Whey protein isolate-Persian gum interaction at neutral pH

Accepted Manuscript Whey protein isolate-Persian gum interaction at neutral pH Hoda Khalesi, Bahareh Emadzadeh, Rassoul Kadkhodaee, Yapeng Fang PII:

S0268-005X(15)30123-5

DOI:

10.1016/j.foodhyd.2015.10.017

Reference:

FOOHYD 3171

To appear in:

Food Hydrocolloids

Received Date: 30 March 2015 Revised Date:

7 September 2015

Accepted Date: 21 October 2015

Please cite this article as: Khalesi, H., Emadzadeh, B., Kadkhodaee, R., Fang, Y., Whey protein isolatePersian gum interaction at neutral pH, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.10.017. 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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Whey protein isolate-Persian gum interaction at neutral pH

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Hoda Khalesi1*, Bahareh Emadzadeh2, Rassoul Kadkhodaee2 and Yapeng Fang3

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1 Department of Food Processing, Research Institute of Food Science and Technology, Mashhad, Iran

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2 Department of Food Nanotechnology, Research Institute of Food Science and Technology, Mashhad, Iran

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3 Glyn O. Phillips Hydrocolloids Research Centre, Hubei University of Technology, Wuhan, China

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*Corresponding author. Hoda khalesi [email protected] Department of food processing, Research Institute of Food Science and Technology (RIFST), Km 12 Quchan highway, Mashhad, Iran. Po Box: 91735-147, Zip code: 9185176933 Tel: +98-51-35425336; Fax: +985135425406 Cell: +989177196055

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Abstract

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A better understanding of Whey protein isolate-Persian gum interaction at neutral pH may

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increases the utilization of Persian gum in foods, pharmaceutical and cosmetic industry. The

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interactions in a neutral system (pH=7) containing whey protein isolate (WPI) and the soluble

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fraction of Persian gum (PG) were studied. The results obtained through Methylene blue

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spectrophotometry, zeta potentiometry, surface tension measurement and the observation of

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phase treatment confirmed the presence of interaction between WPI and PG even when both

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biopolymers were net-negatively charged at neutral pH. The protein-polysaccharide ratio

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influenced the properties of mixed solution. This effect was more significant at the equal amount

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of protein and polysaccharide. The research revealed possible weak interactions in WPI-PG

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mixed system at pH 7.00.

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Key words: Persian gum; Whey protein isolate; interaction 1

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1. Introduction Food technologists have paid considerable attention to the properties of food structure over

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the past decades due to its functional-controlling role in foods and biomaterials. Proteins and

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polysaccharides are natural biopolymers used as functional ingredients to control structure,

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texture, stability, mouthfeel, appearance, shelf life and controlled release (McClements, 2006;

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Perez, Carrara, Sánchez, Rodríguez Patino, & Santiago, 2009; Scholten, Moschakis, & Biliaderis, 2014).

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Combinations of proteins and polysaccharides are often used in numerous technological

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applications, consisting of biological level, food, cosmetics, pharmaceutical industries, etc

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(Dickinson, 1998; Kasran, 2013).

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structure formation, their interaction may find many applications in product development. The

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biopolymer particles may also be able to mimic lipid droplets according to rheological properties

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or its optical property (Jones, Decker, & McClements, 2010). Despite numerous recent studies,

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polysaccharide and protein interaction at neutral pH is one of the most challenging topics to

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comprehend.

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Concerning the ability of cost reduction, the novel function and

Two biopolymers can exist in a single phase system or in a phase separated one (two-phase).

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Biopolymer phase separation can occur through two different physiochemical mechanisms:

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associative (complex coacervation) and segregative separations (thermodynamic incompatibility)

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due to attractive and repulsive interaction, respectively (McClements, 2006; Ghosh &

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Bandyopadhyay, 2012).

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whereas at higher concentrations, it starts the phase separation. There are therefore, two kinds of

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single phase systems at mixed biopolymers: soluble complex and cosolubility. Complexes are

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comprised of proteins and polysaccharide molecules when opposite net charges (or charge

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patches) are present (Amit & Bandyopadhyay, 2012; Thomas, Durand, Chassenieux, & Jyotishkumar,

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At low concentration, two biopolymers can co-exist in a single phase

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2013; Tian, Fang, Nishinari, & Phillips, 2014).

It is worth to be noted that recent researches have

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demonstrated the formation of soluble complexes between proteins and anionic polysaccharides

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even when the pH is neutral or close to neutral, as has been reported in the case of milk protein

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and xanthan gum (Rohart, Jouan-Rimbaud Bouveresse, Rutledge, & Michon, 2015), fish gelatin and

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gum arabic (Yang, Anvari, Pan, & Chung, 2012), whey protein isolate and gum arabic (Klein, Aserin,

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Ishai, & Garti, 2010),

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cellulose (Koupantsis & Kiosseoglou, 2009).

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WPC and k-carrageen (Perez, et al., 2009), Whey protein and carboxymethyl

Whey protein has been the focal point of colloid researches concerning the role of milk

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proteins as structuring agents, emulsifiers, stabilizers and sensory characteristics. β-lactoglobulin

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(β-Lg), as the main protein of WPI, strongly affects its functional and thermal properties.

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Intrinsic factors such as amino acid composition, conformation, molecular size, shape, flexibility,

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net charge, hydrophobicity, substituent chemical groups and sulfhydryl groups affect the

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functionality of WPI. However, temperature, pH and ion concentration, as extrinsic factors can

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also influence its functionalities (Jovanović, Barać, & Maćej, 2005; Kinsella, 1984).

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Persian gum is amongst the highly produced natural hydrocolloids in Iran, which exudates

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from the barks of mountain almond trees (Amygdalus scoparia Spach). Its exudate is recorded in

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traditional Persian herbal medicine references and some therapeutic benefits like appetite

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stimulating, mucus decreasing, teeth pain healer and swollen joints are attributed to it. This

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hydrocolloid is also known as Shiraz, Zedu and Angum gum and about 400 tons Persian gum is

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exported annually from Iran (Abbasi & Rahimi, 2014; Ghasempour, Alizadeh, & Bari, 2012). It

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is an anionic hydrocolloid with low protein and high water absorption capacity. The valuable

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concept about Persian gum is its similar emulsifiering characteristics to gum Arabic which has

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made it as an appropriate candidate for food systems. Arabinose and galactose are the main

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monosaccharides in its chemical structure. So it is classified as arabinogalactan hydrocolloids.

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The molecular weight of this hydrocolloid is reported about 2.59-4.74 × 106 Da with PDI of

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1.04-4.05. Based on HPLC, 1H-NMR and 13C-NMR analyses, the backbone of PG is composed

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of galactose (1→3 linked β-D–galactopyranose) and rhamnose whereas the branches are

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composed of (1→6) linked β-D–Galp and (1→) and/or (1→3) linked α-L-Arabinofuranose

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residues. Persian gum consists of soluble and insoluble fractions and has reasonable surface

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activity (Fadavi, Mohammadifar, Zargarran, Mortazavian, & Komeili, 2014; Golkar, Nasirpour,

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& Keramat, 2014; Jafari, Beheshti, & Assadpour, 2012). Recent studies have shown the

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capability of Persian gum in emulsion system stabilization, edible film formation, low-fat

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yoghurt properties improvement and gelatin replacement in pastilles (Abbasi & Rahimi, 2014;

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Ghasempour, et al., 2012; Jafari, et al., 2012).

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To the best of our knowledge, many researchers have focused on the properties and the

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capabilities of Persian gum recently. However, none of them has considered the interactions

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between Persian gum and WPI. Therefore, in this study, we aim to investigate the behavior and

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the interactions in WPI-PG aqueous systems at neutral pH and low ionic strength.

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2. Materials and methods

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2.1. Materials

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Whey protein isolate (WPI consisted of 90.9% protein) was purchased from Davisco Food

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International (BiPro Unflavoured Whey Protein, Le Sueur, MN, USA). Persian Gum was kindly

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donated by Dena Emulsion Co. (Shiraz, Iran) in intact form which was collected from

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Amygdalus scoparia Spach trees in Fasa city, Fars state, Iran. The white color gum samples were

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milled to a fine powder, sieved to form uniform particles (<500 µm) and stored carefully in air-

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tight polypropylene jars for experiments. Nitrogen, ash, fat and moisture were determined

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according to the Analysis of Association of Official Analytical Chemist (AOAC International,

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2005) in triplicate. Protein content was calculated using the conversion factor 6.25. Carbohydrate

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values were obtained by difference. Chemical composition of the powdered Persian gum in this

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study was 9.656%±0.084 moisture, 1.02% protein (0.1632%±0.02 nitrogen), 1.662%±0.085 ash

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and 87.66% carbohydrate content. The amount of fat was trace. All solutions were prepared

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using deionized water. Sodium azide, Methylene blue and sodium hydroxide were purchased

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from Merck (Germany). Other chemicals were of analytical grade.

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2.2. Sample preparation

Stock dispersion of PG (3% w/w) was prepared and after complete hydration for one night at

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room temperature, it was centrifuged (HERMLE, Z36HK, Germany) at 20000 rpm for 20 min to

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separate the soluble fraction from the insoluble. The supernatant was desired section. The final

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concentration of the PG solution obtained after determining the dry matter of sample. The pH

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adjusted 7± 0.1 by Sodium hydroxide 0.1 N. Whey protein isolate (2% and 16% w/w) was

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dissolved in deionized water at room temperature. The pH of solution was about 7±0.1. They

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stirred for 2h at 250 rpm to achieve complete dissolution. To prevent bacterial contamination,

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0.02 wt% sodium azide was added. Stock solutions were stored at 4–5˚C. For preparing final

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desired concentration of WPI/PG mixed systems, appropriate value of Biopolymer solutions

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were mixed.

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2.3. Spectrophotometric behavior of methylene blue with biopolymer solution

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This method helps to understand the mechanism of interaction between mixed biopolymers

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(Koupantsis, et al., 2009; Michon, Konaté, Cuvelier, & Launay, 2002; Perez, et al., 2009; Rohart,

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et al., 2015; Yang, et al., 2012). Methylene blue (MB) is a cationic chemical compound that has

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a deep blue color in water. The maximum absorption of MB solution is at 664 nm.

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Mixed PG – MB solutions containing 0.001% w/w MB and PG in concentration between

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0.005 – 1.00% w/w were prepared. The change in the ratio between the absorbance obtained at

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664 and 615 nm was determined with the increase in polysaccharide concentration. In the second

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experiment, levels of polysaccharide were increased in a MB–WPI solution containing 0.001%

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w/w MB and 1.00 % w/w WPI at pH 7, and the ratio of the absorbance at 664 and 615 nm was

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followed as a function of PG concentration. The measurements were performed using a

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spectrophotometer (CT-5700, Chrom Tech, Taiwan), 30 min after the preparation of mixed

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system at 22˚C (Perez, et al., 2009).

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2.4. Zeta potential

Zeta potential values of the aqueous solutions composed of WPI, PG or WPI: PG in different

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ratios were determined by dynamic light scattering technique (Zetasizer Nan, Malvern

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Instrument, UK). Samples were prepared at a concentration of 1% Wt. and were diluted to 0.1%

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before experiments. All measurements were performed in triplicate on three different samples of

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the same composition at pH 7 and 25˚ C (Yang, et al., 2012).

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2.5. Surface tension

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Surface tension of WPI, Persian gum solution and their mixtures in various ratios but

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constant concentrations (1% wt) was measured by using a tensiometer (Kruss K100 Tensiometer,

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Germany). All the measurements were carried out for 45 min until a constant value was obtained

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(Klein, et al., 2010). All tests were repeated in triplicate at 22 ± 1 °C.

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2.6. The observation of phase treatment

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Mixed biopolymer solutions with various concentration were prepared in 1:1 wt. ratios of the

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protein and polysaccharide solutions. 10 mL of the biopolymers mixture solution were placed in

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test tubes and centrifuged at 5000 rpm and 25 ˚C for 20 min. afterward, biopolymer mixtures

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were stored overnight at room temperature (Perez, et al., 2009).

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2. 7. Statistical analysis

All experiments were performed in triplicate and are reported as means and standard deviations.

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3. Results and discussion

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3.1. Spectrophotometric behavior of methylene blue with biopolymer solution In general, the absorption behavior of methylene blue (MB) in aqueous phase system is a

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proposed methodology for indicating the interaction between protein-polysaccharide solution

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and formation of their complex (Perez, et al., 2009; Rohart, et al., 2015). The optical density

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spectrum of MB solution versus increasing PG concentration was measured and compared with

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the one obtained for the MB/WPI/PG solution at ambient temperature. In the first stage,

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spectrophotometirc analysis of cationic dye MB and PG solution showed a shift of maximum

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absorption of MB dye towards shorter wavelengths (615 nm). In fact, the intensity of the peak at

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664 nm decreased by increasing the amount of PG concentration, inferring that free MB

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molecules in the system were reduced by electrostatic interaction between the polysaccharide

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and the dye molecules. Therefore, the addition of Persian gum has changed the spectrum. Fig. 1

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shows the value of A664/A615 ratio, declined from 1.52 to 1.47 in MB/PG system. In the second stage, addition of different concentrations of PG to the constant concentration

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of MB/WPI solution (dye-protein, 1% wt.) caused a large increase in the amount of A664/A615

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ratios. When the gum is not implemented in system (at 0% concentration of PG), the amount of

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A664/A615 for MB/WPI is lower than that of MB alone. It seems that WPI has had an interaction

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with MB. Rohart et al (2014) previously reported the possibility of interaction between MB and

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milk proteins. With increasing the amount of gum in constant concentration of MB/WP system,

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the value of A664/A615 became greater. In fact, the amount of free methylene blue increased in the

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system. This result may be due to the weak attractive interaction between WPI and PG. So the

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amount of non-bound MB molecules have increased in system and led to the increasing

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absorption at 664 nm point. In other words, WPI competed with the cationic MB molecules and

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bound preferably to PG. Addition of 0.1% PG to MB aqueous solutions resulted in a sharply

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decrease to 1.483 in A664/A615. However, this ratio showed an increase when 0.1% PG was added

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to MB-WPI aqueous solutions. The difference in A664/A615 value indicates the existence of the

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protein-hydrocolloid interaction which depends on different types of charge and the accessibility

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of the interacting group in biopolymers. The results of this experiment reasonably demonstrate

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that interaction may occur in WPI/PG aqueous phase system at pH 7.

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Based on methylene blue (MB) binding and zeta potential measurements, Koupantsis and

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Kiosseoglou (2009) reported the presence of electrostatic interaction between whey proteins and

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carboxymethyl cellulose molecules at neutral and acidic conditions. Similar results have been

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reported for Xanthan gum and iota-carrageenan with milk protein (Rohart, et al., 2015) and for fish

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gelatin-gum Arabic mixtures (Yang, et al., 2012). It is also important to note that some

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hydrocolloids such as sodium alginate and guar gum did not have any significant influence on

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spectrophotometric behavior of methylene blue (Perez, et al., 2009; Rohart, et al., 2015) as they

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were not able to interact with proteins.

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3.2. Zeta potential

Zeta potential is related to the surface charge of the particle, adsorbed layer at the interface,

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and the nature of the surrounding suspension medium. The zeta potential values of both

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biopolymers were net negatively charged at neutral pH and 25 ºC (Fig. 2). WPI molecules and

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Persian gum alone showed a zeta potential of -26.375 ± 1.034 mV and -32.43 ± 3.36 mV,

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respectively. On the other hand, their mixtures at different ratios showed lower negative charge

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than each of them alone. This interesting phenomenon was higher at 1:1 ratio of mixed

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biopolymers. In fact, this result indicates that WPI-PG charged interaction might occur in

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system, although electrical charge of each biopolymer at pH=7 was negative. Protein had several

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sites that impart positive local charge and those may interact with the gum. So final molecular

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conformation was changed by opposite charges at neutral pH. In other words, this might be due

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to the interactions between the positive charges on the WPI and the negative ones localized on

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PG molecule. Similar profiles were reported for the combination of non-heated WPI and λ-

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carrageenan (Chun, et al., 2014), WPI-gum Arabic system (Klein, et al., 2010) and mixture of pea

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legumin protein (PLA)- Gum Arabic (Klassen, 2010) at pH 7.

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3.3. Surface tension

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Most high-molecular-weight polysaccharides do not have much capability of being adsorbed

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at interfaces. Surface active polysaccharides have recently received considerable interest. The

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surface tension of water as a basic value for comparison of other data was obtained to be 73.06

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mN m−1. 1% WPI and PG solutions reduced the surface tension values to 51.97 and 57.71 mN

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m−1, respectively. Persian gum has emulsifying properties and this property is lower than Arabic

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gum (Abbasi, et al., 2014; Jafari, et al., 2012) maybe due to protein content. In other words, the

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protein content and amino-acid composition of the protein fraction in hydrocolloid affect the

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adsorption behavior and thus the emulsifying activity (Nakauma, et al., 2008). The surface tension

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of two biopolymer mixtures (1 wt.%) at different ratios were higher than that of WPI alone.

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In the case where there is no interaction between biopolymers, the surface tension of the mixed

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solution could be calculated mathematically by the numbers of each biopolymers (Klein, et al.,

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2010).

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been formed between WPI and PG. Figure 3 shows a comparison between the mathematical

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calculated values of surface tension and the measured ones obtained through experimental tests.

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The measured values of surface tension were less than the calculated ones. In addition, at equal

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ratio of protein and polysaccharide, a maximum yield of reduction in surface tension was

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observed. Similar results were obtained for zeta potential measurements. The higher functional

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properties in the combined system may indicate the weak interaction between protein and

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polysaccharide in the system. Previous research showed that the surface tension value of WPI-

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gum Arabic mixture is lower than that the calculated one at 5 wt.% (Klein, et al., 2010). It is

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evident that in comparison to the two biopolymers separately, the protein-polysaccharide mixture

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solution have different functional properties (Corredig, 2009).

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Therefore, according to the measured surface tension values, it seems a combination has

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3.4. The observation of phase treatment The mixtures of two water-soluble biopolymers were separated into phases according to the

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type and the concentrations of the two biopolymers. The phase diagram of WPI- PG aqueous

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solutions (1-8 wt.%) in admixture with PG (0.05 to 1 wt.%) is presented in Fig. 4. WPI and PG

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biopolymer solutions were centrifuged for accelerating the equilibrium of two liquid phases. All

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the studied mixed biopolymer samples were homogeneous and stable. No phase separation

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observed even at 8% (w/w) WPI and 1% (w/w) PG concentration and all the samples were

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compatible at the investigated concentrations. Higher concentration of PG is probably required

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for phase separation. Therefore, the incompatibility was not implemented between the

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biopolymers in this range of concentration.

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Persian gum is an arabinogalactan polysaccharide and contains some proteinaceous material (Fadavi, et al., 2014).

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segregation with the other component and their situation can be much more complex than the

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phase separation of monodisperse systems. According to previous reports, a two-phase zone was

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displayed in mixed system solutions above 0.15% (w/v) λ-carrageenan and 1.5% (w/v) WPI

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(Chun, et al., 2014).

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complexes up to pH 8.00 and 0.05% concentration.

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Yang et al (2012) reported fish gelatin and gum Arabic can form soluble

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Polydisperse and heterogeneous polysaccharide react differently toward

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4. Conclusions

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The interaction between biopolymers at neutral pH has recently become as one of the most

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important and interesting fields of study. The combination of Persian gum, with noticeable

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functional properties, and WPI as one of the major ingredients in many food processes, could be

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considered as a novel system for the improvement of product. No phase separation was observed

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in the studying range of WPI-PG concentration even 48 h after centrifugation. In other words,

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the WPI-PG mixtures tended to be aqueous single homogeneous phase. The results of methylene

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blue spectrophotometric method clearly indicated the presence of more free-methylene blue

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molecules in the WPI/PG system than the PG solution alone. The zeta potential and surface

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tension values of the mixed solutions also confirmed the presence of some kinds of interaction in

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the system. The major insights gained through this research confirm the recent reports about the

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existence of weakly interactions in some protein/gum systems even at a pH where both

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biopolymers are net negatively charged. This phenomenon could be explained by the interaction

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between the protein patches with anionic reactive sites of polysaccharide. We proposed the

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model for an interaction between WPI and PG (Fig. 5) at neutral pH. The behavior of WPI/PG

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combination system can affect the functionalities of these protein and hydrocolloid. The results

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can be applied for designing food microstructure, emulsion and texture. Further research is in

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progress to shed more light on WPI-PG interactions in a wide range of pH .

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The authors would like to thank Dr. Rafe and Dr. Movahedpour. The help and support of

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Acknowledgments

Dena Emulsion Co. is highly appreciated.

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References

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Abbasi, S., & Rahimi, S. (2014). Persian gum. In M. Mishra (Ed.), Encyclopedia of Biomedical Polymers and Polymeric Biomaterials. NewYork: Taylor and Francis Group LLC. Amit, k., & Bandyopadhyay, P. (2012). Polysaccharide-Protein Interactions and Their Relevance in Food Colloids. In D. Karunaratne (Ed.), The Complex World of Polysaccharides (pp. 14): InTech. Chun, J.-Y., Hong, G.-P., Surassmo, S., Weiss, J., Min, S.-G., & Choi, M.-J. (2014). Study of the phase separation behaviour of native or preheated WPI with polysaccharides. Polymer, 55(16), 43794384.

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Corredig, M. (2009). Molecular understanding of the interaction of dairy proteins with other food biopolymers. In M. Corredig (Ed.), Dairy-Derived Ingredients Food and nutraceutical uses (pp. 371-393): Woodhead Publishing. Dickinson, E. (1998). Stability and rheological implications of electrostatic milk protein–polysaccharide interactions. Trends in Food Science & Technology, 9(10), 347-354. Fadavi, G., Mohammadifar, M. A., Zargarran, A., Mortazavian, A. M., & Komeili, R. (2014). Composition and physicochemical properties of Zedo gum exudates from Amygdalus scoparia. Carbohydrate Polymers, 101(0), 1074-1080. Ghasempour, Z., Alizadeh, M., & Bari, M. R. (2012). Optimisation of probiotic yoghurt production containing Zedo gum. International Journal of Dairy Technology, 65(1), 118-125. Golkar, A., Nasirpour, A., & Keramat, J. (2014). b-lactoglobulin-Angum Gum (Amygdalus Scoparia Spach) Complexes: Preparation and Emulsion Stabilization. Journal of Dispersion Science and Technology, 36(5), 685-694. Jafari, S. M., Beheshti, P., & Assadpour, E. (2012). Rheological behavior and stability of D-limonene emulsions made by a novel hydrocolloid (Angum gum) compared with Arabic gum. journal of food engineering, 109(1), 1-8. Jones, O., Decker, E. A., & McClements, D. J. (2010). Thermal analysis of β-lactoglobulin complexes with pectins or carrageenan for production of stable biopolymer particles. Food Hydrocolloids, 24(2– 3), 239-248. Jovanović, S., Barać, M., & Maćej, O. (2005). Whey proteins-Properties and Possibility of Application Mljekarstvo, 55(3), 215-233. Kasran, M. ( 2013). Development of Protein Polysaccharide Complex for Stabilization of Oil-in-Water Emulsions. Guelph, Ontario, Canada. Kinsella, J. E. (1984). Milk proteins: Physicochemical and functional properties. In Critical Reviews in Food Science & Nutrition (Vol. 21). Klassen, D. (2010). Associative Phase Separation in Admixtures of Pea Protein Isolates with Gum Arabic and a Canola Protein Isolate with Carrageenan and Alginate. Saskatchewan university, Saskatchewan. Klein, M., Aserin, A., Ishai, P. B., & Garti, N. (2010). Interactions between whey protein isolate and gum Arabic. Colloids and Surfaces B: Biointerfaces, 79(2), 377-383. Koupantsis, T., & Kiosseoglou, V. (2009). Whey protein–carboxymethylcellulose interaction in solution and in oil-in-water emulsion systems. Effect on emulsion stability. Food Hydrocolloids, 23(4), 1156-1163. McClements, D. J. (2006). Non-covalent interactions between proteins and polysaccharides. Biotechnology Advances, 24(6), 621-625. Michon, C., Konaté, K., Cuvelier, G., & Launay, B. (2002). Gelatin/carrageenan interactions in coil and ordered conformations followed by a methylene blue spectrophotometric method. Food Hydrocolloids, 16(6), 613-618. Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., & Phillips, G. (2008). Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids, 22, 1254–1267. Perez, A. A., Carrara, C. R., Sánchez, C. C., Rodríguez Patino, J. M., & Santiago, L. G. (2009). Interactions between milk whey protein and polysaccharide in solution. Food Chemistry, 116(1), 104-113. Rohart, A., Jouan-Rimbaud Bouveresse, D., Rutledge, D. N., & Michon, C. (2015). Spectrophotometric analysis of polysaccharide/milk protein interactions with methylene blue using Independent Components Analysis. Food Hydrocolloids, 43(0), 769-776.

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Scholten, E., Moschakis, T., & Biliaderis, C. G. (2014). Biopolymer composites for engineering food structures to control product functionality. Food Structure, 1(1), 39-54. Thomas, S., Durand, D., Chassenieux, C., & Jyotishkumar, P. (April 2013). Handbook of Biopolymer-Based Materials. In (Vol. 1, pp. 988): Wiley. Tian, D. Z., Fang, Y. P., Nishinari, K., & Phillips, G. O. (2014). Protein-Polysaccharide interactions:phase behaviour and applications In P. A. Williams & G. O. Phillips (Eds.), Gums and Stabilisers for the Food Industry (17) (pp. 52-63): The Royal Society of Chemistry. Yang, Y., Anvari, M., Pan, C.-H., & Chung, D. (2012). Characterisation of interactions between fish gelatin and gum arabic in aqueous solutions. Food Chemistry, 135(2), 555-561.

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Figure Caption

Fig. 1. Absorbance ratio of aqueous solutions at 664 and 615 nm (A664/A615) containing

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0.001% methylene blue as a function of PG concentration at pH 7.00. Fig. 2. Zeta potential value of WPI-PG mixtures containing different weight ratios and constant total biopolymer concentration (0.1% w/w) at 25˚C.

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Fig. 3. Measured surface tension (gray column) and calculated surface tension (white column) values as a function of WPI: PG wt. The total concentration was constant at 1% wt.

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Fig. 4. A: Phase diagram of WPI- PG solutions at pH=7. B: image of phase behavior for WPI (8% w/w) with various concentrations of PG at pH 7.

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Fig. 5. Proposed model of the interaction between WPI- PG system at pH 7.

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1 1.53 1.52

(MB & WPI)/PG MB/ PG

1.5 1.49 1.48

1.46 0.000

0.200

0.400

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1.47

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A664/A615

1.51

0.800

1.000

1.200

Persian gum concentration (% wt)

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Fig. 1. Absorbance ratio of aqueous solutions at 664 and 615 nm (A664/A615) containing

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0.001% methylene blue as a function of PG concentration at pH 7.00.

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WPI

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3:1 1:1 1:3 (WPI/PG) (WPI/PG) (WPI/PG)

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-5 -10 -15 -20

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Zeta potential (mV)

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WPI, WPI:PG, PG (wt. ratio)

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Fig. 2. Zeta potential value of WPI-PG mixtures containing different weight ratios and constant

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total biopolymer concentration (0.1% w/w) at 25˚C.

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calculated value (predict) Exprimental value (real)

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1:3

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Surface tension (mN m-1)

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1:1

2:1

3:1

4:1

WPI

WPI/PG (Wt. ratio)

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Fig. 3. Measured surface tension (gray column) and calculated surface tension (white column)

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values as a function of WPI: PG wt. The total concentration was constant at 1% wt.

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Fig. 4. A: Phase diagram of WPI- PG solutions at pH=7. B: image of phase behavior for WPI (8% w/w) with various concentrations of PG at pH 7.

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Fig. 5. Proposed model of the mixed WPI- PG system at pH 7.

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- The interaction between WPI and Persian Gum was examined at pH=7. - Lower surface activity was observed for mixture of WPI and Persian gum solution compare to

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- Zeta potential value of their mixture was higher than of each biopolymer solutions.

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- The WPI- PG interaction at pH=7 & low ionic strength may be due to the PG protein part.