Fabrication and Characterization of Novel Water-Insoluble Protein

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Food and Beverage Chemistry/Biochemistry

Fabrication and Characterization of Novel Water-Insoluble Protein Porous Materials Derived from Pickering High Internal Phase Emulsions (HIPEs) Stabilized by Gliadin/Chitosan Complex Particles Fu-Zhen Zhou, Xin-Hao Yu, Tao Zeng, Shou-Wei Yin, Chuan-He Tang, and Xiaoquan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00221 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Agricultural and Food Chemistry

Fabrication and Characterization of Novel Water-Insoluble Protein Porous Materials Derived from Pickering High Internal Phase Emulsions (HIPEs) Stabilized by Gliadin/Chitosan Complex Particles Fu-Zhen Zhou†, Xin-Hao Yu†, Tao Zeng†, Shou-Wei Yin†, ‡*, Chuan-He Tang†, Xiao-Quan Yang†

†

Research and Development Center of Food Proteins, School of Food Science and Engineering; ‡Guangdong

Province Key Laboratory for Green Processing of Natural Products and Product

Safety, South China University of Technology, Guangzhou 510640, P.R. China.

Running title: Development of Pickering HIPEs and porous materials * Corresponding author Yin, S. W. Phone: +86-2087114262. Fax: (+86)-20-87114263. E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Pickering high internal phase emulsions (HIPEs) and porous materials derived from the Pickering

3

HIPEs have received increased attention in various research fields. Nevertheless, non-degradable

4

inorganic or synthetic stabilizers present toxicity risks thus greatly limit their wider applications. In

5

this work, we successfully developed nontoxic porous materials through the Pickering HIPEs

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templating process without chemical reactions. The obtained porous materials exhibited appreciable

7

absorption capacity to corn oil and reached the state of saturated absorption within 3 min. The

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Pickering HIPEs templates were stabilized by gliadin/chitosan complex particles (GCCPs), in which

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the volume fraction of the dispersed phase (90%) was highest in all reported food-grade particles

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stabilized-Pickering HIPEs so far, further contributing to the interconnected pore structure and high

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porosity (> 90%) of porous materials. The interfacial particle barrier (Pickering mechanism) and

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three-dimensional network formed by GCCPs in the continuous phase play crucial roles in

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stabilization of HIPEs with viscoelastic and self-supporting attributes, also facilitating development

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of porous materials with designed pore structure. These materials with favorable biocompatibility

15

and biodegradability possess charming application prospects in foods, pharmaceuticals, materials,

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environments and so on.

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Key words: Pickering high internal phase emulsions, Porous materials, Emulsion templating.

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INTRODUCTION

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Nowadays, Pickering emulsions stabilized by solid particles have received tremendous attention

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in such fields as foods, pharmaceuticals, materials, environments and so on.1-5 The interfacial

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particle layer contributes to its high resistance to Oswald ripening and coalescence as well as other

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unique characteristics and advantages compared to conventional emulsions, i.e., unparalleled

24

stability. Pickering emulsions with the volume fraction of the internal phase higher than 74% are

25

usually referred as Pickering high internal phase emulsions (HIPEs). In conventional HIPEs,

26

considerable fractions of surfactants (5–50% v/v) are required to stabilize the dispersed phase.6

27

Compared to which, surfactant-free Pickering HIPEs usually result in optimized performance of the

28

final products, e.g., avoiding the side effects of surfactants including potential environmental

29

impacts and irritations.7,8 Regrettably, Pickering emulsions tend to phase inversion or oiling-off as

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the internal fraction increasing to a threshold.9-12 Although, a wide range of inorganic particles have

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been proven to be efficient stabilizers of Pickering HIPEs, such as silica particles, titania

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nanoparticles, microgel particles,1,3,12 non-degradable colloidal particles also present toxicity risks

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and greatly limit their wider applications. Comparatively, sparse food grade stabilizers that meet the

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trend of clean labels have been developed for Pickering HIPEs up to now. On a positive note, there

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are still valuable breakthroughs, for instance, HIPEs stabilized by food-grade particles have been

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developed, e.g., cellulose nanocrystals,7 gelatin particle,13 gliadin/chitosan hybrid particles,14

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peanut-protein-isolate microgel particles15. Biological particles-stabilized Pickering HIPEs with

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favorable biosecurity and biocompatibility can be used as advantageous templates or structural

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elements for the preparation of functional materials (e.g., highly porous materials) with various

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applications, which shows good development foreground. Up to now, few works focus on the



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development of natural and nontoxic materials originated from these Pickering HIPEs.

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Recently, porous materials with large surface areas and interconnected pore structure are

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particularly appealing to applications in sorption, microelectronics, biomedical devices, membrane

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processes, catalysis, packaging, and separation science.16-19 Moreover, high internal phase emulsion

45

(HIPE) templating is an attractive route toward well-defined porous materials.20,21 And, stable

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HIPEs usually help to produce porous materials with high porosities and pore interconnections.

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Furthermore, Pickering HIPEs possess many advantages relative to the traditional ones, thus they

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are strong candidates for the preparation of macroporous materials, e.g., particles layer could carry

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functional groups facilitating various further applications. Particularly, polymerization reactions are

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customarily adopted to achieve the target when porous materials with designed structure were

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prepared through the Pickering HIPE templating approach, which known as PolyHIPE. In addition,

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polymerization in dispersed phase usually lead to closed cell polymers with poor

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interconnectivity,22-25 while polymerization in continuous phase result in interconnected and highly

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porous materials after removing the dispersed phase and water.26-28 However, precise control of

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these chemical reactions is also a challenge, not to mention, these reactions or reactants themselves

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with potential toxicity or environment problems are undesirable in applications under certain

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circumstances, e.g., foods require satisfied safety and non-toxicity and scaffolds require favorable

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biocompatibility and biodegradability. Therefore, it is highly desirable to prepare porous materials

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with universal applications through the Pickering HIPE templating process. Although Jiao et al.15

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produced porous materials using peanut-protein-isolate microgel particles stabilized Pickering

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HIPEs as a template, but their work mainly focused on the preparation, little was known about the

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properties and functions of materials. Thus, porous materials with well-defined structure and high


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porosity originated from the food grade particles-stabilized Pickering HIPEs have not been

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systematically reported yet.

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Gliadin is an amphiphilic prolamin-type storage protein in wheat, it features high fraction of

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proline and amidated acidic amino acid residues.29 The same as the other prolamine, it is able to

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self-assemble to form various nano-/micro-structures for drug or bioactive delivery.30-31 In our

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previous research works,14,32-33 gliadin-stabilized Pickering HIPEs with edible oil as internal phase

69

have been successfully produced and the oil fraction was 83%. If the internal phase is easy to

70

remove (e.g., hexane), it is possible to obtain porous materials with well-defined pore structure by

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removing the oil and water, which has wide application prospect in industry but has not been well

72

developed yet. In this work, we demonstrate an attractive strategy to fabricate gliadin-stabilized

73

Pickering HIPEs with hexane fraction as high as 90% within one step, and for the first time, we use

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HIPEs as templates to prepare biodegradable and biocompatible porous materials with well-defined

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pore structure and high porosity by simple freeze-drying, their application as oil absorbents are also

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explored.

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MATERIAL AND METHODS

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Materials. Gluten was bought from Fengqiu Hua Feng powder Co., Ltd (Fengqiu, Henan, China).

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Commercial corn oil was provided by Yihai Kerry group (Guangzhou, China). Chitosan from crab

80

shells (Product 48165), Fluorescent dyes including Nile Red and Rhodamine B solution (0.2% in

81

isopropanol) was obtained from Sigma–Aldrich, Inc. (St. Louis, MO, USA). Other chemicals used

82

were of analytical grade.

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Gliadin Extraction. Gliadin was extracted from gluten powders following our previous

84

method.34 Gluten powders (50 g) were dispersed in 350 mL of ethanol together with 150 mL of



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deionized water at 25 °C for 120 min. Then, the mixture was centrifuged at 8500 rpm for 0.5 h to

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collect supernatant abundant in gliadin. Next, the collections were dialyzed at 4 °C successively

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against deionized water, 0.05 M acetic acid, and deionized water. Gliadin powder was obtained by

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freeze-drying the dialysate.

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Gliadin/Chitosan Complex Particles (GCCPs) Synthesis. GCCPs were prepared following our

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previous method.14 Simply, gliadin (1.25 g) was dissolved in 50 mL of 70% ethanol solution, and

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1.0 g of chitosan was added into 100 ml of 1% acetic acid solution and stirred for 12h. Then, gliadin

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solution (40 mL) was dropwise added into chitosan dispersions (100 mL) under shearing (6000

93

rpm) for 240 s using a T25 homogenizer (IkA, Germany). Next, the dispersion went through a

94

rotary evaporation (RV 10, IkA, Germany) until the volume of the GCCPs dispersions to an ideal

95

value, and gliadin concentrations in GCCPs dispersions were 0.1, 0.5, 1.0, 2.0, and 3.0% (w/w),

96

respectively. Finally, NaOH solution was used adjust pHs of GCCPs dispersions to 5.0.

97

Preparation of Pickering HIPEs. Effect of GCCPs Concentrations. In this part, a fixed oil

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fraction of 80% and a series of GCCPs mentioned-above at pH 5.0 were used to fabricate HIPEs. In

99

brief, hexane (4 mL) was admixed with GCCPs (1 mL) in a glass vial, then sheared using an

100

Ultraturax T10 homogenizer (Janke & Kunkel, Germany) at 20000 rpm for 120 s to form the

101

Pickering HIPEs.

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Effect of Oil Phase Fractions. In this series, the concentrations of GCCPs were 2.0 %, and the

103

volume fractions of hexane were 70%, 80%, and 90% respectively. Pickering emulsions (GCCPEs)

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stabilized by GCCPs were prepared following the above-mentioned operation.

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Droplet Size Distribution of Pickering HIPEs. Particle size distributions of fresh Pickering

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HIPEs were determined by a Malvern Mastersizer 3000 instrument (Malvern Instruments Ltd.,


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Worcestershire, UK) pumping at 2000 rpm. The refractive index of water and hexane were 1.330

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and 1.375, respectively. Surface mean diameter (D3,2) and volume-mean diameter (D4,3) were

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reported.

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Dynamic Oscillatory Measurements. Viscoelastic properties of HIPEs were evaluated by a

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HAAKE RS600 Rheometer (Karlsruhe, Germany) through small amplitude oscillatory

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measurements at 25 °C. Precisely, &O/&P were determined under amplitude and frequency sweep

113

when stress ranged from 0.05 to 100 Pa (frequency = 1Hz) and frequency ranged from 0.05 to 10

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Hz (stress = 1 Pa, within the linear viscoelastic region).

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Confocal Laser Scanning Microscopy (CLSM). Microstructure of Pickering HIPEs was

116

characterized by CLSM technique according to our previous protocol,34 using a TCS SP5 confocal

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microscopy (Leica Microsystems Inc., Germany). GCCPs in Pickering HIPEs were dyed by

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Rhodamine B solution (0.2% in isopropanol). Next, 15 L of colored HIPEs was placed on a groove

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of microscope slide, then covered with glycerol-coated coverslip. The fluorescent dyes in the

120

Pickering HIPEs were excited at 543 nm, the emission signal was collected between 558 and 708

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nm.

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Preparation of Porous Hybrid Materials. The gliadin/chitosan hybrid porous materials

123

(PM-HIPEs) were obtained through solvent evaporation from O/W Pickering HIPEs. Briefly,

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HIPEs prior prepared according to the above-mentioned procedure was poured into a petri dish, and

125

sealed with protective film, then pre-freezing at -40 °C for 12 h, finally, it was freeze-dried for 24 h

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to remove water and hexane preparing porous materials.

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Characterization of Porous Materials. Pore microstructures of the porous materials were

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examined using a Merlin Field emission scanning electron microscope (FE-SEM, Zeiss,



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Oberkochen, German) operating at 10 kV. Lyophilized samples were gently spread on the

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conductive adhesive, before observation, samples were sputtered with a thin layer of gold. The pore

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size and distribution of freeze-dried porous materials were determined by Mercury intrusion method

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using an AutoPore

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U.S.A.). The analysis was performed at pressures range from 0 to 414 MPa, and corresponding pore

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radius range from 3 nm to 1000 S 9 To find a correspondence between intrusion pressures and pore

135

radius values, it was assumed that pores were cylindrical in the calculations, and a surface tension

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value for Hg of 0.485 N/m and contact angle of 130° were used in the Laplace equation.

9510 Hg intrusion porosimeter (micromeritics Instrument Corp, Norcross,

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Oil Absorption by Porous Materials. Firstly, a certain amount of deionized water and excessive

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volume of maize oil were put into a beaker and weighed. Notably, for convenience of observation,

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corn oil was stained by Nile Red in advance. Next, porous samples were immersed in oil until

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equilibrium state was reached at 25 °C, and then samples were took out, the weight of beaker was

141

measured again. The mass absorption capacity (Mabs) was calculated by the following equation:35 =(

142

1

3)

2

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where W1 and W3 was the weight of the beaker before (1) and after (3) absorption, W2 was the

144

weight of samples.

145

Statistics. Statistical analyses were performed using an analysis of variance (ANOVA)

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procedure of the SPSS 13.0 statistical analysis program, and the differences between means of the

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trials were detected by Tukey test (P < 0.05).

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RESULTS AND DISCUSSION

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Development and Appearance of Pickering HIPEs. In this part, Pickering HIPEs or GCCPEs

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were developed using GCCPs as particulate stabilizer for hexane droplet. To clearly investigate the


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emulsifying capacity of GCCPs, GCCPEs were produced at different internal phase (hexane)

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fractions. The photographs of 2% GCCPs-stabilized GCCPEs with hexane fractions ranged from 70

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to 95% are shown in Fig. 1A. Obviously, stable GCCPEs were obtained as the hexane ratios varied

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from 70 to 90% and remained stable after 1 week of storage (Fig. 1B), which proved the successful

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development of Pickering HIPEs stabilized by GCCPs with internal phase up to 90% through a

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one-step preparation. Herein, we first report HIPEs stabilized by GCCPs, in which the fraction of

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the internal phase (hexane) as high as 90%, close to or even higher than the other previous

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studies.13-14,32 Expectedly, GCCPEs underwent distinct creaming and presented a lower water layer,

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also possessed flow character when hexane fractions were 70%, but self-standing in the inverted

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bottle while hexane fractions between 80% and 90% showed a semisolid state, which exhibiting

161

remarkable stability against coalescence as well as creaming. Quite apart from that, HIPEs were

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evenly dispersed in the water but remained intact in the oil (Figure 1S) confirming phase inversion

163

phenomenon did not occurred. In other words, GCCPs are able to stabilize O/W Pickering HIPEs,

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and endowing HIPEs with good stability and self-supporting attribute. Unfortunately, increasing

165

hexane volume factions to 95 %, however, led to complete phase separation (Fig. 1A) due to the

166

fact that emulsifying capacity of GCCPs (2%) had been exceeded.

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To illuminate the effect of particle concentration on HIPEs stability, five representatives of

168

concentration of GCCPs at pH 5.0 were selected to prepare Pickering HIPEs. Visual appearances of

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GCCPs-stabilized Pickering HIPEs with hexane fraction of 80% as a function of particle

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concentrations are shown in Fig. 2A. When GCCPs concentration was 0.1 %, complete

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destabilization of the HIPEs occurred, while the fractions of GCCPs at levels higher than 0.5%,

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homogeneous and stable O/W HIPEs were formed. For 0.5%, maybe, adsorbed GCCPs at interface



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and non-adsorbed GCCPs in aqueous phase were not enough to form a firm three-dimensional

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network surrounding hexane droplet,36 finally caused to the flow of HIPEs in the inverted bottles.

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Gel-like HIPEs clearly showed self-standing properties (Fig. 2B), which confirmed the

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effectiveness of GCCPs as a solid emulsifier to stabilize Pickering HIPEs.

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Droplet size. To investigate the fluctuation of drop size and size distribution in HIPEs while

178

varying hexane fraction and GCCPs concentrations, HIPEs were subjected to size determination

179

using a Malvern Mastersizer, the results are shown in Fig. 3. When GCCPs concentration was 2%,

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there was no significant difference in droplet size, D4,3 of HIPEs with internal phase fractions of

181

70%, 80% and 90% were around 30 S

182

observed in HIPEs with 70% and 80% hexane, while a bimodal distribution having two peaks

183

around 30 and 3.5 S

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diameter (D) is closely related to the oil volume fraction ( V) and area per unit volume (A/V), that

185

is, D = 6 VV/A. At fixed hexane fraction (80%), GCCPs concentration rose from 0.5 to 3.0% lead

186

to a drastic decrease of HIPEs droplet size (Fig. 3B), volume-mean diameter decreased from 43.7 to

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22.4 S

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concentration of GCCPs at 0.5 to 3.0% were falling into size-decaying regime,39 in which the

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interfacial area increase linearly with respect to the particle content and the droplet diameter would

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be inversely proportional to the particle content. These results were coincided with geometrical

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relationship mentioned-above and with findings of Binks et al.,40 Frelichowska et al.

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previous studies.14

(Table 1). Monomodal droplet size distributions were

has appeared at 90%. Based on the geometrical relationship,37,38 the droplet

(Table 1), which was also captured by optical microscope ((Fig. 2S). In expectation,

38

and our

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Rheology of GCCPEs. It is believed that rheological properties have an effect on physical

194

property of the emulsions. To investigate rheology, the GCCPEs with varied hexane fraction and


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GCCPs concentrations were subjected to oscillatory stress and frequency sweep, results are shown

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in Fig. 4. From the picture, before elastic moduli (G') intersect with the corresponding viscous

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moduli (G''), G' were always larger than G'' for all samples, which indicated GCCPEs developed

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exhibiting elasticity-dominated attributes or solid-like property. The crossover points of G' and G''

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during oscillatory stress sweep are usually regarded as rearrangements of structure due to higher

200

stress applied. Higher value of stress at crossover point under test conditions revealed stronger

201

structure of GCCPEs formed at higher GCCPs content or internal phase. While stress continue to

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increase, the value of G'' started exceeded that of G', which indicated viscoelastic solution behavior

203

of GCCPEs. In addition, G' and G'' increased gradually along with GCCPs concentration or hexane

204

fraction at a given stress or frequency, meaning more solid-like or improved in stiffness of GCCPEs

205

at higher GCCPs content or hexane ratio. Obviously, the values of G' were larger than that of G''

206

across the entire frequency range, once again confirmed that increased GCCPs concentration or

207

hexane fraction lead to strengthening solid-like properties. These results were in accordance with

208

appearance of GCCPEs in Fig. 1 and Fig. 2, in which clearly showed HIPEs at 0.5% GCCPs or

209

GCCPEs with internal phase fractions of 70% were in a flow state. According to Dickinson et al.,41

210

the behavior of aqueous continuous phase plays a dominated role in rheology of emulsion with well

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dispersed droplets, and low viscosity aqueous phase in O/W emulsion contribute to liquid-like

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behavior. Maybe, more GCCPs increased the viscosity of water phase to a certain extent due to the

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interactions between gliadin, chitosan and H2O molecule, thus produced GCCPEs with better

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viscoelasticity. And in higher internal phase, compressed packing of droplets leading to a deformed

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shape, meanwhile, formed trapped network structure around the oil drop, facilitating to viscoelastic

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properties of gel-like emulsion. Experiment data from rheological measurements intuitively proves



217

the viscoelastic and self-supporting attributes of GCCPEs prepared, also verified visual appearance

218

shown in Fig. 1 and Fig. 2.

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CLSM. To clearly characterize the microstructure of GCCPEs, better understand the formation

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and stabilization mechanism of HIPEs, further linking the microstructural features to physical

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properties, CLSM technique was used in this section. And CLSM images of the GCCPEs with

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varied hexane fraction and GCCPs concentrations are shown in Fig. 1C and Fig. 2C. In GCCPEs

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with hexane fraction of 70%, droplets with almost perfect round shape were well dispersed and

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sparsely scattered in the field of vision. The average size of which were about 25 S + this result was

225

corroborated by size determination. Interestingly, as hexane fraction increased to 80%, droplets

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became crowded but still keep the round, similar phenomenon in HIPEs stabilized by gelatin and

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gelatin nanoparticles observed by Tan et al. 42 Whereas, deformed shape resulting from compressed

228

packing were observed as hexane fraction up to 90%. Cheerily, red color of GCCPs dispersed

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around black hexane droplet approved again that HIPEs developed were still O/W type and did not

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inverse phase into W/O type even as hexane fraction up to 90%. The CLSM pictures clearly showed

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a downtrend of droplet size as concentration of GCCPs increased (Fig. 2C). Data of size

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determination (Table 1) and microscopy images (Fig. 2S) also supported it. Furthermore, stronger

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particles signals were observed at interface and in continuous phase of the GCCPEs prepared at

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higher GCCPs content. According to previous studies,22,38,42 at hexane fraction of 80%, more

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GCCPs have more chances to adsorb at interface stabilizing larger surface area, thus decreasing the

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droplet size. It is crucial to regulate droplet size of GCCPEs, because the porous materials with

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well-defined pore structure can be obtained using GCCPEs as a template, which has become an

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attractive way nowadays.20,21 Notably, the envelope shell of GCCPs around the hexane droplets


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provided an interfacial mechanical barrier against coalescence. Meanwhile, these adjacent droplets

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were closely packed with each other which were trapped into the three-dimensional network that

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formed by GCCPs in the water phase. The so-called Pickering mechanism and network were

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conducive to the stabilization of GCCPEs, also crucial for the viscoelastic and self-supporting

243

attributes.

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Characterization of the porous materials. In our previous research,14,32 antioxidant Pickering

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HIPEs stabilized by gliadin/chitosan hybrid particles with internal phase (corn oil) of 83% were

246

successfully developed, which can be a comparable and healthy alternative of PHOs. In contrast,

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this paper puts special emphases on developing porous hybrid materials, which were prepared from

248

the high or medium internal phase emulsion templates (referred as PM-HIPEs or PM-MIPEs).

249

Precisely, we obtained porous materials without any chemical reactions merely by removing hexane

250

and water through directly lyophilizing GCCPEs using a freeze dryer. Photographs of PM-HIPEs

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are shown in Fig. 5. Visibly, in the actual appearance of porous material, it appears to be a lump of

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white loose foams, and even distribution of dense macroporous structure at surface was vaguely

253

observed from the magnification image. Therefore, FE-SEM was used to clearly capture the

254

micromorphology of porous material and images are shown in Fig. 6. Macroporous materials with

255

opened-cell and highly interconnected porous structure were formed under trial conditions as

256

GCCPs content ranged from 0.5 to 3.0%, which may result from the insufficient coverage of the

257

droplets.22 Moreover, many small voids on the pore walls were also captured, which may due to the

258

evaporation of small hexane and water droplets. Anyhow, porous hybrid materials with highly

259

interconnected and opened-cell structure has been successfully developed, which validated the

260

feasibility of using GCCPs-stabilized Pickering HIPEs as a compatible template for fabricating



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highly porous foams. Additionally, the microstructural features of PM-HIPEs were highly

262

dependent on the concentration of GCCPs, which play vital roles on the formation of mechanical

263

barrier at interface and 3D network in continuous phase. Compared to CLSM images shown in Fig.

264

2, most section of the porous network structure in PM-HIPEs had collapsed as GCCPs content were

265

0.5 and 1.0%, while they are almost preserved completely after freeze-drying at higher GCCPs

266

content (Fig. 6) since sufficient GCCPs formed firmer network, but at low GCCPs content, the thin

267

particle layers between the adjacent droplets cannot resist against the coalescence during

268

lyophilization,43 which may be the cause of these results. Interestingly, they are all opened and

269

interconnected voids in PM-HIPEs at GCCPs content of 2.0%, whereas, when concentration of

270

GCCPs increased to 3.0%, deformed pore structure appeared in the field of vision, maybe,

271

excessive GCCPs leaded to stronger interactions forming thicker GCCPs barrier around the hexane

272

droplets, which on the contrary increased difficulties in removing of solvent as well as forming

273

interconnecting pores by in situ.25 In order to balance the skeletal and pore structure, HIPEs

274

stabilized by 2% GCCPs with varied hexane fraction were prepared for further research. Turning to

275

the microstructure, SEM images in Fig. 6 showed clear-cut distinctions in PM-HIPEs when the

276

hexane fractions were increased from 70% to 90%: increase of void, the thinning in pore walls from

277

membrane shape into linear skeleton, moreover, high fraction of hexane resulted in the collapse of

278

the void walls. In comparison to the size of hexane droplets in the HIPEs (Fig. 3, Table 1), the

279

corresponding pore size showed by mercury data (Fig. 7) was much smaller. According to Fig. 7A,

280

pores in HIPEs with hexane fractions varied from 70 to 90% were mostly in the range of radius

281

0.015-10 S + meanwhile, a much smaller distribution of pore size ranging from about 10-100 S

282

was also observed. The average pores diameters in PM-HIPEs with 70%, 80% and 90% of the


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internal phase were 1.73, 1.39 and 1.65 S

(Fig. 7B), respectively. Unexpectedly, the raise of

284

hexane fraction did not contribute to upgrading of porosity (about 90%) and shift of the pore

285

diameter to larger sizes. Certainly, water droplets in HIPEs had participated in the formation of

286

pores during freeze-drying procedure.

287

Performance of PM-HIPEs oil absorbents. PM-HIPEs with high porosity and multi-scale pore

288

size shows charming prospects in biomedical, microelectronic, catalytic, environmental applications

289

et al. For instance, oil spills and leakage usually result in serious environmental problems, it is

290

urgent to develop novel high-performance adsorption materials for cleaning up oils. Porous

291

materials were found to be a promising candidate for oil-absorbent applications. Therefore, in this

292

work, 3D ordered macroporous materials with interconnected pores and high porosity were used as

293

an absorbent of oil. Herein, we selected porous hybrid material prepared from the HIPE with

294

hexane fraction of 80% and GCCPs concentration of 2.0% as a representative sample, and tested its

295

absorption capacities for corn oil which was used as a model spilled oil. As shown in Fig. 8,

296

PM-HIPEs absorbed oil from water–oil mixtures quickly and easily, and the PM-HIPEs were

297

floating on the surface of water after reached a state of saturated absorption within 3 min. The

298

absorption capacity of the PM-HIPEs was calculated by dividing the weight of corn oil absorbed in

299

PM-HIPEs by the dry weight of the PM-HIPEs, and surprisingly, the value of which was as high as

300

89.50 g/g, it was close to or even higher than those of oil absorbents or xerogels/organogels

301

reported in the literature.35,44-45 There are many pivotal factors helped to materialize the excellent

302

absorption capacity of the PM-HIPEs, such as 3D interconnected pores with large size, high

303

porosity (90.8%). And certainly, these porous materials can be used widely in industry, e.g., in

304

quick adsorption of plant essential oil from raw materials, in cleanup of oil spills or chemical



305

leakage avoiding environmental pollution.

306

In conclusion. In this work, we successfully fabricated Pickering HIPEs stabilized by GCCPs

307

with internal phase as high as 90%. Notably, the envelope shell of GCCPs around the hexane

308

droplets providing an interfacial mechanical barrier against coalescence, meanwhile, these adjacent

309

droplets were closely packed with each other trapping into the three-dimensional network formed

310

by GCCPs in the water phase, the so-called Pickering mechanism and network were contributed to

311

the stabilization of HIPEs with viscoelastic and self-supporting attributes, also crucial for the

312

structure of porous materials. In addition, for the first time, nontoxic porous hybrid materials with

313

interconnected pore structure and high porosity (>90%) were obtained without any chemical

314

reactions through simple freeze-dried process using GCCPEs as templates. Moreover, PM-HIPEs

315

were used as a typical oil absorbent and displayed remarkable absorption capacity. There is no

316

doubt that these Pickering HIPEs and PM-HIPEs originated from food-grade materials have bright

317

future for applications in foods, pharmaceuticals, materials, environments and so on.

318

ASSOCIATED CONTENT

319

Supporting Information. The Supporting Information is available free of charge. The dispersion

320

of the HIPEs in the oil and water, microscopy images of GCCPs-stabilized HIPEs.

321

AUTHOR INFORMATION

322

Corresponding Author

323

*Phone: (+86)-20-87114262. Fax: (+86)-20-87114263. Email: [email protected].

324

ORCID

325

Shou-Wei Yin: 0000-0001-7373-6316

326

Chuan-He Tang: 0000-0002-8769-2040


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327


ACKNOWLEDGEMENTS

328

This work was supported by The Project granted by the National Natural Science Foundation of

329

China (31471628). We also appreciate the financial support by the Fundamental Research Funds for

330

the Central Universities (SCUT, 2017ZD080).

331

Notes

332

The authors declare no competing financial interest.

333

ABBREVIATIONS

334

HIPEs, high internal phase emulsions; GCCPs, gliadin/chitosan complex particles; GCCPEs,

335

emulsions stabilized by gliadin/chitosan complex particles; CLSM, confocal laser scanning

336

microscopy; PM-HIPEs, porous materials originated from high internal phase emulsions;

337

PM-MIPEs, porous materials originated from medium internal phase emulsions; PM-GCCPEs,

338

porous materials originated from emulsions stabilized by gliadin/chitosan complex particles;

339

FE-SEM, field emission scanning electron microscope;

340

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452 453 454 455 456 457 458


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459

Figure captions

460

Figure 1. Photographs of Pickering emulsions stabilized by 2.0% GCCPs. Panel A: effect of oil

461

phase fraction. Panel B: after 1 week of storage. Panel C: CLSM images of GCCPEs stabilized by

462

2.0% GCCPs with hexane fraction of a: 70%, b: 80%, c: 90%, GCCPs were dyed by Rhodamine B

463

solution and excited at 543 nm.

464

Figure 2. Photographs of HIPEs stabilized by GCCPs at fixed hexane fraction of 80%, Panel A:

465

Effect of particle concentration. Panel B: after 1 week of storage. Panel C: CLSM images of HIPEs

466

with varied GCCPs content: 0.5% (a), 1.0% (b), 2.0% (c), 3.0% (d). GCCPs were dyed by

467

Rhodamine B solution and excited at 543 nm.

468

Figure 3. Particle size distribution of GCCPEs as a function of hexane fraction (A) and of particle

469

concentrations (B).

470

Figure 4. The stress (A, C) and frequency (B, D) dependence of the storage modulus "&O# and loss

471

modulus "&P# of Pickering emulsions as a function of hexane fraction (C, D) and of particle

472

concentrations (A, B).

473

Figure 5. Photographs of porous materials prepared from the HIPE templates, b is the

474

magnification image of a.

475

Figure 6. Visual appearance (A) and SEM images of porous materials prepared from the HIPE

476

templates. PM-HIPEs at fixed hexane fraction of 80% with varied GCCPs content: 0.5% (B), 1.0%

477

(C), 2.0% (G), 3.0% (D); PM-GCCPEs stabilized by 2.0% GCCPs with hexane fraction of E: 70%,

478

G: 80%, F: 90%, and G1 is the magnification image of G.

479

Figure 7. Data for PM-HIPEs at 2.0% GCCPs with varied hexane fraction measured by mercury

480

intrusion porosimeter: Pore size distributions (A), average pore diameter and porosity (B).



481

Figure 8. Absorption and separation of corn oil from water by the porous materials. The corn oil

482

was dyed with Nile Red.

483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502


Page 24 of 34

Page 25 of 34


Table 1 The droplet size of GCCPEs

503

GCCPs concentrations (%)

2.0%

internal phase fractions (%)

D3,2 "S #

70

27.4±0.3

22.7±0.3

80

25.7±0.4

21.5±0.4

90

30.6±0.4

22.5±0.4

43.7±0.3

30.3±0.3

27.8±0.3

23.3±0.3

22.4±0.3

18.3±0.3

0.5% 1.0%

D4,3 "S #

80

3.0% 504 505 506 507




Page 26 of 34

Page 27 of 34




Figure 3


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Figure 4



Figure 5


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Figure 7


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Figure 8

Oil Water




Page 34 of 34

Fabrication and Characterization of Novel Water-Insoluble Protein

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