Effect of protein molecules and MgCl2 in the water phase on the dilational rheology of polyglycerol polyricinoleate molecules adsorbed at the soy oil-water interface

Accepted Manuscript Effect of protein molecules and MgCl2 in the water phase on the dilational rheology of polyglycerol polyricinoleate molecules adsorbed at the soy oil-water interface Qiaomei Zhu, Ce Wang, Nazia Khalid, Shuang Qiu, Lijun Yin PII:

S0268-005X(17)30710-5

DOI:

10.1016/j.foodhyd.2017.06.030

Reference:

FOOHYD 3959

To appear in:

Food Hydrocolloids

Received Date: 24 April 2017 Revised Date:

4 June 2017

Accepted Date: 22 June 2017

Please cite this article as: Zhu, Q., Wang, C., Khalid, N., Qiu, S., Yin, L., Effect of protein molecules and MgCl2 in the water phase on the dilational rheology of polyglycerol polyricinoleate molecules adsorbed at the soy oil-water interface, Food Hydrocolloids (2017), doi: 10.1016/j.foodhyd.2017.06.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Dilational rheological properties of soy oil/water interfacial film stabilized by PGPR with addition of MgCl2 and protein in the aqueous phase 120

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Effect of protein molecules and MgCl2 in the water phase on the dilational rheology of

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polyglycerol polyricinoleate molecules adsorbed at the soy oil-water interface Qiaomei Zhua, Ce Wangb, Nazia Khalidc, Shuang Qiua, Lijun Yina,d*

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Agricultural University, Beijing, China.

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b

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Key Laboratory of Functional Dairy,, College of Food Science and Nutritional Engineering, China

Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China.

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National Institute of Food Science and Technology, University of Agriculture Faisalabad, Pakistan. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food

Science and Nutritional Engineering, China Agricultural University, Beijing, China.

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*

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(Lijun Yin) Tel. /Fax: +86-10-62737424, E-mail: [email protected]

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Affiliation addresses: 17, Qinghua East Road, Haidian, Beijing, 100083, P.R. China

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Abstract

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Corresponding author (

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The objective of this study was to investigate the effect of inorganic salt and protein in the

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aqueous phase on the dilational rheology properties of interfacial film stabilized by the hydrophobic

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emulsifier polyglycerol polyricinoleate (PGPR). The interfacial behavior was investigated using the

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oscillating drop method. With increased PGPR concentration, the interfacial tension tended to

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decrease and reached an equilibrium value of 3.3 mN.m-1, at 1.0% (w/w) PGPR. With 0.01% (w/w)

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PGPR in the oil phase, the presence of whey protein isolate (WPI) increased the dilational elasticity

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modulus of PGPR, but the addition of bovine serum albumin (BSA) decreased the elasticity modulus.

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This was likely due to competitive adsorption of BSA and PGPR at the soy oil/water interface,

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resulting in the desorption of BSA from the interface. At 1.0% (w/w) PGPR, both WPI and BSA

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increased the interfacial dilational elastic modulusand the reason might be that the presence of

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ACCEPTED MANUSCRIPT protein could suppress the diffusion-exchange process of PGPR between bulk phase and interface.

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The addition of MgCl2 may enhance the adsorption of PGPR molecules at the interface and therefore

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increased the dilational modulus.

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Keywords: Drop shape tensiometry; Polyglycerol Polyricinoleate; Protein; MgCl2; Interfacial

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rheology

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1. Introduction

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The continued development of the food industry is strongly dependent on the application of

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emulsion systems. Food products including butter, milk, sauces, salad dressings, and soups are

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examples of either oil-in-water (O/W) emulsion systems, wherein the oil is the dispersed phase and

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water is the dispersion medium or water-in-oil emulsion systems, prepared with water as the

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dispersed phase and oil as the continuous phase. Proteins are important ingredients in food emulsion,

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due to their good emulsifying property (Mitidieri & Wagner, 2002; Chen et al., 2016b) and mixtures

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of protein and emulsifiers are widely used in emulsion products (Chen et al., 2016a). Most food

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emulsion products are complex systems that contain various components including salt, sugar, and

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enzymes. These components may interact with the emulsifier, affecting the stability of emulsion

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systems. Previous study investigated the effect of salt content and emulsifier composition blends on

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the physical properties of salad dressing-type emulsions (Martínez et al., 2007). It was indicated that

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both physical stability and viscoelastic properties of emulsion stabilized by egg yolk or pea protein

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were progressively influenced by salt content.

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Emulsions contain both a dispersed and a continuous phase, and the boundary between the

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phases is the interfacial layer. Protein and emulsifiers can coexist in this interfacial layer, resulting in

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cooperative or competitive adsorption between the emulsifiers and proteins. Both proteins and 2

ACCEPTED MANUSCRIPT surfactants can affect the interfacial layer and the stability of emulsions, via their composition and

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structure (Liu et al., 2017). Previous studies reported that low molecular weight emulsifiers had

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higher surface activity than proteins, allowing the displacement of protein molecules from the

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interface and exhibiting a detrimental effect on emulsion stability (Courthaudon et al., 1991; Euston

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et al., 1995). Cornec et al. (1996) reported that the use of a hydrophilic emulsifier was more effective

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at destabilizing emulsions than a hydrophobic emulsifier. The adsorption behavior of surfactants and

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proteins at the interface is complex and their adsorption property depends on the composition of the

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interfacial film (Patino, Niño, & Sánchez, 2003).

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Salt addition may yield two opposite effects on the stability of emulsion systems. Srinivasan et

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al. (2000) reported that the presence of NaCl could destabilize an emulsion that was stabilized by

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protein, by reducing electrostatic repulsion and altering the structural organization of water

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molecules at the interface. For W/O emulsions, previous researchers reported that increased

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concentration of NaCl could decrease the interfacial elasticity of surfactant Span 80 and Span 83 due

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to the “salting-out” effect, an effect based on electrolyte-nonelectrolyte interaction, in which the

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non-electrolyte become less soluble at higher salt concentration (Opawale & Burgess, 1998).

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Alternatively, salt addition could improve the stability of an emulsion system. Márquez et al. (2010)

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reported that Ca2+ could improve the stability of W/O emulsions as a result of a lower attractive force

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between water droplets and a higher adsorption density of the surfactant. In addition, it has been

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reported that NaCl would interact with PGPR molecules at the soy oil-water interface and therefore

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improve the stability of W/O emulsions (Scherze, Knoth, & Muschiolik, 2006).

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Investigation of interfacial properties is challenging as the compounds adsorbed at the interface

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are complex. Recently, researchers have explored the use of dilational rheology as an efficient 3

ACCEPTED MANUSCRIPT method to study the interfacial adsorption properties of surfactant molecules, hydrocolloid

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macromolecules, and polymer-surfactant complexes (He et al., 2008; Gülseren & Corredig, 2012).

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The details of molecular packing behavior could be determined by exploring the dependence of

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interfacial viscoelasticity on time, disturbance frequency, and concentration of surfactant, polymer or

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their complexes. The interfacial rheological properties of different sorbitan fatty acid ester types

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(Span20, 80, 83 and 85) were investigated and it was reported that the interfacial elasticity was

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affected by the surfactant concentration, temperature and the presence of inorganic salt (Opawale &

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Burgess, 1998). Cao et al. (2014) investigated the equilibrium and dynamic interfacial properties of a

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protein/surfactant mixture at the decane/water interface. The results showed that different types of

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proteins exhibited different interfacial conformations at the liquid interface.Compared with the

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interfacial tension, the interfacial dilational modulus values were more sensitive to the conformation

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of the protein/surfactant mixture. The changes in interfacial dilational rheology parameters indicate

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an alteration in the interfacial area, and this technique is sensitive to the orientation and interaction of

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molecules. The interfacial rheological property can provide information about the adsorption

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properties of a protein/surfactant mixture at the interface, in the presence of other additives in the

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aqueous phase.

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In our previous study, W/O emulsions were prepared using PGPR as the lipophilic emulsifier. It

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was found that the addition of MgCl2 and protein in the water phase could increase the viscosity and

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decrease the particle size of the prepared W/O emulsion, and therefore improved their stability

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against coalescence (Zhu et al., 2015; Zhu et al., 2016). The stabilizing effect was attributed to the

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reduction of interfacial tension (Zhu et al., 2016). However, Lucassen-Reynders (1993) reported that

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surfactants acted by imparting specific dynamic properties to the interface rather than by reducing

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the equilibrium interfacial tension. For long-term stability against coalescence or phase separation,

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the strength of interfacial film was considered to be more important in stabilizing emulsion systems

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than the interfacial tension (Sjoblom, 2005). Rocha et al. (2016) reported that, except for the

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interfacial films, resulting in a relatively stable water in heavy oil emulsions. In addition, Gülseren

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and Corredig (2012) reported that hydrophobic interactions between protein and PGPR lowered the

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equilibrium interfacial tension. However, the reduction in interfacial tension is insufficient to explain

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the effects on stability as emulsions of large surfactant molecule may remain stable even at high

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interfacial tension values. Garti et al. (1994) have reported that protein could interact with surfactants

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via hydrophobic interactions or hydrogen bonding and protein/surfactant might form a complex at

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the oil-water interface,

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protein and surfactant in stabilizing the W/O and W/O/W emulsion system is controversial, and the

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effects of inorganic salt on the interfacial property of emulsion stabilized by PGPR and protein have

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not been reported before.

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which could decrease the overall stability of emulsion systems. The role of

In this study, the interfacial viscoelastic properties of adsorbed film stabilized by PGPR, protein

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and MgCl2 were investigated in order to clarify the stabilizing mechanism of W/O and W/O/W

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emulsion. This study would provide new insights into the interaction between protein and low

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molecular weight emulsifiers, with the presence and absence of inorganic salts. Additionally, it can

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give information about the formulation optimization of W/O and W/O/W double emulsion on base of

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protein-surfactant mixture.

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2. Materials and Methods

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2.1. Materials

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Polyglycerol Polyricinoleate (PGPR) was kindly provided by Taiyo Kagaku Co., Ltd (Tokyo,

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Japan). Bovine Serum Albumin (BSA, 98% pure) was purchased from Roche (Basel, Switzerland).

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Whey Protein Isolate (WPI, 92.0-95.0% pure) was supplied by Glanbia Co. (Idaho, USA).

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Magnesium chloride hexahydrate (MgCl2.6H2O, 99% pure) was purchased from Yixiubogu

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Biotechnology Co. Ltd (Beijing, China). Soybean oil was obtained from a local supermarket without 5

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further purification. All aqueous solutions were prepared using Milli-Q water.

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2.2. Determination of interfacial tension and interfacial viscoelasticity Water/oil emulsion system was prepared by mixing 40 wt.% aqueous phase and 60 wt.% oil

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phases. Protein solutions (concentration varied from 0.1 to 0.7%) were prepared by dispersing WPI

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powder in Milli-Q water with or without 0.1 M MgCl2 and stirred for 8 h at room temperature to

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allow complete dissolution. The oil phase was prepared by dispersing different concentrations of

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PGPR varying from 0.001% to 5% (w/w) in soy oil at 65 °C for 10 min.

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The soy oil was used without further purification in order to investigate the real rheological

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behavior of soy oil/water interface based on commercial oil products. Previous studies reported that

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the interfacial adsorption behavior of protein or surface-active polysaccharide can be affected by the

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impurities in the oil phase, as the surface-active protein or polysaccharide could displace the

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impurities from the oil/water interface (Camino et al., 2009, 2012; Murray, 1997). As a consequence,

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the measurements were carried out using commercial oil to make the soy oil/water interface system

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close to the real products, which was beneficial for their industrial application. The equilibrium

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interfacial tension between the aqueous phase and soybean oil containing PGPR was determined

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using Krüss K100 tensiometry (Krüss, Hamburg, Germany)at room temperature. A Wilhelmy plate

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was used to measure the interfacial tension at the liquid-liquid interface and measurements were

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recorded every 3 s until equilibrium was reached (the waiting time was around 600 s).

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Interfacial rheological parameters were measured by using an oscillating pendant drop

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tensiometer from DataPhysics OCA 20 (DataPhysics Instruments GmbH, Germany). To measure the

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dilational viscoelasticity of interfacial film at the oil-water interface, a stainless steel needle was used

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to inject a water drop of 8 µL into the quartz cuvette containing the oil phase. An image of the drop 6

ACCEPTED MANUSCRIPT was captured by a CCD camera and these images were converted to numerical data by fitting to the

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Laplace equation. The oscillation measurements of the drop were performed at a fixed frequency at

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0.1 Hz and the amplitude was 5% of the original drop volume (∆A/A, 5%), which was within the

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linear viscoelastic region. When the interfacial adsorption was equilibrated, the interfacial film was

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then disrupted by a frequency oscillation that varied from 0.005 to 0.1 Hz. All experiments were

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performed at 25±0.5 oC. In sinusoidal interfacial compression and expansion experiment, the

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interfacial dilational modulus (E), namely complex interfacial modulus, gives a measurement of the

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interfacial resistance to changes in area. According to Gibbs, the dilational modulusis defined as a

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change in the interfacial tension (γ) for a relative surface area change ∆A/A (Lucassen & Van Den

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Tempel, 1972), as described below:

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=

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(1)

where dγ is the interfacial tension (IFT) variation and A is the interfacial area.

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When the interfacial adsorption reaches equilibrium, the interfacial film exhibits some

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viscoelasticity (Wang et al., 2014). This means that when the interfacial adsorption layer undergoes

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periodic compressions and expansions at a given frequency, the relaxation process occurs. To make

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sure that the soy oil/water interface was equilibrated, the interfacial tension was measured as a

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function of time. The interface was assumed to be equilibrated when the trend of interfacial tension

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varied within 0.5 mN/m in 30 min.The dilational modulus can be divided into the real part (storage

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modulus) and the imaginary part (loss modulus), which corresponds to the elasticity (Ed) and

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viscosity (Eη=ωηd), as described in the following equation：

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The storage modulus (Ed) represents the elastic energy stored in the interface, known as 7

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dilational elasticity, and the loss modulus (Eη) accounts for the energy dissipated in the relaxation

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process, expressed as dilational viscosity modulus.

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2.3. Statistical analysis All tests were carried out at least in duplicate and all data analyses were performed with Origin

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9.1 software (Origin Lab Corporation, Northampton, MA, USA).

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3. Result and discussion

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3.1. Dynamic interfacial tension of PGPR adsorbed at the oil-water interface.

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The equilibrium interfacial tension between the soy oil and water phases as a function of PGPR

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concentrations was shown in Fig.1 A. Without PGPR, the equilibrium IFT at the oil-water interface

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was 24.5 mN.m-1. Earlier research by Gülseren and Corredig (2012) measured the IFT at water-oil

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interface was 30.5 mN.m-1. The difference between these results may be due to the presence of

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surface-active molecules in the soy oil. However, according to the Marze (2009), compared with

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unpurified oil, purified oil did not further decrease the interfacial tension and modify the relaxation

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behavior of the oil/water interface, suggesting that unpurified oil did not significantly modify the

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interfacial behavior. The IFT tended to decrease with increasing PGPR concentration and then

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leveled off with an equilibrium value of 3.3 mN.m-1 at 1.0 wt.% PGPR. The critical micelle

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concentration (CMC) of PGPR in soy oil is around 1 wt.% and similar results have been reported by

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previous studies (Marze, 2009; Bus, Groeneweg, & vader Voorst Vader,1990). It is generally

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accepted that a lower IFT value favors emulsification since less energy is required for producing

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smaller droplets, which could improve the stability of the emulsion against coalescence. IFT may

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partially reflect the interfacial properties of the surfactant, and a decrease of IFT value indicates a

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higher adsorption of surfactant molecules at the oil/water interface. With a higher adsorption amount

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of PGPR molecules at soy oil/water interface, the formed interfacial film is able to remain stable

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against the interfacial deformation under periodic compressions and expansions. In order to fully

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characterize the interfacial behavior of PGPR, dilational rheological measurements were performed

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to study the diffusion and reorganization of surfactant molecules at the oil/water interface. The dynamic interfacial elasticity modulus values for different concentrations of PGPR were

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measured with an oscillating pendant drop at a frequency of 0.1 Hz, as shown in Fig.1 B. The results

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indicated that PGPR concentrations played an important role in dominating the viscoelastic

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properties of interfacial film. At low concentrations (below 0.1 wt.%), dilational elastic modulus

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tended to increase over timeand eventually reached a maximum value. However, at higher

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concentrations of PGPR (≥0.5%), there was no obvious response to the interfacial dilation. In

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addition, the dilational elastic modulus exhibited an increasing and then decreasing tendency with an

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increase in PGPR concentration. The dilational elastic modulus value increased from 61.8 mN.m-1 to

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129.6 mN.m-1 when the concentration of PGPR increased from 0.001% PGPR to 0.005%. Above

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0.005% PGPR, the elastic modulus tended to decrease as the PGPR concentration increased. These

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results were consistent with the previous findings that an increase in PGPR concentration reduced the

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interfacial tension and the elastic and viscosity modulus (Márquez et al., 2010). This could be

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explained by the Van den Tempel-Lucassen model that the concentration of PGPR had two effects on

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the dilational viscoelastic properties of interfacial film. On the one hand, the increasing PGPR

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concentration could cause a homogeneous adsorption of the surfactant molecules at the soy oil/water

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interface, strengthening the interaction between surfactant molecules and increasing the dilational

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elasticity modulus. On the other hand, further increasing the surfactant concentration would promote

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the molecular exchange between the bulk phase and the interface. The diffusion of surfactant

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molecules can decrease the interfacial tension gradient and cause a decrease in the dilational modulus.

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Similar results about the initial increase and following decrease of dilational elastic modulus by

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increasing surfactant concentration were reported by Wang et al. (2014). It was reported that

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uponsaturated adsorption of surfactant molecules at the interface, micelles may exist in the bulk oil

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phaseand a further increase in the surfactant concentration would cause the deformation of the

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interfacial film, resulting in a decreasing interfacial elastic modulus. Therefore, at a low

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concentration of PGPR, the interfacial adsorption amount of surfactant molecules determined the

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viscoelastic modulus, and the diffusion exchange of PGPR molecules between the bulk phase and

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interface layer could affect the viscoelastic modulus at higher PGPR concentration.

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3.2. Interfacial dilational rheological properties of WPI and BSA The variations in dilational viscoelasticity of WPI and BSA at the oil/water interface with time

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and frequency were investigated and showed in Fig.2A and B. The changes in interfacial viscoelastic

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of the adsorbed film over time (with a fixed frequency of 0.1 Hz) were thought to be correlated with

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the adsorption and rearrangement of proteins at the interface (Martínez et al., 2009). Compared with

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the trend that occurred with the addition of PGPR, the initial increase in the dilational elasticity

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modulus was slow for BSA and WPI. After 1000 s, arapid increase was observed and the dilational

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modulus of protein

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higher than PGPR (with a maximum value of 130.95 mN/m at 0.005wt.% PGPR), .

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protein molecules, the molecular weight (MW) of PGPR was much smaller and it would quickly

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adsorb at the soy oil/water, reducing the interfacial tension more effectively (data not show). It takes

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long time for protein to reach a complete equilibration of adsorption. In addition, the desorption

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process was slower and previous study reported that no desorption for protein from the interface has

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been found (Bos & van Vliet, 2001). It has been reported that the protein adsorbed at the interface

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could undergo conformational changes and these changes would increase the interactions between

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protein and surface (Ramsden, 1994). Therefore, the adsorption and desorption of protein tended to

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be considerably smaller than that of PGPR and the adsorption could be considered as being

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irreversible. The obtained rheological results also showed that, the interfacial dilational modulus

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increased from 74.2 mN m-1 to 145.0 mN m-1 when the concentration of WPI increased from 0.1 to

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0.7% after 10,000 s. Similar trend was observed when the dilational elasticity modulus was studied

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with the change in frequency as evident from Fig. 2B i.e. with the increase in protein concentration,

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there was an increase in dilational elasticity modulus. WPI is a mixture of different proteins, mainly

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with a maximum value of 146.14 mN/m at 0.7wt.% WPI became significantly

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ACCEPTED MANUSCRIPT containing β–lactoglobulin (β-lg). To the best of our knowledge, few studies have reported the

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interfacial rheological property of WPI. Therefore, it may be possible to compare the viscoelastic

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properties of WPI with β-lg. According to Gülseren and Corredig (2012), in the absence of PGPR,

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the interfacial dilational modulus for β-lg was 38.0 mN m-1 at a concentration of 0.01% and the value

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increased to 84.0 mN m-1 when the concentration increased to 0.1%. The increasing trend in the

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dilational modulus of these two proteins was similar.The dilational modulus of the interfacial film

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increased with prolonged time, indicating that protein molecules at oil-water interface became more

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densely packed and the strength of interfacial film was increased.

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Compared with BSA base film, WPI based interfacial film exhibited a larger dilational

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viscoelastic moduli and this might be due to the formation of an interfacial protein network formed

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by covalent or non-covalent interactions (Dickinson & Matsumura, 1991). The dilational modulus

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for BSA increased from 40.1 mN m-1 to 71.8 mN m-1 at the concentration ranging from 0.1 to 0.7%

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after 10,000 s. These two proteins showed different interfacial activity and stability. When protein

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molecules were adsorbed to the interface, they could undergo a structural rearrangement which

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would maximize the number of contacts between non-polar groups and oils. Whey protein has a

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compact globular structure, and can form stable “skin-like” protein films at the oil-water interface by

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cross-linking (He et al., 2008). In addition, WPI could be more efficiently adsorbed onto the

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oil-water interface, which is consistent with the results that the interfacial tension of whey protein

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and β-lg were lower than that of BSA (Saito et al., 2005). Previous researchers reported that the

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potential adsorption of native BSA at the interface was considerably lower than that of β-lg and soy

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globulins (Ruíz-Henestrosa et al., 2007; Wang & Narsimhan, 2005).

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3.3. Interfacial dilational rheological properties of the protein and surfactant mixture adsorbed

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at the soy oil-water interface.

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The properties of interfacial adsorption film could be reflected by the dilational viscoelastic

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parameters with oscillating frequency. In this study, two concentrations of PGPR (0.01 and 1.0 wt.%) 11

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Corredig, 2012) which reported a moderate reduction in interfacial tension at this concentration. In

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our previous study, it was found that increasing addition of WPI increased the stability of W/O

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emulsions at 1.0% PGPR (Zhu et al., 2015). With the presence of 0.01% PGPR in oil phase, the

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viscoelastic moduli of WPI (Fig.3A, Fig.3B) and BSA (Fig.3C, Fig.3D) with oscillating frequency

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were measured (with an equilibrated adsorption time ranging from 9,000 s to 14, 000 s ). The

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dilational elasticity and viscosity moduli of BSA and WPI at the soy oil-water interface gradually

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increased when the frequency increased (at a range of 0.05~0.1 Hz). At low frequency, the timescale

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of the oscillation was long enough for surfactant and protein molecules to diffuse and rearrange, and

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this could eliminate the interfacial tension gradients caused by interface deformation. Therefore, the

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interfacial tension showed little change and the value of the interfacial viscoelastic modulus was low.

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At a higher frequency, the interfacial area changed rapidly, allowingless time for rearrangements that

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could eliminate interfacial tension gradients. The interfacial film behaved like an insoluble layer,

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resulting in higher dilational viscoelastic values (Wang et al., 2016). In the range of experimental

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frequency, the dilaitonal elasticity modulus was significantly higher than the dilational viscosity

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modulus, suggesting that the adsorbed layer had viscoelastic properties and the formed interfacial

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film was dominated mainly by the dilational elasticity.

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Compared with 0.01wt.% PGPR alone, the addition of WPI in the aqueous phase increased the

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dilational elasticity modulus from 79.1 mN.m-1 to 118.5 mN.m-1 when the concentration of WPI

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increased to 0.7%. This result suggested that the presence of WPI could strengthen the interfacial

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film for the soy oil/water interface. However, the presence of BSA decreased the elasticity modulus

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of the interfacial film and the dilational elastic modulus values varied from 53.8 mN.m-1 to 60.6

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mN.m-1. For non-ionic surfactant at low concentrations, competitive adsorption governed the surface

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behavior. With increasing surfactant concentration, adsorption of the human serum albumin (HAS,

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with a molecular weight of 67 kDa) decreased (Fainerman et al., 2004). The molecular weight of

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BSA (66 kDa) is similar to HAS. PGPR molecules may interfere with the adsorption of BSA at the

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interface, partially displacing proteins from the oil/water interfacial layer. Fig.4 showed the frequency dependence of the interfacial layers at the soy oil-water interface

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(with an equilibrated adsorption time ranging from 7, 000 s to 12, 000s), with 1.0% PGPR in the oil

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phase. At 0.1 Hz, the dilational elastic modulus increased from 17.8 mN m-1 to 21.8 mN m-1 when the

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WPI concentration increased to 0.3%. The dilational elastic modulus of the BSA dispersion at the

294

oil-water interface increased from 18.7 mN m-1 to 24.3 mN m-1 when the concentration of BSA

295

increased to 0.7%. Compared with protein molecules alone (Fig.2B), the addition of PGPR decreased

296

the dilational elasticity modulus of protein molecules, suggesting that PGPR weakened the protein

297

stabilized interfacial film. The change in interfacial tension trend obtained from dynamic modulus

298

test (data not shown) showed that the interfacial tension of PGPR was much lower than that of

299

protein molecules, indicating that PGPR molecules could diffuse more rapidly to the interface. Only

300

a fraction of interface could be covered by the protein segments with other interface regions

301

maintaining high free energy levels (Damodaran, 1997). As it was presented above, the adsorption of

302

protein molecules at the interface was considered as being non-reversible. The dilational modulus

303

depends on the diffusion exchange between the bulk phase and interface (Xia et al., 2006). At 1.0%

304

PGPR, the modulus decreased sharply, indicating that the diffusion-exchange between the bulk phase

305

and interface predominated the rheological characteristic of interfacial film. However the

306

non-reversible adsorption of protein at the interface may suppress the diffusion-exchange process of

307

PGPRand therefore the presence of both BSA and WPI could increase the dilational viscoelasticity

308

modulus of interfacial film stabilized by PGPR. The dilational elastic modulus reflects the strength of

309

protein molecules and intra-protein rigidity to resist the deformation of interfacial film (Pereira,

310

Theodoly, Blanch, & Radke, 2003).

311

of protein could increase the dilational modulus and strengthen the interfacial film stabilized by

312

PGPR and the reinforcement function of protein could explain the stabilizing mechanism of

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In this study, the rheological results showed that the presence

13

ACCEPTED MANUSCRIPT emulsion system containing protein in our previous study (Zhu et al., 2015, 2017). The results

314

indicated that the presence of protein (WPI and BSA) could contribute to the stability of prepared

315

W/O and W/O/W emulsion systems. The stability mechanism of the prepared emulsion system with

316

an addition of protein was attributed to the reduction in the interfacial tension and the formation of

317

complex film with PGPR at the oil-water interface

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When the concentration of PGPR was higher than 0.01% (w/w), the interfacial dilational

319

modulus showed a decreasing tendency with increasing PGPR concentration, as shown in Fig.1.

320

However, Zhu et al. (2016) reported that the stability of W/O emulsion increased with PGPR

321

concentration. The stabilizing effect of PGPR was likely due to the reduction of interfacial tension

322

rather than the viscoelastic modulus of interfacial film. For the PGPR-protein mixture, PGPR

323

concentrations could affect the interfacial adsorption behavior of protein molecules. At low PGPR

324

concentration (0.01%, w/w), the addition of WPI significantly increased the dilational modulus but

325

there was only a slight increase for 1.0% (w/w) PGPR. At low PGPR concentration, protein can

326

merely adsorb on the uncovered portions of the interface, contributing to the overall high elasticity of

327

the interfacial film. At a higher concentration, PGPR molecules would dominate the viscoelastic

328

properties at the interface. Zhu et al. (2015) reported that at 1.0% PGPR, the addition of WPI could

329

further reduce the interfacial tension of the interface which was stabilized by PGPR, suggesting the

330

presence of both protein and surfactant molecules at the interface. Interestingly, protein-PGPR

331

mixtures displayed different results of dilational elasticity with increasing protein concentrations.

332

The interfacial elasticity modulus of the interfacial film formed by the mixture of PGPR-protein

333

began to decrease as the protein concentration was greater than 0.5%. The concentration gradients of

334

protein could affect the degree of unfolding that occurred at the interface. At a lower concentration,

335

protein molecules would probably have more extensive unfolding and rearrangement at the interface

336

(Wüstneck, Moser, & Muschiolik, 1999). In this case, the surface coverage of protein reached the

337

equilibrium and a close-packed interfacial layer was formed as protein concentrations were over

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ACCEPTED MANUSCRIPT 0.5%. A further increase in protein concentration disrupted the protein-protein interactions at the

339

interface, leading to the impairment of interfacial viscoelasticity. This result was consistent with

340

previous research (Tang and Shen, 2015) which reported that the adsorption of BSA at the oil-water

341

interface was determined by protein concentration gradients. Increased BSA concentrations

342

improved the structural rearrangement of adsorbed protein at the interface. Above a certain

343

concentration (>0.5%, w/v), the adsorption rate slowed down due to the presence of energy barrier,

344

the penetration of protein into interface and the structural rearrangements were limited.

345

3.4. Dilational properties of PGPR adsorbed at the interface with MgCl2 in the aqueous phase

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The influence of MgCl2 on the dilational rheology properties of PGPR adsorbed at the soy

347

oil/water interface, at a frequency of 0.1 Hz, was showed in Fig.5. For PGPR at 0.01%, there was a

348

slow initial increase in the adsorbed amount of surfactant, followed by a sharp rise for the interfacial

349

dilational modulus values after 1000 s, due to the increased interfacial adsorption of PGPR

350

molecules. The addition of MgCl2 could significantly increase the interfacial dilational modulus of

351

PGPR, suggesting that MgCl2 could enhance the adsorption of PGPR molecules at the soy oil-water

352

interface.

353

for surfactant to dissolve in the solution, when inorganic salt are added to the water phase

354

(Ben-Yaakov et al., 2011). Therefore, the surfactant molecules would be compelled to move from the

355

bulk phase to the interface, leading to an increase in the effective surfactant concentration in the

356

interface (Hezave et al., 2013). When the concentration of PGPR was above the apparent CMC value,

357

the dilational modulus of PGPR showed a decreased dependency on time. The addition of 0.1 M

358

MgCl2 in the aqueous phase increased the interfacial dilaitonal modulus from 18.7 mN m-1 to 24.8

359

mN m-1. The addition of magnesium salt in the aqueous phase might affect the interactions or

360

reorganization of surfactant molecules. Chattopadhyay et al. (1992) suggested that the addition of

361

inorganic salt could modify the aqueous environment of the polar head groups and enhance the

362

hydrophobic cohesion of adsorbed surfactant molecules through interactions between the polar head

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The dissolution of salt would increase the polarity of solution and make it more difficult

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ACCEPTED MANUSCRIPT group, solvent, and ions. Increased interfacial dilational viscoelasticity modulus decreased the

364

interfacial mobility and the film drainage rate between moving droplets, resulting in a better

365

emulsion stability (Pawlik, Cox, & Norton, 2010). Márquez et al. (2010) elucidated two ways that

366

the inorganic salt can affect the interactions between PGPR molecules. The electrolytes may promote

367

interactions between the hydrophobic chains of PGPR and the cation could act as a bridge to join

368

hydrophilic polyglycerol chains. Therefore, the presence of magnesium salt in W/O emulsions could

369

strengthen the interfacial viscoelastic film.

370

3.5. Frequency dependency of the PGPR/protein mixture adsorbed at the oil/water interface in

371

presence of MgCl2

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Fig. 6 showed the interfacial viscoelastic modulus for PGPR/protein versus oscillating

373

frequency with the addition of 0.1 M MgCl2 in the aqueous phase. The dilational viscoelastic

374

modulus increased dramatically with the frequency,

375

surfactant and protein molecules from the bulk to the interface and the formation of a viscoelastic

376

adsorbed layers at the interface. At 0.01% PGPR, compared with single surfactant, the dilational

377

viscosity and elastic modulus of interfacial film stabilized by both PGPR and protein were more

378

dependent on frequency. According to Wang et al. (2016), for small surfactant molecules, the time

379

required for the diffusion or rearrangement to reach the equilibrium state was much shorter and the

380

characteristic frequency of relaxation process was higher than the experiment frequency. In the

381

presence of protein molecules, the diffusion and rearrangement process was slow, and thus the

382

dilational elasticity of PGPR/protein was more dependent on frequency.

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and the result might be due to the transport of

383

Compared with the rheological viscoelastic property of the PGPR/protein mixture adsorbed at

384

interface (Fig.3), the presence of MgCl2 further increased the dilational elasticity modulus of

385

interfacial film. Ulaganathan et al. (2017) reported that the presence of Ca2+ or Na+ considerably

386

enhanced the adsorption kinetics of β-lg compared to the salt-free system, due to the screening effect

387

of electrolytes. Furthermore, at 1.0% concentration of PGPR, the dilational elastic modulus of 16

ACCEPTED MANUSCRIPT PGPR/protein varied slightly after adding MgCl2 in the aqueous phase. When the PGPR molecules

389

dominated the viscoelastic properties, the increasing concentration of PGPR had no reinforcing effect

390

on the viscoelastic modulus of the interfacial film. The presence of protein and MgCl2 could further

391

increase the dilational modulus by increased adsorption of PGPR molecules or improvement in the

392

interaction with surfactant molecules at the interface.

393

4. Conclusions

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Interfacial dilational rheology is useful in characterizing the properties of interfacial film

395

formed by PGPR, protein molecules and electrolyte additives. The interfacial behavior of PGPR at

396

the oil/water interface was dependent on its concentration. The dilational elastic modulus initially

397

increased and then decreased with continuously increasing PGPR concentration. When the

398

adsorption of PGPR molecules reached saturation at the oil/water interface, the addition of protein

399

could increase the viscoelastic moduli of the interface, due to the interaction between PGPR and

400

protein molecules. The adsorption of protein molecules at the interface was controlled by

401

concentration gradients, and the adsorption of protein at the interface slowed at concentrations above

402

0.5%. In addition, the presence of MgCl2 in the aqueous phase could enhance the dilational modulus

403

of the PGPR molecules and improve the overall stability of the W/O emulsion. Overall, this study

404

demonstrated that additives like inorganic salt and protein molecules could strongly influence the

405

interfacial stability of W/O emulsion systems. This study provides the basis of the regulation and

406

control of interfacial properties to design relatively stable W/O and W/O/W emulsions encapsulating

407

different additives.

408

Acknowledgements

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The author appreciate the financial support from the National Key Technologies R&D Program

410

(No.2016YFD0400804) and the National Science Foundation of China (Project No. 21576072). A

411

financial support was also obtained from the program of China Scholarship Council (CSC,

412

No.201606350115). 17

ACCEPTED MANUSCRIPT 413

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Figure Captions

549

Fig.1. Interfacial tension (A) and dilational interfacial elastic modulus (B) of PGPR (%, w/w) at soy oil/water interface.

550 551 552 553 554

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Fig.2. The change in dilational elasticity modulus for BSA and WPI at 0.1% and 0.7% (w/w), with

time (A) and frequency (B), separately.

555 556 557

Fig.3. Frequency dependence of dilational rheological properties of WPI-PGPR mixture (A, B) and BSA-PGPR mixture(C, D), at 0.01% PGPR.

558 559 560

Fig.4. Dilational elasticity and viscosity as a function of frequency for WPI-PGPR mixture (A, B) and BSA-PGPR mixture (C, D), at 1.0% PGPR

561 562

Fig.5. Interfacial dilational modulus in the presence of PGPR and MgCl2 at the soy oil-water interface. 23

ACCEPTED MANUSCRIPT 563 564 565

Fig.6. Influence of MgCl2 on the dilational elasticity and viscosity of WPI-PGPR mixture and BSA-PGPR mixture at 0.01% PGPR (A, B) and 1.0% PGPR (C, D)

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18

0.001 0.01 0.05 0.1 0.5 1 5

16 14 12 10 8 6

Dilational Elasticity (mN/m)

20

0.001 0.01 0.5 5

50

2 0 0

200

400

600

100

Time (s)

0.005 0.1 1

1000

Time (s)

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150

(A)

22

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100 80 60 40 20 1000

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Dilational Elasticity (mN/m)

（ A）

0.1% WPI 0.7% WPI 0.1% BSA 0.7% BSA

140

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Time (s)

160

0.1% WPI 0.7% WPI 0.1% BSA 0.7% BSA

80 60

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100

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40 20

Fig.2

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Frequency (Hz)

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(A)

100

No WPI 0.1% WPI 0.3% WPI 0.5% WPI 0.7% WPI

12

90 80 70 60

10

No WPI 0.1% WPI 0.3% WPI 0.5% WPI 0.7% WPI

8 6 4 2

50

0

40 0.01

0.01

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60

50

40

12 10 8

(D)

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Dilational Viscosity (mN/m)

14

No BSA 0.1% BSA 0.3% BSA 0.5% BSA 0.7% BSA

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Frequency (Hz)

Frequency (Hz)

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110

Dilational Viscosity (mN/m)

Dilational Elasticity (mN/m)

120

6 4 2

30 0.01

Frequency (Hz)

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0

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ACCEPTED MANUSCRIPT 14

(A)

12

Dilational Viscosity (mN/m)

20 18 16 14

No WPI 0.1% WPI 0.3% WPI 0.5% WPI 0.7% WPI

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8

8 6 4 2

6

0 0.01

0.1

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Frequency (Hz) 26

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(C) Dilational Viscosity (mN/m)

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No BSA 0.1% BSA 0.3% BSA 0.5% BSA 0.7% BSA

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Frequency (Hz)

Fig.4

No BSA 0.1% BSA 0.3% BSA 0.5% BSA 0.7% BSA

(D)

M AN U

18

16

AC C

Dilational Elasticity (mN/m)

20

0.1

Frequency (Hz)

24 22

(B)

No WPI 0.1% WPI 0.3% WPI 0.5% WPI 0.7% WPI

SC

Dilational Elasticity (mN/m)

22

RI PT

24

14 12 10

8 6 4 2 0

0.01

0.1

Frequency (Hz)

ACCEPTED MANUSCRIPT 120

0.01% PGPR 0.01% PGPR+ MgCl2 1% PGPR 1% PGPR+ MgCl2

80

40

20 100

1000

Time (s)

10000

AC C

EP

TE D

M AN U

Fig.5

RI PT

60

SC

Dilational modulus (mN/m)

100

ACCEPTED MANUSCRIPT 110

40

80

MgCl2+0.7% BSA

35

70 60 50

MgCl2+0.7% WPI 30 25

MgCl2+0.1% BSA MgCl2+0.7% BSA

20 15 10

40

5

0.01

0.1

0.01

Frequency (Hz) 18 16

Dilational Viscosity (mN/m)

MgCl2+0.1% WPI MgCl2+0.7% WPI

20

MgCl2+0.1% BSA MgCl2+0.7% BSA

15

10

MgCl2

(D)

MgCl2+0.1% WPI 14

MgCl2+0.7% WPI

SC

(C)

MgCl2+0.1% BSA

12 10 8

MgCl2+0.7% BSA

M AN U

Dilational Elasticity (mN/m)

25

0.1

Frequency (Hz)

30

MgCl2

(B)

MgCl2+0.1% WPI

RI PT

MgCl2+0.7% WPI MgCl2+0.1% BSA

MgCl2

(A) Dilational Viscosity (mN/m)

90

Dilational Elasticity (mN/m)

100

MgCl2 MgCl2+0.1% WPI

6 4

5 0.01

0.1

Frequency (Hz)

AC C

EP

TE D

Fig.6

0.01

0.1

Frequency (Hz)

ACCEPTED MANUSCRIPT

PGPR concentrations affected the dilational viscoelasticity of interfacial film. PGPR interfered with protein/protein interactions at soy oil/water interface. WPI increased the dilational modulus of PGPR molecules adsorbed oil/water

RI PT

interface

AC C

EP

TE D

M AN U

SC

MgCl2 increased the interfacial elasticity of interfacial film stabilized by PGPR.