Effects of heat treatment on the emulsifying properties of pea proteins

Accepted Manuscript Effects of heat treatment on the emulsifying properties of pea proteins Weiwei Peng, Xiangzhen Kong, Yeming Chen, Caimeng Zhang, Yuexi Yang, Yufei Hua PII:

S0268-005X(15)30009-6

DOI:

10.1016/j.foodhyd.2015.06.025

Reference:

FOOHYD 3056

To appear in:

Food Hydrocolloids

Received Date: 7 February 2015 Revised Date:

25 June 2015

Accepted Date: 30 June 2015

Please cite this article as: Peng, W., Kong, X., Chen, Y., Zhang, C., Yang, Y., Hua, Y., Effects of heat treatment on the emulsifying properties of pea proteins, Food Hydrocolloids (2015), doi: 10.1016/ j.foodhyd.2015.06.025. 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.

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Effects of heat treatment on the emulsifying properties of pea

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proteins

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Weiwei Peng*, Xiangzhen Kong, Yeming Chen, Caimeng Zhang, Yuexi Yang, Yufei

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State Key Laboratory of Food Science and Technology, School of Food Science and

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Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province,

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People’s Republic of China

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*Corresponding author: Weiwei Peng

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Tel: 0510-85329091; Fax: 0510-85329091

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E-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract: The effects of heat treatment on the emulsifying properties of pea proteins

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were investigated. Thermal treatment of pea proteins at 95 °C for 30 min increased

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the extent of protein aggregation, and the hydrodynamic diameter increased with the

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increasing of heated protein concentration (ch). Electrophoresis showed that acidic

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and basic (AB) subunits as well as convicilin in unheated pea proteins were involved

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in the formation of polymers linked by disulfide bonds (SS) after heat treatment

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(95 °C, 30 min). At different protein concentrations in the continuous phase (c:

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0.1%–0.5%, w/v) with constant oil fraction of 0.1, emulsions formed by heated pea

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proteins (95 °C, 30 min) showed higher protein adsorption percentage and creaming

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stability than those formed by unheated proteins. Proteins adsorbed at the oil-water

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interface contained higher percentages of vicilin and basic subunit of legumin (leg B)

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in emulsions stabilized by heated pea proteins than in those stabilized by unheated

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proteins. Moreover, increasing c was conducive to the formation of emulsions with

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greater stability against creaming. In addition, emulsion viscosity increased with the

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increasing of ch. These results indicated that the heated pea proteins, as compared to

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the unheated pea proteins, exhibited a greater potential to act as a kind of excellent

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emulsifiers.

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Keywords: pea proteins; emulsion; heat treatment; emulsifying property;

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microstructure

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1. Introduction In recent years, vegetable proteins have been widely used as ingredients in food

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industry because of their relatively low cost and reduced influence on the environment

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(Barac et al., 2010). In addition to soybean seed which has an overwhelming

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advantage in the market, pea seed is one of the commercially available plant protein

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sources (Tian, Kyle, & Small, 1999). Typically, yellow field peas contain 20%–30%

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proteins, 55%–68% starch, and 8%–10% fibers (Aluko, Mofolasayo, & Watts, 2009).

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Starch extraction from peas has been widely studied in the past decades (Sosulski &

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McCurdy, 1987). In addition, pea seeds could be processed by wet milling, salt

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extraction, or acid precipitation to obtain purified protein fractions (Aluko,

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Mofolasayo, & Watts, 2009; Liang & Tang, 2013). Like other legume proteins, pea

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proteins are cholesterol-free and have low fat (Swanson, 1990). The major storage

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proteins in pea seeds are legumin (11S), vicilin (7S), and albumins (2S). The ratio of

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vicilin to legumin is close to 1:2, varying between 1:1.3 and 1:4.2 (Gueguen, 1983).

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Legumin contains more sulfur-containing amino acids than vicilin per unit of protein

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(O’Kane et al., 2004b). Pea protein has a well-balanced amino acid profile and is rich

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in lysine (Schneider & Lacampagne, 2000).

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The emulsifying property of proteins is an important functional feature for the

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food industry (Foegeding & Davis, 2011). Oil-in-water emulsions formed by proteins

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are colloidal systems that serve as a means to deliver nutritional agents, functional

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lipids, and other materials (Jiang, Zhu, Liu, & Xiong, 2014). In recent study (Liang &

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Tang, 2013), the emulsifying abilities of pea protein isolate, legumin, and vicilin at 3

ACCEPTED MANUSCRIPT pH 3.0 were found to be generally better than those at other pH values, whereas all

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proteins exhibited the least emulsifying ability at pH 5.0. Alkaline treatment of pea

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proteins causes structure modifications and enhances the ability of inhibiting

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oxidation in emulsions (Jiang, Zhu, Liu, & Xiong, 2014). The concentration of

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emulsifier (such as proteins) greatly influences the oil droplet size (McClements,

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2004). According to Kim et al. (2005), the protein concentration and order of addition

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significantly affected the flocculation stability of protein-stabilized emulsions. Shao et

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al. (2014) reported that an increase in protein concentration improved the creaming

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stability of emulsions stabilized by soy proteins.

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Heat treatment is often applied to modify the functional properties of food

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ingredients. In many practical applications, protein-stabilized emulsions are subjected

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to thermal processing techniques such as pasteurization and sterilization (McClements,

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2004). Heat treatment above the denaturation temperature usually causes partial

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unfolding and subsequent aggregation of protein (Wang et al., 2012). Heating soy

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protein solutions causes the dissociation of protein subunits, and heating temperature

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influences the aggregation state of proteins (Keerati-u-rai & Corredig, 2009). Protein

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aggregation associated with heat treatment depends highly on ch as well as

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temperature (Cui et al., 2014). The hydrodynamic radius of aggregates induced by

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heat treatment in soy protein dispersions increases with ch (Cui et al., 2014). The

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nanoparticle aggregates of soy protein isolate (SPI) have a great potential to form

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Pickering emulsions (Liu & Tang, 2013). It was also reported that preheating

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dispersions of milk protein concentrate prior to emulsification increased the emulsion

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ACCEPTED MANUSCRIPT creaming stability (Dybowska, 2008). Soy proteins heated at higher concentrations

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generate smaller oil droplet sizes of emulsions and higher surface loads (Cui et al.,

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2014). Moreover, heat treatment of soy protein isolate results in the improvement of

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freeze-thawing stability of oil-in-water emulsions (Palazolo, Sobral, & Wagner, 2011).

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In this work, pea protein samples with different degrees of aggregation were

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obtained from the heat treatment of protein dispersions at different protein

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concentrations. Several properties such as surface hydrophobicity, total free

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sulfhydryl group, interfacial tension, and composition of unheated and heated pea

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proteins were evaluated. The emulsions stabilized by pea proteins were characterized

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in terms of oil droplet sizes, flocculated state of oil droplets, interfacial protein

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concentration and composition, microstructure, creaming stability and rheological

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property.

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2. Materials and methods

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2.1 Materials

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Dry pea [Pisum sativum L.] seeds and soybean oil were obtained from a local

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supermarket in Wuxi (China). All other reagents and chemicals were of analytical

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grade.

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2.2 Preparation of pea proteins

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The pea seeds were freeze-dried and dehulled manually. The dehulled seeds were

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ground into pea flour. The pea flour was defatted five times using hexane with a ratio

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of 1:5 (w/v) at 25 °C. After grinding and passing through an 80 mesh screen (size of

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0.198 mm), the defatted flour was dispersed in 10-fold distilled water and the 5

ACCEPTED MANUSCRIPT suspension was adjusted to pH 9.0 with 2 M NaOH. After stirring for 1 h, the

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suspension was centrifuged at 8000 g for 30 min at 4 °C. The obtained supernatant

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was adjusted to pH 4.5 with 2 M HCl, stored for 2 h at 4 °C and then centrifuged at

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5000 g for 20 min at 4 °C. The protein precipitate was washed twice with distilled

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water and then adjusted to pH 7.0 using 2 M NaOH. The suspension was centrifuged

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at 5000 g for 20 min at 4 °C and the obtained supernatant was dialyzed against

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distilled water for 48 h at 4 °C. Then it was freeze-dried and ground with a motor and

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pestle to pea proteins. The protein content of the pea proteins was 79.07% (w/w) on

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the dry basis as determined by using the micro-Kjeldahl method with a nitrogen

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conversion factor of 5.40.

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2.3 Heat treatment

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Pea protein dispersions of various concentrations (1.0%, 3.0%, and 5.0%, w/v)

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were prepared in the sodium phosphate buffer (10 mM, pH 7.0) and then stored

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overnight at 4 °C to hydrate fully. Sodium azide (0.02%, w/v) was added as an

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antimicrobial agent. The dispersions were centrifuged at 10000 g for 30 min to

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remove insoluble proteins (about 84.90 ± 0.1% (w/w) proteins were retained in the

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supernatants). The supernatants (called as NPP) were heated in a 95 °C water bath for

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30 min and then cooled in an ice bath. The obtained heated supernatants with

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concentrations of 1.0%, 3.0%, and 5.0% (w/v) prior to centrifugation were called HP1,

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HP3, and HP5, respectively. The protein concentrations of the final dispersions were

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determined according to Lowry’s method using bovine serum albumin (BSA) as

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standard (Lowry, Rosebrough, Farr, & Randall, 1951). In order to explore whether

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ACCEPTED MANUSCRIPT heat treatment induces the formation of insoluble aggregates, the heated supernatants

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were centrifuged at 10000 g for 20 min, and the protein concentrations of the

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supernatants were measured. Heat treatment at 95 °C did not significantly decrease

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the solubility of pea proteins. The components of NPP, HP1, HP3, and HP5 were

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analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Section 2.6).

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2.4 High-performance size exclusion chromatography (HPSEC)

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The molecular weight distribution was determined according to the method of

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Cui et al. (2014). Different pea protein dispersions (NPP, HP1, HP3, and HP5) were

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diluted to 1 mg/mL with the sodium phosphate buffer (10 mM, pH 7.0) and passed

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through 0.45 µm filters. HPSEC analysis was performed on a Waters 2690 liquid

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chromatograph system (Waters Chromatography Division, Milford, MA, USA)

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equipped with a Shodex protein KW-804 column (Shodex Separation and HPLC

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Group, Tokyo, Japan). The elution was performed with 50 mM sodium phosphate

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buffer (pH 7.0) containing 0.3 M NaCl at a flow rate of 1 mL/min. Aliquots (10 µL)

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of samples were injected into the column and the absorbance was monitored at 280

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nm. Gel-filtration standard proteins (Bio-Rad, USA), including thyroglobulin, bovine

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γ-globulin, chicken ovalbumin, equine myoglobin, and vitamin B12 (molecular

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weights, in the range 1,350–670,000 Da), were used for calibration. The standard

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curve was established as a linear relationship between the retention time (tR) and the

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logarithm of the molecular weight (MW): log10MW = 4.0789 – 0.2003tR (R2=0.998).

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The molecular weight of the different fractions was based on the retention time and

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calculated using the standard curve. The percentage of each fraction was calculated

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as % area relative to the 100% integrated area of the total spectrum.

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2.5 Dynamic light scattering (DLS) The apparent hydrodynamic diameter (Dh) was determined using the method of

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Wang et al. (2012). The protein samples (NPP, HP1, HP3, and HP5) were diluted to 1

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mg/mL with 10 mM sodium phosphate buffer (pH 7.0) and then passed through 0.45

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µm filters. DLS analysis was carried out at a fixed angle of 173° using a Zetasizer

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Nano-ZS instrument (Malvern Instruments, British) at 25 °C. The appropriate

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viscosity and refractive index parameters were set for each sample. The samples were

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placed in 1 × 1 cm cuvettes. All measurements were carried out at least three times.

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2.6 Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

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SDS-PAGE was conducted according to the method by Laemmli (1970). The

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concentrations of the stacking and separating gels were 5% and 12.5%, respectively.

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Samples were dissolved in the SDS-PAGE sample buffer in the presence (reducing

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conditions) or absence (non-reducing conditions) of 2% (w/v) 2-mercaptoethanol

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(βME). After heating for 3 min in a boiling water bath and centrifuging at 10000 g for

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SDS-PAGE was performed at 15 mA for 2 h. The gel was stained with Coomassie

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Brilliant Blue G-250 and scanned using a computing densitometer (Molecular Imager

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Chemi DocXRS+, Bio-Rad, USA). The intensities of the protein bands were

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integrated using Image Lab software (Bio-Rad, USA). The content of each component

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was determined as a percentage in the sample by comparing the individual band

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intensity with the total intensity of all the bands in a lane.

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2.7 Surface hydrophobicity The surface hydrophobicity of protein samples (NPP, HP1, HP3, and HP5) was

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determined according to Haskard et al. (1998). Protein dispersions were prepared with

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the sodium phosphate buffer (10 mM, pH 7.0) to a serial concentration of

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0.004%–0.02% (w/v). An aliquot (50 µL) of 1-anilino-8-naphthalenesulfonate (ANS-)

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solution (8 mM ANS- in 10 mM phosphate buffer, pH 7.0) was added to 4 mL of each

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dilution. The relative fluorescence intensity was measured at 390 nm (excitation) and

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470 nm (emission) using an F7000 fluorescence spectrophotometer (Hitachi Co.,

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Japan). The slit width of both was 5 nm. The index of surface hydrophobicity was

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expressed as the initial slope of the plot of fluorescence intensity versus protein

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concentration.

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2.8 Interfacial tension

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The interfacial tension between soybean oil and the sodium phosphate buffer (10

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mM, pH 7.0) or protein samples (NPP, HP1, HP3, and HP5) was determined

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according to the Wilhelmy plate method (Sakuno, Matsumoto, Kawai, Taihei, &

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Matsumura, 2008). The protein samples (NPP, HP1, HP3, and HP5) were diluted to

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0.3% (w/v) with the sodium phosphate buffer (10 mM, pH 7.0). A dynamic contact

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angle meter and tensiometer (DCAT21, Data-Physics Instruments GmbH, Germany)

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was used as the detecting system. The soybean oil was purified with Florisil (60–100

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mesh, Sigma-Aldrich).

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2.9 Total free sulfhydryl group (SH)

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The determination of total free sulfhydryl group was carried out according to the 9

ACCEPTED MANUSCRIPT method of Van der Plancken et al. (2005), with a few modifications. The protein

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samples (NPP, HP1, HP3, and HP5) were diluted to 2 mg/mL with the sodium

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phosphate buffer (10 mM, pH 7.0) containing 6 M urea. Aliquots (80 µL) of Ellman’s

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reagent (5′, 5-dithiobis (2-nitrobenzoic acid)) were added to 2.5 mL of diluted

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samples. The absorbance was measured at 412 nm after 30 min. For the reagent blank,

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the protein samples were replaced with 10 mM sodium phosphate buffer (pH 7.0)

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containing 6 M urea, and the solution mixed with 80 µL of Ellman’s reagent. The total

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free SH contents were calculated as follows:

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µM SH/g protein = (A412 − A412r ) / (εC) ×100000 (1)

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where A412 is the absorbance at 412 nm, A412r is the absorbance at 412 nm for the

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reagent blank, ε is the extinction coefficient, which was taken as 13600 M-1cm-1, and

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C is the sample concentration.

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2.10 Emulsion preparation

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Each of the pea protein dispersions (NPP, HP1, HP3, and HP5) was diluted to

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three different concentrations (0.1, 0.3, and 0.5% (w/v)) using the sodium phosphate

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buffer (10 mM, pH 7.0) which contained 0.02% (w/v) sodium azide as an

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antimicrobial agent. Mixtures of 90% (v/v) pea protein dilutions and 10% (v/v)

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soybean oil were initially emulsified using a high-shear homogenizer (FA25 model,

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Fluko Equipment Co., Ltd., Shanghai, China) at 10000 rpm for 1 min, and then

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homogenized at 40 MPa with three passes using a high-pressure homogenizer

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(AH2010, ATS Engineering, Inc., Shanghai, China). Emulsions were stored at 4 °C

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for further analysis.

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2.11 Determination of droplet size and flocculation index (FI) The droplet size and particle distribution in the emulsions were determined by

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the Mastersizer 2000 Laser Particle Size Analyzer (Malvern Instruments, Malvern,

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UK). The relative refractive index of the emulsion was taken as 1.107, that is, the

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ratio of the refractive indices of soybean oil and the phosphate buffer (1.472 and

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1.330, respectively). Distilled water or 1.0% (w/v) sodium dodecyl sulfate (SDS)

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solution was used as the dispersant, and the emulsions were diluted twenty times with

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the same dispersant immediately before the determinations. The measured droplet size

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is reported as the volume-average diameter (d43) and as the surface-average diameter

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(d32). All experiments were carried out at least three times.

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The percentage of flocculation index (FI, %) for the emulsions was calculated as follows:

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FI(%) = [(d43 in water) / (d43 in 1% SDS) − 1.0] ×100 (2)

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2.12 Optical microscopy

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The microstructure of freshly formed 0.1% (w/v) emulsions was analyzed by

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using a microscope (CX31, Olympus Co., Tokyo, Japan) equipped with

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charge-coupled-device (CCD) camera (VIS500, Vihent, Inc., Shanghai, China).

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Aliquots (0.2 mL) of the emulsions were diluted in 1.8 mL of distilled water. Aliquots

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(20 µL) of diluted emulsions were placed on microscopy glass slides with cover slips.

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The magnification was 40 (objective) × 10 (eyepiece). Observations were carried out

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at ambient temperature.

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2.13 Determination of percentage of adsorbed proteins (AP) and interfacial protein

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concentration (Γ) Percentage of adsorbed proteins and interfacial protein concentration were

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determined according to the method described by Liang et al. (2014), with a few

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modifications. Emulsions were centrifuged at 10000 g for 30 min at room temperature

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inducing the separation of a cream layer at the top and an aqueous phase at the bottom

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of the tube. The aqueous phase was carefully withdrawn using a syringe and passed

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through a 0.45 µm filter. The protein concentration of the aqueous phase was

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determined with the Lowry method using BSA as the standard. The Γ (mg/m2) was

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calculated as follows:

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Г (mg/m2 ) = (d32 in SDS)(CINI − CSER)(1 − Ф) / (6Ф) (3)

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where CINI and CSER refer to the initial protein concentration of samples used for

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the emulsion preparation and the protein concentration of the aqueous phase after

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centrifugation, respectively; Φ is the volume fraction of the oil phase.

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The AP% was calculated as follows: AP (%) = (C INI − C SER ) × 100 / C INI (4)

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2.14 Interfacial protein composition

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The components of proteins adsorbed or unadsorbed at the oil-water (O/W)

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interface of freshly formed 0.5% (w/v) emulsions were analyzed by SDS-PAGE

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according to Keerati-u-rai et al. (2009), with a few modifications. Emulsions were

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centrifuged at 10000 g for 30 min. The cream phase was carefully removed from the

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top, dried on the filter paper, and suspended in the sodium phosphate buffer (10 mM,

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pH 7.0) to the original volume fraction (0.1). The aqueous phase was withdrawn using 12

ACCEPTED MANUSCRIPT a syringe and passed through a 0.45 µm filter. Samples of the cream and aqueous

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phase were dissolved in an equal volume of the SDS-PAGE sample buffer in the

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presence (reducing conditions) or absence (non-reducing conditions) of 2% (w/v)

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2-mercaptoethanol. An aliquot (10 µL) of each sample was loaded into a cell and

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electrophoresed.

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2.15 Rheology measurement

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The rheological properties of emulsions were measured using a controlled-stress

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rheometer AR1000 (TA Instrument, New Castle, UK), equipped with parallel plate

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geometry (PP50, 50 mm diameter and 1 mm gap). The temperature was kept constant

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at 25 °C. Viscosity measurements (η) were taken in a steady-rate flow mode by

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increasing the shear rate from 0.1 to 100 s-1. The power law was used to fit the data

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(Ruan, Chen, Kong, & Hua, 2014), and is represented as follows:

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η = τ/γ = kγn−1 (5)

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where η is the apparent viscosity (Pa·s), τ is the shear stress (Pa), γ is the shear rate (s-1), k is the consistency index (Pa·sn), and n is the flow behavior index.

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2.16 Visual observation of creaming and determination of creaming index (CI)

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Fresh emulsions (3 mL) were added into sample bottles (1.8cm internal diameter

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× 4.0cm height) and then stored vertically at ambient temperature. The heights of

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emulsions (Ht) and aqueous phase (Hs) were recorded at different times for 14 days.

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The creaming index (CI, %) was calculated as follows:

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CI (%) = ( H s / H t ) ×100 (6)

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2.17 Statistics 13

ACCEPTED MANUSCRIPT All experiments were conducted at least three times. Data reported are mean

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values ± standard deviations. The data were analyzed using SPSS for Windows

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(version 13.0, SPSS Inc. Chicago, IL) following an analysis of variance (ANOVA)

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one-way linear model. The means were compared by a least significant difference

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(LSD) test with a confidence interval of 95%.

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3. Results and discussion

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3.1 Characterization of unheated and heated pea proteins

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3.1.1 HPSEC and Hydrodynamic diameter (Dh)

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Fig. 1 shows the elution profiles of unheated pea proteins and the soluble parts of

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heated pea proteins. The elution profile of unheated pea proteins (NPP) showed two

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major eluting peaks at 9–10 and 12–13 min, corresponding to molecular weights of

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about 150 kDa and 37.6 kDa, respectively. In addition, a small fraction of aggregates

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(6–7min) formed by the freeze-dried process appeared in the NPP elution profile. The

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elution profiles of the soluble parts in heated pea proteins showed two main eluting

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peaks at about 5.5 and 11 min. As revealed by the analysis of area distribution, the

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percentages of aggregates (eluted at about 5–6 min) in HP1, HP3, and HP5 were 15%,

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31%, and 40%, respectively. These results suggested that partial unheated pea proteins

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developed into soluble heat-induced aggregates with high molecular weights. Heating

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whey protein isolate (WPI) at higher temperature can cause the peak of protein

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aggregates to shift to higher molecular weights (Mahmoudi, Axelos, & Riaublanc,

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2011).

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Hydrodynamic diameter of proteins obtained from DLS experiments is 14

ACCEPTED MANUSCRIPT summarized in Table 1. The hydrodynamic diameter of the heated pea proteins was

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found to be higher than that of unheated pea proteins. Furthermore, the hydrodynamic

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diameter also increased with ch. The hydrodynamic radius of soy proteins also

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increased as the ch increased (Cui et al., 2014).

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3.1.2 SDS-PAGE

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The components of the unheated and heated pea proteins were analyzed by

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electrophoresis. As shown in Fig. 2, unheated pea proteins consist of multiple

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components which could be mainly ascribed to legumin and vicilin. The 75-kDa band

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could be ascribed to convicilin, which was denoted as the α-subunit of vicilin because

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of its high degree of homology with vicilin (O’Kane et al., 2004a). The bands of 50

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kDa, 31–34 kDa, and 10–12 kDa could be ascribed to vicilin. Vicilin is a trimeric

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protein composed of ~50 kDa, ~47 kDa, ~34 kDa, and ~30 kDa subunits and minor

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components with molecular weights less than 19 kDa (Koyoro & Powers, 1987).

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There are no disulfide bonds (SS) between different polypeptides of vicilin. Moreover,

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the subunits with the molecular weights of 38 kDa and ~15 kDa can be ascribed

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respectively to the acidic subunit (leg A) and basic subunit (leg B) of legumin.

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Legumin has six subunit pairs, which consists of an acidic and a basic subunit linked

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by a single disulfide bond.

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Unheated pea proteins have acidic and basic (AB) subunits under non-reducing

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conditions. In addition, heat treatment induced an increase in the intensity of bands at

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the top of the stacking gel and a decrease in the intensity of bands of convicilin. These

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findings indicate that AB subunits and convicilin in unheated pea proteins were 15

ACCEPTED MANUSCRIPT involved in the formation of polymers linked by disulfide bonds after heat treatment.

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Moreover, in the presence of βME, pea protein aggregates were dissociated into

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subunits, and no differences were observed among the lanes. Wang et al. (2012)

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proposed that the presence of intermolecular disulfide bonds in heated soy proteins

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were caused by oxidation and/or SH-SS interchange reactions, since soy protein

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isolate (SPI) aggregates formed at 90 °C were dissociated into subunits under the

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reducing condition. Furthermore, the differences among heat-treated pea proteins

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(HP1, HP3, and HP5) were not significant under both non-reducing and reducing

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conditions.

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3.1.3 Surface hydrophobicity and Interfacial tension

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The surface hydrophobicity (H0) values and interfacial tension of unheated and

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heated pea proteins are summarized in Table 1. Surface hydrophobicity influences the

343

ability of the protein to adsorb to the interface and is closely correlated with the

344

emulsifying properties (Mahmoudi, Axelos, & Riaublanc, 2011). The H0 values of

345

heated pea proteins were higher than those of unheated proteins, and the H0 value of

346

HP5 was almost two times that of NPP. It is well known that heat treatment can

347

expose hydrophobic groups buried in globular proteins as a result of partial unfolding

348

(Sorgentini, Wagner, & Anon, 1995). In addition, the surface hydrophobicity of

349

heat-treated soy proteins was reported to be higher than that of unheated proteins

350

(Wang et al., 2012). Moreover, pea proteins heated at higher concentrations showed

351

higher H0 values.

352

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Adsorption of proteins could reduce the interfacial tension at the O/W interface. 16

ACCEPTED MANUSCRIPT The interfacial tension between soybean oil and unheated pea proteins was 26.72 ±

354

0.39 mN·m-1. The reduction in interfacial tension reduced the energy required for

355

emulsification (Amine, Dreher, Helgason, & Tadros, 2014). A slight reduction of

356

interfacial tension was induced by heat treatment of pea proteins. The interfacial

357

tension of soybean oil-heated protein dispersions decreased with the increasing of ch.

358

3.1.4 Total free sulfhydryl group (SH)

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The total free sulfhydryl groups were determined to evaluate the effects of ch on

360

the covalent interactions of pea proteins. As shown in Table 1, heat treatment resulted

361

in a significant (p < 0.05) reduction in total free SH contents of unheated pea proteins.

362

This result can be explained by the oxidation and/or conversion of sulfhydryl groups

363

into disulfide bonds (Wang et al., 2012). Moreover, the total free SH contents of

364

heated pea proteins increased with increasing ch. The observation was probably due to

365

the cleavage of the disulfide bonds and/or the inhibition of disulfide bond formation

366

(Wang et al., 2012).

367

3.2 Characteristics of emulsions

368

3.2.1 Oil droplet sizes in the emulsions

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The d43 values (in 1% SDS or in water) of unheated and heated pea

370

protein-stabilized emulsions are shown in Table 2. The d43 values determined using 1%

371

SDS as the dispersing solvent reflect the individual oil droplet size, since the presence

372

of 1% SDS may disrupt flocculation of oil droplets. At any c value, d43 values (in 1%

373

SDS) of heated protein emulsions were smaller than those of unheated protein

374

emulsions. The underlying reason for this observation might be the more effective 17

ACCEPTED MANUSCRIPT packing and higher diffusion (or initial adsorption) of the heated proteins at the O/W

376

interface (Liu & Tang, 2015). It was reported that a heat pretreatment of soy proteins

377

(95 °C, 15 min) resulted in a significant (p < 0.05) decrease in d43 (in 1% SDS) values

378

of the emulsions when compared to unheated proteins (Shao & Tang, 2014).

379

Furthermore, emulsions stabilized by pea proteins heated at higher concentrations

380

showed smaller d43 values (in 1% SDS). Similar observations have been reported for

381

soy proteins (Cui, Chen, Kong, Zhang, & Hua, 2014). Besides, the d43 values (in 1%

382

SDS) decreased progressively with increasing c from 0.1% to 0.5% (w/v). This is

383

presumably due to the stabilization of a larger interfacial area by a higher protein

384

concentration in the continuous phase, which is consistent with the smaller oil droplet

385

size (Liu & Tang, 2013). Roesch et al. (2002) reported that oil droplet size in soy

386

protein-stabilized emulsions decreased with the protein concentrations, a result that

387

was also found by Liu et al. (2013)

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On the other hand, at any c value, heat treatment resulted in significant (p < 0.05)

389

increases in the d43 values (in water) of emulsions, which represent the size of flocs.

390

This can be expected, since heat treatment increased the extent of protein aggregation,

391

accompanied by increase of surface hydrophobicity, thus attractive interactions (e.g.,

392

hydrophobic interactions) between protein-coated oil droplets increased (Lakemond et

393

al., 2000). Keerati-u-rai et al. (2009) found that heated soy proteins (95 °C, 30 min)

394

stabilized emulsions with large oil droplets (> 10 µm). In addition, Shao et al. (2014)

395

reported that d43 values (in water) of preheated soy protein-stabilized emulsions were

396

larger than those in unheated protein-stabilized emulsions.

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ACCEPTED MANUSCRIPT 397

3.2.2 Flocculated state of oil droplets in fresh emulsions In order to evaluate the flocculated state of oil droplets in fresh emulsions,

399

droplet size distribution (Fig. 3) and flocculation index (Table 2) were determined. In

400

the presence of 1% SDS, all emulsions showed a unimodal distribution profile and the

401

location of the peak varied with the heat treatment and applied c (Fig. 3A'-C').

402

Unheated protein-stabilized emulsions presented a monomodal distribution in water,

403

and the FI values were relatively low, indicating a low extent of droplet flocculation

404

in the emulsions (Puppo et al., 2011). In this case, the electrostatic repulsion between

405

oil droplets played an important role (Shao & Tang, 2014). On the contrary, emulsions

406

prepared with HP1, HP3, and HP5 showed a bimodal or trimodal distribution in water

407

(Fig. 3A-C), FI values of which were much higher than those of unheated protein

408

emulsions, suggesting the occurrence of bridging flocculation between oil droplets.

409

Preheating is reported to significantly increase the flocculation index in soy

410

protein-stabilized emulsions (Shao & Tang, 2014). The flocculation in heated

411

protein-stabilized emulsions may be induced by hydrophobic interactions among

412

proteins adsorbed at the surface of the oil droplets. The bridging flocculation in heated

413

protein-stabilized emulsions resulted in the large d43 values in water.

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The flocculated state of fresh emulsions varied also with applied c. For

415

emulsions prepared with unheated proteins, the FI values decreased from 2.35 ± 0.07

416

to 0.11 ± 0.01 as c increased from 0.1% to 0.5% (w/v) (Table 2). In contrast, for

417

heated protein-stabilized emulsions, the FI values increased as c increased from 0.1%

418

to 0.3% (w/v), whereas a further increase in concentration caused a decrease in FI. 19

ACCEPTED MANUSCRIPT This finding indicates that hydrophobic interactions between the adsorbed proteins

420

contributed to the flocculated state of the emulsion oil droplets. The decrease in FI

421

could be interpreted as a result of inhibited flocculation (Liu & Tang, 2013).

422

3.2.3 Microstructure of emulsions

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The optical microscopy images of freshly formed 0.1% (w/v) emulsions are

424

shown in Fig. 4. For emulsions prepared with unheated pea proteins, a part of oil

425

droplets were in their individual form, the rest remained flocculated. A different

426

scenario was observed in heated protein emulsions where the majority of the oil

427

droplets were presented in clusters, which produced large flocculated particles. The

428

results are consistent with the d43 values (in water) and the FI data. At the same time,

429

individual oil droplets in emulsions formed by heated proteins were found to be

430

smaller than those in unheated protein emulsions. Nevertheless, for the emulsions

431

formed by HP1, HP3, and HP5, the differences were not significant.

432

3.2.4 Interfacial protein adsorption

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The percentage of adsorbed proteins (AP) is shown in Table 2. For any type of

434

pea protein emulsions, the AP values decreased progressively with applied c.

435

Similarly, Li et al. (2011) reported that emulsions prepared with soy proteins,

436

unheated or heated at 95 °C for 30 min, exhibit a decrease in AP values with

437

increasing protein concentrations. At the same c, the AP values of emulsions formed

438

by heated proteins were higher than those of unheated protein emulsions. This is

439

consistent with our previous study showing that the adsorbed protein percentage of

440

heated soy protein emulsions was higher than that of unheated protein emulsions (Li,

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20

ACCEPTED MANUSCRIPT Kong, Zhang, & Hua, 2011). Heat treatment caused an increase in surface

442

hydrophobicity of proteins, enhancing their potential adsorption at the O/W interface

443

(Keerati-u-rai & Corredig, 2009). Emulsions prepared with proteins heated at higher

444

concentrations possessed larger AP values. With the increase in ch, heat treatment

445

increased the size of protein aggregates and the surface hydrophobicity of pea proteins.

446

The higher hydrophobic interactions contributed to the protein adsorption at the O/W

447

interface for the larger protein aggregates.

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Interfacial protein concentrations of emulsions are presented in Table 2. It can be

449

seen that the interfacial protein concentration of all emulsions ranged from 2 to 4

450

mg/m2. For emulsions stabilized by unheated and heated proteins, the interfacial

451

protein concentration increased with the increasing of c. When c was higher, more

452

proteins could be adsorbed at the O/W interface (per unit interfacial area) (Shao &

453

Tang, 2014). The interfacial protein content of emulsions formed by HP1 and HP3

454

was found to be larger than that of the NPP emulsion; however, the interfacial protein

455

content of HP5 emulsions was smaller. Liang and Tang (2013) reported that heat

456

treatment caused little influence on the interfacial protein content of native protein

457

emulsions. This behavior is presumably due to the combination of the larger

458

interfacial area, caused by small droplet size formed during emulsification, and the

459

increased protein adsorption percentage.

460

3.2.5 Interfacial protein composition

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461

Both non-reducing and reducing SDS-PAGE analyses were conducted in order to

462

investigate whether heat treatment induced differences in the composition of adsorbed 21

ACCEPTED MANUSCRIPT and non-adsorbed proteins. As shown in Fig. 5, when compared with non-adsorbed

464

proteins in heated protein emulsions, most of the heat-induced aggregates adsorbed at

465

the O/W interface during emulsification. A detailed analysis of the band intensities

466

showed that, under non-reducing conditions, the percentages of vicilin in the adsorbed

467

protein layer for emulsions stabilized by NPP, HP1, HP3, and HP5 were 15.2%,

468

20.6%, 29.6%, and 30.8%, while the percentages of vicilin for NPP, HP1, HP3, and

469

HP5 prior to emulsification were 28.4%, 28.7%, 28.4%, and 28.3%, respectively. In

470

the heated protein emulsions, most vicilin was adsorbed at the O/W interface, while in

471

unheated protein emulsions it remained in the aqueous phase. Moreover, under

472

non-reducing conditions, the percentages of leg B in the adsorbed protein layer for

473

emulsions formed by NPP, HP1, HP3, and HP5 were 8.9%, 10.2%, 11.5%, and 12.8%,

474

respectively, whereas the percentages of leg B before emulsification were 6.5%, 6.0%,

475

5.7%, and 5.5%, respectively. These findings indicate that, heat treatment led to an

476

increase in the percentages of vicilin and leg B in the adsorbed protein layer compared

477

to that in the unheated pea proteins.

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463

Leg A and leg B were recovered after adding βME to the samples of both

479

unheated and heated pea protein emulsions. For unheated protein emulsions, this

480

recovery is caused by the dissociation of AB subunits. In the heated pea protein

481

emulsions, βME interrupted the disulfide bonds in the aggregates of adsorbed proteins.

482

Moreover, under reducing conditions, the percentages of vicilin in non-adsorbed

483

proteins for emulsions formed by NPP, HP1, HP3, and HP5 were 28.3%, 24.4%,

484

24.0%, and 22.8%, respectively, which is consistent with the detailed analysis of the

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478

22

ACCEPTED MANUSCRIPT 485

adsorbed proteins in the emulsions.

486

3.2.6 Rheological properties The rheological properties of the emulsions were investigated. Fig. 6 shows the

488

flow properties of the emulsions, including the consistency index (k) and the flow

489

behavior index (n). The emulsions showed pseudoplastic behavior and possessed

490

shear thinning properties, which can also be deduced from the low n values (n < 1).

491

The consistency indices of the heated pea protein emulsions were higher than those of

492

the unheated protein emulsions, and emulsions prepared with proteins heated at higher

493

concentrations possessed larger k values. This indicates that heat treatment increased

494

emulsion viscosity, presumably because the extent of flocculation of oil droplets

495

increased with ch. Moreover, increases in c resulted in a higher viscosity for emulsions

496

of any types.

497

3.2.7 Creaming stability

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487

The creaming stability is reflected by the creaming index. Fig. 7 presents

499

changes in the percentage of creaming index for all emulsions upon storage for up to

500

2 weeks. In general, the creaming behavior varied with the heat treatment and applied

501

c. At protein concentrations of 0.1% and 0.3%, the emulsions formed by unheated and

502

heated pea proteins rapidly developed into two layers in one day; yet, the creaming

503

rate slowed down in the following days. At protein concentration of 0.5%, no

504

creaming behavior was detected after a storage period of 7 days. For all emulsions,

505

the creaming behavior improved progressively with increasing c. The protein

506

concentration dependence of the creaming behavior was also reported in previous

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23

ACCEPTED MANUSCRIPT studies. For emulsions prepared with pea proteins at pH 3.0 (Liang & Tang, 2014),

508

soy protein nanoparticles (Liu & Tang, 2013), and chitin nanocrystal particles

509

(Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis, 2011), increased particle

510

concentration resulted in a decrease in the creaming index.

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507

At a fixed c and storage time, the creaming index of the heated protein emulsions

512

was lower than that of the unheated protein emulsions. Furthermore, emulsions

513

stabilized by proteins heated at higher concentrations showed a lower creaming index.

514

This observation indicates that heat treatment of pea proteins prior to emulsification

515

improved the creaming stability of emulsions. Similar observations were also reported

516

in previous studies. Heat pretreatment of soy proteins decreased the creaming index of

517

emulsions significantly when compared with that of unheated proteins (Shao & Tang,

518

2014). The height of the serum layer of emulsions prepared with denatured egg yolk

519

was also smaller than that of native yolk emulsions (Guilmineau & Kulozik, 2006).

520

The flocculated oil droplets in heated protein-stabilized emulsions resulted in the

521

formation of gel-like network and increased emulsion viscosity (Shao & Tang, 2014;

522

Liu & Tang, 2013). The movement of the oil droplets could be held back due to the

523

increased emulsion viscosity. Reasonably, the improvement of creaming stability

524

could be attributed to the gel-like network.

525

4. Conclusions

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526

The present work showed that the oil droplet size, flocculated state, and

527

creaming stability of pea protein emulsions were closely associated with heat

528

treatment and applied c. Pea proteins with different degrees of aggregations closely 24

ACCEPTED MANUSCRIPT depend on ch and influence the adsorption behavior of pea proteins. Heat treatment of

530

pea proteins results in inter-droplet hydrophobic interactions in emulsions, increasing

531

the droplet flocculation and creaming stability. These findings would be helpful for

532

the development of pea protein-stabilized emulsion products.

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529

533 534

Acknowledgments

The work was financially supported by National Natural Science Foundation of

536

China (No. 21276107), The National Great Project of Scientific and Technical

537

Supporting Programs funded by Ministry of Science & Technology of China during

538

the 12th five–year plan (No. 2012BAD34B04–1) and 863 Program (Hi-tech research

539

and development program of China, NO. 2013AA102204–3) and “Doctor Candidate

540

Foundation of Jiangnan University (JUDCF10025)”.

543 544 545 546

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ACCEPTED MANUSCRIPT Table captions Table 1: Hydrodynamic diameter (Dh), surface hydrophobicity (H0), interfacial tension (σ), and total free sulfhydryl group (SH) content of unheated and heated pea proteins (NPP, HP1,

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HP3, and HP5). Table 2: Mean particle size values (d43, in water or 1 % SDS), flocculation index (FI), percentage of adsorbed protein (AP) and interfacial protein concentration (Γ) in the fresh

SC

emulsions stabilized by unheated and heated pea proteins (NPP, HP1, HP3, and HP5) at

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different protein concentrations (0.1%, 0.3%, and 0.5% (w/v)).

ACCEPTED MANUSCRIPT Table 1 Dh (nm)

H0

σ (mN·m-1)

Total SH (µM/g)

NPP

64.18 ± 0.12d

2650 ± 14d

26.72 ± 0.39a

8.69 ± 0.09a

HP1

84.96 ± 0.21c

4590 ± 18c

25.76 ± 0.35ab

1.78 ± 0.05d

HP3

137.55 ± 0.35b

5191 ± 20b

25.02 ± 0.20b

2.98 ± 0.07c

HP5

216.65± 0.64a

5275 ± 21a

24.60 ± 0.26b

6.37 ± 0.08b

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Samples

Means with different letters in the same column are significantly different at the 5%

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level.

ACCEPTED MANUSCRIPT Table 2 d43 in water (µm)

d43 in SDS (µm)

FI

AP (%)

Γ (mg/m2)

NPP

9.88 ± 0.34b

2.95 ± 0.14a

2.35 ± 0.07d

66.40 ± 0.56b

2.41 ± 0.03b

HP1

19.35 ± 1.41a

2.06 ± 0.06b

8.39 ± 0.08b

96.20 ± 1.70a

2.75 ± 0.06a

HP3

18.02 ± 1.13a

2.06 ± 0.04b

7.75 ± 0.10c

97.30 ± 0.99a

2.49 ± 0.04b

HP5

21.28 ± 1.58a

2.05 ± 0.06b

9.38 ± 0.06a

97.90 ± 0.28a

2.36 ± 0.04b

NPP

2.45 ± 0.21c

1.30 ± 0.07a

0.88 ± 0.03d

49.63 ± 0.52c

2.62 ± 0.04b

HP1

10.28 ± 0.81b

0.98 ± 0.03b

9.49 ± 0.04c

73.43 ± 0.66b

2.77 ± 0.06a

HP3

12.49 ± 0.72b

0.93 ± 0.03b

12.43 ± 0.08b

73.90 ± 0.28b

2.73 ± 0.04a

HP5

16.10 ± 1.27a

0.92 ± 0.01b

16.50 ± 0.10a

77.33 ± 0.95a

2.74 ± 0.04a

NPP

1.29 ± 0.15c

1.16 ± 0.06a

0.11 ± 0.01d

44.28 ± 0.73c

3.10 ± 0.06b

HP1

1.56 ± 0.07bc

0.93 ± 0.01b

0.68 ± 0.03c

62.54 ± 0.51b

3.50 ± 0.08a

HP3

1.67 ± 0.04ab

0.82 ± 0.01c

1.04 ± 0.08b

63.88 ± 0.23ab

3.11 ± 0.06b

HP5

1.90 ± 0.14a

0.53 ± 0.02d

2.58 ± 0.06a

65.42 ± 0.69a

2.85 ± 0.05c

Samples

EP

TE D

0.5% (w/v)

SC

M AN U

0.3% (w/v)

RI PT

0.1% (w/v)

Means with different letters in the same column at the same protein concentration are

AC C

significantly different at the 5% level.

ACCEPTED MANUSCRIPT Figure captions Fig. 1. HPSEC profiles of unheated and heat-treated pea proteins (NPP, HP1, HP3, and HP5). Fig. 2. Reducing and non-reducing SDS-PAGE profiles of unheated and heat-treated pea

RI PT

proteins (NPP, HP1, HP3, and HP5). Fig. 3. Typical droplet size distribution profiles of fresh emulsions stabilized by NPP (■); HP1 (●); HP3 (▲); HP5 (★) at protein concentrations of 0.1% (w/v) (A, A’), 0.3% (w/v) (B, B’) and

SC

0.5% (w/v) (C, C’), as determined with water (A-C) or 1% SDS (A’-C’).

M AN U

Fig. 4. Optical microscopy images of emulsions stabilized by NPP (A), HP1 (B), HP3 (C), and HP5 (D) diluted with deionized water at protein concentration of 0.1% (w/v). The bar accounts for 10µm.

Fig. 5. Reducing and non-reducing SDS-PAGE profiles of adsorbed and non-adsorbed protein

TE D

in emulsions prepared with unheated and heated pea proteins (NPP, HP1, HP3, and HP5) at protein concentration of 0.5% (w/v).

Fig. 6. Shear-rate dependence of viscosity for emulsions stabilized by NPP (■); HP1(▲); HP3

EP

(●); HP5 (★) at protein concentrations of 0.1% (w/v) (A), 0.3% (w/v) (B) and 0.5% (w/v)

AC C

(C). k is the consistency index (mPa·sn), and n is the flow behavior index. Fig. 7. Creaming index (CI, %) of the emulsions prepared by NPP(■); HP1(△); HP3(★); HP5 (○) at protein concentrations of 0.1 (w/v) (A), 0.3% (w/v) (B), and 0.5% (w/v) (C), upon

storage up to 14 days. (illustrations were typical visual images of the unheated and heated pea protein-stabilized emulsions after storage of 2 weeks)

1

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 2

ACCEPTED MANUSCRIPT Fig. 3 18

A

30

A'

16

25

Volume fraction (%)

Volume fraction (%)

14 12 10 8 6 4

20

15

10

0

0 0.1

1

10

100

0.1

1000

1

Size (µm)

16

16

14

14

Volume fraction (%)

20

B' 18

10 8 6 4

12

100

1000

SC

12

Size (µm)

10 8 6 4

M AN U

Volume fraction (%)

20

B 18

10

RI PT

5 2

2

2

0

0 0.1

1

10

100

Size (µm)

0.1

1000

1

10

100

1000

100

1000

Size (µm)

16

C 14

C' 14

12

Volume fraction (%)

10

8

4 2

8 6

TE D

6

4 2

0

0

0.1

1

10

100

EP

Size (µm)

AC C

Volume fraction (%)

12

10

1000

0.1

1

10

Size (µm)

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 4

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 5

ACCEPTED MANUSCRIPT Fig. 6 A

1

Viscosity (Pa·s)

0.1

n

n

k (mPa·s )

NPP

0.65 ± 0.01

3.37 ± 0.01

HP1

0.72 ± 0.02

7.02 ± 0.01

HP3

0.72 ± 0.02

8.64 ± 0.02

HP5

0.57 ± 0.01

13.09 ± 0.02

d c b a

1E-3

1E-4 1

10

100

-1

Shear rate (s ) 1

0.01

1E-3

1E-4 1

k (mPa·s )

0.51 ± 0.01

5.75 ± 0.01

HP1 HP3

0.54 ± 0.01 16.58 ± 0.02c 0.55 ± 0.01 17.34 ± 0.02b

HP5

0.45 ± 0.01 30.62 ± 0.03a

d

M AN U

Viscosity (Pa·s)

0.1

n

n NPP

SC

B

RI PT

0.01

10

100

-1

Shear rate (s )

TE D

1

C

Viscosity (Pa·s)

0.1

n

n

k (mPa·s )

NPP

0.76 ± 0.01

5.80 ± 0.01

HP1 HP3

0.59 ± 0.01 43.71 ± 0.02c 0.55 ± 0.01 58.52 ± 0.03b

HP5

0.52 ± 0.02 96.15 ± 0.03a

d

EP

0.01

AC C

1E-3

1E-4 1

10

-1

Shear rate (s )

100

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 7

ACCEPTED MANUSCRIPT Highlights: 1. Heat treatment at higher protein concentrations induced larger-sized aggregates. 2. Heat treatment of pea proteins increased the emulsion creaming stability.

RI PT

3. Heat treatment increased vicilin and legumin B in the adsorbed protein layer.

AC C

EP

TE D

M AN U

SC

4. Protein concentration was positively related to emulsion viscosity.