Fabrication and Characterization of Quinoa Protein Nanoparticle

Jinan University, Guangzhou 510632 , China. J. Agric. Food Chem. , Article ASAP. DOI: 10.1021/acs.jafc.8b00225. Publication Date (Web): April 17, ...
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Food and Beverage Chemistry/Biochemistry

Fabrication and Characterization of Quinoa Protein Nanoparticles Stabilized Food-grade Pickering Emulsions with Ultrasound Treatment: Interfacial Adsorption/Arrangement Properties Xin-Sheng Qin, Zhigang Luo, and Xichun Peng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00225 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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Fabrication and Characterization of Quinoa Protein Nanoparticles Stabilized Food-grade Pickering Emulsions with Ultrasound Treatment: Interfacial Adsorption/Arrangement Properties

Xin-Sheng Qin,† Zhi-Gang Luo,*,†,‡ and Xi-Chun Peng§



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

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ABSTRACT: The natural quinoa protein isolate (QPI) was largely reflected in the

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nanoparticle form at pH 7.0 (about 401 nm), and the ultrasound at 20 min progressively

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improved the contact angle (wettability) and surface hydrophobicity of the nanoparticles.

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Ultrasound process also modified the type of intra-particle interaction, and the internal forces

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of sonicated particles were largely maintained by both disulfide bonds and hydrophobic

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interaction forces. In emulsion system, the ultrasound progressively increased the

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emulsification efficiency of the QPI nanoparticles, particularly at high protein concentration

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(c>1%, w/v), and higher emulsion stability against coalescence. As compared with the natural

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QPI stabilized emulsions, the 20 min-sonicated emulsions exhibited higher packing and

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adsorption of at the protein interface. The microstructure of emulsions is occurred bridging

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flocculation of droplets at low c (≤1%, w/v), while the amount of protein particles could be

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high enough to cover the droplets surface at high c (>1%, w/v) with hexagonal array model

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arrangement. Thus, these results illustrated that both natural and sonicated QPI nanoparticles

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could be performed as effective food-grade stabilizer for Pickering emulsion, however, the

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sonicated QPI nanoparticles exhibited much better emulsifying and interfacial properties.

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KEYWORDS: quinoa protein nanoparticles, food-grade, Pickering emulsions, ultrasonic

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treatment, interfacial property

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INTRODUCTION

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Emulsion droplets can be stabilized by either forming physical screen via surface-active

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colloidal particles, or reducing interfacial tension through surfactants, or by forming spatial

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interfacial films via hydrocolloids.1 Stabilization of emulsions realized by the first sort is

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defined as Pickering emulsions.2 To show as a Pickering emulsion stabilizer, surface-active

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particles could maintain steady in water and oil systems, and have a suitable wettability

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(contact angle).3 There is increasing interest in Pickering emulsions, because of their

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superiority of “surfactant-free”, favorable restores to flocculation, functionality in

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environmental responsive emulsions of high internal phase emulsions.4 In recent years,

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synthetic or inorganic micro/nano-particles, such as carbon nanotubes, SiO2 and laponite clay,

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seem to be most promising to apply for Pickering emulsion stabilizer.5 However, the usage of

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these stabilizer are restricted by their non-compatibility or non-biodegradability. Hence, the

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developments of biodegradable food-grade particles based Pickering emulsion are of

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importance in foods and pharmaceutics fields, e.g., controlled release of some embedded

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material, and reinforcement of stability.6 The food-grade Pickering stabilizer could be

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probably classified into three types: (i) protein-type, e.g., preheated soy protein, whey protein,

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water-insoluble zein and pea protein; (ii) polysaccharide-type, e.g., starch nanocrystals,

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hydrophobically-modified starch, cellulose and chitin; (iii) miscellaneous, e.g., lipid and

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flavonoids.3 Many particles generally need a further chemical modification to facilitate their

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emulsifying properties, and moreover, the creating emulsions have large droplet sizes.7 For

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example, the formation of starch particle stabilized Pickering emulsion need to 3

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hydrophobically-modify using chemical reagent such as octenyl succinic anhydride (OSA).3

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Thus, it would be an interesting problem to find an effective and cheap food-grade

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nanoparticles to stabilize the Pickering emulsion.

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Quinoa is an Andean cereal of South America that has been planting all over the world

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recently for their abundant nutritional property, mainly owing to the high content of proteins

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that ranges from 12% to 23%.8 Quinoa protein isolate (QPI) is one of the most promising and

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readily available quinoa products due to their high content of essential amino acids. It largely

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consists of 11S globulins (20–25 kDa for basic polypeptides and 30–40 kDa for acid

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polypeptides) and 2S albumins (8–9 kDa), which in proportion as probably 75% of the overall

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protein amount of QPI.9 Our previous work had indicated that the natural QPI was largely

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reflected in the nanoparticle form at pH 7.0. Compared with the stabilizers, the QPI

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nanoparticles have several advantages: (i) they are functional and nutritional food stuff; (ii)

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they can perform surface hydrophobic and/or hydrophilic property; (iii) they do not demand

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any chemical surface modification to facilitate the particle wettability and hydrophobicity; (iv)

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they are compatible with emulsification techniques (high-speed shear/ultrasound/high pressure

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homogenizer). In conclusion, QPI nanoparticles show good surface active nature with the

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potentiality of providing as a promising and effective stabilizer for food-grade Pickering

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emulsions.10

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The ultrasound process is not only correlated to particle surface property, but also affects

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the emulsion efficiency. 11,12 Ultrasound technology applied waves at a frequency >16 kHz

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over the damage of human hearing. The acoustic cavitation phenomenon of high intensity 4

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ultrasound (10–100 W/cm2 of power, 16–100 kHz of frequency) is utilized to destroy physical

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bonds or facilitate chemical reactions in ingredients, thereof cavitation bubbles are quickly

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produced and fiercely collapsed.13 The bubbles combination generates excessive pressure and

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temperature that leads to turbulence and high shear stress waves in the sonicated cavitation

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region.14 Ultrasound treatment led to decreased size but improved solubility and surface

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hydrophobicity (Ho) of the soy protein isolate aggregates solutions.15-17

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To date, no work had explored in the emulsifying nature, adsorption and arrangement at

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the interface of QPI nanoparticles stabilized Pickering emulsion. The objective of this work

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was to elucidate the promising potential of QPI nanoparticles to act as an effective food-grade

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Pickering-type emulsion stabilizer. Firstly, we explored the effect of the ultrasound process on

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the hydrodynamic diameter, contact angle, surface hydrophobicity and interactive forces of

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QPI nanoparticles. Secondly, the effect of protein concentration (c; 0.25–6%, w/v) and/or corn

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oil fraction (φ; 0.2–0.7) on the interfacial properties and stability of QPI nanoparticles

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stabilized Pickering emulsion was determined by z-average diameters (Dz), microstructure,

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creaming stability and adsorption/arrangement of surface particles. The correlation of varied c

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and/or φ was further studied to preferable achieve the stabilization mechanism of QPI

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nanoparticles stabilized Pickering emulsion.

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MATERIALS AND METHODS

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Materials. QPI (80% protein) was acquired from Yuanye Biological Technology Co., Ltd.,

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China. Corn oil was acquired from Yihai Kerry limited, China. Potassium Sorbate was

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acquired from Kemiou Chemical Reagent Co., Ltd., China. Coomassie brilliant blue G-250,

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1-anilino-8-naphthalene-sulfonate (ANS), bovine serum albumin (BSA), DL-Dithiothreitol

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(DTT), urea, sodium dodecyl sulfate (SDS), Nile Blue and Nile Red were acquired from

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Solarbio Science & Technology Co., Ltd, China. All chemical reagents were of analytical

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grade.

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Fabrication of QPI Nanoparticle Dispersions with Ultrasound Treatment. The QPI

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dispersions (0.25–6%, w/v) were fabricated by immediately adding different ratio QPI

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powders in distilled water under magnetic agitation for 2 h at 25 °C. Potassium Sorbate

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(0.01%, w/v) served as an antimicrobial reagent. The protein dispersions were deployed to pH

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7.0 using HCl or NaOH and stored at 4 °C for proteins hydration. The dispersions were treated

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using a 100 W ultrasonic homogenizer model KH-250DE (Kunshan Ultrasonic Instrument

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CO., LTD., China) at 25 °C for 20 min. And the blank control samples were conducted

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without ultrasound treatment.

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z-average Diameter. The z-average diameters (Dz) of QPI nanoparticles were evaluated

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employing a Nano-ZS Zetasizer analyzer (Malvern Instruments, UK) by dynamic light

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scatting (DLS) method. The QPI nanoparticle dispersions were diluted in deionized water at a

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proportion of 1:5 (v/v) after stored at 4 °C overnight. Sample was measured at 25 °C and 1.33

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of refractive index in triplicate. 6

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Surface Hydrophobicity (Ho). Surface hydrophobicity of non- or sonicated QPI samples

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was measured based on the method of Qin et al.18 with the fluorescence probe ANS. The QPI

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sample was attenuated to the concentration with the range of 0.1–0.0005 mg/mL with 0.01 M

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phosphate buffer (pH = 7.0). Then, 20 µL of 8.0 mM ANS solutions was added to 3 mL of

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each attenuated sample, and avoid light reaction for 20 min. The fluorescence intensity (FI)

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was determined at 390 nm (excitation wavelength) and 400–750 nm (emission wavelength)

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employing F-7000 fluorescence spectrophotometer (Hitachi Ltd., Japan). The excitation and

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emission slit widths were taken to 5 nm, and the temperature was maintained at 25 °C. Ho was

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calculated as the slope of the FI as the protein concentration (mg/mL).

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Evaluation of Intra-particle Interaction. The intra-particle interaction was assessed by

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comparing the turbidity of QPI dispersions (c = 0.25%, w/v) described by Zhang and others.19

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The blank and ultrasound treated suspensions were mixed in four solvents: A) distilled water

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(DW), B) 6.0 M urea, C) 0.5% SDS (w/v), D) 30 mM DTT, or their combinations with an

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equal bulk. The suspensions turbidity was determined at 595 nm employing a 722SP visible

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spectrophotometer (Shanghai Lengguang Technology CO., LTD., China) with BSA as the

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standard by the Bradford method. The Coomassie brilliant blue were determined as blank

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controls.

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Contact Angle (θ). The θ of freeze-dried QPI samples was measured using OCA 40 micro

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analyzer (Data Physics Corporation, Germany) loaded with a high-speed video camera. The

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sonicated protein samples were formed into 2 mm slice with a hydraulic press. The slice was

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immersed in the oil phase and placed into a glass vessel, and a glob of Milli-Q water (3 µL) 7

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was dripped on the superficies of the slice with corn oil employing a high-precision syringe.

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Then, the contact surface image was obtained with a high-speed camera.

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Preparation of QPI Stabilized Pickering Emulsion. The QPI Pickering emulsions were

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fabricated after the fabrication of QPI nanoparticle dispersions (0.25–6%, w/v) with

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ultrasound treatment. And the emulsions were conducted with variation in corn oil fraction (φ)

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from 0.2 to 0.7, while dispersed with a T25 digital Ultra homogenizer (IKA Inc., Germany) at

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20000 rpm for 1 min. The emulsions formed freshly were immediately applied to

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emulsification determination, or stored for 7 days for further emulsion physical stability

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determination.

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Droplet Size Distribution. The surface- or volume- mean diameter (D32 and D43) and

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droplet size distribution of various QPI stabilized Pickering emulsions, were measured

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employing a Malvern Mastersizer analyzer 2000 (Malvern Instruments, UK).13 The samples

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were scattered in deionized water at 2500 rpm of pump speed and 0.001 absorption index. The

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comparative refractive index of the emulsions was set as 1.095, due to the proportion of the

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refractive index of corn oil (1.467) to that of deionized water (1.33). All determinations were

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taken the average of three determinations.

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Creaming Index (CI) of Emulsion Stability. CI of emulsions formed at varying cases used

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to evaluate the stability commonly. 20 mL of each emulsion sample was transferred to a scale

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tube (10 cm height × 2 cm diameter) and then depot at room temperature. Height of the serum

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(Hs) and total height (Ht) of emulsions were measured before or after storage (7 days). The

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proportion of CI% was defined as ( Hs / Ht ) × 100%. 8

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Microstructure of Emulsions. Optical Microscopy. The microstructure of QPI

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nanoparticles stabilized emulsions was measured using a BH2 light microscope analyzer

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(Olympus Co. Ltd., Japan) outfitted with a photographic camera. A 50 µL aliquot of each

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emulsion was taken and transferred onto a glass slide, then coated with a coverslip. The

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measurements were performed at room temperature with 500 magnification lens, as described

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by Gao et al.20

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Confocal Laser Scanning Microscopy (CLSM). The microstructure of protein adsorbed to

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QPI Pickering emulsion interface was observed by CLSM using TCS-SP5 microscope

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analyzer (Leica Microsystem Inc. Germany). Emulsions were pre-stained with Nile Blue A

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and Nile Red during the emulsion fabrication process. Then, samples were transferred onto a

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glass slide and coated with a coverslip. Silicone oil was used to avoid water evaporation. The

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samples were explored by an argon/krypton laser at an excitation wavelength of 488 and 633

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nm (HeNe laser) with a 100 magnification lens, respectively.

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Percentage of Adsorbed Proteins (AP%). The amount of QPI proteins adsorbed to the

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interface of the droplets was evaluated according to Isaschar-Ovdat et al.21 with some

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modifications. A 5 mL QPI Pickering emulsion was centrifuged for 10 min at 4000 rpm

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(Hitachi Ltd., Japan). Two layers were obtained after centrifugation: the aqueous layer on

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bottom, the creamed oil droplet layer of the emulsion at the top of the tube. The clarified

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aqueous phase was extracted by injector and then colated using a 0.45-mm aqueous film. Cf is

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the protein concentration in the filtered aqueous layer with the Bradford method using BSA

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reagent. Co is the initial protein concentration in the protein dispersions. The AP% of the

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emulsion was calculated as follows:

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AP% = (Co – Cf) × 100%/Co

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Surface Particle Coverage at the Interface of Droplets. Limited coalescence process

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occurs in Pickering emulsions where solid particles are inconvertible adsorbed at the droplets

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interface. When the matrix is conducted with a small number of solid particles, the particles is

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inadequate to protect the newly formed droplets. After the homogeneity, the total particle

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amount of droplets interface is reduced because of the droplets coalescence. Since the solid

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particles are inconvertibly anchored, the droplet coalescence course stops when the interface is

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competent coated.22,23 Particles arrangement at the oil-water interface could be indicated from

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the surface particle coverage (SC%), which is defined as the proportion between the droplets

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interfacial area (Sint) and the total particle area (Seq). The total particle area can be

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approximately calculated from their equatorial area: Seq = nparticlesπ(Dz/2)2, where nparticles was

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the total amount of solid particles and Dz was the hydrodynamic diameter. The nparticles was

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calculated by the adsorbed proteins (AP%) at the interface. The Sint is directly connected to

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D32 (Sint = 6Vd/D32), where Vd is the oil capacity. Hence, the surface particle coverage SC% =

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Seq/Sint. The SC% can be deduced from the slope of (1/D32) against (Seq/Vd).

(1)

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Statistical Analysis. The determinations presented as the average of three samples ±

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standard deviations. The data statistics were determined employing SPSS software (p < 0.05)

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by ANOVA.

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RESULTS AND DISCUSSION

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Characterization of QPI Nanoparticles by Ultrasound Treatment. The size, surface

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hydrophobicity and wettability of particle in the natural and sonicated (at 20 min) QPI

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dispersions at c = 0.25% (w/v) were measured by dynamic light scattering, fluorescence probe

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and oil-in-water contact angle. Figure 1 shows the Dz of QPI dispersions changed remarkably

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with the increasing ultrasound time. The Dz of the natural QPI nanoparticles was 401 nm,

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while it significantly decreased to 207 nm for 20 min-sonicated sample. The results illustrated

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that the ultrasound treatment markedly reduced particle size because of the micro-steaming

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and turbulent forces by cavitation effect. A similar result about the effect of ultrasound on the

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protein particles has been obtained by Gao et al. which was well consistent with the fact that

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the ultrasound treatment usually led to rapidly production and violently sink of cavitation

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vesicles.20,24

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The surface characteristics of Pickering stabilizer are very important for their emulsifying

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property. Such as organic and inorganic particles, including starch granules, microcrystalline

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cellulose, or solid lipid nanoparticles, this particles generally need surface hydrophobic

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modification.4,5,7 Surface hydrophobicity (Ho) also plays a vital part in the conformation,

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function and stability of proteins. Figure 2 displays the typical intrinsic fluorescence emission

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profiles of the natural and 20 min-sonicated QPI nanoparticles. The principle of fluorescence

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probe ANS is as follows: the fluorescence spectra of QPI samples are very susceptive to the

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hydrophobic Trp or Tyr residues chromophores in the polar environment.25 The Ho values are

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calculated by the spectra and slope of FI as protein concentration in Figure 2. Ho values of the 11

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natural and sonicated QPI samples are also summarized and presented in Figure 1. The Ho of

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the 20 min-sonicated QPI dispersion displayed a remarkable increase contrasted with the

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natural sample (Ho increased from 22 to 44). These results were attributed to the cavitation

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phenomenon of ultrasound, which could induce unfolding of the QPI nanoparticles, lead to the

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exposure of hydrophobic regions from the interior to the exterior of QPI nanoparticles.26

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The wettability of the solid particles plays an important part for the fabrication of

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Pickering-stabilized emulsions. The wettability of particles was usually determined by the

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contact angle θ. The θ value close to 90° can favor the packing behavior of particles at the

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droplet interface.6 For a spherical particle, the energy obtained for separating the solid

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particles from oil/water interface (∆E), which count as follows: ∆E = πR2γ(1±cosθ)2, where γ

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is the particle surface tension. The particles at the droplet interface can be inconvertible

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adsorbed to oil droplets if 30° 1%, w/v), the number of QPI particles could be

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sufficient to stabilize the droplets surface. The sonicated QPI stabilized emulsions with better

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emulsification against flocculation might be due to the greater intra-particle hydrophobic

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interactions and more effective adsorption at the droplets interface. A similar limitation of

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flocculation by increasing c has been obtained for the heat induced soy glycinin nanoparticles

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stabilized Pickering emulsion.28

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Creaming Index (CI) of Emulsion Stability. All the QPI stabilized emulsions formed at

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c = 0.25–6% with different φ values of 0.2–0.7 after 0 and 7 days storage, gradually to divide

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into two layers: the creamed layer on the upside and the serum layer at the underpart (Figure

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5A). In these Pickering emulsions, the storage of 7 days was sufficient for creaming, and a

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further store time did not increase the creaming index any more. Figure 5B shows the

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creaming index of QPI nanoparticles stabilized emulsions changed with the applied c and φ

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after 7 days storage. The observations suggested that the application of the increasing c or φ

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could enhance the creaming stability of the QPI Pickering emulsions against oiling off. The

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increase in c resulted in a prominent reduction in CI%, indicating the limitation of creaming

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by improving the particle concentration. Analogical data about the effect of c on creaming

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stability have been obtained for the Pickering emulsions stabilized by zein, chitin nanocrystal

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particles and soy glycinin.28,32-33 These results might be due to the increasing c reduced the

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droplet size.16 Another possible reason for the improvement of creaming stability might be 15

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attributed to the formation of a thicker tier at the surface of oil droplets at higher c.32 It was

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also obtained that the physical creaming stability of the emulsions increased upon with

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different φ values of 0.2–0.7 at a specific c value. The observations were in accordance with

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Liu and Tang,28 which revealed that high φ increased the particle amount of per interfacial

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area adsorbed at the droplets surface.

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Percentage of Adsorbed Proteins (AP%). In the protein particles stabilized Pickering

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emulsions, the physical creaming stability and interfacial property is not only contacted with

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attractive and repulsive inter-droplet interactions, but also related to inter-protein interactions

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including unadsorbed and adsorbed protein at the interface.17 To further elaborate the

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significance of inter- protein interactions for QPI stabilized emulsions, we determined the

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number of adsorbed proteins at the droplets interface of the natural and 20 min-sonicated QPI

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stabilized emulsions (Figure 6). Under all the investigated conditions, the AP% data of

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sonicated QPI stabilized emulsions were obviously larger than that of natural QPI stabilized

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emulsions. From Figure 6, the AP% of QPI stabilized emulsions progressively improved with

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the increasing c. At higher c values, more protein particles were adsorbed at the droplet

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interfaces, thus formed a thicker interfacial protein layer. With the increasing φ, the AP%

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values of 20 min-sonicated QPI stabilized emulsions were in the range of 56–80.96%. This

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conclusion was consistent with the creaming stability, illustrating that the high φ provided

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larger interface area for protein to adsorb.

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Nanoparticle Packing at the Interface and Surface Particle Coverage (SC). The

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limited coalescence phenomenon of Pickering emulsion has been successfully used to supply 16

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an deep understanding of packing, adsorption and arrangement of solid particles at the droplet

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interface. The percentage of surface particle coverage (SC%) can be examined by the slope of

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the reciprocal surface diameter (1/D32) against the total particle area (Seq) divided by the oil

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capacity (Vd). The simulation of (1/D32) of the natural and 20 min-sonicated QPI stabilized

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Pickering emulsions as a control of the (Seq/Vd) is displayed in Figure 7A. The results revealed

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that all the tested emulsion performed a good linear relation between (1/D32) and (Seq/Vd) with

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a coefficient factor R2 of 0.988-0.993. Similar evolutions have been found for Pickering

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emulsions stabilized by heat pretreated soy glycinin nanoparticles, whey protein microgel

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particles, kafirin nanoparticles and quinoa starch granules.6,7,28,31 The SC% and QPI particle

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center-to-center distance at the droplet interface (Dc-to-c) values of the natural and 20

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min-sonicated QPI stabilized emulsions are summarized in Table 2. It can be observed that the

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natural and sonicated QPI nanoparticles performed a relatively low SC% about 48.67–54.43%

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compared to theoretical hexagonal array at interface (SC% =

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nanoparticles exhibited high coating availability and efficient coverage at the droplets

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interface. Ultrasound treatment progressively increased the SC% from 48.67 to 54.43%, thus

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suggesting that the sonicated QPI nanoparticles would be more effective to serve as Pickering

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stabilizer. We can still approximately calculate the Dc-to-c at the interface between two

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neighboring particles according to square or hexagonal array models (Figure 7B and Table

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2).31 The 20 min-sonicated QPI nanoparticles performed strong interactive forces between

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adsorbed proteins, which led to large inter-particle space of particles at the interface.

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According to comparison, the hexagonal array model appears to be more suitable for the 17

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arrangement of 20 min-sonicated QPI nanoparticles at the droplets interface than the square

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array model.

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We demonstrated the QPI nanoparticles could be used as effective food-grade stabilizers

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of O/W Pickering emulsion. The supposed scheme for the formation of QPI nanoparticles

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stabilized food-grade Pickering emulsions with ultrasound treatment is shown in Figure 8.

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Ultrasound treatment considerably reduced the QPI particle size, and revealed the hydrophobic

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groups from the interior to the outside of the QPI nanoparticles. The sonicated QPI stabilized

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emulsions exhibited better emulsification efficiency and stronger inter-particle structure, and

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the higher stability against coalescence and coacervation. Compared with the natural QPI

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stabilized emulsions, the 20 min-sonicated emulsions exhibited higher packing and adsorption

346

of protein particles at the droplets interface. The microstructure of QPI Pickering emulsions

347

are occurred bridging flocculation of droplets at low c (≤1%, w/v), while the number of

348

particles could be high sufficient to fully cover the droplets surface at high c (>1%, w/v) with

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hexagonal array model arrangement. Thus, the results would likely be of major significance

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not only for the progress of QPI stabilized food-grade Pickering emulsions, but also for

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facilitating the bioactive ingredients (e.g. polyphenols, carotenoid) applied in food and

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pharmaceutical delivery products with health effects.

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AUTHOR INFORMATION

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Corresponding Authors

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* Fax: +86-20-87113848. Tel.: +86-20-87113845. E-mail: [email protected]

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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Financial support from the China Postdoctoral Science Foundation (2016M590787,

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2017T100616), Key Project of Science and Technology of Guangdong Province of China

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(2017B090901002), and the National Natural Science Foundation of China (21376097,

366

21576098).

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REFERENCES

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(1) Dickinson, E. Food emulsions and foams: stabilization by particles. Curr Opin Colloid & Interface Sci.

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fabrication, characterization and research trends. Trends Food Sci & Technol. 2016, 55, 48–60.

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(4) Oh, B. H.; Bismarck, A.; Chan-Park, M. B. High internal phase emulsion templating with

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self-emulsifying and thermoresponsive chitosan-graft-PNIPAM-graft-oligoproline. Biomacromolecules

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2014, 15(5), 1777–1787.

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(5) Lam, S.; Velikov, K. P.; Velev, O. D. Pickering stabilization of foams and emulsions with particles of

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biological origin. Curr Opin Colloid & Interface Sci. 2014, 19(5), 490–500.

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(6) Xiao, J.; Wang, X. A.; Gonzalez, A. J. P.; Huang, Q. Kafirin nanoparticles-stabilized Pickering

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emulsions: Microstructure and rheological behavior. Food Hydrocolloids 2016, 54, 30–39.

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(7) Rayner, M.; Timgren, A.; Sjöö, M.; Dejmek, P. Quinoa starch granules: a candidate for stabilising

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food-grade Pickering emulsions. J. Sci Food Agric. 2012, 92(9), 1841–1847.

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(8) James, L. E. A. Quinoa (Chenopodium quinoa Willd.): composition, chemistry, nutritional, and

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functional properties. Adv Food Nutr Res. 2009, 58, 1–31.

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(9) Ruiz, G. A.; Xiao, W.; van Boekel, M.; Minor, M.; Stieger, M. Effect of extraction pH on heat-induced

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aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd). Food

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Chem. 2016, 209, 203–210.

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(10) Kaspchak, E.; de Oliveira, M. A. S.; Simas, F. F.; Franco, C. R. C.; Silveira, J. L. M.; Mafra, M. R.;

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Igarashi-Mafra, L. Determination of heat-set gelation capacity of a quinoa protein isolate (Chenopodium

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quinoa) by dynamic oscillatory rheological analysis. Food Chem. 2017, 232, 263–271.

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(11) Dickinson, E. Use of nanoparticles and microparticles in the formation and stabilization of food

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emulsions. Trends Food Sci & Technol. 2012, 24(1), 4–12.

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(12) Liu, F.; Tang, C. H. Emulsifying properties of soy protein nanoparticles: influence of the protein

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concentration and/or emulsification process. J. Agric. Food Chem. 2014, 62(12), 2644–2654.

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(13) Qin, X. S.; Luo, S. Z.; Cai, J.; Zhong, X. Y.; Jiang, S. T.; Zhao, Y. Y.; Zheng, Z.

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Transglutaminase-induced gelation properties of soy protein isolate and wheat gluten mixtures with high

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intensity ultrasonic pretreatment. Ultrason. Sonochem. 2016, 31, 590–597.

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(14) Chemat, F.; Khan, M. K. Applications of ultrasound in food technology: processing, preservation and

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extraction. Ultrason. Sonochem. 2011, 18(4), 813–835.

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(15) Arzeni, C.; Martínez, K.; Zema, P.; Arias, A.; Pérez, O. E.; Pilosof, A. M. R. Comparative study of

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high intensity ultrasound effects on food proteins functionality. J. Food Eng. 2012, 108(3), 463–472.

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(16) Tang, C. H.; Liu, F. Cold, gel-like soy protein emulsions by microfluidization: emulsion characteristics,

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rheological and microstructural properties, and gelling mechanism. Food Hydrocolloids 2013, 30(1), 61–72.

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(17) Liu, F.; Tang, C. H. Cold, gel-like whey protein emulsions by microfluidisation emulsification:

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Rheological properties and microstructures. Food Chem. 2011, 127(4), 1641–1647.

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(18) Qin, X. S.; Chen, S. S.; Li, X. J.; Luo, S. Z.; Zhong, X. Y.; Jiang, S. T.; Zhao, Y. Y.; Zheng, Z.

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Gelation Properties of Transglutaminase-Induced Soy Protein Isolate and Wheat Gluten Mixture with

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Ultrahigh Pressure Pretreatment. Food Bioprocess Technol. 2017, 10(5), 866–874.

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complex interface and stability of oil-in-water (O/W) emulsion prepared by soy lipophilic protein

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nanoparticles. J. Agric. Food Chem. 2013, 61(32), 7838–7847.

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(21) Isaschar-Ovdat, S.; Rosenberg, M.; Lesmes, U.; Fishman, A. Characterization of oil-in-water

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emulsions stabilized by tyrosinase-crosslinked soy glycinin. Food Hydrocolloids 2015, 43, 493–500.

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Matter 2014, 10(36), 6963–6974.

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emulsions stabilized by soft microgels: influence of the emulsification process on particle interfacial

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organization and emulsion properties. Langmuir 2013, 29(40), 12367–12374.

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(24) Gülseren, I.; Güzey, D.; Bruce, B. D.; Weiss, J. Structural and functional changes in ultrasonicated

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properties and emulsion oxidative stability of soy protein isolate at pH 3.0 and 7.0. Food Hydrocolloids

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gelation behavior of soybean protein isolate with high intensity ultrasonic pre-treatments. Ultrason.

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emulsifying properties and adsorption/arrangement at interface. Food Hydrocolloids 2016, 60, 606–619.

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(29) Schmitt, C.; Moitzi, C.; Bovay, C.; Rouvet, M.; Bovetto, L.; Donato, L.; Leser, M. E.; Schurtenberger,

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(31) Destribats, M.; Rouvet, M.; Gehin-Delval, C.; Schmitt, C.; Binks, B. P. Emulsions stabilised by whey

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protein microgel particles: towards food-grade Pickering emulsions. Soft Matter 2014, 10(36), 6941–6954.

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(32) Tzoumaki, M. V.; Moschakis, T.; Kiosseoglou, V.; Biliaderis, C. G. Oil-in-water emulsions stabilized

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Figure Captions

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Figure 1. z-average diameter (Dz, histogram), surface hydrophobicity (Ho, alignment), and

461

contact angle (θ) of natural and sonicated (20 min) QPI nanoparticles at c = 0.25%.

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Figure 2. Typical intrinsic fluorescence emission profiles of the natural (A) and 20

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min-sonicated (B) QPI nanoparticles at c = 0.25%.

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Figure 3. Effect of various solvents (DW, 6.0 M urea, 0.5% SDS, 30 mM DTT) on the

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turbidity of particles in the natural and 20 min-sonicated QPI dispersions at c = 0.25%.

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Figure 4. (A) Droplet size distribution and representative optical microscopy images (500×

467

magnification) of QPI stabilized Pickering emulsions formed at φ = 0.5 with varying c values

468

of 0.25–6%. (B) CLSM images microstructure of the 20 min-sonicated QPI nanoparticles

469

stabilized emulsion at φ = 0.5.

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Figure 5. (A) Visual observation of the QPI stabilized Pickering emulsions prepared at c =

471

0.25–6% with varying φ values of 0.2–0.7 after 0 and 7 days storage. (B) Creaming index (%)

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of emulsions stabilized by the QPI nanoparticles formed at c = 0.25–6% with varying φ values

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of 0.2–0.7 after 7 days storage.

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Figure 6. Percentage of adsorbed protein (%) for the natural and 20 min-sonicated QPI

475

stabilized emulsions, prepared by varying c values (at a specific φ value of 0.5, histogram), or

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at varying φ values (at a specific c value of 6%, alignment).

477

Figure 7. (A) Simulation of the average inverse surface diameter (1/D32) of the natural and 20

478

min-sonicated QPI Pickering emulsions as a function of the total particle area (Seq) normalized

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by the oil volume (Vd). (B) Graphic scheme of QPI nanoparticles packing and arrangement at

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interface in a square or hexagonal array.

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Figure 8. Supposed mechanism for the formation of food-grade Pickering emulsions

482

stabilized by the QPI nanoparticles with ultrasound treatment.

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Table 1. The D43 and D32 values of droplets in the QPI Pickering emulsions, formed at c = 0.25– 6% with varying φ values of 0.2–0.7 by ultrasound treatment. Parameter φ

D43 (µm) 0.25

1

2

6

0.25

1

2

6

0.2

79.128a

61.692b

50.253c

42.95d

11.253a

8.689b

8.187c

8.044c

0.5

121.687a

104.588b

89.302c

62.006d

85.238a

68.799b

54.167c

39.262d

0.7

127.099a

123.016b

120.964c

120.76c

89.322a

85.463b

84.353b

81.2d

20 min

0.2

53.839a

48.294b

47.495c

35.395d

8.195a

7.782b

8.375b

6.59d

-sonication

0.5

113.552a

102.461b

85.875c

55.065d

75.957a

68.974b

13.428c

10.242d

0.7

120.55a

117.169b

112.383c

111.408c

82.487a

81.532b

78.051c

77.675c

natural

c

D32 (µm)

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

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Table 2. The SC% and particle center-to-center distance at interface (Dc-to-c) values of the natural and 20 min-sonicated QPI stabilized emulsions. QPI

Dz (nm)

SC%

Dc-to-c (nm)

Nanoparticles

Square array 

Hexagonal array

4 Dz

√3Dz 6

natural

401.37

48.67

50.97

54.79

20 min-sonication

207.57

54.43

24.93

26.79

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Figure 1

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Figure 2

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Figure 3

120

a

A

Control 20 min-sonication

B

b

Turbidity (µ g/mL)

100

80

C c

60

D

40 d

20 DW

urea

SDS

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Figure 4

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Figure 5

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Creaming Index (%)

B 80

0.25% 1% 2% 6%

60

40

20

0.2

0.5 ϕ

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Figure 6

0.5

0.2

0.7

40

60

75 Control 20 min-sonication

80

50

100

25

0

0.25

2

1

Protein concentration (%, w/v)

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120

Percentage of Adsorbed Proteins (%)

Percentage of Adsorbed Proteins (%)

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Figure 7

A

200 Control 20 min-sonication

150 2

-1

1/D32 (cm )

Y=0.34242X-0.14363 (R =0.9884)

100

2

Y=0.30623X+4.5794 (R =0.99255)

50

0 0

200

400 -1

Seq/Vd (cm )

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Figure 8

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