Fabrication and Characterization of Quinoa Protein Nanoparticle

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Food and Beverage Chemistry/Biochemistry

Fabrication and Characterization of Quinoa Protein NanoparticleStabilized Food-Grade Pickering Emulsions with Ultrasound Treatment: Effect of Ionic Strength on the Freeze-thaw Stability Xin-Sheng Qin, Zhigang Luo, Xichun Peng, Xuanxuan Lu, and Yu-Xiao Zou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02407 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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Journal of Agricultural and Food Chemistry

Fabrication and Characterization of Quinoa Protein Nanoparticle-Stabilized Food-Grade Pickering Emulsions with Ultrasound Treatment: Effect of Ionic Strength on the Freeze-thaw Stability

Xin-Sheng Qin,† Zhi-Gang Luo,*, †,‡,§ Xi-Chun Peng,∥ Xuan-Xuan Lu,⊥ and Yu-Xiao Zou*,#

†

School of Food Science and Engineering, South China University of Technology, Guangzhou

510640, China ‡

Food Nutrition and Human Health Overseas Expertise Introduction Center for Discipline

Innovation (111 Center), Guangzhou 510640, China §

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product

Safety, South China University of Technology, Guangzhou 510640, China ∥

Department of Food Science and Engineering, College of Science and Engineering, Jinan

University, Guangzhou 510632, China ⊥

Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Rd.,

New Brunswick, NJ 08901, USA #

Sericultural & Agri-Food Research Institude Gaas, Guangdong Academy of Agricultural

Sciences, Guangzhou 510610, China

1

ACS Paragon Plus Environment


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ABSTRACT: The development of multilayered interfacial engineering on the emulsion

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freeze-thaw properties has recently attracted widespread attention, because of the essential

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freeze-thaw storage process in some emulsion-matrix food products. In this research, we

4

studied the role of salt concentration on the freeze-thaw properties of quinoa protein (QPI)

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nanoparticles stabilized Pickering emulsions. The QPI nanoparticles (particle concentration

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c=2%, w/v) with increasing particle size and surface hydrophobicity (H0) were fabricated by

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ultrasound treatment at 100 W for 20 min, by varying the NaCl addition (salt concentrations,

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0-500 mM). The sonicated QPI nanoparticles with increasing salt concentrations showed

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higher β-sheet structure contents and stronger hydrophobic interactions, which were attributed

10

to the decreasing charged groups and particle aggregation by electrostatic interactions.

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Compared with the sonicated QPI nanoparticles stabilized Pickering emulsions (c=2%, oil

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fraction φ=0.5) without salt accretion, the emulsions with salt accretion exhibited better

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freeze-thaw properties after three times freeze-thaw circulations, which might be mainly

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caused by the generation of gel-like three-dimensional structure and multilayered network at

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the droplets interface with smaller droplet sizes. Increasing the salt concentration

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progressively enhanced the freeze-thaw properties of sonicated QPI nanoparticles stabilized

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Pickering emulsions probably due to the inhibit formation of ice crystal by the “salting-out”

18

effects. The results of this study would provide great significance to investigate the role of salt

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concentration in the freeze-thaw properties of protein stabilized Pickering emulsions.

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KEYWORDS: quinoa protein, Pickering emulsions, interaction force, salt concentration,

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freeze-thaw properties 2


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Introduction

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Recently, there is an increasing prospect in fabrication and properties of food-grade

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granules-stabilized Pickering emulsions in the food and pharmaceutical fields, by reason of

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their promising development to delivery and encapsulation of bioactive ingredients.1–5 Protein

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nanoparticles show excellent interfacial adsorption and emulsifying properties in

27

emulsion-based products. In addition, the particles have favorable nutrition, function and well

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compatible with environment.6 Quinoa protein (QPI) exhibits comprehensive variety of

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essential amino acids which extracted from quinoa products.7 Our previous work found that

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the QPI nanoparticles possess favorable interfacial packing and adsorption in Pickering

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emulsion as a food-grade stabilizer.8

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Freeze-thaw properties is a crucial peculiarity for some emulsion-matrix food products

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that ask for frozen before to storage and transportation, e.g. mayonnaise, some beverages and

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sauces.9 The freeze-thaw properties of emulsion-matrix foods depended on their component

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(such as lipids, thickener, salts, cryoprotectants and emulsifiers) and machining course

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(refrigeration, reposit, homogenization and thawing methods).9 When emulsion products are

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refrigeration, the oil and water portions could crystallize, and influence the properties and

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stability of the refrigerated emulsion. When the refrigerated emulsions are thawed, they

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usually destabilized in oil separation, aggregation and coacervation. The instability of

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freeze-thaw emulsions comes from two reasons: refrigeration and melt of oil and water

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portions, and remarkable variations in salt concentrations, pH, seepage pressure and

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viscoelasticity.10 A few tactics applying interfacial assembly project have been certified to 3



43

enhance the freeze-thaw properties of emulsion. For example, the Pickering emulsions might

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possess outstanding freeze-thaw properties, since the composition of multilayered structure at

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the interface of droplets would provide a remarkable steric repulsion towards coalescence.11

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Numerous studies have investigated the freeze-thaw properties of Pickering emulsions.

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Marefati et al.12 found that the modified starch particles stabilized Pickering emulsion showed

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favorable freeze-thaw properties. This could be attributed to the composition of a strong and

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dense tier of granules between droplets against coalescence. In addition, Xu et al.13 obtained

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that the soybean protein hydrolysate stabilized emulsions emulsified with additional

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microcrystalline cellulose also showed improved freeze-thaw properties. This could be related

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to the steric repulsive stabilization between the droplets. Recently, Zhu et al.14 studied the role

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of protein particle volume (c) or oil fraction (φ) on the freeze-thaw properties of the soy (SPI)

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and/or whey protein particles stabilized emulsions at a constant salt strength (300 mM). The

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consequences indicated that the reinforcement of freeze-thaw properties was closely connected

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to the strengthening of gel-like three-dimensional structure. Although the protein-stabilized

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Pickering emulsion, as affected by the c, φ, pH and emulsification process has been well

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recognized, however, it still remains uncertain how the salt concentration affects the Pickering

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emulsions freeze-thaw properties.

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The presence of salts could generate a restrain role on the aqueous crystallization in the

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course of the refrigeration procedure, which could be beneficial to the freeze-thaw properties

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of O/W Pickering emulsions.9 Liu and Tang15 confirmed that the NaCl addition (0-500 mM)

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observably facilitated the emulsification properties and interfacial adsorption of heated soy 4


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glycinin nanoparticle-stabilized Pickering emulsion by electrostatic screening. The

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improvement was mainly related to the improved movement and packing at the surface of oil

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droplets.16 Therefore, it is logical to speculate that the freeze-thaw properties of the

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protein-stabilized emulsion may be enhanced by the salt electrostatic screening effects. On the

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other hand, our recent work has confirmed that as contrast with the essential QPI nanoparticles

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stabilized Pickering emulsions, the QPI nanoparticles with ultrasound pretreatment stabilized

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Pickering emulsion showed preferable emulsifying and interfacial characterizations, thus

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maintaining a superior stability of Pickering emulsions.8

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Based on the above consideration, the current study was to determine the role of salt

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concentrations (NaCl, 0-500 mM) on the stability and freeze-thaw properties of the QPI

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nanoparticles with ultrasound pretreatment stabilized food-grade Pickering emulsions.

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Primarily, the role of salt concentrations variation (NaCl, 0-500 mM) on the sonicated QPI

76

nanoparticles properties (prepared at c=2%) was represented in ζ-potential, particle size

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distribution, secondary structure, interactive forces of tertiary and quaternary structure, and

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surface hydrophobicity. Then, the role of salt concentrations on the dynamic rheological

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viscoelasticity of the fresh Pickering emulsions (0 days), prepared at c=2% (w/v) and φ=0.5,

80

besides their freeze-thaw properties, was represented by droplet size, visual observations and

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microstructure. Finally, to ascertain whether the salt concentrations played a crucial effect in

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the freeze-thaw properties of sonicated QPI-stabilized Pickering emulsion, the ice crystal

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generation and thawing of these resultant emulsions were determined employing differential

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scanning calorimetry measurement. 5



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Materials and Methods

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Materials. Quinoa protein (QPI, ≥ 80% protein content) was provided by Yuanye Co., Ltd.,

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Shanghai, China. Corn oil was purchased from Yihai Kerry limited, China. Glycine, KBr,

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ANS, Tris, DTNB, TCA and Na2EDTA were obtained from Aladdin Industrial Corporation,

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Shanghai, China. Potassium sorbate was obtained from Kemiou Co., Ltd., Tianjin, China. All

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chemical agentia were analytically pure.

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Fabrication of Sonicated Quinoa Protein Nanoparticle Solutions. The QPI solutions

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(c=2%, w/v) were fabricated by directly dissolving QPI grains in distilled water with constant

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stirring at 25 °C overnight for proteins hydration. Potassium sorbate (formed at 0.01%, w/v)

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was appended as the antibacterial solvent. The pH of QPI solutions was modulated to 7.0

95

using 1 M NaOH solutions. Then, the salt concentrations of QPI solutions were regulated to

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100, 200, 300, 400, 500 mM using NaCl solutions. And the blank control sample was

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conducted without NaCl addition. Finally, the QPI solutions were treated with KH-250DE

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ultrasonic machine (Kunshan Ultrasonic, Co., Ltd., Jiangsu, China) at 100 W for 20 min prior

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to further particle characterization.

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Properties of Sonicated Quinoa Protein Particles. Particle Size. The particle size Dz

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(z-average diameters) of sonicated QPI particles was analyzed by dynamic light scatting

102

technique utilizing a Malvern Nano-ZS Zetasizer.17 The QPI nanoparticle solutions were

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thinned in distilled water after kept at 25 °C for complete protein hydration. All samples of

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refractive index were setting at 1.33.

6


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ζ-potential. ζ-potentials of sonicated QPI nanoparticles were determined utilizing a Malvern

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Nano-ZS Zetasizer, in connection with a MPT-2 multifunctional testplate.17 A 1 mL sample of

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each thinned specimen was titrated into a test DTS 1060C electrophoresis cell at 25 °C.

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Fourier Transform Infrared Spectroscope (FTIR) Test. The FTIR spectrum of the sonicated

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QPI nanoparticles were measured employing a 70 FTIR spectrometer (Bruker Daltonics Inc.,

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Germany) attached with a deuterated triglycine sulphate (DTGS) detector employing the

111

method discussed by Qin et al.18 A 1–2 mm slice was molded using a pressure device

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including 1 mg of lyophilized powders and moderate KBr reagent. Totally 64 cycles were

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carried from 4000 to 400 cm−1 at the speed of per step 4 cm−1 resolution ratio. The secondary

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structural contents variations of sonicated QPI nanoparticles in the 1600-1700 cm-1 amide I

115

band were simulated employing Omnic 8.2 and PeakFit 4.0 analysis software. The protein

116

secondary structure were analyzed after deal with infrared spectrum (1600-1700 cm-1) by

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baseline correction, Gaussian deconvolution, second derivative fitting, and then calculated the

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proportion of secondary structure according to each peak area.

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Surface Hydrophobicity (H0). H0 of sonicated QPI nanoparticles was explored using the

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ANS fluorescence mark on the basis of the technique of Qin et al.19 The protein concentration

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was thinned to the scope of 0.1–0.0005 mg/mL with 0.01 M pH 7.0 phosphate buffers (PBS)

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according to Bradford method. After that, a 30 µL aliquot of ANS solutions (8.0 mM in PBS)

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was appended to every thinned solution (3 mL) and stored for 15 min in the dark. The relative

124

fluorescence

intensity

(RFI)

was

explored

using

7


F-7000

Hitachi

fluorescence


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spectrophotometer at constant excitation wavelength values of 390 nm and emission

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wavelength values of 470 nm. H0 was figured as the slope function of the RFI as the c values.

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Test of Sulfhydryl perssad (SH) and Disulfide Bond (SS) Quantities. The SH bonds and SS

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bonds quantities of sonicated QPI nanoparticles were explored according to Ellman’s reaction.20

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For free SH content (SHF) determination, samples (0.1 g) were dispersed in 5 mL of pH 8.0

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Tris-glycine buffer (including Tris, glycine, and Na2EDTA) containing 8 M urea. Next, 0.05 mL

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of Ellman’s agent (DTNB in Tris-glycine buffer, w/v) was appended, and absorbance was

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explored utilizing a 722SP visible spectrophotometer at 412 nm (Lengguang Co, Ltd., Shanghai,

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China) after stored at 25 °C for 15 min. For total SH quantity (SHT) determination, 0.1 g of the

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lyophilized powders was appended to β-ME (mercapto ethanol) solutions (0.02 mL) and

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Tris-glycine buffer including 10 M urea solutions (1 mL). The dispersion was stored at 25 °C for

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1 h. After another 1 h of reaction with TCA solution (10 mL, 12%). And the dispersion was

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centrifuged at 3000 rpm for 5 min. The sediment was washed and centrifuged two times by 12%

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TCA solution. The final sediment was dispersed in Tris-glycine buffer including 8 M urea (3

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mL). Next, Ellman’s agent (0.03 mL) was appended, and absorbance was detected at wavelength

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412 nm after stored at 25 °C for 8 min. The pure Tris-glycine buffer was used as a comparative

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group. The quantities of SHF, SHT and SS were calculated by the formulas as shown below:

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SH (µmol/g protein) = A412 × D/C × 73.53

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SS (µmol/g protein) = (SHF – SHT)/2

144 145

where C is the QPI contents, A412 is the 412 nm absorbance values, and 73.53=106/(1.36 × 104) (1.36 × 104 is the molar extinction coefficient). 8


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Fabrication of Fresh QPI-Stabilized Pickering Emulsion. All the sonicated QPI

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nanoparticles stabilized Pickering emulsions at increasing salt concentrations of 0-500 mM,

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were fabricated at constant c (2%, w/v) and φ (0.5) values. Firstly, the corn oil phase and QPI

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nanoparticles aqueous phase were operated with an IKA T25 Ultra Turrax homogenizer at

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22000 rpm for 5 min. Next, the rough emulsion samples were homogenized through a high

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pressure homogenizer (Guangzhou juneng nano & bio technology, China) for five cycles at 80

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MPa.

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Freeze-thaw Circulation. Each freeze-thaw circulation of emulsions including a

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refrigerating course at -20 °C for 24 h in a freezer, and a melting course at 30 °C for 2 h in an

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oven till all the refrigeration aqueous and oil phase were absolutely melted. After that, the

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emulsions were return to refrigeration for another circulation of freeze-thaw treatment

157

mentioned above, till repeat three times circulation.

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Characterization and Stability of Fresh and Freeze-thawed Pickering Emulsions.

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Rheological Measurements. The rheological properties of QPI nanoparticles stabilized fresh

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Pickering emulsions (0 day) with various salt concentrations (NaCl, 0-500 mM) were

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determined using a HAAKE RheoStress 600 models (Thermo Fisher Scientific, U.S.A.) with 1

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mm gap and 40 mm diameter parallel board at room temperature.21 For the flow properties

163

measurement, the steady shear viscous properties of the Pickering emulsions was conducted

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using shear rate from 0.5 to 300 s-1. For the dynamic oscillatory measurement, a temperature

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sweep mode was performed at identical 0.05% strain and an increasing frequency (0.1-100

166

Hz). The G′ values (storage modulus) and G′′ values (loss modulus) were observed. 9



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Droplet Size of Emulsions. Droplet sizes of QPI nanoparticles stabilized fresh and

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freeze-thawed Pickering emulsion were determined by 2000 Malvern Mastersizer analyzer.

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The refractive values of 1.095 and 1.333 were employed as proposed for dispersed emulsion

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phase and water phase, respectively. The volume-mean diameter (D43) values were recorded.

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Optical Microscopy of Emulsions. The microstructures of QPI nanoparticles stabilized fresh

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and freeze-thawed Pickering emulsions were evaluated employing an Olympus BH2 light

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microscope equipped with a digital recorder.22 A small amount Pickering emulsion was put on

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a glass slice with a cover glass. The prepared samples were incubated at room temperature

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with cedar wood oil at the magnification of 500-fold.

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Differential Scanning Calorimetry (DSC). The thermal behavior (ice crystal generation

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and thawing) of QPI nanoparticles stabilized fresh and freeze-thawed Pickering emulsions was

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determined employing an 8000 DSC Instruments (PerkinElmer, U.S.A.).23 Accurately 10 mg

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of emulsions was weighed in aluminum lipid vessel. Setting temperatures were reduced from

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40 to -40 °C with a 10 °C /min varied rate, and then rose to 40 °C at the same rate. A sealed

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empty pan was used as control.

182 183

Statistics. The results recorded as the mean values of five times with variance difference. The statistical analyses were explored using ANOVA model of SPSS software version 19.0.

184 185 186 187 10


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Results and Discussion

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Fabrication and Properties of Sonicated Quinoa Protein Nanoparticles. Particle

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Sizes and ζ-potentials. Figure 1 shows the particle sizes (z-average diameters, Dz) and

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ζ-potentials of sonicated QPI nanoparticles at varying salt concentrations (0-500 mM).

192

Compared with the control sample without salt accretion, the Dz values of samples with

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varying salt concentrations were relatively higher, which ranged from 120.6 to 169.8 nm. The

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results confirmed that the electrostatic screening with salt accretion facilitated the protein

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aggregation with greater sizes by “salting-out” effects. However, it can be still observed that

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the changing tendency of the Dz was highly relied on the salt concentrations. Firstly, the Dz

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remarkably increased from 120.6 to 169.8 nm with the increasing salt concentrations from 0 to

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200 mM. The progressive increase in Dz indicated the improved inter-particle interaction of

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the proteins at low salt concentrations. The progressive decrease in Dz with salt concentrations

200

increasing from 200 to 500 mM might be related to the formation of smaller aggregates of the

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sonicated QPI nanoparticles at higher salt concentrations. Xu et al.16 also found that the

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smaller aggregates of soy protein particles gathered together to form larger aggregates with

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monomodal particle size distribution at 100 mM salt concentrations.

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Furthermore, salt accretion significantly decreased the magnitude of ζ-potentials of

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sonicated QPI nanoparticles. The variation of ζ-potentials values could by reason of the

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differences in density and number of charged perssad on the surface of QPI protein particles.

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In fact, the reduction of ζ-potentials revealed that the salt appended to the QPI solution phase

208

can decrease the density of electrostatic screening.24 Thus, the decrease of charged groups 11



209

resulting in protein particles aggregation or association. These consequences were in

210

accordance with the particle size measurement.

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Secondary Structure. FTIR measurement was applied to illustrate secondary structural

212

content changes of protein configuration. The FTIR spectrum (4000-400 cm-1) of sonicated

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QPI nanoparticles with different salt concentrations is displayed in Figure 2. The amide I

214

region, which is attributed to the C=O tensile oscillation of polypeptide bonds, generally

215

applied to calculate protein secondary structure contents. The absorption peaks were observed

216

for the secondary structure, such as 1610–1640 and 1670–1690 cm−1 wavenumbers for

217

β-sheets; 1660–1670 and 1690–1700 cm−1 wavenumbers for β-turns; 1650–1660 cm−1

218

wavenumbers for α-helices, and 1640–1650 cm−1 wavenumbers for random coils.19 The

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proportions of secondary structure components of sonicated QPI nanoparticles with different

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salt concentrations are summarized in Table 1. With increasing salt concentrations, the

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sonicated QPI nanoparticles showed improve in β-sheets (from 32.55% to 42.79%) and

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β-turns (from 17.42% to 25.16%), while reduction in α-helices (from 28.03% to 18.48%) and

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random coils (from 22% to 13.57%). The decrease of α-helix structures suggested that salt

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concentrations led to gradual extending and aggregation of sonicated QPI nanoparticles.

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Moreover, the increase of QPI β-sheet structure contents exposed the hydrophobic bonds and

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free SH bonds embedded in the interior of the QPI protein molecules, therefore facilitating the

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increase in surface hydrophobicity. However, the increase of QPI β-turn structure contents

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may be on account of the transformation of random coils into more ordered structures (β-turn

229

structures). It could be deduced that salt accretion changed the secondary structure contents of 12


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sonicated QPI nanoparticles and improved the intramolecular interactive forces due to protein

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aggregation.25

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Interactive Forces of Tertiary and Quaternary Structure. Protein surface hydrophobicity

233

reflects the amount of hydrophobic bonds on the exterior of protein molecules. Thus, the H0

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values are closely connected with the configuration and properties of proteins.26 Figure 3

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reveals the H0 values of sonicated QPI nanoparticles with varying salt concentrations of 0-500

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mM. The H0 of all sonicated QPI nanoparticles under salt accretion was significantly higher

237

than the 0 mM salt concentration sample. Furthermore, the sonicated QPI nanoparticles

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conducted at higher salt concentrations possessed more H0 values. The results might be due to

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the high salt concentrations converted the free water to bound water in protein solutions,

240

which could alter the conformational structure and interactive forces of protein molecules. The

241

observations confirmed that more hydrophobic bond bunches in the internal of sonicated QPI

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nanoparticle molecules transformed to protein’s outside as a result of salt revulsive protein

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aggregation (“salt-out” effect).27 Thus, it can be reasonably hypothesized that protein

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aggregation or unfolding of sonicated QPI nanoparticles might be much more favorable for the

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exposure of hydrophobic groups.

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Our previous work confirmed that the internal tertiary and quaternary structure of

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sonicated QPI nanoparticles were largely sustained by both hydrophobic interactive forces and

248

disulfide bonds.8 Therefore, the effect of salt concentrations on these two interactive forces

249

were studied, and the consequences are displayed in Figure 3. The results observed that the

250

free SH group contents of the sonicated QPI nanoparticles improved markedly with salt 13



251

accretion, while the disulfide bond contents remain basically unchanged. So it could be cause

252

that the hid SH groups of sonicated QPI nanoparticles become dissociated in the meanwhile

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when spatial structure and hydration layer were broken due to saltaccretion.28 However, the

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salt is not sufficient to cause the fracture of protein polymers, so it doesn’t affect the disulfide

255

bond content. From previous research, Shimada and Cheftel29 obtained that free SH bonds

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from disulfide bonds via oxidation effects (free SH bonds→S-S bonds) during the formation

257

of heat-induced SPI gels. The free SH groups explored to the outside, and the soy protein

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isolate particles could easily form disulfide bonds during salt accretion. In conclusion, the

259

diverse consequences might be attributed to Shimada and Cheftel explored the disulfide bond

260

variation in intermolecular soy protein isolate gel system, while our research investigated the

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disulfide bond content in sonicated QPI nanoparticles internal structure.

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Characterization and Freeze-thaw Properties of Pickering Emulsions. Droplet Size.

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From the visual observation (Figure 4), it can be observed that all fresh Pickering emulsions

264

were highly steady against flocculation formed at various salt concentrations, suggesting good

265

physical stability. After freeze-thaw circulations, the emulsions formed at 0-500 mM salt

266

concentrations destabilized at varying levels depending on the salt concentrations and

267

circulation numbers. The QPI nanoparticles stabilized Pickering emulsions without salt

268

accretion exhibited great oiling off, coalescence and creaming by the freeze-thaw circulations

269

up to 3, indicating its low freeze-thaw properties. However, salt accretion (0-500 mM)

270

significantly enhanced the freeze-thaw properties of QPI nanoparticles stabilized Pickering

271

emulsions after three times freeze-thaw circulations. Table 2 depicts the D43 values of QPI 14


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nanoparticles stabilized fresh and freeze-thawed Pickering emulsion at various salt

273

concentrations. All the QPI nanoparticles stabilized fresh Pickering emulsions had similar

274

droplet sizes. With the applying of freeze-thaw circulations, the droplet size of the

275

QPI-stabilized emulsions without salt accretion was much larger than that of salt accretion.

276

The salt accretion enhanced the sonicated QPI nanoparticles emulsifying efficiency. When the

277

charge of sonicated QPI nanoparticles is neutralized by salt accretion, the energy rampart for

278

their adsorption will be decreased, thus facilitating the emulsifying performance and their

279

capacity on the droplets interfacial packing and adsorption.30 The formation of multilayered

280

structure and compact adsorption at the surface of droplets of QPI-stabilized Pickering

281

emulsions with salt accretion would provide a prominent steric repulsion towards coalescence

282

with smaller droplet size and higher freeze-thaw properties.11

283

Optical Microscopy. For the sake of further study the freeze-thaw properties mechanism

284

of QPI-stabilized Pickering emulsions, the microstructure of these emulsion were investigated

285

by optical microscopy (Figure 5). The micrographs obtained that the microstructures of QPI

286

nanoparticles stabilized Pickering emulsions with salt accretion were basically steady with the

287

freeze-thaw circulations. In reverse, the emulsion without salt accretion exhibited droplet

288

coalescence after freeze-thaw circulation. Interestingly, the QPI nanoparticles stabilized

289

Pickering emulsions at higher salt concentrations (400-500 mM) exhibited larger droplet sizes,

290

these results could be explained by the less coalescence stability to a limited extent.14 Zhu et

291

al.28 (2017) indicated that the difference in the coalescence degree of Pickering emulsion

292

might be largely due to the difference in ζ-potential and H0 values of the nanoparticles. 15



293

Together with the emulsion droplet size and visual observations, the results definitely

294

indicated that the salt accretion prominent facilitated the physical and freeze-thaw properties

295

of the QPI-stabilized Pickering emulsions against oil separation and coalescence.

296

Rheological Measurements. The rheological properties of these QPI nanoparticles

297

stabilized fresh Pickering emulsions at varied salt concentrations (0-500 mM) were

298

corroborated by the steady shear viscosity and dynamic viscoelastic measurements, as

299

displayed in Figure 6. The QPI nanoparticles stabilized fresh Pickering emulsions without salt

300

accretion exhibited a great flow property independent on the shear rate. However, all the QPI

301

nanoparticles stabilized fresh Pickering emulsions with salt accretion showed a shear-thinning

302

behavior (Figure 6A), with viscosity reduced significantly when shear rate rose from 0.5 to

303

300 s-1, indicating the degradation of sonicated QPI nanoparticles stabilized droplets during

304

the shearing. These results confirmed that the salt accretion favored the generation of gel-like

305

three-dimensional structure in QPI nanoparticles stabilized Pickering emulsions.15 In combine

306

with the interactive forces data, the results recommended that hydrophobic interactive forces

307

played a crucial effect on gel-like emulsion formation. The viscoelastic characterizations

308

verified the gel-like network formation in QPI nanoparticles stabilized Pickering emulsions at

309

salt concentrations of 100-500 mM. Figure 6B illustrated a much larger G′ values than the G′′

310

values of all the fresh Pickering emulsions in the whole 0.1-100 Hz frequency process,

311

confirming these emulsions formed elastic gel-like network.18 The uncharged and aggregated

312

sonicated QPI nanoparticles due to the salt electrostatic screening effect tend to generate a

313

gel-like three-dimensional structure around the interface of emulsion droplets.30,31 16


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314

Crystallization behaviour of DSC Analyses. Figure 7 depicts the effect of salt

315

concentrations (0-500 mM) on the ice crystal generation and thawing in the QPI nanoparticles

316

stabilized

317

(40 °C→-40 °C→40 °C). The exothermic event peak temperature of all the QPI nanoparticles

318

stabilized Pickering emulsions is around -15～-21 °C during cooling phase, whereas in the

319

heating phase, the endothermic event peak temperature is around 2.5～5.0 °C. It can be

320

obtained that the QPI nanoparticles stabilized Pickering emulsions with salt accretion resulted

321

in reduce in freezing point from -15 to -21 °C. These results indicated that salt accretion

322

confined the formation of ice crystal.32 The slight difference of freeze temperature between

323

QPI nanoparticles stabilized Pickering emulsions with 0 and 100 mM salt concentrations

324

reflected that the difference of freeze-thaw properties in these emulsions was largely because

325

of the difference in emulsion network and rheological properties, rather than the difference in

326

salt accretion.33 On the other hand, the varying volume of salt concentrations progressively

327

enhanced the freeze-thaw properties of QPI nanoparticles stabilized emulsions could be

328

mainly because of the inhibit generation of ice crystal by the “salting-out” effects.34

fresh

Pickering

emulsions

during

a

cooling-heating

circulation

329

In this study, we demonstrated the salt accretion (0-500 mM) progressively improved the

330

stability and freeze-thaw properties of the QPI nanoparticles stabilized Pickering emulsions.

331

The supposed mechanism for the effect of salt concentrations on the freeze-thaw properties of

332

food-grade sonicated QPI-stabilized Pickering emulsions is displayed in Figure 8. The salt

333

accretion observably advanced the particle size and H0 values of sonicated QPI nanoparticles,

334

which could be attributed to the decrease of charged groups and particle aggregation by 17



335

electrostatic interactions (“salting-out” effects). The sonicated QPI nanoparticles with salt

336

accretion showed higher β-sheets structure contents and stronger hydrophobic interactions.

337

Compared with the QPI nanoparticles stabilized Pickering emulsions without salt accretion,

338

the emulsions with salt accretion exhibited better freeze-thaw properties during three times

339

freeze-thaw circulations, which might be mainly caused by the generation of gel-like

340

three-dimensional structure and multilayered network at the droplets interface with smaller

341

droplet sizes. However, the increasing volume of salt concentrations (0-500 mM)

342

progressively enhanced the freeze-thaw properties of QPI nanoparticles stabilized Pickering

343

emulsions could be mainly because of the inhibit generation of ice crystal by the “salting-out”

344

effects. Therefore, the results would provide great significance for the role of salt

345

concentrations in the freeze-thaw properties of food-grade protein-stabilized Pickering

346

emulsions.

347 348 349 350 351 352 353 354 355 18


Page 18 of 36

Page 19 of 36


356

Author Information

357

Corresponding Authors

358

* Phone: (+86-020)87113845. Fax: (+86-020)87113848. E-mail: [email protected] (Z.-G.

359

L)

360

* E-mail: [email protected] (Y.-X. Z)

361

Funding

362

Financial support from the National Natural Science Foundation of China (21576098,

363

21376097), Key Project of Science and Technology of Guangdong Province of China

364

(2017B090901002), 111 Project (B17018), and China Postdoctoral Science Foundation

365

(2016M590787, 2017T100616).

366 367 368 369 370 371 372 373 374 375 376 19



377

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Page 25 of 36


479

Figure Captions

480

Figure 1. Particle size (Dz, alignment) and ζ-potentials (histogram) of sonicated QPI

481

nanoparticles at different salt concentrations (0-500 mM).

482

Figure 2. The full FTIR spectrum (4000-400 cm-1) of sonicated QPI nanoparticles with

483

different salt concentrations.

484

Figure 3. H0 values, free SH and disulfide bond contents of sonicated QPI nanoparticles with

485

varying salt concentrations of 0-500 mM.

486

Figure 4. Visual observation of the QPI nanoparticles stabilized fresh and freeze-thawed

487

Pickering emulsions at various salt concentrations of 0-500 mM.

488

Figure 5. Representative optical microscopy images of QPI nanoparticles stabilized fresh and

489

freeze-thawed Pickering emulsions at various salt concentrations of 0-500 mM.

490

Figure 6. (A) Apparent viscosity (shear rate at 0.5-300 s-1) of the QPI nanoparticles stabilized

491

fresh Pickering emulsions at various salt concentrations of 0-500 mM; (B) G′ and G′′ values

492

(frequency at 0.1-100 Hz) of the QPI nanoparticles stabilized fresh Pickering emulsions at

493

various salt concentrations of 100-500 mM.

494

Figure 7. DSC thermograms and freeze temperature (°C) of the QPI nanoparticles stabilized

495

fresh Pickering emulsions at various salt concentrations of 0-500 mM.

496

Figure 8. Supposed mechanism for the salt concentrations on the freeze-thaw properties of

497

food-grade Pickering emulsions stabilized by sonicated QPI nanoparticles.

25



Page 26 of 36

Table 1 Percentages of secondary structure components of the sonicated QPI nanoparticles at various salt concentrations of 0-500 mM. Sample

Secondary structure composition (%) α-helix

β-sheet

β-turn

Random coil

0 mM

28.03 ± 0.12a

32.55 ± 0.17f

17.42 ± 0.34f

22.00 ± 0.16a

100 mM

23.06 ± 0.04b

38.63 ± 0.05d

21.82 ± 0.38d

16.49 ± 0.63d

200 mM

20.94 ± 0.51e

40.18 ± 0.80b

23.02 ± 0.19b

15.86 ± 0.51e

300 mM

18.48 ± 0.28f

42.79 ± 0.09a

25.16 ± 0.73a

13.57 ± 0.06f

400 mM

21.52 ± 0.08d

39.14 ± 0.61c

22.31 ± 0.88c

17.03 ± 0.19c

500 mM

22.73 ± 0.27c

37.98 ± 0.07e

21.72 ± 0.71e

17.57 ± 0.53b

Values represent the means ± standard error (n = 3). Different letters in the same row indicate significant differences (p < 0.05).

26


Page 27 of 36


Table 2. Particle sizes (D43) of the QPI nanoparticles stabilized fresh and freeze-thawed Pickering emulsion droplets at various salt concentrations of 0-500 mM. Parameter Type

µ

D43 (µm) 0 mM

100 mM

200 mM

300 mM

400 mM

500 mM

Fresh

28.064f

31.726d

32.105a

31.968b

31.933c

30.716e

1 circulation

59.35a

42.572c

41.53e

40.774f

42.114d

43.679b

2 circulations

117.169a

55.722d

53.967e

53.257f

57.329c

58.015b

3 circulations

242.159a

62.98c

62.57e

59.365f

62.799d

63.816b

Values represent the means ± standard error (n = 3). Different letters in the same row indicate significant differences (p < 0.05).

27



Page 28 of 36

Figure 1

-40 A

180

B

a

ξ-potential

Dz

165

-30 C

150 D

-20 b

135 c d

F

-10

120

e f

105 0 0

100

200

300

Salt Concentrations (mM)

28


400

500

Dz (nm)

ξ -potentials

E

Page 29 of 36


Figure 2

500 mM 400 mM 300 mM 200 mM

Absorbance

100 mM 0

Amide I region -1 (1600-1700 cm )

500

1000

1500

2000

2500

-1 Wavenumber (cm )

29


3000

3500

4000


Page 30 of 36

Figure 3

10 Ho Free SH content ------ Disulfide bond content

a

10000

Ho

e

D

A

B

C

4

E

f F

2

0

4000 0

100

200

300

Salt Concentrations (mM)

30


400

500

µmol/g protein

6

d

8000

6000

8

c

b

Page 31 of 36


Figure 4

31



Figure 5

32


Page 32 of 36

Page 33 of 36


Figure 6

A

1000

Apparent viscosity (Pa⋅s)

100

10

0 100 mM 200 mM 300 mM 400 mM 500 mM

1

0.1

0.01 1

10 -1 Shear rate (s )

100

Modulus (Pa)

B 1000 Storage (G') 100 mM 200 mM 300 mM 400 mM 500 mM Loss (G'') 100 mM 200 mM 300 mM 400 mM 500 mM

100

10 0.1

1

10 Frequency (Hz) 33


100


Page 34 of 36

Figure 7

0

30

100 mM 200 mM 300 mM

20

400 mM

Heat Flow (W/g)

(II) Heating (-40°C→40°C)

500 mM

10

0

-10

-20 -40

-30

-20

u

freeze temperature

0

-15.417

100 mM

-16.225

200 mM

-16.292

300 mM

-16.938

400 mM

-17.442

500 mM

-21.184

(I) Cooling (40°C→-40°C)

-10 0 10 Temperature (°C)

34


20

30

40

Page 35 of 36


Figure 8

35



272x205mm (150 x 150 DPI)


Page 36 of 36

Fabrication and Characterization of Quinoa Protein Nanoparticle

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