Covalent Interaction between Rice Protein Hydrolysates and

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Food and Beverage Chemistry/Biochemistry

Covalent interaction between rice protein hydrolysates and chlorogenic acid: Improving the stability of oil-in-water emulsions Xin Pan, Yong Fang, Lingling Wang, Yi Shi, Fei Pei, Peng Li, Ji Xia, Wenfei Xiong, Xinchun Shen, Qiuhui Hu, and MINHAO XIE J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06898 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 24, 2019

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Journal of Agricultural and Food Chemistry

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Covalent interaction between rice protein hydrolysates and

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chlorogenic acid: Improving the stability of oil-in-water

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emulsions

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Xin Pan, Yong Fang*, Lingling Wang, Yi Shi, Minhao Xie, Ji Xia, Fei Pei, Peng Li,

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Wenfei Xiong, Xinchun Shen, Qiuhui Hu

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College of Food Science and Engineering, Nanjing University of Finance and

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Economics/Key Laboratory of Grains and Oils Quality Control and Processing,

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Collaborative Innovation Center for Modern Grain Circulation and Safety, Nanjing

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210023, China

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*Corresponding author: Yong Fang

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E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT

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Protein hydrolysates, as surfactants, can scavenge radicals, but their poor distributions

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at the oil-water interface limit their storage stability. Therefore, we studied covalent

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interaction between rice protein hydrolysates and chlorogenic acid under alkaline

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conditions to improve the physical and oxidative stability of oil-in-water emulsions.

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Turbidity and particle size measurements demonstrated the formation of

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hydrolysates-chlorogenic acid complexes, and their covalent interaction resulted in

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the decrease and redshift of the fluorescence intensity. The emulsifying activity of the

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hydrolysates could be effectively improved after the covalent interaction with 0.025%

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chlorogenic acid. The modified emulsions possessed a notable physical stability

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according to the least changes in size (0.08 μm) and ζ-potential (3.34 mV) of the

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emulsion (P > 0.05). Moreover, the covalent interaction endowed modified emulsions

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with high oxidative stability to effectively inhibit lipid oxidative deterioration during

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storage. The adsorption of hydrolysates to the emulsion interface was increased by the

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adequate addition of chlorogenic acid, resulting in the oil droplet being surrounded by

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a thicker interfacial film. The covalent interaction between the protein hydrolysates

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and chlorogenic acid could be used to construct natural emulsion systems with a

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higher physical and oxidative stability during storage.

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KEYWORDS

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Rice; protein hydrolysates; chlorogenic acid; covalent interaction; emulsion; stability

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INTRODUCTION

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Systems of oil-in-water emulsion play an important role in improving texture,

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appearance, stability, taste and nutrition attributes of common food products, such as

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milk, ice cream and coffee.1, 2 However, emulsion systems are thermal sensitive, often

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resulting in creaming, aggregation and flocculation during storage. To improve the

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shelf-lives and resistances to the environmental stresses of commercial products, it

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was necessary to add stabilizers including emulsifiers, thickening and gelling agents.3

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Emulsifiers are considered significant components to stabilize emulsions for

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functional attributes during long storage. Many emulsifiers now used to prepare stable

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emulsions are synthetic surfactants (e.g., Tween-80 and Spans), or animal-based

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emulsifiers, (e.g., whey protein and sodium caseinate).4 Nevertheless, food

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manufacturers are increasingly interested in developing label-friendly natural

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alternatives instead of synthetic surfactants, and using plant proteins instead of animal

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proteins due to consumer demand for products with all-natural and clean labels.5

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Recently, plant protein and their hydrolysates, such as zein hydrolysates 6 and

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black bean protein hydrolysates,7 are potential emulsifiers due to their ability to

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stabilize emulsion and to prevent oxidation.8 However, these common plant-based

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proteins are not suitable for applications in food products with ‘‘clean” labels due to

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potential allergenicity. Hirano et al.9 reported that rice proteins hardly had any

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sequence similarity to known allergens in databases according to identification by

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mass-based proteomics. Moreover, our unpublished study found that emulsions

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containing trypsin hydrolysates from rice protein (4% degree of hydrolysis) were 3



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more stable than other enzyme hydrolysates against a series of environmental stresses,

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showing their potential for use as label friendly plant-based emulsifiers. Generally,

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the stability of hydrolysates emulsions during storage was associated with

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physicochemical properties of hydrolysates (i.e., size, charge, hydrophobicity), and

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the physicochemical properties affected the interface distribution of hydrolysates in

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emulsions.10 Then, protein hydrolysates are more effective in the aqueous phase and

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the interface than in the lipid phase of emulsions, leading to limited oxidative

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stability.11 To further improve lipid oxidation stability of emulsions, plenty of

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methods have been reported, such as controlling storage conditions, adding

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antioxidants, and engineering the interface.4 Currently, adding antioxidant to

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oil-in-water protein emulsions has attracted interest to improve the storage stability

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due to its ease of operation. Phenolic compounds, as common antioxidants, easily

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bound to proteins, and this binding might improve functional properties of interface

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proteins in emulsions.12 Cao and Xiong13 reported that the addition of chlorogenic

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acid in protein emulsions effectively inhibited lipid oxidation due to the interaction

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between proteins and phenolic compounds, resulting in the increase of phenolic

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compound concentrations at the oil-water interface.

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Phenolic compounds are also readily oxidized into quinones by polyphenol

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oxidase and oxygen, binding with proteins by covalent interaction. The reducing or

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antioxidant ability of the oxidized phenolics might be partially impaired due to

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oxidation.14 However, these quinones could act as protein cross-linker. The oxidised

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phenolic compounds can react with the nucleophilic groups of several amino acids 4


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such as tryptophan, cysteine, methionine, histidine, tyrosine and proline, thus

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inducing the cross-linking via those reactive groups.15 In addition, You et al.16 found

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that catechin covalently bounding to lysine (residues 327) and glutamic acid (residues

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186) in ovotransferrin could improve the antioxidant activity of ovotransferrin.

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Moreover, Intarasirisawat, Benjakul, & Visessanguan 17 found that oxidized tannic

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acid, acting as a protein cross-linker in protein-stabilized emulsions, could improve

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the storage stability and oxidative stability of skipjack roe protein hydrolysate

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emulsions. However, knowledge of the effect of oxidized polyphenols on the

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interfacial distribution of protein in two-phase systems to improve the stability of the

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emulsion is still unclear.

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The objective of this study was to investigate the covalent interaction between

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chlorogenic acid and rice protein hydrolysates by dynamic light scattering and

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fluorescence. Additionally, the stability of emulsions prepared complexes of

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chlorogenic acid and rice protein hydrolysates during storage was studied by

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observing the zeta potential, droplet diameters, microstructures, peroxide values, and

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2- thiobarbituric acid-reactive substances. Moreover, the effect of chlorogenic acid on

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the interface distribution of protein in emulsion was assessed by the partition of

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protein and by fourier transforms infrared spectroscopy (FTIR) of the emulsions to

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elucidate the possible mechanism of oxidised phenolic compounds in improving

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stability of rice protein hydrolysates emulsions.

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MATERIALS AND METHODS

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Materials. Rice protein powder (90%, w/w, dry basis) from rice endosperm were

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purchased from Pioneer Biotech Co., Ltd. (Xi’an, Shan’xi Province, China). Trypsin

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(≥ 2500 U/mg), chlorogenic acid and Nile Red were purchased from Sigma (St. Louis,

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MO). Soybean oil was purchased from Luhua Co., Ltd. (Shandong province, China).

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Other reagents were of analytical grade.

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Preparation of rice protein hydrolysates. The trypsin hydrolysates of rice protein

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were obtained at a degree of hydrolysis of 4% (which produced strong surface

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hydrophobicity from unpublished tests), prepared according to the method described

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elsewhere.18 In brief, rice protein powder (10 g) was dissolved in 150 mL of distilled

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water and stirred for 0.5 h. Before hydrolysis, the rice protein solutions were adjusted

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to 50 °C, pH 8.0. The mass ratio of enzyme to protein was 1 : 250 (w/w), and trypsin

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(0.024%, w/v) was added into the mixed solutions to obtain a mixture containing

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900,000 U of trypsin and rice protein at a concentration of 6% (w/v). After 0.5 h of

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hydrolysis, the mixed solutions were heated to 90 °C for 10 min to terminate the

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hydrolysis reaction and promptly cooled to ambient temperature. Finally, the pH was

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adjusted to 7.0 in the mixture, followed by centrifuging at 12,000 × g for 15 min and

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freeze-drying.

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Interactions between protein hydrolysates and chlorogenic acid.

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Turbidity and particle size measurements. Rice protein hydrolysates (2.5%, w/v)

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mixed with different concentrations of chlorogenic acid (0 ~ 0.125%, w/v) were

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dissolved in phosphate buffer at pH 9. After 24 h of shaking at room temperature, the 6


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absorbance of the mixture was recorded at 600 nm as a measure of the turbidity. The

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particle size and polydispersity index of the complexes were determined according to

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dynamic light scattering (ZS-90, Malvern Instruments, Worcestershire, UK).

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Experiments were conducted in triplicate.

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Fluorescence spectroscopy. Fluorescence measurements were conducted according to

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the method of reported by Wang, et al.19 with minor modification, using a

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fluorescence spectrophotometer (F-7000, Hitachi, Tokyo, Japan). The fluorescence

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intensity of the complexes was determined at emission wavelength from 310 to 400

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nm and an excitation wavelength of 280 nm.

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Preparation of complex of rice protein hydrolysates and chlorogenic acid. The

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conjugation was performed by mixing different concentrations of chlorogenic acid

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and rice protein hydrolysates as described above. Then, the free chlorogenic acid was

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removed according to the method described by You et al (2014).16 The complexes

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were transferred into dialysis tube with a 3000 molecular weight cutoff at 4 °C for 72

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h with distilled water for dialysis, followed by freeze-drying. The contents of covalent

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polyphones bounds in complexes prepared with different concentrations of

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chlorogenic acid were calculated to 13.19 nmol/mg sample, 19.08 nmol/mg sample,

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and 23.21 nmol/mg sample by the Folin-Ciocalteu method.20

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Emulsions preparation. Emulsions were obtained by the method described by Qiu,

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et al.21A mixture with a 10% oil phase (soybean oil, w/w) and a 90% (w/w) aqueous

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phase of freeze-dried complex solution (2.5%, w/v) was homogenized using a

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high-shear mixer (T18, IKA, Staufen, Germany) at 10,000 × g for 3 min. The coarse 7



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emulsions were transported into a high-pressure homogenizer (DSJ-20L, ATS

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Engineering Inc, Shanghai, China) and further homogenized at the pressure of 60 Mpa

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for 2 cycles. Sodium azide (0.01%, w/v) was utilized to prevent microbial growth.

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The measurements of size and ζ-potential in the emulsions were performed at day 1

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and 7.

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Emulsions characterization.

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Particle size and ζ-potential measurement. The particle size, polydispersity index and

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ζ-potential of the emulsions were examined using dynamic light scattering. The

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distilled water was added to the emulsions to avoid multiple scattering before

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measurement. All the measurements were performed in triplicate.

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Emulsifying activity. The emulsifying activity index and the emulsifying stability

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index were tested according to Molina, et al.22 with minor modifications. Fresh

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emulsion (50 μL) was pipetted from the bottom of breaker at 0 and 30 min, followed

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by dissolution with 5 mL of sodium dodecyl sulphate solution (0.1%, w/w). The

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emulsifying activity index (EAI) and the emulsifying stability index (ESI) were

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measured by the absorbance at 500 nm at 0 (A0) and 30 (A30) min, expressed as:

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EAI (m2/g) = 2T × C

A0 × N × Ф × 10000

A30

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ESI (%) =

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where T= 2.303, N is the dilution factor, c is the weight of protein per unit

A0

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volume (g/mL), and Ф is the oil volumetric fraction.

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Oxidative stability. The oxidative stabilities of the emulsions were analysed by

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peroxide value and the 2- thiobarbituric acid-reactive substances in emulsions for 7 8


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days at 25 °C. The measurements of peroxide value were performed by sodium

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thiosulphate titration in the AOCS standard procedure according to the method

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reported by Ahn et al.23 The emulsion (100 uL) was mixed with 10 mL of glacial

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acetic/chloroform (3:2, v/v) and saturated solution of KI (0.5 mL), followed by

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stirring for 30 s. After reacting for 3 min in the dark, 30 mL of the distilled water was

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added to terminate the reaction, and 0.5 mL of starch solution (0.5%, w/v) was

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incorporated as an indicator. Then, the peroxide value was measured by titration of 2

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mM sodium thiosulphate solution against above the mixtures. The 2- thiobarbituric

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acid-reactive substances in emulsions during storage was measured according to Mei,

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et al.24 The thiobarbituric acid solution (2 mL) was added into emulsions (1 mL) and

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the mixed solutions was put in boiling distilled water for 15 min, followed by

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centrifugation at 5,000 × g for 20 min at the ambient temperature. Then, the amount

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of 2- thiobarbituric acid-reactive substances in emulsions was calculated from a

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malonaldehyde standard curve prepared with 1,1,3,3-tetraethoxypropanesolution at an

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absorbance of 532 nm.

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Fluorescence microscopy. The microstructures of the emulsions were observed using

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fluorescence microscopy at day 1 and 7. Before imaging, 20 μL of Nile Red solution

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(0.1% w/w, ex/em wavelength = 488-530/575-580 nm) was added to the emulsions (1

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mL). Then, 50 μL of the mixture was placed on a microscope slide and covered by a

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cover slip, followed by the acquisition of images by digital images processing

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software.

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Partition of protein in emulsion. The measurement of the partition of the protein 9



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hydrolysates emulsion was performed according to the procedure reported by Liu, et

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al.25 The serum and cream layers of the emulsions containing different chlorogenic

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acid concentrations were obtained by centrifuging at 15,000×g for 30 min, followed

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by filtering of the serum layer by a 0.22 mm syringe filter. The Lowry’s method26 was

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used to measure the hydrolysates concentration in the aqueous phase. Then, the

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partition coefficient (PC) for the hydrolysates in the continuous phase was determined

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by the method reported by Huang, et al.27, and calculated by the following equation:

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PC =

Vw V1

Wt

× (Ww -1)

where Vw is the volume of hydrolysates solution, V1 is the volume of oil, Wt is

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the total amount of protein concentration (mg/mL), and Ww is the protein

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concentration of the aqueous phase (mg/mL). The percent of the protein in the

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aqueous phase was calculated as (Ww/Wt) × 100%.

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FTIR. FTIR measurements were performed using a Fourier transform infrared

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spectrometer (Tensor 27, Bruker Optics Inc., Karlsruhe, Germany) equipped with an

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attenuated total reflectance (ATR) accessory. The cream layer of the emulsions were

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freeze-dried and the powder (10 mg) was mixed with potassium bromide (1 g),

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followed by pressing in a tableting machine as test sample. A droplet of soybean oil

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was placed on the ATR crystal as blank sample. Then, spectra in the range of 4000

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cm-1 – 400 cm-1 were recorded for an average of 32 scans.

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Statistical analysis. Analysis of variance (ANOVA) was conducted using the SAS

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Software (Version 9.4, SAS Institute, Cary, NC). The means were compared by

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Duncan’s test at a significance level of P < 0.05. 10


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RESULTS and DISCUSSION

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Effect of chlorogenic acid concentrations on the formation of complexes

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Turbidity

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Fig. 1A shows that the turbidity of the complexes between chlorogenic acid and rice

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protein hydrolysates increased with increasing concentration of the chlorogenic acid.

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Turbidity formation, a complicated process, could be affected by several elements e.g.

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particle diameter, colour, and particle interactions.28 In addition, the turbidity of the

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mixture did not initially change substantially at the concentrations of chlorogenic acid

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less than 0.075% (P > 0.05) and some obvious mixtures aggregates were observed at

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concentrations of chlorogenic acid higher than 0.100%. The increase in turbidity

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might be due to the interactions between the hydrolysates and a higher concentration

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of chlorogenic acid, leading to the micro-aggregation of the complex.

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Sizes

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To provide some further information regarding the complexes, the particle sizes and

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polydispersity index values of complexes were measured (Fig. 1B). When the

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concentration of chlorogenic acid was 0.075%, the particle size was 603 nm and the

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particle size values significantly differed from (P < 0.05) complexes with a higher

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chlorogenic acid concentration. In addition, the polydispersity index of the complexes

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was around 0.85 at concentration of chlorogenic acid higher than 0.075%, indicating

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obvious aggregations. Le and Renard29 found that polyphenols could bridge proteins

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at appropriate mass ratio by multidentate ligands and polyphenols improved the 11



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achievement of the metastable dispersions of particles. Furthermore, increasing the

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amount of polyphenols could result in phase separation. In our study, chlorogenic

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acid-rice protein hydrolysates complexes were soluble at concentrations of

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chlorogenic acid less than 0.075% based on nephelometry data and dynamic light

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scattering analysis. Therefore, all subsequent experiments were controlled at a

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concentration of chlorogenic acid less than 0.075%.

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Fluorescence measurements

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Fig. 2 gives the maximum emission wavelength of rice protein hydrolysates

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containing different concentrations of chlorogenic acid. The fluorescence intensity

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significantly decreased and the maximum emission was redshifted from 347 nm to

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363 nm with higher chlorogenic acid concentrations. These differences in

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fluorescence intensity might be caused by the participation of aromatic amino acids

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(e.g. tyrosine and tryptophan) in the covalent reaction of the rice protein hydrolysates

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with the chlorogenic acid. A similar result was reported in which covalent bonding

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between gelatine and gallic acid or catechin led to the redshifted fluorescence.30

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Moreover, the redshift of the emission maximum of complexes showed the

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conformational changes at the tertiary structures of hydrolysates due to the shift of the

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emission maximum reflecting the changes in the polarity of the tyrosine and

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tryptophan residues.31 Generally, the redshift of the spectrum resulted from the

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exposure of tyrosine and tryptophan residues to the solvent, whereas a blue shift is a

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consequence of transferring tyrosine and tryptophan residues into a more hydrophobic

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environment.32 Therefore, the redshift here indicated that tyrosine and tryptophan 12


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residues were in a more hydrophilic environment due to the tertiary structural change

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of protein hydrolysates. The intensity of the maximum fluorescence emission in the

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complexes was decreased and redshifted, indicating conformational changes. Then,

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these changes could result in the unfolding and denaturation of rice protein

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hydrolysates.33

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Effect of chlorogenic acid concentrations on the stability of rice protein

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hydrolysate emulsions

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Emulsifying properties

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As shown in Table 1, the emulsifying activity index of emulsions first increased and

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then decreased at higher concentration of chlorogenic acid. The emulsifying activity

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index of the emulsions containing 0.025% chlorogenic acid reached to maximum

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values (25.03 m2/g) and it significantly differed from the others (P < 0.05). Then, the

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emulsifying stability index exhibited the same tendency and emulsions containing

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0.025% chlorogenic acid could significantly enhance the emulsifying stability index

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of hydrolysates emulsions from 89.23 to 93.57% (P < 0.05). It has been reported that

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the emulsifying properties depend on the protein–protein and protein–lipid

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interactions.34 The appropriate chlorogenic acids might change the protein-protein

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interaction and this change decreased the free energy to reduce the interfacial tension

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of the oil-in-water interface.35 Then, the decrease in the emulsifying properties of the

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protein hydrolysates emulsions containing a higher chlorogenic acid concentration

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might be attributed to the obvious aggregation (Fig. 1). Similarly, Cao, et al.36 13



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reported that higher concentration levels of (−)-epigallocatechin-3-gallate decreased

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the emulsifying activity of porcine myofibrillar protein due to the extensive

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aggregation.

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Physical stability

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Fig. 3 shows the size and ζ-potential values of emulsions prepared with protein

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hydrolysates containing different chlorogenic acid concentrations. After 7 days of

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storage, the polydispersity index of emulsion containing chlorogenic acid at a

283

concentration of 0.025% were less than 0.2 during storage, showing a uniform size

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distribution.37 In addition, there was no significant (P > 0.05) change in the sizes of

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the hydrolysates emulsions containing chlorogenic acid at a concentration of 0.025%

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from 0.24 μm to 0.32 μm. Intarasirisawat, Benjakul, & Visessanguan11 reported that

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quinone resulted from the oxidation of the polyphenols under alkaline conditions and

288

created a dimer in a side reaction. Then, polypeptides might form covalent C-N or

289

C-S bonds with phenolic rings and their amino or sulphydryl side chains could also

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react with the dimer. Hence, the addition of chlorogenic acid could induce some

291

cross-linking of protein hydrolysates and it might increase the physical stability of the

292

film surrounding the oil droplets. Additionally, the increases in particle size of the

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other emulsions with higher chlorogenic acid concentrations were notable (P < 0.05),

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and the polydispersity index of these emulsions was higher than 0.5 after storage.

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These changes during storage indicated that the instability of the emulsions might be

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due to the aggregation mechanism.

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The ζ-potentials of the hydrolysates emulsions with chlorogenic acid at different

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concentrations was examined in Fig. 3B to further clarify their storage stability. The

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hydrolysates emulsion without chlorogenic acid showed approximately -27.70 mV at

300

pH 9.0. Then, the hydrolysates emulsions incorporated with 0.025% chlorogenic acid

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exhibited minimum potential (-30.77 mV) and it significantly differed from the others

302

(P < 0.05). The charge change can be attributed to the fact that chlorogenic acid

303

might adjoin some positively charged amino acid residue or lead inter-/intra

304

molecular cross-linking, masking negatively charged residues. Additionally,

305

ζ-potential was related to the stability of the emulsions, and an absolute value of

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charge higher than 30 mV was considered to possess more electrostatic stability than

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emulsions with an absolute value of charge lower than 30 mV.38 These observations

308

might also demonstrate the changes in the particles during storage.

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Oxidative stability

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Fig. 4 gives the peroxide value and the 2- thiobarbituric acid-reactive substances to

311

evaluate the oxidative stability of the emulsions containing hydrolysates and

312

chlorogenic acid at different mass ratios. The peroxide value of the emulsions

313

exhibited an increasing tendency during storage, the control was 3.69 mmol/kg oil on

314

day 1 and the amount grew quickly during storage, reaching a value twice that of the

315

initial emulsions after 7 days of storage. In comparison, the peroxide value of the

316

emulsion with 0.025% chlorogenic acid changed from 3.39 mmol/kg oil to 5.21

317

mmol/kg oil after 7 days of storage, and it always showed the lowest peroxide value.

318

This result occurred because phenolic compounds binding to protein could more 15



319

effectively inhibit lipid oxidation than free phenolics due to the free forms being more

320

prevalent in the aqueous phase.39 However, the emulsions with 0.075% chlorogenic

321

acid could not effectively inhibit lipid oxidation according to the change in peroxide

322

values and the 2- thiobarbituric acid-reactive substances. The addition of higher

323

amounts of chlorogenic acid might result in the aggregates of protein (Fig. 1) and

324

phase separation of emulsions. Then, chlorogenic acid might concentrate in aqueous

325

phase and might not enter the oil-water interface, showing lipid oxidation occurred at

326

the interface. Additionally, the emulsions with 0.025% chlorogenic acid significantly

327

reduced the 2-thiobarbituric acid-reactive substances levels, which might be due to a

328

small amount of chlorogenic acid absorbing to the interface of the emulsions and

329

possessing excellent radical scavenging activity.

330

Microstructures

331

Fig. 5 gives the microstructures of the emulsions containing different concentrations

332

of chlorogenic acid during 7 days of storage as observed by fluorescence microscopy.

333

After a day of storage, microscopy images of the hydrolysates emulsions with 0.075%

334

chlorogenic acid exhibited larger particulate clusters or clumps of oil droplets, while

335

the other emulsions showed smaller oil droplets. After emulsions were storaged for 7

336

days, the flocculation was found in the hydrolysate emulsion without chlorogenic

337

acid, and the oil droplets in the emulsions with 0.075% chlorogenic acid were further

338

aggregated. It was noted that the smaller changes in the droplet sizes of the emulsions

339

with 0.025% chlorogenic acid took place during storage (Fig. 3A), demonstrating that

340

0.025 % chlorogenic acid binding with hydrolysates can enhance the storage stability 16


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of emulsions.

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Effect of chlorogenic acid concentrations on the proteins of emulsions

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Partition of protein in emulsions

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Table 1 shows the protein in the aqueous phases of hydrolysate emulsions with

345

different concentrations of chlorogenic acid. The proteins in the aqueous phase of

346

emulsions without chlorogenic acid were 91.02%, and it was significantly decreased

347

to 86.72% at a concentration of 0.025% chlorogenic acid. Lee, et al.40 found that

348

emulsions with more interface proteins around the oil droplet formed a more flexible

349

membrane resulting in higher physical stability. In addition, as the chlorogenic acid

350

concentration increased, the protein in the aqueous phase of the hydrolysates emulsion

351

increased to 92.61%. This phenomenon showed that chlorogenic acid improved the

352

hydrolysates capacity to adsorb to the surface of the oil droplet in a competitive

353

manner. The polypeptide originally adsorbing on the interface was partially replaced

354

by moderate chlorogenic acid or chlorogenic acid binding to hydrolysates indirectly

355

adsorbed to the surface of the oil droplet. Elias, Kellerby and Decker 8 found that the

356

main oxidation occurred at the interface of the oil droplets containing concentrated

357

lipid hydroperoxides. The partitioning of protein hydrolysates showed that the

358

complexes possessed a stronger physical barrier and it could enhance the physical

359

(Fig. 3) and oxidative stability (Fig. 4) of the emulsions during storage.

360

FT-IR

361

As presented in Fig. 6, the spectra of the cream layer of rice protein hydrolysates

362

emulsions showed major bands at 3441 cm-1 (amide A, representative of N-H 17



363

stretching coupled with hydrogen bonding), 1653 cm-1 (amide I, representative of C-O

364

stretching/hydrogen bonding coupled with COO-) and 1548 cm-1 (amide II,

365

representative of C-N stretching coupled with NH bending modes). The functional

366

groups of soybean oil were substantially identical to these the rice protein

367

hydrolysate, resulting in similar peaks of FT-IR. However, the C=C stretching (1650

368

cm-1) of soybean oil was not found at hydrolysates, and it can be due to the fact weak

369

peak of C=C stretching might be easily be covered by the amide I.41 Additionally, the

370

FT-IR of the cream layer in the emulsions with 0.025% chlorogenic acid was

371

measured to confirm the interaction between the interface hydroysates and

372

polyphenols. The analysis of the amide bands was important for the secondary

373

structures of protein due to its sensitivity to hydrogen bonding, dipole-dipole

374

interactions, and the geometry of the polypeptide backbone. 42 Generally, spectra of

375

the complexes showed different amide I (1656 cm−1) and amide II (1545 cm−1) bands

376

and it exhibited different secondary structures of the protein.26 Moreover, none new

377

peaks in the spectra also indicated the interaction between hydrolysates in the cream

378

layer and polyphenols.

379

In conclusion, covalent interaction between hydrolysates and chlorogenic acid

380

could improve the physical and oxidative stability of hydrolysates emulsions. The

381

addition of a moderate amount of chlorogenic acid improved the adsorption of protein

382

onto the oil-water interface during storage, resulting in the interfacial membrane being

383

more compact in the emulsion. Hence, our study suggested that the combination of

18


Page 18 of 32

Page 19 of 32


384

hydrolysates with chlorogenic acid has excellent application potential for improving

385

the stability of a natural emulsion system during a long storage.

386

ACKNOWLEDGEMENTS

387

This work was financially supported by the National Key Research and Development

388

Program of China (Grant no. 2017YFD0401105), the National Youth Talent in Grain

389

Area Support Program of China, and the Priority Academic Program Development of

390

Jiangsu Higher Education Institutions (PAPD).

391

CONFLICTS OF INTEREST

392

The authors declare no competing financial interest.

393

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519

Figure captions: Figure 1. Influence of chlorogenic acid concentrations on the (A) turbidity, (B) particle size of complexes formed with rice protein hydrolysates. (RPH, rice protein hydrolysates; CA, chlorogenic acid; PDI, polydispersity index). Figure 2. Effect of chlorogenic acid concentrations on quenching of rice protein hydrolysates fluorescence. (RPH, rice protein hydrolysates; CA, chlorogenic acid). Figure 3. Droplet size (A) and ζ- potential (B) of emulsions prepared with rice protein hydrolysates emulsions containing different chlorogenic acid concentrations after room temperatures storage 1 and 7 days. Values are given as the means ± SD; different lowercase letters (a-d) differ significantly (P < 0.05) among the emulsions prepared with rice protein hydrolysates emulsions containing different chlorogenic acid concentrations, while * indicated significant difference (P < 0.05) among emulsions after storage for 1 and 7 days. (PDI, polydispersity index) Figure 4. Formation of peroxide values (POV) (A) and thiobarbituric acid-reactive substances (TBARS) (B) of the oil-in-water emulsions containing rice protein hydrolysates and chlorogenic acid at different concentrations at ambient temperature for 7 days. Figure 5. Fluorescence microscopy of the oil-in-water emulsions containing rice protein hydrolysates and chlorogenic acid at different concentrations magnification; 400×. A, emulsions containing rice protein hydrolysates alone; B, rice protein hydrolysates emulsions containing 0.025% chlorogenic acid; C, rice protein hydrolysates emulsions containing 0.050% chlorogenic acid; D, rice protein hydrolysates emulsions containing 0.075% chlorogenic acid. Figure 6. The FTIR spectra of cream layer in emulsions prepared with RPH and RPH-CA complex with an addition of 0.025% CA. (RPH, rice protein hydrolysates; CA, chlorogenic acid). 24


Page 24 of 32

Page 25 of 32


Figure 1

(A) 0.8 start to precipitate

0.7

Turbidity

0.6 0.5 0.4 corresponding to CA concentration of 0.0075%

0.3 0.2 0.000

3000

0.050 0.075 0.100 CA concentration (%, w/v)

0.125

1.2 Size PDI

1.0 0.8

Size(nm)

2000

0.6

1000

0.4

350 300

0.2

250

0.0

200 0.000

0.025

0.050

0.075 0.100 CA concentration (%, w/v)

25


0.125

PDI

(B) 4000

0.025


Page 26 of 32

Figure 2 3500

RPH RPH+0.025% CA RPH+0.050% CA RPH+0.075% CA

Fluorescence intensity

3000 2500 2000 1500 1000 500 0

320

340

360

380

Wavelength (nm)

26


400

Page 27 of 32


Figure 3 day 1 day 7

1.2

PDI

(B)

1.2

0.8

0.3 0.0

b* b

b

a

0.0

c b

-potential (mv)

b*

0.6

0.025%

0.050%

Chlorogenic acid (%, w/v)

0.050%

0.075%

-10 -15 -20

0.075%

a*

-25 -30

0

0.025%

-5

0.9 0.4 0.2

Chlorogenic acid (%, w/v)

0

0

a*

PDI

Mean Particle Diameter (m)

(A) 1.5

-35

27


a* b* a

b

c d

c

day 1 day 7


Page 28 of 32

Figure 4

(A) 12

RPH RPH+0.025CA RPH+0.050CA RPH+0.075CA

POV(mmol/kg oil)

10 8

a b a

6

a

a

4

a

a

b

a a

b

a

b

c c

b

d

c

c

b

2 0

0

1

3

7

5

Storage time (days)

TBARS (mmol/ kg oil)

(B) 1.6

RPH RPH+0.025CA RPH+0.050CA RPH+0.075CA

1.2

a

a

b

c

a a

0.8

b

b c c

a

b a a a

b c

d

d

d

0.4

0.0

0

1

3 5 Storage time (days)

28


7

Page 29 of 32


Figure 5 RPH+0.050% CA

RPH+0.025% CA

RPH

RPH+0.075% CA

Day 1

5 μm

5 μm

5 μm

5 μm

5 μm

5 μm

5 μm

Day 7

29


5 μm


Page 30 of 32

Figure 6

Transmittance (%)

RPH RPH+0.025% CA Soybean oil

3441.13

1548.99

1653.121460.96

3009.59 2853.76

734.17

1745.09 1545.04 1462.41 731.22

3423.08

1656.11

3008.49 2855.86

1747.17 1650.18

1465.40 2856.37 2925.65

3500

722.90

1748.20

3000 2000 1500 Wavelength (nm)

1000

500

30


Page 31 of 32


Table 1. Emulsifying properties and partition of proteins in the aqueous phase versus at the interfacial membrane in oil-in-water emulsions prepared with rice protein hydrolysates containing different chlorogenic acid (CA) concentrations.

CA (%,w/w) EAI(m2/g)

ESI(%)

Proteins in the

Partition coefficient

aqueous phase (%)

(aqueous phase)

0

23.64±0.76b

89.23±0.15b

91.02±1.18a

0.89

0.025

25.03±0.66a

93.57±1.45a

86.72±1.02c

1.37

0.050

22.30±0.27c

87.87±0.81b

88.97±0.62b

1.12

0.075

21.23±0.47c

83.87±0.58c

92.61±1.32a

0.72

Values are given as means ± SD from triplicate determinations. Different letters in the same column mean significant difference at P < 0.05. (EAI, emulsifying activity index, ESI, emulsifying stability index).

31



Page 32 of 32

Rice protein hydrolysates

OH OH OH HO

+ HO

HO

OH

OH HO HO

HO OH OH

-

OH

OH OH HO HO OH

HO

-

HO

OH

HO

OH

-

HO HO OH

+

+

OH

HO

OH

OH

OH

HO

HO

HO

HO

OH

OH

OH

OH

HO

HO

OH HO

HO OH

OH

OH HO HO

OH HO

OH HO HO

HO

Homogenization Chlorogenic acid

The covalent interaction between rice protein hydrolysates and chlorogenic acid could improve adsorption of protein onto the oil-water interface and a thicker interfacial film around oil of rice protein hydrolysates emulsions, resulting in a higher storage stability.


Covalent Interaction between Rice Protein Hydrolysates and

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