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Particle size distribution

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Dynamic Light Scattering (DLS) technique was applied to obtain the information

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of zein particles. Particle distributions, hydrodynamic diameter and specific surface

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area (SSA) of zein particles were measured at 25.0 ± 0.1°C by a laser nano-particle

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size analyzer (BT-90, Baite Co. Ltd, China). Zein solutions (3%, w/v) were filtered

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through a prerinsed (0.2-µm) filter and equilibrated for 5 min before measurement.

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The pH and electric conductivity

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The pH and electric conductivity were determined by pH meter (PB-10, Sartorius

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Scientific Instruments Co., Limited, China) and conductivity meter (DDS-307, Rex

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Instruments Factory, China), respectively. Zein solutions (3%, w/v) were measured at 6

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a constant room temperature of 25 ± 0.1°C. All measurements were performed in

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triplicate.

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Viscosity

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The apparent viscosity vs. shear rate (s-1) of sample was determined by an DV-II

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viscometer (Brookfield, Middleboro, MA) equipped with a cone (1°, 40 mm diameter)

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and plate geometry at 25± 0.1°C. 25 mL of zein solutions (3%, w/v) after

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discriminative ACP treatment conditions was evaluated for each test. The

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determination of viscosity was conducted at a shear rate of 30 ~ 80 s-1 when the

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system stabilized.

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Surface hydrophobicity index (So)

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Surface hydrophobicity of zein solutions (10mg/mL) in 0.01 mol/mL sodium

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phosphate buffer (pH 7) was measured by using 1-anilinonaphthalene-8-sulfonic acid

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(ANS) as a fluorescence probe which was according to the method described by Kato

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and Nakai.22 The fluorescence spectrophotometer (RF-5301, SHIMADZU, Japan)

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was used to obtain the relative fluorescence intensity at the excitation and emission

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wavelengths of 337 and 501 nm with a corresponding slit width of 5 and 3 nm,

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respectively. The So was calculated from the initial slope of the plot of fluorescence

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intensity versus protein concentration.

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Content of free sulfhydryl (free-SH) group and disulfide bond

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Free-SH group concentration in zein solutions was measured consulting to the

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description of Handa et al. 23 2.0 mL Zein solution was diluted with 4.0 mL 0.1

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mol/mL Tris-glycine buffer (pH 8) and 0.04 mL Ellman’s reagent. After incubation at 7

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25°C for 5min, the absorbance was measured by spectrophotometer (756PC UV,

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Ruipu T6 spectrophotometer, China) at 412 nm compared with the blank without

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adding zein. The total sulfhydryl (total-SH) groups content was measured according

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to the description of Beveridge et al. with slight modifications.24 2.0 mL zein solution

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was added by 0.1 mL of β-mercaptoethanol and 4.0 mL of Urea-GuHCl and the

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mixture was incubated for 1.0 h at 25 °C. After an additional 1.0 h incubation with

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10 mL of 12% trichloroacetic acid (TCA), the tubes were centrifuged at 5000 × g for

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10 min. The precipitate was twice resuspended in 5.0 mL 12% TCA and centrifuged

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to remove the β-mercaptoethanol. Afterwards, the precipitate was dissolved in 10 mL

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8 mol/mL urea in Tris-Gly buffer (pH 8.0) and the color was developed with 0.04 mL

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of Ellman’s reagent. Absorbance was measured at 412 nm compared with blank

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sample without zein. The concentration of free-SH and the total-SH groups was

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calculated according to eq.(1) and the disulfide bonds concentration was calculated

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according to eq.(2). SH (

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µmol (73.53 × A 412 × D) )= g c

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Where A412 = absorbance of zein solution or reagent blank, D = dilution multiple,

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C = concentration of protein solution (mg/mL).

SS ( 150

µmol TotalSH − FreeSH )= g 2

(1)

(2)

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Where TotalSH = concentration of total SH groups (µmol/g), FreeSH = concentration of

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free SH groups (µmol/g).

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Scanning electron microscope (SEM) 8

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The microtopography of zein aggregations was characterized by scanning

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electron microscope (SU1510, Hitachi, Japan). Zein solution was dropped on the

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center of the mica and quickly dried in the fume hood. Before scanning, samples were

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sprayed a gold layer for 5min in vacuum and placed in electron microscope with an

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acceleration voltage of 5.0 KV to capture images.

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Ultraviolet Absorption Spectroscopy

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UV absorption spectroscopy is considered as an indicator to provide protein

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structures information. The UV spectra of native zein and ACP treated zein were

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performed by lambda 25 UV/vis Spectrometer (Perkin Elmer, USA) with a scanning

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range of 195 ~ 300 nm at a speed of 200 nm/min.

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Fluorescence spectra

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The emission fluorescence spectroscopic technique was employed to provide

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unique information about the sensitivity of intrinsic fluorescence to the

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microenvironment change around proteins.25 The fluorescence spectra of zein

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solutions

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SHIMADZU, Japan) according to the method of Sun et al.25 The emission spectra

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were collected in the range of 290 ~ 450 nm at a excitation wavelength of 280 nm

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with a scanning speed of 100 nm/min. Both the excitation and emission slit widths

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were 3 nm.

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Fourier Transform Infrared Spectroscopy (FTIR)

were

recorded

by

fluorescence

spectrophotometer

(RF-539/PC,

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The attenuated total reflectance (ATR) mode was chose to characterize the

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plasma treated samples and the spectroscopy was carried out by Thermo Nicolet iS 50 9

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FTIR spectrometer from 4000 to 900 cm-1. The amide I band (1600 -1700 cm-1) was

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further analyzed by Omnic software (version 8.0, Thermo Nicolet Inc, Waltham, MA,

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USA) and Peakfit software (version 4.12, SPSS Inc., Chicago, IL, USA) to calculate

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the secondary structure components of zein.

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Film-forming properties of zein films

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The mechanical property, thermal property and surface hydrophilicity of zein

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films were characterized according to our published method by electronic

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universal-testing machine (Shenzhen Reger Instrument Co., Ltd., China), DSC-600A

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differential scanning calorimetry (Shimadzu Inc., Japan), and JY-82 Video Contact

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Angle Meter (Chengde DingSheng Co., Ltd., China), respectively.26 All values

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conducted at least five replications.

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Statistical analysis

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The statistical analysis was performed using SPSS 18.0 software. The significant

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differences between zein samples were statistically analyzed by Turkeys multiple

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comparison test at 5% significance level. All figures were presented by Origin 8.0

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software.

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RESULTS AND DISCUSSION

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Particle size distribution

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It has been widely reported that zein form aggregates containing small globules

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in aqueous ethanol.27 The particle size distribution, average particle diameter and

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specific surface area (SSA) of zein were given in Figure 2 and Table 1, respectively. 10

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From Figure 2, the typical monomodal particle size distribution was observed for all

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samples. The average diameters varied in a range of 177.16 ± 3.07 nm to 231.25 ±

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2.38 nm (Table 1) which was in good agreement with the published results between

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150 and 550 nm.28 It could be observed that the particle size distribution showed a

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downward tendency to smaller size with the increasing ACP treatment intensity.

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Meanwhile, the distribution curves exhibited narrower and sharper peaks after ACP

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treatment which indicated more uniform particle size of zein aggregations. As shown

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in Table 1, the average diameter of zein micelles decreased significantly (p