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Emulsifying Properties of Polysaccharide Conjugates Prepared from Chin-brick Tea Xiaoqiang Chen, Yuntian Zhang, Yu Han, Qian Li, Li Wu, Jia Zhang, Xiaoling Zhong, Jianchun Xie, Shengrong Shao, Yinjun Zhang, and Zhengqi Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03161 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Journal of Agricultural and Food Chemistry

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Emulsifying Properties of Polysaccharide Conjugates Prepared from

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Chin-brick Tea

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Xiaoqiang Chen*1,2,Yuntian Zhang§1, Yu Han§1, Qian Li1, Li Wu3, Jia Zhang1,

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Xiaoling Zhong1, Jianchun Xie2, Shengrong Shao1, Yinjun Zhang4, Zhengqi Wu1

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1National

"111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China 2Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU),Beijing 100048,China 3Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan 430060, China 4College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China

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§Equal

contribution * Corresponding author Tel: (+86) 27-87950483 Fax: (+86) 27-87950483 E-mail address: [email protected]

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ABSTRACT: Chin-brick tea polysaccharide conjugates (TPC-C) were prepared to

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study their emulsion capabilities. The interfacial tension and the effects of some

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factors, such as storage time, metal ion concentrations (Na+, Ca2+), pH (2.0-8.0), and

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heat treatment (70-100 °C) on the emulsions stabilized by TPC-C were studied. The

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interfacial tension of TPC-C (10.88 mN/m) was lower than that of gum arabic (15.18

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mN/m) at the concentration of 0.08%. As TPC-C concentration increased from 0.1

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wt% to 3.0 wt%, the mean particle diameter (MPD) (d32) of emulsions stabilized by

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TPC-C decreased from 1.88 μm to 0.16 μm. Furthermore, at a concentration of 0.5

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wt% or higher, the MPD (d32) of emulsions stabilized by TPC-C at 25 °C and 60 °C

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for 10 days were between 0.20 μm and 0.50 μm. In the tested pH conditions from 2.0

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to 8.0, the MPD (d32) of emulsions stabilized by 2.0 wt% TPC-C was less than 0.20

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μm. At Na+ concentration conditions between 0.10 mol/L to 0.50 mol/L, the MPD (d32)

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of emulsions were between 0.19 μm to 0.20 μm, and the zeta potential values varied

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from -34.10 mV to -32.60 mV. However, with increasing Ca2+ concentrations from

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0.01 mol/L to 0.05 mol/L, the MPD (d32) of emulsions were between 0.20 μm to

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21.65 μm, and the zeta potential raised sharply from -34.10 mV to -28.46 mV. The

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emulsions stabilized by TPC-C have decent storage stability after high temperature

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heat treatment. Overall, tea polysaccharide conjugates (TPC) strongly stabilized the

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emulsions, which support their new application as natural emulsifiers.

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KEYWORDS: tea polysaccharide conjugates; Chin-brick tea ; emulsifier; stability

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Introduction

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Oil-in-water emulsions are widely utilized in many food products, including milk,

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yogurt, dressings, desserts, ice cream, sauces, and beverages. Because they are

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thermodynamically unstable systems, they require the addition of emulsifiers acting

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on the interface to provide kinetic stability by reducing the interfacial tension and

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generating repulsive interactions (steric or electrostatic).1 At present, emulsifiers used

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in food, cosmetics, and pesticide products are primarily synthetic, including fatty acid

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monoglycerides, sucrose esters, Tween-80, Span, and polysorbates.2,3 With the

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growing desire to consume healthy foods, the development of natural emulsifiers has

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received much attention.4 To date, protein-based emulsifiers have

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utilized in the food industry due to their abundance and good emulsifying ability.

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However, the stability of emulsion stabilized by protein-based emulsifiers is easily

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influenced by high ionic strength, elevated temperature, and pH values close to their

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isoelectric point.5,6

been widely

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Polysaccharide-based emulsifiers are also commonly used in the food industry as

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natural emulsifiers. Tea produced from the buds and leaves of Camellia sinensis L.

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has been widely utilized in dietary and medicinal applications for thousands of years.7

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There are six major categories of teas: green, white, yellow, Oolong, black, and dark

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teas.8 Chin-brick tea, a dark tea similar to Pu'er tea, is primarily produced in Hubei,

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China. Chin-brick tea is prepared from sundried green tea by commonly used methods,

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such as pile-fermentation, sieving, shaping, and drying. The pile-fermentation process

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lasts for several weeks and has greatly improved the quality and functional

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components in tea.9 Tea polysaccharide conjugates (TPCs), an important bioactive

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compounds in six categories of tea, have received increased attention because of their

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various health-promoting activities, including hypoglycemic, immunomodulatory, and

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anti-oxidation.10,11,12 TPCs are macromolecules containing a small amount of

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covalently bound protein. It is thought that the binding protein in TPC is coated by the

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carbohydrate chain and forms a hydrophobic "core",13-16and this polysaccharide and

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protein conjugate could induce an emulsification function to TPC. To date, it is

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difficult to find

reports on the emulsification ability of TPC.

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Therefore, the objectives of this investigation were to quantify the emulsifying

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properties and emulsion stability of polysaccharide conjugates from Chin-brick tea

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(TPC-C). To achieve this objective, emulsions stabilized by different levels of TPC-C

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were generated by high speed blender and microfluidizer. Droplet sizes, zeta potential,

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microstructure, and interfacial properties of the oil-in-water emulsions were measured.

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This work could provide valuable information about the potential utilization of TPC

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as a natural emulsifier.

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MATERIALS AND METHODS

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Materials. Chin-brick tea was purchased from Yichang, Hubei province, China.

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The monosaccharide standards (D-glucuronic acid, D-galacturonic acid, L-fucose,

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D-arabinose, D-mannose, D-xylose, D-fructose, L-rhamnose, D-galactose, D-ribose,

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and D-glucose), gum arabic (GA), and nile red were purchased from Sigma-Aldrich

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(St. Louis, MO, USA). All other reagents used were of analytical grade and were

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obtained from either Sigma-Aldrich (St. Louis, MO, USA) or Aladdin (Shanghai,

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China). Double distilled water was used to prepare all solutions and emulsions.

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Polysacchride conjugates prepared from Chin-brick tea. Chin-brick tea (1.0 kg)

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was ground using a blender. The processed material was extracted by hot water (1:16

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m/v) under 95 °C for 3.3 h with continuous stirring. After centrifugation at 5000 rpm

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for 15 min, the supernatant was collected and concentrated via a rotary evaporator at

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50 °C under reduced pressure. Three volumes of ethanol were added to the

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concentrated solution to precipitate the polysaccharide overnight at 4 °C. The

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obtained polysaccharide was re-dissolved in double distilled water, was concentrated

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again to remove the residual ethanol, and then lyophilized to give TPC-C.10

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Chemical properties of TPC-C. The monosaccharide composition, amino acid

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composition, and relative molecular weight of tea polysaccharide conjugates were

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determined according to the reported methods. The sample was derivatized with

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acetic anhydride, and the monosaccharide fraction was determined by gas

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chromatography. Gel Permeation Chromatography-Multi angle light scattering (GPC-

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MALS) was used to analyze the molecular weight distribution of the TPC-C. After the

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sample was hydrolyzed by HCl, the amino acid composition was determined by the

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external standard method of Hitachi L-8900 Amino Acid Auto Analyzer.17 The pH of

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0.1-3.0 wt% TPC aqueous solution was determined by pH meter (Mettler

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Toledo,Switzerland).

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Interfacial tension measurements. The interfacial tension of TPC-C solutions

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with different concentrations was measured at MCT oil-water interfaces using a Drop

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Tensiometer (Teclis, France).15 The interfacial tension was measured by performing

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an online automatic analysis of the oil drop profile using the Laplace equation:

p 

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2

r

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Where γ is the specific surface free energy, p is the additional pressure and r is the

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radius of curvature.

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Ten microliters of oil droplet was delivered using a micro syringe (Exmire, Japan)

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through a U-shaped stainless steel needle (internal diameter 0.56 mm) into an optical

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glass cuvette containing 5 mL TPC-C solutions maintained at 25 °C. The

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measurement was continued at 25 °C for 12000 s. The concentrations of TPC-C

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ranged from 0.02 wt% to 0.10 wt% at an interval of 0.02, because the polysaccharide

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conjugate solution was not transparent enough at concentrations above 0.10 wt%, and

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the measurement results were not reliable. The GA solution with a concentration of

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0.08 wt% was used as a control. During the detection process, the entire system

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should be balanced to avoid external vibration interference measurements.

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Emulsion stabilized by different concentration of TPC-C.

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i) Emulsion preparation. Emulsions were produced by homogenizing 8.0 wt%

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MCT with 92.0 wt% phosphate buffer solution (PBS, pH 7.0, 0.01M) containing 0.1 -

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3.0 wt% TPC-C using a high-speed shearer (25,000 rpm) for 3 min (T18, IKA,

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Germany). Next, the solution was passed through a high-pressure homogenizer

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(Microfluidics M-110L, USA) at 75 MPa for three cycles. Sodium azide (0.02 % w/v)

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was added to final emulsions to prevent microorganism growth. The concentration

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gradient of TPC-C was set to 0.10 wt%, 0.25 wt%, 0.50 wt%, 1.00 wt%, 2.00 wt%, and

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3.00 wt%.

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ii) Particle size (d32) and zeta potential measurements. The particle size

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distribution of emulsions was determined using static light scattering (Mastersizer

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2000, Malvern Instruments Ltd., UK). Emulsions were dispersed in phosphate buffer

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solutions at the same pH until an obscuration rate of 10-20% was obtained. The mean

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particle size was reported as the surface-weighted mean diameter (d32). The zeta

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potential of emulsions was measured using Zetasizer Nano-ZS (Malvern Instruments,

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UK). Prior to analysis, samples were diluted with 10 mM phosphate buffer at the

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same pH to avoid multiple scattering effects. All tests were carried out at 25 °C and

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repeated in triplicate. It is worth noting that the sample was vortexed for 5 s before

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each measurement.

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Influencing factors of emulsion stability

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i) Effect of storage time on TPC-C emulsion stability. The emulsions stabilized

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by 2.0 wt% TPC-C (pH 7.0) were prepared and distributed into different beakers as

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test samples. These samples were stored at 25 °C and 60 °C, while their zeta potential

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and MPD (d32) were determined daily during the storage of 10 days as the methods

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described above. Their emulsion appearance was observed daily until the 21st day.

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ii)

Effect of metal ions on the stability of TPC-C emulsions. The emulsions

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stabilized by 2.0 wt% TPC-C containing 0.10, 0.20, 0.30, 0.40, and 0.50 mol/L NaCl

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and 0.01, 0.02, 0.03, 0.04, and 0.05 mol/L CaCl2 (pH 7.0) were prepared. The

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emulsion samples were placed in glass test tubes and then incubated at 25 °C for 10

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days. The stability of the emulsions was characterized by observing their appearance,

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measuring the particle size distribution, mean particle diameter (d32), and zeta

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potential. The measurement methods were performed as described above.

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The microstructure of TPC-C emulsions treated by metal ions was determined by

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laser scanning confocal microscope (LSCM) with a SIM scanner for simultaneous

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laser stimulation and observed with a 100 × oil immersion objective (Olympus Corp.,

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Tokyo, Japan). The oil phase of emulsions was dyed by nile red solution (1 mg/mL

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ethanol). The excitation and emission wavelength for nile red were 488 nm and 520

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nm, respectively. All the microstructure images were acquired and analyzed using

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Olympus viewer (Olympus Fluoview Ver.3.1 Viewer).

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iii) Effect of pH on the stability of TPC-C emulsion. The emulsions stabilized by

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2.0 wt% TPC-C (pH 7.0) were distributed into different beakers, and then each

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sample was adjusted to a different pH value ranging from 2.0 to 8.0 at an interval of

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1.0 using HCl or NaOH solutions. The emulsion samples were placed in glass test

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tubes and then incubated at 25 °C for 10 days. The stability of the emulsions was

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characterized by observing their appearance, measuring their particle size distribution,

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mean particle diameter (d32), zeta potential, and the microstructure. These

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measurement methods were performed as described above.

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iv) Effect of thermal treatment on TPC-C emulsion stability.

The emulsions

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stabilized by 2.0 wt% TPC-C (pH 7.0) were prepared and distributed into different

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beakers, and then each sample was bathed at 70 °C, 80 °C, 90 °C, and 100 °C for 30

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min. After heat treatment, these TPC-C emulsions were cooled to room temperature

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and placed at 25 °C for 24 h. The particle size distribution and zeta potential were

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measured. Their mean particle diameter (d32) and appearance were investigated during

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the storage of 10 days at 25 °C. The measurement methods were as described above.

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Statistical analysis. The statistical analysis was carried out using the statistical

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software Statistical Package for Social Sciences 21.0 (SPSS Inc., 2012). The

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comparisons between groups were analyzed by Dunnett’s two-tailed t-test after

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one-way ANOVA.

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RESULTS

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Chemical properties of TPC-C. GPC-MALLS results indicated that the average

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molecular weight of TPC-C was 31580 Da. The monosaccharide and uronic acid

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compositions of polysaccharides from Chin-brick tea were analyzed by complete acid

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hydrolysis. The results indicated that the monosaccharide compositions were found to

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be arabinose, rhamnose, galactose, glucose, xylose, mannose, and galacturonic acid in

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the molar ratio of 1.00, 1.30, 2.65, 0.85, 0.39, 1.40, and 2.15, respectively. The free

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amino acids (Cf.a.a) were not detected in TPC-C. The amino acid composition of

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TPC-C protein moiety were Asp, Glu, Ser, Gly, Thr, Arg, Ile, Phe, Val, Tyr, Pro, Ala,

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Leu, and Lys with the mass content (mg/g) of 7.12, 9.00, 5.72, 5.12, 4.34, 3.77, 2.19,

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2.88, 3.72, 2.72, 2.96, 4.17, 3.36, and 1.29, respectively. The total amount of these

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amino acids (Ca.a.) were 5.84%. The content of the protein moiety of TPC-C was

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calculated using the formula: (Ca.a. − Cf.a.a) × 110/128 with 5.02% of TPC-C. 13

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Interfacial tension of TPC-C. An effective emulsifier must be surface-active and

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have the capacity to reduce the oil-water interfacial tension. Thus, the interfacial

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tension of emulsifiers is closely related to the formation and stabilization of

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emulsions.18,19 Therefore, in order to investigate the potential emulsifying activity of

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TPC-C, we measured the dynamic interfacial tension of TPC-C at different

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concentrations (0.02 - 0.10 wt%) and 0.08 wt% GA in solution with an MCT oil

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droplet (Figure 1). As shown in Figure 1A, the interfacial tension decreased rapidly at

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the beginning of adsorption, indicating that TPC-C can quickly adsorb onto the oil-

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water interfaces. In addition, the interfacial tension decreased as the TPC-C

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concentrations increased, with the highest concentration of TPC-C having the lowest

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interfacial tension. As shown in Figure 1B, at the concentration of 0.08%, the

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interfacial tension of TPC-C (10.88 mN/m) was lower than that of GA (15.18 mN/m),

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which indicated that TPC-C can more quickly adsorb onto the oil-water interface and

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more effectively decrease the interfacial tension. Reports have suggested that the

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formation of emulsion is dependent on the ability of emulsifiers to absorb onto the

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oil-water interface and to undergo a conformational rearrangement to form a

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viscoelastic film surrounding the oil droplets.20 Therefore, TPC-C has the potential to

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form emulsions as a natural emulsifier.

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Effect of TPC-C concentration on the emulsion formation. A well- established

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polysaccharide-based emulsifier, GA (5.0 wt%), was used as a control to compare to

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the emulsifying capability of TPC-C. An oil-in-water emulsion was generated by

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passing oil (8.0 wt%) and aqueous phase (92.0 wt%) through a high-pressure

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homogenizer at 75MPa. The mean particle diameter (MPD) (d32) and particle size

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distribution of emulsions stabilized by different concentrations of TPC-C and GA

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were measured.

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Particle size of emulsions plays an important role in their application.21 As shown

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in Figure 2A, the emulsion stabilized by 5.0 wt% GA had the widest particle size

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distribution, followed by 0.1 wt% TPC-C; both stabilized emulsions had bimodal

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distributions. The particle size distribution of the emulsions stabilized by 0.25-3.0

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wt% TPC-C exhibited unimodal distribution. The higher the concentration of TPC-C,

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the smaller the particle size. If there was insufficient emulsifier molecules to cover the

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oil droplet surface, the emulsions were more susceptible to bridging flocculation.22,23

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Large droplet surface areas can be covered by increasing concentrations of TPC-C

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within the homogenizer, leading to the quick formation of a smaller droplet size and

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the reduction of droplet re-coalescence, indicating that TPC-C possesses excellent

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emulsifying properties.1

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As shown in Figure 2B, the MPD (d32) of emulsions stabilized by TPC-C

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decreased with the increasing concentrations. Specifically, the MPD (d32) value of

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emulsions decreased from 1.88 to 0.16 µm as the TPC-C concentration increased from

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0.1 to 3.0 wt%. This phenomenon may occur, because more emulsifiers can cover a

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larger droplet surface area, leading to smaller droplets sizes within the homogenizer.

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Additionally, oil droplet surfaces can be covered more quickly, thereby decreasing

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droplet re-coalescence during homogenization.24,25 The MPD (d32) value of emulsion

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stabilized by GA was 8.68 µm, which was significantly higher than that of the

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emulsions stabilized by TPC-C. This suggested that the emulsifying capacity of

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TPC-C was more effective than that of the commercial polysaccharide-based

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emulsifiers and that TPC-C can be used as a novel emulsifier.

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Zeta potential is an indicator of the emulsion stability induced by the electrostatic

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repulsive interactions.1 As shown in Figure 2B, the surface charges of droplets in

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TPC-C-stabilized emulsions were above -35.0 mV. In addition, the negative surface

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charge of emulsion droplets decreased with increasing concentrations of TPC-C.

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Because TPC is weakly acidic, the pH of the solution decreases slightly as the

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concentration of TPC increases (Figure 2C), resulting in a slight decrease in the zeta

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potential. Previous studies have also reported that the zeta potential of emulsion

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droplets decreases with a reduction of emulsion pH.26,27

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Emulsion storage stability. Emulsion storage stability is imperative for the shelf

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life of commercial food and beverage products. In order to further investigate the

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potential of TPC-C as a natural emulsifier, we measured the storage stability of

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emulsions stabilized by TPC-C and GA at 25 °C and 60 °C for 10 days.

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As shown in Figure 3, there was no significant change in the mean particle

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diameter of a droplet in emulsion stabilized with TPC-C (concentrations of 0.5 to 2.0

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wt%) at 25 °C for up to 10 days of storage. Particle size distribution analysis showed

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a unimodal distribution at higher TPC-C concentrations during storage, indicating that

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TPC-C possess excellent emulsion-stabilizing properties (Shown in Figure 4). In

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addition, visual observation results showed that TPC-C-stabilized emulsions were still

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stable, and no visible cream layers were observed on the top of the emulsions when

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the TPC-C concentration was above 0.5 wt% after 10 days of storage. GA-stabilized

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emulsions formed visible white-creaming layers on the top after 24 h of storage. The

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relatively high stability of emulsions stabilized by TPC-C at certain concentrations

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maybe due to their small droplet size; the creaming rate is proportional to the square

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of the droplet diameter, and the rate of gravitational separation decreases with a

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reduction in droplet size.1

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No significant difference was observed in the d32 values of emulsions stabilized by

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TPC-C at 0.5, 1.0 and 2.0 wt% at 60 °C after 10 days of storage (Shown in Figure 3).

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As shown in Figure 5, there were no significant changes in the microstructures of

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droplets in emulsions at higher TPC-C concentration during 10 days of storage.

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Interestingly, the particle size distribution of emulsions stabilized by TPC-C

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(concentrations from 0.5 to 2.0 wt%) remained unimodal throughout storage.

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Furthermore, negligible or no creaming was observed in emulsions at higher TPC-C

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concentrations due to the small droplet size in these systems. Nevertheless,

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GA-stabilized emulsions significantly separated into two phases, with a white cream

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layer on top and a relative clear phase at the bottom. The above results indicated that

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the emulsion stabilized by TPC-C had excellent long-term stability; thus, TPC-C

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could be used as a natural emulsifier in commercial food and beverage applications.

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Effects of metal ions on the stability of TPC-C emulsion. Na+ has a small impact

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on the particle size distribution, zeta potential, and MPD (d32) of 2.0 wt% TPC-C

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stabilized emulsions. As the concentration of Na+ increased from 0 to 0.5 mol/L, the

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particle size distribution of the emulsions showed single peaks that almost overlapped

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each other. All MPD (d32) values were lower than 0.30 μm (Shown in Figure 6A). The

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absolute values of the zeta potential of the emulsions slightly decreased from 34.10 to

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32.60 mV (Shown in Figure 6B). These emulsions maintained stability during storage

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for 10 days at 25 °C (As captured in Figure 7). Calcium ion concentrations above 0.03

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mol/L have a destructive effect on TPC-C stabilized emulsions. Calcium ions cause

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droplet aggregation and demulsification during the storage for 10 days at 25 °C

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(Shown in Figure 7). As the concentration of calcium ions increased from 0 to 0.05

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mol/L, the MPD (d32) of the emulsions also increased from 0.2 to 21.65 μm (Shown

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in Figure 8A,and Figure 9), and the absolute values of the zeta potentials of the

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emulsions decreased linearly from 34.10 to 28.46 (Shown in Figure 8B). The linear

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equation was Y=11.98X-32.26, R2=0.99, where Y is the Zeta potential, and X is the

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concentration of calcium ions. The increased calcium ion concentration neutralizes the

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negative charges on the surface of TPC-C and reduces the absolute value of the zeta

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potential in a concentration- dependent manner.

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Effects of pH on the stability of TPC-C emulsion. The effect of pH on the stability

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of TPC-C emulsions is shown in Figure 10 and Figure 11. The size of the emulsion

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droplets did not change significantly under different acidity and alkalinity (pH2-8),

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and the emulsion had a single peak distribution with particle sizes of less than 0.20

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μm (shown in Figure 10A, 11B). The zeta potential values ranged from -2.95 to -35.4

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when the pH value increased from 2 to 8 (shown in Figure 10B), which suggests that

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the emulsion will be more stable in a higher pH solution environment. At a pH greater

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than 4.0, the absolute value of the zeta potential of the TPC-C emulsions was higher

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than 25 mV. As shown in Figure 11B, the size of the emulsion droplets did not change

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significantly under different acidity and alkalinity, and was less than 0.20 μm.

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Thermal stability of TPC-C emulsion. In order to better promote the commercial

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application of emulsions, the thermal stability of emulsions during production,

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processing, transportation, and storage should be studied. The effect of heat treatment

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at 70-100 °C on the stability of TPC-C emulsions was investigated. The emulsion

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stabilized by 2.0 wt% TPC-C was stored at 60 °C for 10 days to maintain good

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stability as a control. All the particle size distributions of TPC-C emulsions

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heat-treated at different temperatures overlap with that of the control, have a single

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peak distribution, and maintained a small particle size (Figure 12A). The zeta

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potential values of the heat-treated emulsions were measured on the first, seventh, and

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tenth days of storage at 25 °C (as shown in Figure 12B). The zeta potential values of

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the heat-treated emulsions at different temperatures were close and maintained at

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approximately -32.5 mV. The magnitude of the change in the absolute values of the

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zeta potential was 0.30-1.96 during storage for 10 days, indicating that the high

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temperature heat treatment did not destroy the electrostatic repulsion and steric

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hindrance between the droplets in the emulsions. The average particle size d32 of the

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TPC-C emulsions treated at different temperatures was not significantly changed with

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the increase of storage days, and was maintained near 0.2 μm (Figure 12C). The

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TPC-C emulsions did not appear to be layered at 25 °C by the 10th day after heat

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treatment of 70-100 °C (Figure 12D), and they were apparently indistinguishable from

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the unheated control. The TPC-C emulsions maintained storage stability after high

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temperature heat treatment.

336 337

DISCUSSION

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The emulsification properties of TPC have been discovered in this work, which will

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greatly enhance its development and application value. At present, emulsifiers used in

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food industry, such as fatty acid monoglycerides and sucrose esters, are primarily

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synthetic.3, 4 However, with the increasing concern about healthy foods, the

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development of natural emulsifiers has received much attention.2 Many current

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studies on natural emulsifiers focus on protein or its complex with exogenous

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polysaccharides.28 A complex in which a polysaccharide is covalently or

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non-covalently bound to a protein is typically used as an emulsion stabilizer to

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synergistically exert the emulsification activity of the protein and the stability of the

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polysaccharide over a wider range of pH and ionic strength.30 TPC is composed of a

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polysaccharide and a covalently bound protein, which allows it to have a decent

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emulsification properties without the need to be attached to external proteins. The

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polysaccharide moiety provided pH stability to the TPC-C stabilized emulsions .

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Calcium ions, unlike sodium ions, destroyed the emulsion stability. The Ca2+-induced

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protein aggregation developed because of three effects: 1) electrostatic shielding, 2)

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crosslinking of adjacent anionic molecules by forming protein-Ca2+-protein bridges,

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and 3) ion-specific hydrophobic interaction.29,30 In this study, the absolute value of the

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zeta potential of the emulsion decreased in a calcium ion concentration-dependent

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manner. The positively charged calcium ions neutralize the negative charge on the

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surface of the TPC-C, which resulted in cross-linking between the molecules and

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emulsion aggregation .

359 360

Funding This research was financially supported by the National Natural Science Foundation

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of China (grant number 31871813) and the Beijing Advanced Innovation Center for

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Food Nutrition and Human Health (grant number 20161012).

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Notes The authors declare no competing financial interest.

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REFERENCE (1) McClements, D. J.; Gumus, C. E. Natural emulsifiers-Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci. 2016, 234, 3-26. (2) Krog, N.; Sparsø, F.; Friberg, S.; Larsson, K.; Sjöblom, J. Food emulsifiers: their chemical and physical properties. Food Emulsions. 2004. (3) Wang, F.; Marangoni, A. Advances in the application of food emulsifier α-gel phases: saturated monoglycerides, polyglycerol fatty acid esters, and their derivatives. J Colloid Interface Sci, 2016, 483, 394-403. (4) Mcclements, D. J.; Bai, L.; Chung, C. Recent advances in the utilization of natural emulsifiers to form and stabilize emulsions. Annual Review of Food Science and Technology. 2017, 8(1), 205-236. (5) Dickinson, E. Interfacial structure and stability of food emulsions as affected by protein–polysaccharide interactions. Soft Matter. 2008, 4(5), 932. (6) McClements, D. J.; Gumus, C.E. Natural emulsifiers - Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci. 2016, 234, 3-26. (7) Nie, S.P.; Xie, M.-Y. A review on the isolation and structure of tea polysaccharides and their bioactivities. Food Hydrocolloids. 2011, 25(2), 144-149. (8) Zhu, Y. F.; Chen, J. J.; Ji, X. M.; Hu, X.; Ling, T. J.; Zhang, Z. Z.; Bao, G. H.; Wan, X. C. Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea. Food Chem. 2015, 170, 110-117. (9) Lu, S. F.; Zheng, P. C.; Liu, P. P.; Wang, S. P.; Teng, J.;Feng, L.; Gong, Z.M. Research Progress on Qingzhuan Tea. Acta Tea Sinica. 2018, 59 (3),162 -167. (10) Chen, X. Q.; Fang, Y. P.; Nishinari, K.; We, H.; Sun, C. C.; Li, J. R.; Jiang, Y. W. Physicochemical characteristics of polysaccharide conjugatesprepared from fresh tea leaves and their improving impaired glucose tolerance. Carbohydrate Polymers. 2014, 112, 77-84. (11) Chen, X. Q.; Zhang, Z. F.; Gao, Z. M.; Huang, Y.; Wu, Z. Q. Physicochemical properties and cell-based bioactivity of Pu’erh teapolysaccharide conjugates. International Journal of Biological Macromolecules. 2017, 104, 1294– 1301. (12) Chen, X. Q.; Ye, Y.; Cheng, H.; Jiang, Y. W.; Wu, Y. L. Thermal Effects on the Stability and Antioxidant Activity of an Acid Polysaccharide Conjugate Derived from Green Tea. J. Agric. Food Chem. 2009, 57, 5795–5798. (13) Chen, X. Q.; Du, Y.; Wu, L.; Xie, J. C.; Chen, X. L.; Hu, B. B.; Wu, Z. Q.; Yao, Q. F.; Li, Q. Effects of Tea Polysaccharide Conjugates and Metal Ions on Precipitate Formation by Epigallocatechin Gallate and Caffeine, the Key Components of Green Tea Infusion. J. Agric. Food Chem 2019, 67(13), 3744-3751. (14) Chen, X. Q.; Xie, J. C.; Huang, W.; Shao, S. R.; Wu, Z. Q.; Wu, L.; Li, Q. Comparative analysis of physicochemical characteristics of green tea polysaccharide conjugates and its decolored fraction and their effect on HepG2 cell proliferation. Industrial Crops and

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Products. 2019, 131, 243-249. (15) Chen, X. Q.; Shao, S. R.; Xie, J. C.; Yuan, H.; Li, Q.; Wu, L; Wu, Z. Q.; Yuan, H. B.; Jiang, Y. W. Analysis of Protein Moiety of Polysaccharide Conjugates Water-extracted from Low Grade Green Tea. Chem. Res. Chin. Univ. 2018, 3(44), 691-696. (16) Chen, X. Q.; Song, W.; Zhao, J.; Zhang, Z. F.; Zhang, Y. T. Some physical properties of protein moiety of alkali-extracted tea polysaccharide conjugates were shielded by its polysaccharide. Molecules 2017, 22(6), 914 (17) Chen, X. Q.; Lin, Z.; Ye, Y.; Zhang, R.; Yin, J. F.; Jiang, Y. W.; Wan, H. T.; Suppression of diabetes in non-obese diabetic (NOD) mice by oral administration of water-soluble and alkali-soluble polysaccharide conjugates prepared from green tea. Carbohydrate Polymers, 2010, 82, 28–33. (18) Dickinsion, E. Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids. 2003, 17, 25-39 (19) Jiang, J.; Jin, Y.; Liang, X.; Piatko, M.; Campbell, S.; Lo, S. K.; Liu, Y. Synergetic interfacial adsorption of protein and low-molecular-weight emulsifiers in aerated emulsions. Food Hydrocolloids. 2018, 81, 15-22. (20) Uruakpa, F. O.; Arntfield, S.D. Emulsifying characteristics of commercial canola protein– hydrocolloid systems. Food Research International. 2005, 38(6), 659-672. (21) Xu, X.; Zhong, J.; Chen, J.; Liu, C.; Luo, L.; Luo, S.; Wu, L.; McClements, D. J. Effectiveness of partially hydrolyzed rice glutelin as a food emulsifier: Comparison to whey protein. Food Chem. 2016, 213, 700-707. (22) Dickinson, E. Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids. 2009,.23(6), 1473-1482. (23) Mc, C.; David, J. Enhanced delivery of lipophilic bioactives using emulsions: a review of major factors affecting vitamin, nutraceutical, and lipid bioaccessibility. Food & function, 2018, 9, 22-41. (24) Dickinson, E. Strategies to control and inhibit the flocculation of protein-stabilized oil-in-water emulsions. Food Hydrocolloids. 2019, 96, .209-223 (25)Tisserand, C.; Brambilla G.; Meunier G.; Parker A. Predicting the long-term stability of depletion-flocculated emulsions by static multiple light scattering (SMLS). Journal of Dispersion Science and Technology. 2019, 1-8. (26) Ma, F.; Zhang, Y.; Yao, Y.; Wen, Y.; Hu, W.; Zhang, J.; Liu, X.; Bell, A. E.; Tikkanen-Kaukanen, C. Chemical components and emulsification properties of mucilage from Dioscorea opposita Thunb. Food Chem. 2017, 228, 315-322. (27) Nakauma, M.; Funami, T.; Noda, S.; Ishihara, S.; Al-Assaf, S.; Nishinari, K.; Phillips, G. O. Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids. 2008, 22(7), 1254-1267. (28) Krstonosic, V.; Dokic, L.; Nikolic, I.; Milanovic, M. Influence of xanthan gum on oil-in-water emulsion characteristics stabilized by OSA starch. Food Hydrocolloids. 2015, 45, 9-17. (29) Ju, Z. Y.; Kilara,A. Aggregation induced by calcium chloride and subsequent thermal gelation of whey protein isolate, J Dairy Sci ,1998, 81, 925-931.

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(30) Wang, C. H.; S. Damodaran. Thermal gelation of globular proteins: influence of protein conformation on gel strength. J. Agric. Food Chem. 1991, 39, 434-442.

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Figure Captions Figure 1. Interfacial tension at different concentrations of TPC-C (A) and 0.08 wt% GA (B) at 25 °C . Figure 2. Particle size distribution (A), the mean particle diameter (d32) and zeta potential of emulsions stabilized by different concentrations of TPC-C at 25 °C (B); pH of different concentrations of TPC-C aqueous solution at 25 °C (C). Figure 3. The mean particle diameter (d32) of emulsions stabilized by different concentrations of TPC-C during 10 days of storage at 25 °C (A) and 60° C (B). Figure 4. Particle size distribution of emulsions stabilized by different concentrations of TPC-C during 10 days storage at 25 °C (A) and 60 °C (B). Figure 5. Appearance changes were observed on day 0 and day 10 (A) and day 21 and day 31 (B) for different concentrations of TPC-C emulsions and 5.00% GA emulsion. (The numbers 1, 2, 3, 4, 5, 6, and 7, in pictures represent 0.10% TPC-C emulsion, 0.25% TPC-C emulsion, 0.5% TPC-C emulsion, 2.0% TPC-C emulsion, 3.0% TPC-C emulsion, and 5.00% GA emulsion, respectively. Figure 6. Particle size distribution (A) and zeta potential (B) of emulsions stabilized by 2.0 wt% TPC-C with different concentrations of NaCl. Figure 7. Appearance change of emulsions stabilized by 2.0 wt% TPC-C with different metal ion concentrations over 10 days at 25 °C. The numbers 1-5 in pictures represent the concentration of NaCl from 0.10 to 0.50 mol/L, respectively. The numbers 6-10 in pictures represent the concentration of CaCl2 from 0.01mol/L to 0.05 mol/L, respectively. Figure 8. Particle size distribution (A) and zeta potential (B) of emulsions stabilized by 2.0 wt% TPC-C with different concentrations of CaCl2. Figure 9. Microscopic appearance of 2.0 wt% TPC-C emulsions stained with nile red dye and observed by using LSCM under different metal ion concentrations on the 10th day of storage at 25 °C. The ionic strength of the solution was prepared by NaCl (0.10-0.50 mol/L) and CaCl2 (0.01-0.05 mol/L). Figure 10. Particle size distribution (A) and zeta potential (B) of the emulsions stabilized by 2.0 wt% TPC-C under different pH conditions on the 10th day at 25 °C. Figure 11. Appearance (A) and 10th-day microscopic appearance (B) of emulsions stabilized by 2.0 wt% TPC-C under the conditions of pH 2-8 and 25 °C, stained with nile red dye and observed using LSCM. Figure 12. Particle size distribution (A) and zeta potential (B) of 2.0 wt% TPC-C stabilized emulsion under the thermal treatment at 70-100 °C during 24 h storage at 25 °C, MPD (d32) (C) and appearance (D) during the 10 day storage at 25 °C .The emulsion stabilized by 2.0 wt% TPC-C was stored at 60 °C as an untreated control.

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Figure 1.

A 0.10% TPC-C 0.08% TPC-C 0.06% TPC-C 0.04% TPC-C 0.02% TPC-C

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28 26 24 22 20 18 16 14 12 10 0

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Figure 2. 5.00% GA 0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C 3.00% TPC-C

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Figure 3. 0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C

A 3.5

D[3,2]-25C

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Figure 4. 40

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Figure 6.

A Fresh emulsion 0.10 mol/L NaCl 0.20 mol/L NaCl 0.30 mol/L NaCl 0.40 mol/L NaCl 0.50 mol/L NaCl

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Figure 9.

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