Isothermal titration calorimetric and spectroscopic studies of β-lactoglobulin-water-soluble fraction of Persian gum interaction in aqueous solution

Food Hydrocolloids 55 (2016) 108e118

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Isothermal titration calorimetric and spectroscopic studies of b-lactoglobulin-water-soluble fraction of Persian gum interaction in aqueous solution Mohammad Hadian a, Seyed Mohammad Hashem Hosseini a, *, Asgar Farahnaky a, Gholam Reza Mesbahi a, Gholam Hossein Yousefi b, c, Ali Akbar Saboury d a

Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran d Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 June 2015 Received in revised form 5 October 2015 Accepted 3 November 2015 Available online 12 November 2015

In this work, the interaction between b-lactoglobulin (b-lg) and water-soluble fraction of Persian gum (WPG) was studied under the effects of extrinsic parameters including pH, protein to polysaccharide mixing ratio (MR 8:1e1:4), total biopolymer concentration (TC 0.1e0.6% (w/w)), ion type (Naþ and Ca2þ), ionic strength (0e100 mM) and temperature (25, 40 and 55  C). Soluble complexes were formed at a pH above protein's isoelectric point (pI). MR and TC had significant effects on the values of critical pH41 (formation of insoluble complexes) and pHopt (maximum optical density). A decrease in MR (at a constant TC) and in TC (at a constant MR) shifted the values toward more acidic domains; while, the critical pHc (formation of soluble complexes) remained constant. The effect of divalent ions (Ca2þ) in preventing the complex coacervation was more than that of monovalent ones (Naþ). Increasing the ionic strength had significant effect in decreasing the interaction. In this study, the effects of temperature on the thermodynamic parameters were obvious. Biopolymers binding enthalpy as obtained by isothermal titration calorimetry (ITC) was independent from the temperature in the studied range. Analysis of the temperature effect showed that electrostatic interaction, hydrogen bonding and hydrophobic interactions were involved in the complexation process. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Persian gum b-lactoglobulin Complex coacervation ITC Ionic strength

1. Introduction Proteins and polysaccharides play important roles both in macro and micro scales of food systems. Individual biopolymers can be used as emulsifiers, foaming agents, carriers for other molecules, stabilizers, thickeners, and gelling agents (Corredig, Sharafbafi, & Kristo, 2011; Dickinson, 1998, 2008; Goh, Sarkar, & Singh, 2009; Livney, 2010; Tolstoguzov, 2003). The key factors influencing the physicochemical properties of these macromolecules in dispersion include molar mass, molecular conformation, polydispersity, charge density, concentration, pH, ionic strength, temperature, solvent quality and nature of intra- or inter-molecular interactions Abbreviations: b-lg, b-lactoglobulin; PG, Persian gum; WPG, Water-soluble fraction of Persian gum; MR, Protein to polysaccharide mixing ratio; TC, Total biopolymer concentration; GDL, Glucono-delta-lactone; OD, Optical density; ITC, isothermal titration calorimetry; pI, Isoelectric point. * Corresponding author. E-mail address: [email protected] (S.M.H. Hosseini). http://dx.doi.org/10.1016/j.foodhyd.2015.11.006 0268-005X/© 2015 Elsevier Ltd. All rights reserved.

(Goh et al., 2009). The physicochemical properties of the proteins and polysaccharides depend not only on the molecular parameters of the individual biopolymers but also on the nature of interactions between the protein and polysaccharide molecules (Goh et al., 2009). Therefore, tailor-made functionalities such as microencapsulation (Ahmadi, Nasirpour, Sheikhzeinodin, & Keramat, 2015; Jun-xia, Hai-yan, & Jian, 2011; Yang, Gao, Hu, Li, & Sun, 2015), nanoencapsulation (Hosseini, Emam-Djomeh, Van der Meeren, & Sabatino, 2015; Ron, Zimet, Bargarum, & Livney, 2010), interfacial (emulsion and foam) stabilization (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012; Dickinson, 2009; Liszka-Skoczylas, _  ski, 2014; Schmitt et al., 2005), texturizing Ptaszek, & Zmudzi n such as fat replacing (Laneuville, Paquin, & Turgeon, 2005) and formation of novel electrostatic (mixed) gels (van den Berg, van Vliet, van der Linden, van Boekel, & van de Velde, 2007; Picard, Giraudier, & Larreta-Garde, 2009; Le & Turgeon, 2015) can often be introduced into a system by using non-covalent (electrostatic

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and hydrophobic) interactions as well as hydrogen bonding between biopolymer mixtures. The interaction between biopolymers is of increasing interest in soft-condensed matter research (de Vries & Cohen Stuart, 2006; Schmitt, Aberkane, & Sanchez, 2009). In aqueous solutions, proteins and polysaccharides undergo two different types of phase separation phenomena including thermodynamic incompatibility (segregative phase separation) and thermodynamic compatibility (associative phase separation or complex coacervation) depending mainly on the electrical charges of both biopolymers as affected by extrinsic (pH, ionic strength, mixing ratio, total biopolymer concentration, temperature, pressure, shearing and acidification method) and intrinsic (biopolymer charge density, charge distribution, molecular weight and molecular conformation) parameters (Schmitt et al., 2009; Schmitt & Turgeon, 2011; Turgeon & Laneuville, 2009). These phase behaviors arise from long- or short-range interactions between the biomacromolecules themselves, and also possibly because of different affinities between the biomacromolecules and the solvent (Schmitt et al., 2009). In thermodynamic incompatible systems, two non-interacting macromolecular species mutually segregate into two different distinct immiscible aqueous phases, one phase mainly rich in one biopolymer (e.g. protein) and the other phase mainly rich in the other biopolymer (e.g. polysaccharide) (Dickinson, 1998; Goh et al., 2009; Turgeon, Beaulieu, Schmitt, & Sanchez, 2003; Turgeon & Laneuville, 2009; Ye, 2008); while in thermodynamic compatible systems, molecules spontaneously attract each other and the complexation phenomenon results in the formation of a solventrich (biopolymer depleted) phase and a phase rich in both biopolymers (Doublier, Garnier, Renard, & Sanchez, 2000; Ould Eleya & Turgeon, 2000). The protein and polysaccharide in the biopolymer rich phase are held together mainly by electrostatic forces and can appear as coacervates, complexes (either soluble or insoluble) and gels (Turgeon & Laneuville, 2009). This phenomenon which occurs at a pH between the proteins' isoelectric point (pI) and the pKa of the polysaccharide is involved in the structuring of many biological systems. The interactions between different proteins and polysaccharides under the effect of different parameters have been extensively investigated in previously published works. This large spectrum of protein-polysaccharide pairs can be found in excellent reviews available in this field (Benichou, Aserin, & Garti, 2002; Bouyer et al., 2012; Cooper, Dubin, Kayitmazer, & Turksen, 2005; Corredig et al., 2011; daSilva, Lund, Jonsson, & Akesson, 2006; de Kruif & Tuinier, 2001; de Kruif, Weinbreck, & de Vries, 2004; Dickinson, 1998; Dickinson, 2008; Doublier et al., 2000; Goh et al., 2009; Grinberg & Tolstoguzov, 1997; Kizilay, Kayitmazer, & Dubin, 2011; Lapitsky, 2014; McClements, 2006; Rodríguez Patino & Pilosof, 2011; Schmitt, Sanchez, DesobryBanon, & Hardy, 1998; Schmitt et al., 2009; Schmitt et al. 2011; Turgeon et al., 2003; Turgeon & Laneuville, 2009; Turgeon, Schmitt, & Sanchez, 2007; van der Sman, 2012; Ye, 2008). Among different types of proteins, the interactions of b-lactoglobulin (b-lg) with different polysaccharides have been well studied because of its specific characteristics. This small globular protein is the main protein in the whey fraction of milk from ruminants and some nonruminants and is a member of the lipocalin protein family because of its ability to bind small hydrophobic molecules. Bovine b-lg has a relative molecular weight of 18.4 kDa and its 162-amino acid polypeptide chain folds up into an eight-stranded anti-parallel bbarrel. A ninth b-strand flanks the first strand (Fox, 2009). The compact globular conformation of b-lg is stabilized by two intramolecular disulfide bridges. The quaternary structure (association properties) of the protein varies among monomers, dimers or oligomers depending on the pH, temperature, concentration and ionic strength as a result of a delicate balance among hydrophobic,

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electrostatic and hydrogen-bond interactions (Gottschalk, Nilsson, Roos, & Halle, 2003; Sakurai & Goto, 2002). At pH 5e8, b-lg exists as a dimer, at pH 3e5 the dimers associate to form octamers, and at extreme pH values (<2 or >8) most protein exists as monomers. At pH > 9, the molecule is irreversibly denatured (Harnsilawat, Pongsawatmanit, & McClements, 2006). Persian gum (also known as Zedo gum, Shirazi gum, Angum gum and Farsi gum) is an exudate gum which is naturally secreted from the barks of mountain almond trees (Amygdalus scoparia Spach) that mainly grow in central parts of Iran. Persian gum contains two different fractions, a water-soluble and a water-insoluble. During recent years, some limited studies have been performed to make a deeper insight into the physicochemical properties and structureefunction relationships of this gum. Jafari, Beheshti, and Assadpour (2012) studied the rheological behavior and the stability of D-limonene emulsions stabilized by Angum gum and/or gum Arabic. Angum gum revealed higher viscosities than gum Arabic. Therefore, its ability to stabilze the emulsions was better than gum Arabic. The rheological behavior of Angum-gum-stabilized emulsions followed HerscheleBulkley model. In another research, Jafari, Beheshti, and Assadpour (2013) studied the emulsification properties of Angum gum and gum Arabic for use as a flavor (D-limonene) encapsulating material in spray drying encapsulation. Increasing the amount of gum Arabic decreased the emulsion droplet size, while, increasing the amount of Angum gum resulted in larger droplet sizes. However, no significant differences were reported in droplet size (Jafari et al., 2013). Taking into account the smallest particle size, the optimum levels of biopolymer and flavor oil in Angum gum-emulsified dispersion were 2 and 5%, respectively. Persian and tragacanth gums were used to stabilize the orange peel essential oil nanoemulsions prepared by high intensity ultrasound (Mirmajidi Hashtjin & Abbasi, 2015). The soluble fraction of native gums applied at low concentration showed significant effect on stability, particle size as well as rheology of nanoemulsion. Abbasi and Mohammadi (2013) utilized different (soluble and insoluble) fractions of Persian gum and gum tragacanth (individually and in different combinations) to stabilize milk-orange juice mixture against serum separation. Persian gum, its soluble fraction and a mixture of soluble fraction of Persian gum and that of gum tragacanth (80:20) could effectively prevent serum separation at concentration level of 2.20, 1.00 and 0.37%, respectively, by adsorbing onto casein micelles and hence producing electrostatic and steric repulsions (Abbasi & Mohammadi, 2013). In this work, Persian gum and its soluble fraction made considerable changes in the zeta potential (Abbasi & Mohammadi, 2013) indicating the anionic character of the polysaccharide. Golkar, Nasirpour, Keramat, and Desobry (2015) studied the emulsifying properties of Angum gum covalently bonded to b-lg. Emulsifying properties of emulsions containing b-lg:Angum gum (1:1) conjugates were studied with the advancement of Maillard reaction. Dry-heating time had no significant (p > 0.05) effect on the emulsion activity index; however, after two weeks emulsion stability index was significantly (p < 0.05) increased. b-lg-Angum gum conjugates (1:1, 1:2, and 2:1) exhibited much better emulsification performance than individual Angum gum and gum Arabic at the same emulsifier (1.5% w/w) to oil (40% v/ v) ratio. Golkar, Nasirpour, and Keramat (2015) studied the complexation between b-lactoglobulin and un-fractioned Persian gum under the effect of biopolymer mixing ratio (1:2, 1:1 and 2:1) and pH using spectrophotometric and particle size analyses as well as microscopic observation. Biopolymer complexes were then used to stabilize oil in water emulsions. Maximum solubility was observed at pHs between pHc (formation of soluble complexes), and pH41 (formation of insoluble complexes). After 48 h of storage, the stabilized emulsions were not phase separated and the droplet size distribution remained constant (Golkar, Nasirpour, et al., 2015).

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Fadavi, Mohammadifar, Zargarran, Mortazavian, and Komeili (2014) characterized the composition and physicochemical properties of three types of Zedo gum exudates. Although the anionic character of Persian gum (especially that of soluble fraction) has been confirmed in previous studies (Abbasi & Mohammadi, 2013; Golkar, Nasirpour, et al., 2015); these researchers reported that monosaccharide analysis using GCeMS resulted in the identification of arabinose and galactose as the main sugars. Fadavi et al. (2014) also reported that no differences in functional groups were observed between different types of Persian gum using FTIR spectroscopy. It seems that the properties (including the chemical structure) of Persian gum are similar to those of polysaccharides extracted from Prunus amygdalus (almond) gum. Bouaziz et al. (2015a) investigated antioxidant and antimicrobial activities as well as chemical structure of low molecular weight oligosaccharides prepared by enzymatic hydrolysis of almond gum. Analyses showed that the most prominent residues were galactose and arabinose with traces of xylose, rhamnose, glucose and mannose. Analysis of the glycosyl linkage positions using gas chromatography-mass spectrometry revealed a main chain composed of galactose units [/ 3)-Gal-(1 /] branched mainly with arabinose residues [Ara-(1 /]. High antioxidant and antimicrobial capacities of oligosaccharides resulted in significant inhibitions (p < 0.05) of lipid oxidation and microbial growth in ground beef meat incorporated with oligosaccharides (Bouaziz et al., 2015a). In another research project, Bouaziz et al. (2015b) demonstrated the bioactivity of almond (P. amygdalus) gum polysaccharides. Characterization of polysaccharides using highperformance liquid chromatography revealed an average molecular weight of 99.3 kDa. The monosaccharide composition was analyzed using gas chromatography-mass spectrometry. Complex hetero-polysaccharides were found to be mainly composed of galactose, arabinose, xylose, mannose, rhamnose and glucuronic acid in mass ratio of 45:26:7:10:1:11, respectively. The acidic nature of the polysaccharide was attributed to the presence of glucuronic acid (Bouaziz et al., 2015b). In another research, Mahfoudhi, Chouaibi, Donsì, Ferrari, and Hamdi (2012) investigated chemical composition and functional properties of gum exudates from the trunk of the almond tree (Prunus dulcis). Arabinose, xylitol, galactose and uronic acid (46.8:10.9:35.5:6.0 mass ratio, respectively) with traces of rhamnose, mannose and glucose were reported as the monosaccharides present in the polysaccharide chain. The most stable and homogeneous olive oil in water emulsion was prepared with an 8% w/w aqueous almond gum solution at a pH between 5.0 and 8.0 (Mahfoudhi et al., 2012). It seems that the molecular weights of polysaccharides present in Persian gum (extracted from Amygdalus scoparia Spach) is extremely larger than those of present in another species of almond tree; since the maximum allowed concentration to obtain a homogenous dispersion was around 2.5% w/ w. Fadavi et al. (2014) reported larger molecular weights ranging from 2600 to 4700 kDa, depending on the gum type classified as white, yellow and red. To our knowledge, the complex coacervation between b-lactoglobulin (b-lg) and water-soluble fraction of Persian gum (WPG) has not yet been studied. Therefore, the main objective of the current work is characterizing the interaction between b-lg and WPG in aqueous solution under the effects of extrinsic parameters including pH, protein to polysaccharide mixing ratio (MR), total biopolymer concentration (TC), ion type, ionic strength and temperature. 2. Materials and methods 2.1. Materials

b-lg-rich whey protein isolate (Bipro) was obtained from

Davisco Foods International Inc. (Eden Prairie, MN, USA). The powder composition (% w/w) was 93.3% protein (N  6.38), 4.9% moisture, and 1.8% ash. The mineral composition of b-lg (% w/w) was 0.004% Mg2þ, 0.034% Ca2þ, 0.918% Naþ and 0.013% Kþ. Powdered white Persian gum (PG, average molecular weight of 4.7  103 kDa) was a gift from Dena Emulsion Company (Shiraz, Iran). The powder composition (% w/w) of un-fractioned PG was 0.5% protein (N 6.38), 6.70% moisture, and 3.63% ash. After fractionation, the mineral composition (% w/w) of the water soluble fraction was 0.11% Mg2þ, 1.6% Ca2þ, 0.01% Naþ and 0.09% Kþ. Sodium azide (as a preservative, minimum purity 99.5%), citric acid, glucono-delta-lactone (GDL) and sodium hydroxide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Analytical grade hydrochloric acid, sodium chloride and calcium chloride were obtained from Merck Co. (Darmstadt, Germany). Ethanol (96% (v/v)) used for PG fractionation was obtained from Zakaria Jahrom Co. (Jahrom, Iran). Double-distilled water (DDW) was used to prepare all solutions. In this study, all materials were used directly from the sample containers without additional purification taking into account their purity. 2.2. PG fractionation PG dispersion (2.5% (w/w)) was prepared by dispersing known amounts of PG in DDW under magnetic stirring at 250 rpm at room temperature for 6 h. The dispersion was then left for 12 h at 4  C to ensure complete hydration process and then centrifuged at 11,000 g for 1 h at 25  C followed by separation of supernatant and the pellet. To precipitate the WPG present in the supernatant, ethanol was added until its concentration reached to 70% (v/v). The precipitates were then freeze-dried and milled to obtain a fine powder. 2.3. Preparation of stock solutions WPG (0.5% (w/w)) and b-lg (1 and 3% (w/w)) stock solutions were prepared by dispersing into DDW including 0.03% (w/w) sodium azide. The dispersions were then stirred at 250 rpm at room temperature for 18 h to ensure complete hydration process. 2.4. Turbidimetric analysis WPG-b-lg aqueous mixtures were prepared by mixing the appropriate amounts of WPG and b-lg stock solutions and DDW (if necessary). Turbidimetric analysis as a function of pH was performed to study the influence of protein to polysaccharide mixing ratio (MR, 8:1e1:4), total biopolymer concentration (TC, 0.1e0.6% (w/w)), ion type (Naþ and Ca2þ) and ionic strength (0e100 mM). The optical density of WPG- b-lg mixtures was measured using a UV/visible spectrophotometer (Rayleigh UV-9200, Beijing Rayleigh Analytical Instrument Corporation, Beijing, China) at 600 nm in glass cells (1 cm optical path length) during either a step-wise acid titration using HCl (of 0.1, 0.4 and 2 M concentrations depending on the required pH level) or in situ acidification using GDL (0.15% (w/ w)). During acidification by HCl, a 3 min magnetic stirring for each pH level was applied to obtain uniform pH during mixing process. In situ acidification using GDL was only performed to determine the effect of ion type and ionic strength. In this experiment DDW was used as blank. Control samples containing either WPG or b-lg at their corresponding concentrations were also prepared and treated in the same manner. Critical pH values (indicating important events during acidification) including pHc (formation of soluble complexes), pH41 (formation of insoluble complexes) and pHopt (maximum optical density) were obtained graphically as the intersection point of two curve tangents according to the method

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described by Weinbreck, de Vries, Schrooyen, and de Kruif (2003). 2.5. Isothermal titration calorimetry (ITC) In order to study the thermodynamic parameters resulting from WPG-b-lg interactions under the effect of different temperatures (25, 40 and 55  C), ITC measurements were performed using a VPITC calorimeter (Microcal Inc., Northampton, MA, USA). Sodium citrate buffer solution (5 mM, pH 4.3) was used for preparing WPG and b-lg dispersions. The buffer was used to eliminate the experimental errors arising from pH mismatch. The b-lg dispersion (2 mg/ ml) was filtered through a 0.22-mm low protein binding polyether sulphone (PES) syringe filter (MS®, TX, USA) to obtain aggregate free b-lg dispersion. The concentration of b-lg dispersion (monomeric equivalent) was then measured by UV/visible light spectroscopy using specific extinction coefficient of 17,600 M1 cm1 at 278 nm and amounted to 103 mM. The sodium citrate buffer solution was used as blank. The dispersions were degassed under vacuum for 3 min. Portions of 10 ml (except for the first injection which was 5 ml) of WPG dispersion (1.33 mM) was injected sequentially into the titration cell (V ¼ 1.408 ml) initially containing either aggregate free b-lg dispersion or buffer solution. Each injection lasted 20 s with an interval of 180 s between consecutive injections. The stirring speed was set at 310 rpm. The heat of dilution from the blank titration of WPG dispersion into sodium citrate buffer was measured and subtracted from raw data to determine corrected enthalpy changes. The low concentrations of the biopolymer solutions supplied a low viscosity at any point of titration, which did not affect the mechanical stirring of the microcalorimeter. Data analyses were performed using Microcal Origin software (v.7.0). The “One Sets of Sites” binding model (assuming the existence of one independent binding site for each protein molecule) was used to fit binding isotherms. Thermodynamic parameters including binding stoichiometry (N), affinity constant (K), enthalpy (DH) and entropy (DS) changes were calculated by iterative curve fitting of the binding isotherms. The Gibbs free energy change (DG) was calculated from the equation (DG ¼ DH  TDS). 2.6. Statistical analysis Measurements were performed at least two or three times using freshly prepared samples and analyzed by ANOVA using SPSS software (version 20). Results were reported as means and standard deviations. Comparison of means was carried out using Duncan's multiple range tests at a confidence level of 0.05. 3. Results and discussion 3.1. Turbidimetric analysis 3.1.1. Effect of pH It has been proved that pH could play a key role in electrostatic complexation through affecting the ionization degree of the functional groups of proteins and polysaccharides (Weinbreck, Nieuwenhuijse, Robijn, & de Kruif, 2003). The variation in optical density (OD) at different pH values for b-lg/WPG mixture as compared to individual b-lg and WPG dispersions is shown in Fig. 1. In the absence of protein, the WPG dispersion remained transparent during pH reduction in the studied range; indicating that the polysaccharide molecules were not able to form particles large enough to scatter light strongly which is due to the electrostatic repulsion between similarly charged polysaccharide chains. The blg dispersion revealed a different pattern as a broad peak within pH range of 4e5 with a maximum value around protein's pI; which is

Fig. 1. Phase diagrams showing critical pH values as a function of pH for (C) WPG dispersion (0.1% (w/w)), (£) b-lg dispersion (0.2% (w/w)), and (-) WPG- b-lg aqueous mixture (TC 0.3% (w/w), MR 2). pHc: formation of soluble complexes, pH41: formation of insoluble complexes, pHopt: maximum optical density, pH42: dissolution of complexes. The insets are phase contrast optical micrographs obtained at different stages; a: co solubility, b: formation of insoluble complexes, c: higher-order aggregation and bulk phase separation.

due to the self-association of the protein molecules around its pI. The driving force for protein aggregation around the pI is probably a combination of hydrophobic attraction, van der Waals attraction and some electrostatic attraction between positive groups on one protein molecule and negative groups on another (Harnsilawat et al., 2006). OD decreased as pH shifted to higher or lower values. Same results have been shown by Mounsey, O'Kennedy, Fenelon, and Brodkorb (2008). Fig. 1 also shows the kinetic of associative phase separation within mixed b-lg/WPG systems (TC 0.3% (w/w), MR 2:1). Soluble complexes were formed at a pHc around 5.30. Weinbreck, Nieuwenhuijse, Robijn, and de Kruif (2004) and Hosseini et al. (2013b) reported pHc values of 5.5 and 5.35 for different mixtures of whey protein isolate and non-gelling carrageenan (comprised mainly l-carrageenan) and a mixture of blg-sodium alginate, respectively. According to Turgeon and Laneuville (2009), this transition occurs at the molecular level (i.e. complexation begins between a single polysaccharide chain and a defined amount of protein). Formation of soluble complexes occurred at a pHc above the pI of the b-lg (~4.7e5.2) (Santipanichwong, Suphantharika, Weiss, & McClements, 2008) which is thought to be due to the ability of the globular proteins for charge regulation around the pI resulting from their electrical capacitance properties (Dickinson, 2008) and/or due to the presence of positive patches (localized regions with higher charge density) on the surface of b-lg as a result of low ionic strength conditions which inhibit charge screening (Turgeon & Laneuville , 2009; Weinbreck, de Vries, et al., 2003). b-lg has several charged patches (basic peptides 1e14, 41e60, 76e83 and 132e148, the latter being part of the a-helix), which are sensitive to complexation with polyanions above the protein's pI (Girard, Turgeon, & Gauthier, 2003a). When the pH decreased further, the critical pH41 was reached. At this point, more and more protein molecules attached to the polysaccharide chain (due to an increase in charge density of the protein) until electroneutrality was attained yielding neutral interpolymer complexes that tend to precipitate (Turgeon & Laneuville, 2009). Diminishing the net charges on the macromolecular reactants reduces both the hydrophilicity and the solubility of the resultant complex (Tolstoguzov, 1997). This step appears as an intermediate process before the system undergoes extensive higher-order aggregation and bulk phase separation (Laneuville, Sanchez, Turgeon, Hardy, & Paquin, 2005). The highest amount of b-lg-WPG interactions (pHopt) occurred at pH 4.38 with maximum optical density of 2.174. At pH < pHopt, the turbidity of

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the b-lg-WPG complexes was reduced as a result of protonation of the acidic functional groups of WPG where another critical point in the curve could be recognized, known as the pH42. It is worth mentioning that some authors have found other critical pH values, notably for protein conformational changes following binding or for morphological changes in the coacervates droplets in the blactoglobulinegum Arabic system using a multimethodological approach (Mekhloufi, Sanchez, Renard, Guillemin, & Hardy, 2005). 3.1.2. Effect of MR MR is among the most important parameters in the interactions between the two biopolymers. Fig. 2 (a, b and c) illustrates the changes in critical pH values (pHc, pH41 and pHopt, respectively) versus MR at three different TC including 0.1, 0.3 and 0.6% (w/w). The results showed that different MRs made no significant changes in the values of pHc, demonstrating this critical pH is independent from the mixing ratio. This phenomenon is due to the transition at the molecular level. Formation of soluble complexes at pHc is a local interaction, not influenced by events elsewhere along the polymer chain or events on the other chains, and is therefore not affected by

Fig. 2. The variations in the values of critical pHs including (a) pHc, (b) pH41 and (c) pHopt as a function of MR (8:1e1:4) at three different TC including (A) 0.1, (-) 0.3 and (:) 0.6% (w/w).

chain length, macromolecular concentrations or mixing ratio but ionic strength (Antonov, Mazzawi, & Dubin, 2010). Thus, as soon as protein molecules mix with the polysaccharide ones, soluble complexes start forming, independently from the initial mixing ratio (Schmitt et al., 2009; Turgeon & Laneuville, 2009). At a constant TC, decreasing in MR shifted the other critical pHs (pH41 and pHopt) to lower values, indicating these critical pHs are strongly ratio dependent as they corresponds to full saturation of the polysaccharide chains by protein molecules (Kaibara, Okazaki, Bohidar, & Dubin, 2000; Schmitt et al., 2009; Turgeon & Laneuville, 2009). Increasing the amount of polysaccharide (or decreasing the MR) resulted in the formation of smaller complexes that remained charged over a wider pH range and required lower pH values to attain electroneutrality. Therefore, the decrease in critical pHs was more obvious in lower MRs (<2). 3.1.3. Effect of TC Generally, electrostatic complexation and coacervation can occur at a wide range of the total biopolymer concentration (TC), starting at extremely low concentrations (102 mg/ml), providing sufficiently low ionic strength conditions (<200 mM), compared to the concentration needed for a segregative phase separation (~4% (w/w) under the absence of protein aggregates) (Tolstoguzov, 1986). At very high biopolymer concentrations, when the polysaccharide or the protein is in excess in the solution and when the mixture composition reaches the concentration of the complexed or coacervated phase, an auto-suppression of the interaction occurs, due to the entropic factors favoring complex coacervation (Turgeon & Laneuville, 2009; Weinbreck, de Vries, et al., 2003). The effect of TC on critical pH values is shown in Fig. 2 (a, b and c). In this work the highest TC was 0.6% w/w. We could not prepare mixtures of higher concentrations because of the limited solubility of WPG. So, the autosuppression phenomenon was not observed during the current study. In this study, the critical pHc was independent from TC. The critical pH41 was relatively independent from TC. Nevertheless, significant differences between the values of the critical pH41 at different TCs were observed in MR below 2. Weinbreck, de Vries, et al., 2003 reported that pHc and pH41 are independent from TC below 0.5% (w/w). However, for higher TC, pH41 has been reported to shift to higher values. In this study the shift in pH41 in a ratio dependent manner was also observed. There was a similar pattern for the changes in critical pHopt. 3.1.4. Effect of ion type and ionic strength An important driving force for the complex coacervation is the electrostatic entropy gain induced by the release of the biopolymer counterions upon complex formation. Since ion type and ionic strength can affect the counterion release phenomenon, both parameters have significant effects on associative phase separation. The effects of ion type and ionic strength on the ODs of b-lg-WPG complexes (TC, 0.3% (w/w), MR, 2:1) are shown in Fig. 3(a and b). Increasing in the ionic strength decreased the OD indicating the preventive effects of the salt in complexed biopolymer systems. According to Schmitt et al. (2009), when the ionic strength of the system increases, two major energetically detrimental effects can occur: 1) the screening of the charges of the macromolecules due to small ion-pairing and 2) the overall equivalence of the counterion concentration in the bulk phase and in the neighborhood of the biopolymer chains. The former effect reduces the number of protein molecules able to interact with the polysaccharide chain, whereas the second one suppresses the energetic advantage of forming a complex as already predicted by the Veis and Aranyi model (Veis & Aranyi, 1960). Moreover, salt addition to the solvent modifies its dielectric constant which is detrimental as far as

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Fig. 3. Effects of salt type (a: NaCl and b: CaCl2) and ionic strength on the optical density of b-lg-WPG aqueous mixture (TC: 0.3% (w/w), MR: 2) as a function of pH.

electrostatic interactions are concerned. The preventive effects of divalent ions (Ca2þ) were more than those of monovalent ones (Naþ), hence the suppression of the complex coacervation occurred at lower ionic strength values (Fig. 3b), which is due to double electrostatic entropy gain upon releasing two monovalent ions compared to a single divalent ion (Schmitt et al., 2009). Fig. 4(a and b) shows the effects of ion type and ionic strength on the values of the different critical pHs (pHc, pH41 and pHopt). The addition of mono- and divalent ions shifted critical pH values towards more acidic values in order to compensate the partial screening of the charges induced by the added microions (i.e. the charge density of proteins needs to be increased so as to reach the same level of soluble complex formation or charge neutralization between proteins and polysaccharides) (Schmitt et al., 2009). In order to confirm the effect of overall equivalence of the counterion concentration in the bulk phase and in the neighborhood of the biopolymer chains on preventing the complex coacervation, a b-lg-WPG mixture containing a defined amount of NaCl (100 mM) at a given pH (4.4) was diluted with the solvent of similar pH. The results are shown in Fig. 5. An increase in OD was observed during dilution, which can be due to the decrease in the concentration of the bulk ions, favoring again counterion release and hence complex formation.

Fig. 4. Changes in the values of critical pHs ((A) pHc, (-) pH41 and (:) pHopt) under the effect of salt type (a: NaCl and b: CaCl2) as a function of ionic strength.

Fig. 5. The increase in optical density during diluting a b-lg-WPG mixture (pH 4.4) containing a defined amount of NaCl (100 mM) demonstrates the requirement of counterion concentration gradient between the bulk phase and the neighborhood of the biopolymer chains on the complex coacervation. Dilution was performed with the solvent of similar pH.

3.2. ITC results As a calorimetry technique, ITC is very useful to determine energetic and binding parameters in the complexation process of biopolymers by titrating one binding partner with another while

measuring the heat released or absorbed in a reaction chamber. During last decade, ITC has been successfully applied in a growing number of studies concerning biopolymer mixtures (Girard, Turgeon, & Gauthier, 2003b; Schmitt et al., 2005; Harnsilawat

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et al., 2006; Guzey & Mcclements, 2006; de Souza, Bai, do Pilar Gonçalves, & Bastos, 2009; Aberkane, Jasniewski, Gaiani, Scher, & Sanchez, 2010; Aberkane et al., 2012; Hosseini et al., 2013a,b). Thermograms (heat flow vs. time profiles) arising from the titration of b-lg with WPG at 25, 40 and 55  C and pH 4.3 are shown at the top of Fig. 6 (a, b and c, respectively). The area under each peak corresponds to the heat exchange within the calorimeter cell containing b-lg after each WPG injection. Generally, the injection profiles were exothermic and decreased regularly to a state of thermodynamic stability after 19th, 20th and 25th injections of

WPG at 25, 40 and 55  C, respectively. After attainment the electroneutrality of the soluble complexes, the endothermic peaks could be observed because of the dilution heat of the citrate buffer. The relatively disordered pattern (fluctuations) at 55  C may be due to the effects of high temperature on partial aggregation of protein molecules. Exothermicity is mainly related to the nonspecific electrostatic neutralization of the opposite charges carried by the two biopolymers demonstrating the enthalpic contribution of complex coacervation (Girard et al., 2003b; Schmitt et al., 2005), while its regular decrease is attributed to a reduction in free protein

Fig. 6. Thermograms (top panels) and binding isotherms (bottom panels) corresponding to the titration of the b-lg dispersion (103 mM) with WPG dispersion (1.33 mM), separately dispersed in sodium citrate buffer solution (5 mM, pH 4.3), obtained at three different temperatures including (a) 25  C, (b) 40  C and (c) 55  C.

M. Hadian et al. / Food Hydrocolloids 55 (2016) 108e118

molecules remaining in the reaction chamber after successive injections resulted in a reduction in the released energy. Girard et al. (2003b) and Hosseini et al. (2013a,b) reported exothermic sequence for b-lg interaction with pectin, sodium alginate and k-carrageenan, respectively; while Aberkane et al. (2010, 2012) and Nigen, Croguennec, Renard, and Bouhallab (2007) reported an exothermic-endothermic sequence as indicative for the other energetic contributions such as the liberation of water molecules and ions, conformational changes of biopolymers, the aggregation of protein-polysaccharide complexes as well as two different structuration steps involved in the complexation. To describe thermodynamic parameters, the binding isotherms (obtained by integrating the isotherm peaks and subtraction of the heats of dilution of polysaccharides into buffer solution) were fitted using the one site binding model and plotted against WPG:b-lg molar ratio (Fig. 6 a, b and c, bottom panel). The first injection was not considered for analysis. The calculation gives a relatively typical sigmoidal saturation curve, which can be taken into account as a progressive binding of the protein molecules present in the reaction cell to the binding sites along the WPG backbone. Thermodynamic parameters including binding stoichiometry (N), affinity constant (Ka), enthalpy (DH) and entropy (TDS) contributions and Gibbs free energy change (DG) calculated for the interaction between WPG and b-lg at three different temperatures are shown in Table 1. At different studied temperatures, the binding enthalpy was negative and favorable, whereas the binding entropy was negative and unfavorable. The enthalpic contribution is regulated by the mixing ratio and the nature and density of charges carried by biopolymers. Based on the Langevin dynamics simulation, Ou and Muthukumar (2006) reported that the complexation process between weakly charged polyelectrolytes is driven by a negative enthalpy due to the coulombic interaction between two oppositely charged components, while counterion release entropy plays only a minor role. The unfavorable entropic effects originate mainly from the loss in biopolymer conformational freedom after complexation (Dickinson, 2008). At 25  C, b-lg and WPG interacted with an extremely high affinity constant and a strong enthalpy change. Using ITC, we have calculated for the first time that about 862 protein molecules were involved in the interaction process with WPG by assuming a molecular weight of 4700 kDa as reported by Fadavi et al. (2014). Schmitt et al. (2005) and Aberkane et al. (2010) reported the values of 86 and 90 for binding stoichiometry upon complexation of b-lg with Acacia gum (MW ~ 540 kDa) at pH 4.2, respectively. The variations can be attributed to the differences in charge density, biopolymer's structure and molecular weight. The interaction between b-lg and WPG at 40  C occurred with higher enthalpy change, lower affinity constant and lower binding stoichiometry (i.e. the increased number of protein molecules bound per polysaccharide chain). As can be seen in Fig. 6, exothermicity decreased by increasing the temperature from 25 to 40  C; however the software provided significantly higher enthalpy change value at 40  C. It seems that during increasing the temperature from 25 to 40  C, the role of hydrophobic interactions became more significant in the complexation process and these observations may partly be related to their effects. The hydrophobic interaction is comprised of

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an enthalpy component due to the change in the overall strength of the forces (e.g., hydrogen bonding and Van der Waals forces) when two or more non-polar groups associate, and an entropy component due to the change in the structural organization of the water molecules surrounding the non-polar groups (McClements, Decker, Park, & Weiss, 2009). Hydrophobic bonding is promoted by conformational and structural modifications of biopolymers, mostly by unfolding of polymeric chains favoring exposure of more additional hydrophobic regions (Goh et al., 2009). Hydrophobic interactions tend to increase in strength when the temperature is increased, and decrease in strength when the dielectric constant of the aqueous phase is decreased (e.g., by adding alcohol) (Dickinson, 1998; McClements et al., 2009). Therefore, the temperature increase may have significant effects on protein and polysaccharide conformational changes, resulting uncovering more binding sites and thus stoichiometry binding would increase for this reason. Another explanation is that the increase in temperature may increase the flexibility of biopolymers. Flexible molecules are able to form more contacts (junction zones) with the other oppositely charged molecules by adopting configurational adjustments which maximized interactions. The interaction between b-lg and WPG at 55  C occurred with the lowest enthalpy change, a middle affinity constant and the highest binding stoichiometry (i.e. the lowest amount of protein molecules bound per polysaccharide chain). These observations could be related to the effects of high temperature on hydrogen bond breakage as well as protein aggregation and/or denaturation. Hydrogen bonds tend to decrease in strength as the temperature is increased. Formation of the protein aggregates at high temperature could reduce the binding stoichiometry since protein molecules could bind in more condensed forms. It is very difficult to assign a precise molecular change in the system to the observed enthalpy change because the overall measured signal is a consequence of enthalpy changes associated with different molecular phenomena such as conformational changes, possible aggregation of molecules, electrostatic interactions, various kinds of associationedissociation processes and counter ion binding-dissociation (Guzey & Mcclements, 2006; Harnsilawat et al., 2006). Hence, the enthalpy value measured could have been either overestimated or underestimated. The unfavorable entropic contribution (TDS) was relatively in the same range as the favorable enthalpic contribution (DH) (Fig. 7), indicating that any change in enthalpy is accompanied by a similar change in entropy, that is, entropy-enthalpy compensation occurred (Aberkane et al., 2012; Hosseini et al., 2013a,b). The changes in Gibbs free energy were negative for all polysaccharides indicating the spontaneous nature of the interactions. The significant difference in Gibbs free energy change at 25  C can be attributed to the fact that the loss in polysaccharide conformational freedom after association (i.e. unfavorable entropic contribution or TDS) is more prominent for flexible molecules than for inflexible ones. As mentioned previously, polysaccharide chains have more flexibility at higher temperatures. In order to provide a deeper insight into the effects of temperature on complex coacervation, molar heat capacity change (DCp) was calculated from the slope of the binding enthalpy (DH) vs.

Table 1 Thermodynamic parameters of binding between b-lg and WPG at different temperatures in 5 mM sodium citrate buffer (pH 4.3). Temperature (K)

N  105 (mol Ps/mol Pr)

Ka (M1  107)

DH (kcal mol1)

TDS (kcal mol1)

DG (kcal mol1)

298.15 313.15 328.15

116b 83c 188a

9.65a 1.79c 2.92b

3932b 5053a 2269c

3906b 5042a 2258c

26a 11b 11b

Different letters in each column indicate significant differences (p < 0.05).

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during the initial biopolymer complex formation but large-scale aggregation or coacervation would be mainly driven by hydrogen bonding or hydrophobic interactions, depending on the temperature. 4. Conclusion

Fig. 7. Entropic (TDS) contribution versus enthalpic (DH) contribution illustrating the occurrence of entropy-enthalpy compensation.

temperature relationship (Fig. 8). This indicator is highly sensitive to the interactions between macromolecule residues and solvent molecules (Aberkane et al., 2010; Schmitt et al., 2009). DCp originates from changes in the degree of surface hydration in the free and complexed molecules, and to a lesser extent from changes in molecular vibrations (Jelesarov & Bosshard, 1999). The heat capacity of binding is useful in predicting the surface-exposed polar and nonpolar surface area change upon ligand binding onto macromolecules (Baker & Murphy, 1997). The curve fitting was obviously not so good for the temperature range studied indicating that DCp was not temperature dependent; however, it was generally large and positive as a typical signature of charge neutralization reactions. This mainly arises because less charged groups are in contact with the solvent after electrostatic complex formation. In addition, a positive DCp with a favorable DH parameter (i.e. DH<0) at all studied temperatures, would be indicative of a significant contribution of hydrogen bonding (Gonçalves, Kitas, & Seelig, 2005; Turgeon et al., 2007). Aberkane et al. (2010) reported the DCp values of 28.5 and 8.3 kcal mol1 K1 for the first and the second structuration stages during b-lg-gum Arabic interaction. Increasing the temperature from 25 to 40  C led to a negative DCp indicating the role of hydrophobic interactions. Therefore, electrostatic interactions, hydrogen bonding and hydrophobic interactions play important roles in b-lg-WPG interactions. It has been suggested that hydrogen bonding would be involved, particularly at pHs above the proteins' pI (Girard, Turgeon, & Gauthier, 2002) and is only favored when the charge densities are low. According to Nigen et al. (2007) electrostatic interactions could be mostly important

Fig. 8. Binding enthalpy (DH) as a function of temperature (K) showing the temperature independency at the studied range; the slope is molar heat capacity change (DCp).

Complexes and coacervates resulting from proteins and polysaccharides associative interactions are promising tools to make tailor-made food microstructures. The present work showed that extrinsic parameters including pH, protein to polysaccharide mixing ratio, total biopolymer concentration, ion type, ionic strength and temperature can affect the interaction between blactoglobulin and water-soluble fraction of Persian gum. The results confirmed that the soluble fraction has an anionic character which can be used to interact with the proteins below their pI. This interaction may have potential applications in the formation of delivery systems, texturizing, interfacial stabilization and development of novel gels especially in foods of acidic pH. Since the polysaccharide used in the current work was of high molecular weight, exploring protein interaction with a mixture of polysaccharides of different molecular weights and vice versa would be interesting. Moreover, physical and chemical modifications of protein as well as hydrolysis prior to complexation are the other important areas of research. Acknowledgment The authors are thankful to Shiraz University for financial support (Grant number 93GCU1M194065). References Abbasi, S., & Mohammadi, S. (2013). Stabilization of milkeorange juice mixture using persian gum: efficiency and mechanism. Food Bioscience, 2, 53e60. Aberkane, L., Jasniewski, J., Gaiani, C., Hussain, R., Scher, J., & Sanchez, C. (2012). Structuration mechanism of b-lactoglobulin e acacia gum assemblies in presence of quercetin. Food Hydrocolloids, 29, 9e20. Aberkane, L., Jasniewski, J., Gaiani, C., Scher, J., & Sanchez, C. (2010). Thermodynamic characterization of acacia gum-b-lactoglobulin complex coacervation. Langmuir, 26, 12523e12533. Ahmadi, N., Nasirpour, A., Sheikhzeinodin, M., & Keramat, J. (2015). Microencapsulation of ubiquinone using complex coacervation for functional yoghurt. Food Science and Biotechnology, 24, 895e904. Antonov, M., Mazzawi, M., & Dubin, P. L. (2010). Entering and exiting the proteinpolyelectrolyte coacervate phase via nonmonotonic salt dependence of critical conditions. Biomacromolecules, 11, 51e59. Baker, B., & Murphy, K. (1997). Dissecting the energetics of a proteineprotein interaction: the binding of ovomucoid third domain to elastase. Journal of Molecular Biology, 268, 557e569. Benichou, A., Aserin, A., & Garti, N. (2002). Protein-polysaccharide interactions for stabilization of food emulsions. Journal of Dispersion Science and Technology, 23, 93e123. van den Berg, L., van Vliet, T., van der Linden, E., van Boekel, M. A. J. S., & van de Velde, F. (2007). Breakdown properties and sensory perception of whey proteins/polysaccharide mixed gels as a function of microstructure. Food Hydrocolloids, 21, 961e976. Bouaziz, F., Helbert, C. B., Romdhane, M. B., Koubaa, M., Bhiri, F., Kallel, F., et al. (2015a). Structural data and biological properties of almond gum oligosaccharide: application to beef meat preservation. International Journal of Biological Macromolecules, 72, 472e479. Bouaziz, F., Koubaa, M., Helbert, C. B., Kallel, F., Driss, D., Kacem, I., et al. (2015b). Purification, structural data and biological properties of polysaccharide from Prunus amygdalus gum. International Journal of Food Science and Technology, 50, 578e584. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J.-L., & Agnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 436, 359e378. Cooper, C. L., Dubin, P. L., Kayitmazer, A. B., & Turksen, S. (2005). Polyelectrolyteeprotein complexes. Current Opinion in Colloid & Interface Science, 10, 52e78. Corredig, M., Sharafbafi, N., & Kristo, E. (2011). Polysaccharideeprotein interactions in dairy matrices, control and design of structures. Food Hydrocolloids, 25, 1833e1841.

M. Hadian et al. / Food Hydrocolloids 55 (2016) 108e118 Dickinson, E. (1998). Stability and rheological implications of electrostatic milk proteinepolysaccharide interactions. Trends in Food Science & Technology, 9, 347e354. Dickinson, E. (2008). Interfacial structure and stability of food emulsions as affected by proteinepolysaccharide interactions. Soft Matter, 4, 932e942. Dickinson, E. (2009). Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids, 23, 1473e1482. Doublier, J. L., Garnier, C., Renard, D., & Sanchez, C. (2000). Proteinepolysaccharide interactions. Current Opinion in Colloid & Interface Science, 5, 202e214. 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, 10, 1074e1080. Fox, P. F. (2009). Milk: an overview. In A. Thompson, M. Boland, & H. Singh (Eds.), Milk proteins: From expression to food (pp. 1e54). New York: Elsevier. Girard, M., Turgeon, S. L., & Gauthier, S. F. (2002). Interbiopolymer complexing between b-lactoglobulin and low- and high-methylated pectin measured by potentiometric titration and ultracentrifugation. Food Hydrocolloids, 16, 585e591. Girard, M., Turgeon, S. L., & Gauthier, S. F. (2003a). Quantification of the interactions between b-lactoglobulin and pectin through capillary electrophoresis analysis. Journal of Agricultural and Food Chemistry, 51, 6043e6049. Girard, M., Turgeon, S. L., & Gauthier, S. F. (2003b). Thermodynamic parameters of blactoglobulin-pectin complexes assessed by isothermal titration calorimetry. Journal of Agricultural and Food Chemistry, 51, 4450e4455. Goh, K. K. T., Sarkar, A., & Singh, H. (2009). Milk proteinepolysaccharide interactions. In A. Thompson, M. Boland, & H. Singh (Eds.), Milk proteins: From expression to food (pp. 347e376). New York: Elsevier. Golkar, A., Nasirpour, A., & Keramat, J. (2015). b-Lactoglobulin-Angum gum (amygdalus scoparia spach) complexes: preparation and emulsion stabilization. Journal of Dispersion Science and Technology, 36, 685e694. Golkar, A., Nasirpour, A., Keramat, J., & Desobry, S. (2015). Emulsifying properties of angum gum (Amygdalus scoparia Spach) conjugated to beta-lactoglobulin through Maillard-type reaction. International Journal of Food Properties, 18, 2042e2055. Gonçalves, E., Kitas, E., & Seelig, J. (2005). Binding of oligoarginin to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide. Biochemistry, 44, 2692e2702. Gottschalk, M., Nilsson, H., Roos, H., & Halle, B. (2003). Protein self-association in solution: the bovine b-lactoglobulin dimer and octamer. Protein Science, 12, 2404e2411. Grinberg, V. Y., & Tolstoguzov, V. (1997). Thermodynamic incompatibility of proteins and polysaccharides in solutions. Food Hydrocolloids, 11, 145e158. Guzey, D., & Mcclements, D. J. (2006). Characterization of b-lactoglobulin-chitosan interactions in aqueous solutions: a calorimetry, light scattering, electrophoretic mobility and solubility study. Food Hydrocolloids, 20, 124e131. Harnsilawat, T., Pongsawatmanit, R., & McClements, D. J. (2006). Characterization of b-lactoglobulinesodium alginate interactions in aqueous solutions: a calorimetry, light scattering, electrophoretic mobility and solubility study. Food Hydrocolloids, 20, 577e585. Hosseini, S. M. H., Emam-Djomeh, Z., Razavi, S. H., Moosavi-Movahedi, A. A., Saboury, A. A., Atri, M. S., et al. (2013b). b-Lactoglobulin-sodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound. Food Hydrocolloids, 32, 235e244. Hosseini, S. M. H., Emam-Djomeh, Z., Razavi, S. H., Moosavi-Movahedi, A. A., Saboury, A. A., Mohammadifar, M. A., et al. (2013a). Complex coacervation of blactoglobulin e k-Carrageenan aqueous mixtures as affected by polysaccharide sonication. Food Chemistry, 141, 215e222. Hosseini, S. M. H., Emam-Djomeh, Z., Sabatino, P., & Van der Meeren, P. (2015). Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds. Food Hydrocolloids, 50, 16e26. 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, 1e8. Jafari, S. M., Beheshti, P., & Assadpour, E. (2013). Emulsification properties of a novel hydrocolloid (Angum gum) for d-limonene droplets compared with Arabic gum. International Journal of Biological Macromolecules, 61, 182e188. Jelesarov, I., & Bosshard, H. R. (1999). Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. Journal of Molecular Recognition, 12, 3e18. Jun-xia, X., Hai-yan, Y., & Jian, Y. (2011). Microencapsulation of sweet orange oil by complex coacervation with soybean protein isolate/gum Arabic. Food Chemistry, 125, 1267e1272. Kaibara, K., Okazaki, T., Bohidar, H. B., & Dubin, P. L. (2000). pH-Induced coacervation in complexes of bovine serum albumin and cationic polyelectrolytes. Biomacromolecules, 1, 100e107. Kizilay, E., Kayitmazer, A. B., & Dubin, P. L. (2011). Complexation and coacervation of polyelectrolytes with oppositely charged colloids. Advances in Colloid and Interface Science, 167, 24e37. de Kruif, C. G., & Tuinier, R. (2001). Polysaccharide protein interactions. Food Hydrocolloids, 15, 555e563. de Kruif, C. G., Weinbreck, F., & de Vries, R. (2004). Complex coacervation of proteins and anionic polysaccharides. Current Opinion in Colloid & Interface Science, 9, 340e349. Laneuville, S. I., Paquin, P., & Turgeon, S. L. (2005). Formula optimization of a low-fat food system containing whey protein isolate-xanthan gum complexes as fat

117

replacer. Journal of Food Science, 70, S513eS519. Laneuville, S. I., Sanchez, C., Turgeon, S. L., Hardy, J., & Paquin, P. (2005). Small-angle static light-scattering study of associative phase separation kinetics of blactoglobulin-xanthan gum mixtures under shear. In E. Dickinson (Ed.), Food colloids: Interaction, microstructure and processing (pp. 443e465). Cambridge: The Royal Society of Chemistry. Lapitsky, Y. (2014). Ionically crosslinked polyelectrolyte nanocarriers: recent advances and open problems. Current Opinion in Colloid & Interface Science, 19, 122e130. Le, X. T., & Turgeon, S. L. (2015). Textural and waterbinding behaviors of b-lactoglobulin-xanthan gum electrostatic hydrogels in relation to their microstructure. Food Hydrocolloids, 49, 216e223. _  ski, D. (2014). The effect of hydrocolLiszka-Skoczylas, M., Ptaszek, A., & Zmudzi n loids on producing stable foams based on the whey protein concentrate (WPC). Journal of Food Engineering, 129, 1e11. Livney, Y. D. (2010). Milk proteins as vehicles for bioactives. Current Opinion in Colloid & Interface Science, 15, 73e83. Mahfoudhi, N., Chouaibi, M., Donsì, F., Ferrari, G., & Hamdi, S. (2012). Chemical composition and functional properties of gum exudates from the trunk of the almond tree (Prunus dulcis). Food Science and Technology International, 18, 241e250. McClements, D. J. (2006). Non-covalent interactions between proteins and polysaccharides. Biotechnology Advances, 24, 621e625. McClements, D. J., Decker, E. A., Park, Y., & Weiss, J. (2009). Structural design principles for delivery of bioactive components in nutraceuticals and functional foods. Critical Reviews in Food Science and Nutrition, 49, 577e606. Mekhloufi, G., Sanchez, C., Renard, D., Guillemin, S., & Hardy, J. (2005). pH-induced structural transitions during complexation and coacervation of b-lactoglobulin and acacia gum. Langmuir, 21, 386e394. Mirmajidi Hashtjin, A., & Abbasi, S. (2015). Nano-emulsification of orange peel essential oil using sonication and native gums. Food Hydrocolloids, 44, 40e48. Mounsey, J. S., O'Kennedy, B. T., Fenelon, M. A., & Brodkorb, A. (2008). The effect of heating on b-lactoglobulin-chitosan mixtures as influenced by pH and ionic strength. Food Hydrocolloids, 22, 65e73. Nigen, M., Croguennec, T., Renard, D., & Bouhallab, S. (2007). Temperature affects the supramolecular structures resulting from a-lactalbumin-lysozyme interaction. Biochemistry, 46, 1248e1255. Ould Eleya, M. M., & Turgeon, S. L. (2000). The effects of pH on the rheology of blactoglobulin/k-carrageenan mixed gels. Food Hydrocolloids, 14, 245e251. Ou, Z., & Muthukumar, M. (2006). Entropy and enthalpy of polyelectrolyte complexation: Langevin dynamics simulations. Journal of Chemical Physics, 124, 154902e154911. Picard, J., Giraudier, S., & Larreta-Garde, V. (2009). Controlled remodeling of a protein-polysaccharide mixed gel: examples of gelatin-hyaluronic acid mixtures. Soft Matter, 5, 4198e4205. Rodríguez Patino, J. M., & Pilosof, A. M. R. (2011). Proteinepolysaccharide interactions at fluid interfaces. Food Hydrocolloids, 25, 1925e1937. Ron, N., Zimet, P., Bargarum, J., & Livney, Y. D. (2010). Beta-lactoglobulin-polysaccharide complexes as nanovehicles for hydrophobic nutraceuticals in nonfat foods and clear beverages. International Dairy Journal, 20, 686e693. Sakurai, K., & Goto, Y. (2002). Manipulating monomeredimer equilibrium of bovine b-lactoglobulin by amino acid substitution. Journal of Biological Chemistry, 277, 25735e25740. Santipanichwong, R., Suphantharika, M., Weiss, J., & McClements, D. J. (2008). Coreshell biopolymer nanoparticles produced by electrostatic deposition of beet pectin onto heat-denatured b-lactoglobulin aggregates. Journal of Food Science, 73, N23eN30. Schmitt, C., Aberkane, L., & Sanchez, C. (2009). Protein-polysaccharide complexes and coacervates. In G. O. Phillips, & P. A. Williams (Eds.), Handbook of hydrocolloids (2nd ed., pp. 420e476). Florida: CRC Press. Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure and technofunctional properties of protein-polysaccharide complexes: a review. Critical Reviews in Food Science and Nutrition, 38, 689e753. Schmitt, C., da Silva, T. P., Bovay, C., Rami-Shojaei, S., Frossard, P., Kolodziejczyk, E., et al. (2005). Effect of time on the interfacial and foaming properties of blactoglobulin/acacia gum electrostatic complexes and coacervates at pH 4.2. Langmuir, 21, 7786e7795. Schmitt, C., & Turgeon, S. L. (2011). Protein/polysaccharide complexes and coacervates in food systems. Advances in Colloid and Interface Science, 167, 63e70. daSilva, F. L. B., Lund, M., Jonsson, B., & Akesson, T. (2006). On the complexation of proteins and polyelectrolytes. Journal of Physical Chemistry B, 110, 4459e4464. van der Sman, R. G. M. (2012). Soft matter approaches to food structuring. Advances in Colloid and Interface Science, 176e177, 18e30. de Souza, H. K. S., Bai, G., do Pilar Gonçalves, M., & Bastos, M. (2009). Whey protein isolateechitosan interactions: a calorimetric and spectroscopy study. Thermochimica Acta, 495, 108e114. Tolstoguzov, V. B. (1986). Functional properties of protein-polysaccharide mixtures. In J. R. Mitchell, & D. A. Ledward (Eds.), Functional properties of food macromolecules (pp. 385e415). London: Elsevier Applied Science. Tolstoguzov, V. B. (1997). Proteinepolysaccharide interactions. In S. Damodaran, & A. Paraf (Eds.), Food proteins and their applications (pp. 171e198). New York: Marcel Dekker. Tolstoguzov, V. B. (2003). Some thermodynamic considerations in food formulation. Food Hydrocolloids, 17, 1e23. Turgeon, S. L., Beaulieu, M., Schmitt, C., & Sanchez, C. (2003).

118

M. Hadian et al. / Food Hydrocolloids 55 (2016) 108e118

Proteinepolysaccharide interactions: phase-ordering kinetics, thermodynamic and structural aspects. Current Opinion in Colloid & Interface Science, 8, 401e414. Turgeon, S. L., & Laneuville, S. I. (2009). Protein þ polysaccharide coacervates and complexes: from scientific background to their application as functional ingredients in food products. In S. Kasapis, I. T. Norton, & J. B. Ubbink (Eds.), Modern biopolymer science (pp. 327e363). New York: Elsevier. Turgeon, S. L., Schmitt, C., & Sanchez, C. (2007). Proteinepolysaccharide complexes and coacervates. Current Opinion in Colloid & Interface Science, 12, 166e178. Veis, A., & Aranyi, C. (1960). Phase separation in polyelectrolyte systems. I. Complex coacervates of gelatin. Journal of Physical Chemistry, 64, 1203e1210. de Vries, R., & Cohen Stuart, M. A. (2006). Theory and simulations of macroion complexation. Current Opinion in Colloid & Interface Science, 11, 295e301. Weinbreck, F., de Vries, R., Schrooyen, P., & de Kruif, C. G. (2003). Complex

coacervation of whey proteins and gum Arabic. Biomacromolecules, 4, 293e303. Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & de Kruif, C. G. (2003). Complex formation of whey proteins: exocellular polysaccharide EPS B40. Langmuir, 19, 9404e9410. Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & de Kruif, C. G. (2004). Complexation of whey proteins with carrageenan. Journal of Agricultural and Food Chemistry, 52, 3550e3555. Yang, X., Gao, N., Hu, L., Li, J., & Sun, Y. (2015). Development and evaluation of novel microcapsules containing poppy-seed oil using complex coacervation. Journal of Food Engineering, 161, 87e93. Ye, A. (2008). Complexation between milk proteins and polysaccharides via electrostatic interaction: principles and applications e a review. International Journal of Food Science and Technology, 43, 406e415.