Stabilization of Water-in-Water Emulsions by Polysaccharide-Coated

Article pubs.acs.org/Langmuir

Stabilization of Water-in-Water Emulsions by Polysaccharide-Coated Protein Particles Rilton A. de Freitas,*,†,‡ Taco Nicolai,† Christophe Chassenieux,† and Lazhar Benyahia† †

IMMM, Polymères, Colloïds, Interfaces Department, UMR CNRS Université du Maine, 72085 Le Mans cedex 9, France BioPol, Chemistry Department, Federal University of Paraná, 81531-980 Curitiba, Paraná, Brazil

‡

S Supporting Information *

ABSTRACT: The phase diagram of mixtures of xyloglucan (XG) and amylopectin (AMP) in aqueous solution is presented. Water-in-water emulsions prepared from mixtures in the two-phase regime were studied in detail, and the interfacial tension was determined. It is shown that the emulsions can be stabilized by addition of β-lactoglobulin microgels (βLGm), but only at pH ≤ 5.0. Excess βLGm preferentially entered the AMP phase at pH > 5.0 and the XG phase at lower pH. The inversion was caused by adsorption of XG onto βLGm that started below pH 5.5. It is shown that modification of the surface of particles by coating with polysaccharides is a potential lever to control stabilization of water-in-water emulsions.

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INTRODUCTION Mixing aqueous solutions of two incompatible polymers leads to the formation of water-in-water emulsions. Contrary to oil-inwater emulsions, these emulsions cannot be stabilized by amphiphilic molecules, because the interface between the two water phases is expressed only on length scales larger than the correlation length of the polymer solutions. However, it has been observed, recently, that water-in-water emulsions can in some cases be stabilized by colloidal particles that spontaneously accumulate at the interface.1−10 Particle stabilization of oil-inwater emulsions has been investigated in detail.11,12 The free energy (ΔG) required to remove a particle from the interface depends on its radius (R), the contact angle with the interface (θ), and the interfacial tension (γ) as follows: ΔG = πR2γ(1 − |cos θ|)2

water-in-water emulsions. For instance, coalescence of droplets fully covered by latex particles has been observed leading to expulsion of particles from the interface,1 whereas the same emulsion could be stabilized by protein particles even though they were smaller.2 Not only was the chemistry of the particles found to be important for stabilization, but also the conditions of pH and ionic strength, even if in all cases thermal energy was not sufficient to allow the particles to leave the interface spontaneously.3 In one investigation it was found that even the softness of the particles can make a difference for the stability, because it allowed the particles to partially interpenetrate.9 These findings indicate that interaction between the particles at the interface is of crucial importance. Another remarkable feature for water-in-water emulsions is that the stability depends on which phase is continuous.3 One potential application of stabilized water-in-water emulsions is to develop novel fat-free food products. For this reason we have investigated water-in-water emulsions formed by mixing aqueous solutions of two food-grade polysaccharides: xyloglucan (XG) and amylopectin (AMP). In an attempt to stabilize these emulsions we added protein particles formed by heating aqueous solutions of the milk protein β-lactoglobulin (βLG), which had been found earlier to stabilize emulsions formed by mixtures of poly(ethylene oxide) and dextran.2 Mixtures of starch with nonstarch polysaccharides have been studied extensively in the past as the presence of the latter can influence starch gelatinization and retrogradation. In some of these mixtures, phase separation occurs,13 which appears to be

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The contact angle depends on the interfacial tension between the particles and each phase (γpi): cos θ = (γp1 − γp2)/γ. If |γp1 − γp2| > γ, the free energy is reduced when a particle enters the interface. The strength of the interaction increases strongly with increasing particle size, and, for large colloidal particles, it is orders of magnitude larger than the kinetic energy. Therefore, particle stabilized emulsions, so-called Pickering emulsions, are very stable. It was recently demonstrated that the driving force for the particles to stay in the water-in-water interface is the same as for the oil/water interface even though the interfacial tension is orders of magnitude smaller.1 It has become clear, however, that there are marked differences between the two systems. Notably, whereas for oil/water emulsions the presence of particles at the interface invariably leads to stabilization, this is not the case for © XXXX American Chemical Society

Received: October 9, 2015 Revised: January 12, 2016

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microgels (βLGm; 0−0.5 wt %), at different pH between 7.0 and 3.0. The pH was adjusted to within 0.1 units by adding aliquots of 0.01 mol· L−1 HCl. The samples were mixed using a minishaker. No significant differences were observed when different speeds were used or even when the samples were hand shaken. Five parts per million of sodium azide was added to avoid bacterial growth. Size Exclusion Chromatography. The concentration of the polysaccharides in each phase was determined using size exclusion chromatography (SEC) with a Tosho G6000PW column. The light scattering intensity was detected with a Dawn EOT (Wyatt technology, Santa Barbara, CA, USA), and the refractive index was measured using a Shodex RI 71 (Showa Denko K.K., Tokyo, Japan). A volume of 100 μL of the XG or AMP solutions was injected using an automatic injection system (Autoinjector 234, Gilson, Middleton, WI, USA). The refractive index increment used was 0.113 mL/g16 and 0.146 mL/g15 for XG and AMP, respectively. The system was eluted with 0.1 mol·L−1 NaNO3 at pH 7, with a flow rate of 1 mL·min−1. The data were analyzed using the software ASTRA 6.1.1. Confocal Laser Scanning Microscopy. Confocal laser scanning microscopy (CLSM) images were obtained with a Leica TCS-SPs microscope (Leica Microsystems Heidelberg, Germany). Images of 512 × 512 pixels were obtained using water immersion objectives (HCXPL APO 63X NA = 1.2 and 20X NA = 0.7). All the CLSM images were processed using ImageJ software v1.48. The polysaccharides were visualized by fluorescently labeled polymers. The protein particles were labeled by adding 5 ppm of rhodamine B to the solutions. No effect of labeling on the phase behavior or the structure of the emulsions was observed.

related to the ratio between the fractions of linear amylose and of highly branched AMP. Freitas et al.14 reported that segregative phase-separation occurred in mixtures of XG and gelatinized waxy corn starch, which is composed almost exclusively of AMP. However, the phase diagram of this mixture as a function of the XG and AMP concentrations has not yet been determined. Here we will show the phase diagram of XG/AMP mixtures as well as the interfacial tension between the two polysaccharide phases as a function of the tie-line length (TLL). The TLL can be determined from the composition of the phases: TLL = [(ΔXG)2 + (ΔAMP)2]1/2, where ΔXG is the concentration of the XG-rich phase and ΔAMP is the concentration of the AMP-rich phase. We will report on the stability and the structure of the emulsions in the presence of protein particles as a function of the pH. The particles were found to enter the interface and stabilize the emulsions only for pH ≤ 5.0. It will be shown that stabilization of the emulsions was induced by spontaneous coating of the protein particles with XG that started below pH 5.5.

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MATERIAL AND METHODS

Materials. AMP has a α-D-glucopyranose (1 → 4) backbone that is highly ramified due to the presence of α-D-glucopyranose (1 → 6). The AMP used for this study was obtained by purification of corn starch as described in ref15. Corn starch purchased from Sigma-Aldrich (batch S9679) was dissolved in a mixture of dimethyl sulfoxide (DMSO):water (95:5 v:v %), under stirring, for 16 h. After, the solution was centrifuged using an Allegra Beckman coulter, Rotor R030, at 104g for 30 min, at 20 °C. The AMP was precipitated from the supernatant by adding 3 volumes of ethanol 99.5%. The precipitate was recovered by filtration using a glass filter G3 and washed successively with ethanol 99.5%, acetone, and diethyl ether. The precipitate was dried overnight under a hood and subsequently dried for 12 h in a vacuum oven. The polymer was characterized by light scattering and chromatography yielding a weight-average molar mass Mw = 1.3 × 107 g/moL, dispersity Đ = 1.4, and radius of gyration Rg = 71 nm. XG has a backbone of (1 → 4) β-D-glucopyranose, with substitutions at O-6 by α-D-xylopyranose, and substituted at O-2 by β-Dgalactopyanose. The XG used for this study was obtained by aqueous extraction at 25 °C of pooled and milled Hymenaea courbaril seeds (Jatobá) as described in ref 16. The viscous extract was centrifuged at 105 g for 20 min, and the polysaccharide in the supernatant was precipitated with two volumes of 96% ethanol, washed with 99.5% ethanol and with 1 volume of acetone. The characteristics of the polymer were Mw = 2.9 × 106 g/moL, Đ = 1.3, and Rg = 76 nm.16 β-LG (Biopure, Lot JE 001−8−415) was purchased from Davisco Foods International, Inc. (Le Sueur, MN, U.S.). Protein particles were prepared and characterized as previously described in ref 2. Briefly, aqueous solutions of 40 g/L β-LG (18.2 kg/moL) at pH 6.9 were heated in the presence of 5.3 mM of CaCl2 at 85 °C for 12h. Dynamic light scattering showed that, after heating, 80% of the proteins had formed microgels with a z-average hydrodynamic radius Rh = 250 nm, Mw = 9.2 × 109 g/moL, density ρ = 0.22 g/mL, and zeta potential ζ = −41 ± 3 mV at pH 6.9. The residual proteins formed small strands or denatured monomers and dimers. XG was labeled with rhodamine isothiocyanate (XG-RITC) and AMP was labeled with fluorescein isothiocyanate (AMP-FITC) following the method of ref 17. Briefly, 0.5 g of polysaccharides was dissolved in 10 mL of DMSO, containing a few drops of pyridine. RITC or FITC (0.05 g) was added together with 20 mg of dibutylindilaurate as a catalyst. The solution was stirred for 2 h at 95 °C. After heating, the polysaccharides were precipitated with 4−5 volumes of ethanol, filtered, and the precipitate was sequentially washed with ethanol several times, until no free dye was visible. The polymer was washed with acetone and dried under a vacuum at room temperature. Sample Preparation. The emulsions were prepared by mixing aqueous solutions of AMP (0−10 wt %), XG (0−2 wt %), and β-LG

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RESULTS AND DISCUSSION Phase Diagram. Mixtures of AMP and XG were prepared in the absence of protein particles over a range of concentrations between 0 and 10 wt % and 0−2 wt %, respectively. Above critical polymer concentrations, phase separation was observed, which was slow due to the high viscosity so that it could be monitored as a function of time with CLSM (see Figure 1). The images taken at different times show the typical features of spinodal decomposition. A video of the process can be found in the Supporting Information (Video S1). Phase separation led to the formation of an AMP-rich bottom layer and a XG-rich top layer. In all mixtures that were studied, the phase volumes reached steady state within 24 h if no particles were added. Mixtures were checked for phase separation both visually and using CLSM. The concentration of XG and AMP in each phase was determined by SEC. The phase diagram of AMP/XG mixtures including a number of tie lines is shown in Figure 2. It appears that, whereas the concentration of XG in the AMP-rich phase was negligible at higher polysaccharide concentrations, approximately 0.5 wt % AMP remained in the XG-rich phase even at high XG concentrations. The critical point of the mixture was situated at 0.125 wt % of XG and 0.625 wt % of AMP. The tie-lines show that the AMP phase is much more concentrated in polysaccharides than the XG phase. Interfacial Tension. The interfacial tension (γ) between the XG-rich and AMP-rich phases was determined by droplet relaxation measurements in the same manner as was reported by Balakrishnan et al.1 for dextran/PEO mixtures.18 Figure 3 shows that γ increased with the length of the tie-lines (TLL) expressed in terms of wt % following a power law: γ ∝ TLL5.7±0.8. Power law dependence was earlier reported for dextran/PEO mixtures, but the exponent was lower for that system (γ ∝ TLL3.7), which means that the interfacial tension between XG and AMP solutions is larger than that between dextran and PEO solutions at the same polymer concentrations. B

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Figure 2. Phase diagram of aqueous mixtures of XG and AMP. The solid line indicates the binodal and the dotted lines indicate tie-lines. Circles indicate the concentrations of each phase for mixtures with initial composition indicated by ×. The dashed line indicates the phase inversion. The triangles correspond to the compositions of the emulsions that were studied in detail. The inset shows a zoom at low XG and AMP concentrations.

Figure 1. CLSM images of an AMP-XG mixture containing 1.25 wt % fluorescently labeled AMP (gray phase in pictures) and 0.25 wt % XG at different times after mixing and placing on the microscope as indicated. The scale bar represents 100 μm. A movie of the spinodal decomposition is provided as Supporting Information as Video S1.

Water-in-Water Emulsions. In the following, we will focus on emulsions of the AMP-rich phase dispersed in the continuous XG-rich phase. In the first instance, water-in-water emulsions were prepared by mixing 0.5 wt % XG and 0.5 wt % AMP in the presence of 0.1 wt % βLGm with Rh = 250 nm at different pH between 7.0 and 3.0. At these conditions, the volume fraction of the AMP-rich phase was ΦAMP = 0.2 and CLSM images taken immediately after mixing showed droplets of the AMP phase in a continuous XG phase; see Figure 4. In these mixtures, only the protein particles were fluorescently labeled. For pH > 5.0 microgels did not accumulate at the interface and partitioned almost exclusively to the AMP phase. It appears that for pH > 5.0, contact between βLGm and the XG phase is not only much less favorable than contact with the AMP phase, which explains the strong partitioning, but it is even less favorable than contact between the XG phase and the AMP phase. Therefore, there is no reduction of the free energy if the microgels are localized at the AMP-XG interface. As a consequence, macroscopic phase separation occurred at the same rate as in the absence of microgels (see Figure 6). Therefore, images taken after 24 h for pH > 5.0 showed the homogeneous AMP bottom phase containing the microgels. The observation that for pH ≤ 5.0 a layer of βLGm was formed at the droplet interface, implies that the interfacial tension between the particles and one or both polysaccharide phases was modified by the pH decrease. Migration of the microgels to the

Figure 3. Interfacial tension (γ) of XG/AMP mixtures as a function of the length of the tie-line (TLL) mean ± standard deviation on the basis of three independent measurements. The solid line represents a linear least-squares fit.

interface was slower at lower pH and was not clearly visible during the first few hours at pH 4.0 and pH 3.0. Also, partitioning of excess microgels between the phases was slower. Closer inspection shows that the microgels formed a relatively dense layer at the interface, but they were not close packed and remained disordered (see Figure 5). As a consequence of the formation of a microgel layer, the emulsions were stable in this pH range. At all pH, a few large clusters of βLGm can be seen in the images, which we believe are caused by depletion effects. The macroscopic evolution of the mixtures with time after mixing is shown in Figure 6. For pH ≥ 5.5, macroscopic phase separation was complete after 24 h. The top XG-rich layer was clear and contained almost no microgels. At pH 5.0, slow sedimentation of stable AMP droplets was observed, but at lower pH, sedimentation was not yet significant after 3 days. C

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Figure 5. Close-up of a section of the CLSM image shown in Figure 4 at pH 5.0 after 24 h. For some droplets, the focus is at the interface layer, while for others it is closer to the center of the droplets. Individual microgels can be distinguished at the interface in both views. The scale bar represents 10 μm.

Figure 4. CLSM images of mixtures of 0.5 wt % AMP and 0.5 wt % XG at different pH in the presence of fluorescently labeled βLGm (0.1 wt %) at different times after mixing. The scale bar represents 50 μm. The bright dots correspond to clusters of microgel.

Similar behavior was observed for a mixture on the same tieline (1.1 wt % AMP and 0.381 wt % XG) with a larger volume fraction of the AMP phase (ΦAMP = 0.36; see Figure 7). Again particle stabilized emulsions were formed only at pH ≤ 5.0. However, larger AMP droplets were formed in this case that slowly sedimented. The CLSM images were made of the bottom of the emulsion and show sedimented densely packed AMP droplets separated by a thin layer of XG. The weak interfacial tension allowed the droplets to deform so that the volume fraction of the XG phase between the sedimented droplets became very small. For this mixture it can be clearly observed that excess βLGm preferred to be in the AMP phase for pH ≥ 5.0 and preferred the XG phase for pH < 5.0. The effect of varying the microgel concentration was assessed by investigating emulsions containing 0.01, 0.05, 0.1, or 0.5 wt % microgels at pH 5.0, 4.5 or 3.5 (see Figure 8). Particle stabilized emulsions were formed even at the lowest microgels concentration, but 0.01 wt % βLGm was not enough to stabilize all of the AMP phase. At 0.05 wt %, almost all of the AMP phase could be stabilized and at 0.5 wt % a large amount of excess βLGm could be observed. In this case, βLGm was still mainly located in the AMP phase at pH 5.0 and partitioned to the XG phase at pH 4.5 and lower. A more detailed investigation of the

Figure 6. Mixtures of 0.5 wt % AMP and 0.5 wt % XG at different pH in the presence of fluorescently labeled βLGm (0.1 wt %) as a function of time after mixing.

influence of the pH showed that the critical pH is very close to pH 5.0 and that the transition is sharp. This means that the observed difference in behavior at pH 5.0 is most likely caused by small differences in the pH that are within the experimental error. Screening of electrostatic interactions by adding 50 mM NaCl did not cause major changes in the behavior of the emulsions (see Supporting Information). However, it led to faster sedimentation of the AMP droplets.

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DISCUSSION We have found that emulsions of AMP droplets in a continuous XG phase can be stabilized by addition of β-LG microgels below a critical pH of 5.0. Above this pH, the microgels do not enter the interface, but are almost exclusively situated in the AMP phase. This observation contrasts with that of emulsions of PEO droplets in a continuous dextran phase for which the same microgels entered the interface at pH 7.0. For the latter mixture, excess βLGm was mainly situated in the dextran phase, but a significant fraction went to the PEO phase. We may tentatively D

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all pH, and CLSM images did not show large scale aggregation of the microgels. In neat AMP solutions, βLGm was insoluble in the same pH range as in pure water. This remarkable observation suggests specific interaction between XG and βLGm in this pH range. Indeed, a more detailed investigation that will be reported elsewhere showed that XG adsorbs at the surface of the microgels for pH ≤ 5.5. Adsorption of anionic polysaccharides unto β-LG microgels close to and below the iso-electric point has been reported in the literature20 and was explained in terms of attraction between positively charged groups on the protein and negative charged groups on the polysaccharide. However, XG is a neutral polysaccharide so that the adsorption cannot be explained in this manner. On the other hand, XG contains hydrophobic segments, which might interact with the proteins, but it is not clear why this interaction only leads to adsorption at low pH. Adsorption of XG on the microgels can be visualized by fluorescently labeling XG. Figure 9 shows images at two different

Figure 7. CLSM images of mixtures of 1.1 wt % AMP and 0.38 wt % XG at different pH in the presence of fluorescently labeled βLGm (0.1 wt %) as a function of time after mixing. The scale bar represents 100 μm.

Figure 9. CLSM images of a mixture of 1.1 wt % AMP and 0.38 wt % fluorescently labeled XG at pH 5.0 in the presence of βLGm (0.1 wt %) taken 24 h after mixing. In panel a, the scale bar represents 100 μm and in panel b it represents 10 μm. Panel c shows a schematic representation of the interaction of βLGm with XG and its behavior at the interface, at different pHs.

scales of a dense layer of sedimented AMP droplets formed by a mixture containing 1.1 wt % AMP and 0.38 wt % fluorescently labeled XG at pH 5.0 in the presence of 0.1 wt % βLGm. In Figure 9b one can distinguish clearly the microgels adsorbed at the interface or embedded in the XG layer. Since the proteins were not labeled in this case, the fact they can be observed implies that XG chains were adsorbed onto the microgels. Images taken as a function of time show that diffusion of microgels to the interface and partitioning of excess βLGm can be very slow. Figure 1 shows that shortly after mixing neat polysaccharide solutions, the system is homogeneous on length scales larger than a few microns and that the size of the AMP domains increased quickly. It is clear that, in mixtures containing microgels, phase separation of XG and AMP led rapidly to the formation of small naked AMP droplets and that only during

Figure 8. CLSM images of mixtures of 1.1 wt % AMP and 0.38 wt % XG at different pH in the presence of fluorescently labeled βLGm (0.1 wt %) taken 24 h after mixing. The scale bar represents 100 μm.

conclude from this comparison that particles will enter the interface only if they partition at least to a small extent into both phases. This conclusion is corroborated by the observation that when the particles showed an increased affinity with the XG phase at lower pH, they did enter the interface spontaneously. The critical pH below which βLGm entered the interface and partitioned preferentially to the XG phase is close to the isoelectric point of β-LG. In fact, in pure water, βLGm is insoluble between pH 4.4 and pH 5.6.18,19 However, when βLGm was added to neat XG solutions, it remained in solution at E

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(3) Nguyen, B. T.; Wang, W.; Saunders, B. R.; Benyahia, L.; Nicolai, T. pH-responsive water-in-water Pickering emulsions. Langmuir 2015, 31 (12), 3605−11. (4) Firoozmand, H.; Murray, B. S.; Dickinson, E. Interfacial Structuring in a Phase-Separating Mixed Biopolymer Solution Containing Colloidal Particles. Langmuir 2009, 25 (3), 1300−1305. (5) Hanazawa, T.; Murray, B. S. The Influence of Oil Droplets on the Phase Separation of Protein-Polysaccharide Mixtures. Food Hydrocolloids 2014, 34, 128−137. (6) Dewey, D. C.; Strulson, C. A.; Cacace, D. N.; Bevilacqua, P. C.; Keating, C. D. Bioreactor droplets from liposome-stabilized all-aqueous emulsions. Nat. Commun. 2014, 5, 4670. (7) Murray, B. S.; Phisarnchananan, N. The effect of nanoparticles on the phase separation of waxy corn starch+ locust bean gum or guar gum. Food Hydrocolloids 2014, 42, 92−99. (8) Poortinga, A. T. Microcapsules from self-assembled colloidal particles using aqueous phase-separated polymer solutions. Langmuir 2008, 24 (5), 1644−1647. (9) Hanazawa, T.; Murray, B. S. Effect of Oil Droplets and Their Solid/ Liquid Composition on the Phase Separation of Protein−Polysaccharide Mixtures. Langmuir 2013, 29 (31), 9841−9848. (10) Vis, M.; Opdam, J.; van’t Oor, I. S.; Soligno, G.; van Roij, R.; Tromp, R. H.; Erné, B. H. Water-in-Water Emulsions Stabilized by Nanoplates. ACS Macro Lett. 2015, 4, 965−968. (11) Binks, B. P.; Horozov, T. S. Colloidal Particles at Liquid Interfaces; Cambridge University Press: Cambridge, U.K., 2006. (12) Aveyard, R.; Binks, B. P.; Clint, J. H. Emulsions stabilised solely by colloidal particles. Adv. Colloid Interface Sci. 2003, 100, 503−546. (13) BeMiller, J. N. Pasting, paste, and gel properties of starchhydrocolloid combinations. Carbohydr. Polym. 2011, 86 (2), 386−423. (14) Freitas, R. A.; Gorin, P. A. J.; Neves, J.; Sierakowski, M. R. A rheological description of mixtures of a galactoxyloglucan with high amylose and waxy corn starches. Carbohydr. Polym. 2003, 51 (1), 25−32. (15) Bello-Pérez, L. A.; Roger, P.; Baud, B.; Colonna, P. Macromolecular Features of Starches Determined by Aqueous Highperformance Size Exclusion Chromatography. J. Cereal Sci. 1998, 27, 267−278. (16) de Freitas, R. A.; Spier, V. C.; Sierakowski, M. R.; Nicolai, T.; Benyahia, L.; Chassenieux, C. Transient and quasi-permanent networks in xyloglucan solutions. Carbohydr. Polym. 2015, 129, 216−23. (17) de Belder, A. N.; Granath, K. Preparation and properties of fluorescein-labelled dextrans. Carbohydr. Res. 1973, 30, 375−378. (18) Ding, P.; Wolf, B.; Frith, W. J.; Clark, A. H.; Norton, I. T.; Pacek, A. W. Interfacial tension in phase-separated gelatin/dextran aqueous mixtures. J. Colloid Interface Sci. 2002, 253 (2), 367−76. (19) Schmitt, C.; Bovay, C.; Vuilliomenet, A.-M.; Rouvet, M.; Bovetto, L.; Barbar, R.; Sanchez, C. Multiscale characterization of individualized beta-lactoglobulin microgels formed upon heat treatment under narrow pH range conditions. Langmuir 2009, 25, 7899−909. (20) Jones, O. G.; McClements, D. J. Recent progress in biopolymer nanoparticle and microparticle formation by heat-treating electrostatic protein-polysaccharide complexes. Adv. Colloid Interface Sci. 2011, 167 (1−2), 49−62.

growth of the droplets grew by fusion microgels entered the interface. When the droplets reached a certain average size further growth was inhibited by the layer of microgels at the interface. Subsequently, the microgels partitioned between the two phases, which requires diffusion over length scales of tens of microns through the viscous polysaccharide solutions, which can take hours to complete.

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CONCLUSION Segregative phase separation occurs in mixtures of xyloglucan and amylopectin. Water-in-water emulsions of AMP droplets in a continuous XG phase can be stabilized by addition of protein particles for pH ≤ 5.0. At higher pH, β-LG microgels have a much stronger affinity for the AMP phase than the XG phase and therefore do not enter the interface between the two incompatible polysaccharide solutions. Below pH 5.5, XG binds to βLGm, which inhibits large scale aggregation of the microgels close to the isoelectric point of the proteins. It also leads to an increase of the affinity of the microgels for the XG phase and as a consequence they enter the interface and stabilize the emulsions. When the volume fraction of AMP is high, large AMP droplets are formed that sediment into a close packed layer in which they are separated by thin layers of the XG phase. Addition of 50 mM NaCl does not have a major impact on the behavior of the mixtures. The capacity of particles to stabilize water-in-water emulsions can be modified by adsorption of polysaccharides onto the surface, thereby forming core−shell particles. In this manner, the property of particle stabilized waterin-water emulsions can be modified using the same particles, by changing their surface and thus their interaction with each other and the two phases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03761. Movie of the spinodal decomposition (AVI) Photos and CLSM images of emulsions of 1.1 wt% AMP and 0.381 wt% XG, at different pH, using 0.1 wt% of βLGm and 5 ppm of Rhodamine, in the presence of 50 mM of NaCl (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +55 41 33613260. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Brazilian funding agencies CNPq (Conselho Nacional de Pesquisa, Process No. 477275/20125). Rilton Alves de Freitas has a postdoctoral scholarship from CNPq (246301/2013-9).

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REFERENCES

(1) Balakrishnan, G.; Nicolai, T.; Benyahia, L.; Durand, D. Particles trapped at the droplet interface in water-in-water emulsions. Langmuir 2012, 28 (14), 5921−6. (2) Nguyen, B. T.; Nicolai, T.; Benyahia, L. Stabilization of water-inwater emulsions by addition of protein particles. Langmuir 2013, 29 (34), 10658−64. F

DOI: 10.1021/acs.langmuir.5b03761 Langmuir XXXX, XXX, XXX−XXX