Self-Assembly of Î²-Lactoglobulin and Acacia Gum in Aqueous Solvent

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Self-Assembly of β-Lactoglobulin and Acacia Gum in Aqueous Solvent: Structure and Phase-Ordering Kinetics C. Sanchez,*,† G. Mekhloufi,† C. Schmitt,†,‡ D. Renard,§ P. Robert,§ C.-M. Lehr,| A. Lamprecht,| and J. Hardy† Laboratoire de Physico-Chimie et Ge´ nie Alimentaires, ENSAIA-INPL, 2 avenue de la Foreˆ t-de-Haye BP176, 54505 Vandoeuvre-le` s-Nancy Cedex, France; INRA, Centre de Recherches Agro-Alimentaires, Unite´ de Physico-Chimie des Macromole´ cules, BP 71627, 44316 Nantes Cedex 03, France; Department of Biopharmaceutics and Pharmaceutical Technology, University of Saarland, P.O. Box 151150, D-66041 Saarbru¨ cken, Germany Received July 15, 2002. In Final Form: October 8, 2002 Complex coacervation in β-lactoglobulin (BLG)/acacia gum (AG) dispersions has been studied at pH 4.2 using time-resolved confocal scanning laser microscopy (CSLM) and small angle static light scattering (SALS). Two BLG/AG dispersions differing in the extent of charge neutralization were used in these experimentssBLG(1)/AG(1) and BLG(2)/AG(1) with a BLG/AG weight ratio of 1 and 2, respectively. A few seconds after mixing BLG and AG, both dispersions showed the presence of vesicular and unstable multivesicular coacervates (apparent diameters da ) 1-15 µm). In BLG(1)/AG(1) dispersions, the unneutralized anionic polysaccharide was located at the surface of vesicles, providing steric and electrostatic stabilization of particles. Sedimentation of vesicles occurred gradually and led to a great number of small stable coacervates (da < 5 µm) in the focal plane after ∼30 min. In BLG(2)/AG(1) dispersions, coalescence of coacervates and rapid sedimentation of insoluble particles onto the observation slide were observed. After ∼15 min, an almost continuous layer of adsorbed coacervates was observed. Interactions between coacervates and precipitation led to the formation of rough surfaces. SALS experiments showed that the turbidity of BLG(1)/AG(1) dispersions was low and did not evolve markedly as a function of time. The scattered light intensity functions I(q) versus q displayed initially a correlation peak located at a length scale R (2π/qmax) of about 10 µm. Neither the maximum intensity Imax nor R changed significantly as a function of time. A transient increase of Imax was however observed, suggesting a delayed emergence of coacervates. BLG(2)/AG(1) dispersions were initially very turbid and unstable. The scattered light intensity functions displayed initially a correlation peak that moved to smaller wave vectors. The length scale R increased from 14 to 50 µm within 3000 s and followed the power law R ∼ tR with an R exponent value of ∼0.5, slightly larger than the 0.2-0.3 value characteristic of a purely diffusion-controlled growth of particles. During the same time interval, interfaces sharpened but remained fractal. At longer coarsening times, a new correlation peak appeared that slowly moved toward smaller q (R ∼ 0.2). Interfaces became rough or fractal. The evolution of scattering patterns during the two growth processes was compatible both with late stage spinodal decomposition and nucleation and growth. Polydispersity of biopolymers, different rates of coarsening, and existence of an interfacial length scale did not allow dynamic scaling of data.

Introduction Complex coacervation between oppositely charged proteins and polysaccharides was discovered by Tiebackx in 1911.1 He observed opalescence or precipitation mixing together acacia gum and gelatin dispersions in an acetic acid solution. This phase separation phenomenon was extensively studied by Bungenberg de Jong and co-workers in the 20s to 40s, and most of our present knowledge stems from their studies.2 The word “coacervation” was coined by Bungenberg de Jong and Kruyt3 and derives from the Latin “acervus” ) aggregation (a heap) and the prefix “co” (together) to signify the preceding union of the colloidal * Corresponding author. E-mail: Christian.Sanchez@ ensaia.inpl-nancy.fr. Phone: + 33 3 83 59 57 88. † Laboratoire de Physico-Chimie et Ge ´ nie Alimentaires, ENSAIAINPL. ‡ Present address: Nestle ´ Research Center, Department of Food Science, Vers-chez-les-Blanc, CH-1000, Lausanne 26, Switzerland. § INRA, Centre de Recherches Agro-Alimentaires, Unite ´ de Physico-Chimie des Macromolećules. | University of Saarland. (1) Tiebackx, F. W. Z. Chem. Ind. Kolloide 1911, 8, 198. (2) Bungenberg de Jong, H. G. In Colloid Science; Kruyt, H. R., Ed.; Elsevier: Amsterdam, 1949; p 232. (3) Bungenberg de Jong, H. G.; Kruyt, H. R. Proc. Koninkl. Nederland. Akad. Wetenschap. 1929, 32, 849.

particles. Colloidal particles refer to the liquid droplets, called coacervates, primarily induced by demixing. This discovery was an important historical event, since it was suggested that coacervates could play a role in the appearance of life on Earth.4 However, despite much research efforts since almost one century ago, coacervates remain the most esoteric of the colloidal systems.5 A survey of the abundant literature shows clearly that pH, ionic strength, type of ions, protein-to-polysaccharide ratio, total biopolymer concentration, size, shape, charge density, and flexibility of macromolecules are important parameters controlling the extent of phase separation as determined at equilibrium.2,6-16 The actual perception of the mechanism of complex coacervation is schematically (4) Oparin, A. I. The Origin of Life; Dover Publications: New York, 1953. (5) Menger, F. M.; Peresypkin, A. V.; Caran, K. L.; Apkarian, R. P. Langmuir 2000, 16, 9113. (6) Ledward, D. A. In Protein Functionality in Food Systems; Hettiarachchy, N., Ziegler, G. R., Eds.; Marcel Dekker: New York, 1994; p 225. (7) Burgess, D. J. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davis, R., Schulz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; p 281. (8) Xia, J.; Dubin, P. L. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davis, R., Schulz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; p 247.

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as follows: primary Coulombic interactions between proteins and polysaccharides, or between polymers in general, induce the formation of soluble interpolymeric complexes that interact to form electrostatically neutral insoluble complexes. Insoluble complexes then aggregate and precipitate, forming a dispersed phase of complex coacervates.7,8,12,17-22 Coacervates coarsen with time and sediment, forming the coacervated phase. Significant contributions in the last 10 years concern the discovery of critical pH values corresponding to structural and morphological changes in polymer/protein dispersions.23-26 The global picture has been mainly established with mixtures equilibrated at different selected pHs, macromolecular ratios, or ionic strengths. However, complex coacervation is intrinsically a kinetic process, and outof-equilibrium conditions also need to be considered. This is probably the more challenging facet of the phenomenon. Among some important issues, the supposed transition between aggregated complexes and coacervates together with the coarsening behavior of coacervates from their formation toward equilibrium needs to be considered from a mechanistic and structural point of view. Better knowledge of phase-ordering kinetics and structural transitions during complex coacervation should improve our understanding of important biological processes based on self-assembly of biological macromolecules such as DNA/histones collapse,27 gene replication,28 elastogenesis,26,29 antigen-antibody reactions,30 channeling of enzymes,31 or cytoplasmic organization.32 (9) Piculell, L.; Bergfeldt, K.; Nilsson, S. In Biopolymer Mixtures; Harding, S. E., Hill, S. E., Mitchell, J. R., Eds; Nottingham University Press: Nottingham, 1995; pp 13-36. (10) Tolstoguzov, V. B. In Functional Properties of Macromolecules; Mitchell, J. R., Ledward, D. A., Eds.; Elsevier: London, 1986; pp 385415. (11) Tolstoguzov, V. B. Food Hydrocolloids 1991, 4, 429. (12) Tolstoguzov, V. B. In Food Proteins and their Applications; Damodara, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997; p 171. (13) Syrbe, A.; Bauer, W. J.; Klostermeyer, H. Int. Dairy. J. 1998, 8, 179. (14) Braudo, E. E. In Gums and Stabilizers for the Food Industry; Williams, P. A., Williams, G. O., Eds.; Royal Society of Chemistry: Cambridge, 1998; p 169. (15) Schmitt, C.; Sanchez, C.; Desobry-Banon, S.; Hardy, J. Crit. Rev. Food Sci. Nutr. 1998, 38, 689. (16) Doublier, J.-L.; Garnier, C.; Renard, D.; Sanchez, C. Curr. Opin. Colloid Interface Sci. 2000, 5, 202. (17) Overbeek, J. T. J.; Voorn, M. J. J. Cell. Comput. Physiol. 1957, 49, 7. (18) Veis, A.; Aranyi, C. J. Phys. Chem. 1960, 64, 1203. (19) Tainaka, K.-I. J. Phys. Soc. Jpn. 1979, 46, 1899. (20) Tsuchida, E.; Abe, K. In Interactions between Macromolecules in Solution and Intermacromolecular Complexes; Springer Verlag: Berlin, 1982; Chapter 4, p 77. (21) Mattison, K. W.; Wang, Y.; Grymonpre´, K.; Dubin, P. L. Macromol. Symp. 1999, 140, 53. (22) Wang, Y.; Kimura, K.; Jaeger, W.; Dubin, P. L. Macromolecules 2000, 33, 3324. (23) Park, J. M.; Muhoberac, B. B.; Dubin, P. L.; Xia, J. Macromolecules 1992, 25, 290. (24) Mattison, K. W.; Brittain, I. J.; Dubin, P. L. Biotechnol. Prog. 1995, 11, 632. (25) Mattison, K. W.; Dubin, P. L.; Brittain, I. J. J. Phys. Chem. B 1998, 101, 3830. (26) Kaibara, K.; Okazaki, T.; Bohidar, H. B.; Dubin, P. L. Biomacromolecules 2000, 1, 100. (27) Tribet, C.; Porcar, I.; Bonnefont, P. A.; Audebert, R. J. Phys. Chem. B 1998, 102, 1327. (28) Radler, J. O.; Koltover, L.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810. (29) Wu, W. J.; Vrhovski, B.; Weiss, A. S. J. Biol. Chem. 1999, 274, 21719. (30) Tsuchida, E. J. Macromol. Sci., Pure Appl. Chem. 1994, 31, 1. (31) Ova´di, J.; Srere, P. A. In Microcompartmentation and phase separation in cytoplasm; Walter, H., Brooks, D. E., Srere, P. A., Eds.; Academic Press: San Diego, CA, 2000; p 255. (32) Pagliaro, L. In Microcompartmentation and phase separation in cytoplasm; Walter, H., Brooks, D. E., Srere, P. A., Eds.; Academic Press: San Diego, CA, 2000; p 303.

Sanchez et al.

We recently determined the initial structure and stability as a function of time of coacervated β-lactoglobulin/acacia gum/water ternary systems at pH 4.2 and 1 wt % total biopolymer concentration.33 β-Lactoglobulin and acacia gum were selected because the former is a model globular protein for which secondary and tertiary structures are well-known, while the latter displays complex coacervation with a large number of proteins. At a protein to polysaccharide weight ratio of 2, multivesicular coacervated structures (apparent diameters da ∼ 10-25 µm) and strong coalescence appeared within 2 min after mixing biopolymers. Small coacervates were also present (da ∼ 1-2 µm). Particles grew in size, as determined by diffusing wave spectroscopy, and sedimentation was important, since dispersions lost ∼60% of their inital turbidity after 20 min. At a weight ratio of 1, multivesicular and small coacervates were also present. Mixed dispersions lost 15% turbidity after 20 min. Particle size did not change significantly as a function of time. In the present paper, we extended the out of equilibrium approach using time-resolved confocal scanning laser microscopy (CSLM) and small angle static light scattering (SALS). Specific objectives of the study were to clarify structural transitions and phase-ordering kinetics during complex coacervation between β-lactoglobulin and acacia gum in aqueous solvent. The paper is structured as follows: the Introduction is followed by an Experimental Section describing the biopolymers used, the sample preparation, and the techniques used. In the time-resolved SALS section, a short theoretical background of the use of the technique to follow phase separation phenomena is given. The Results section is followed by a general Discussion. A Summary finally concludes the paper. Experimental Section Biopolymers Used: β-Lactoglobulin and Acacia Gum. Physicochemical properties of individual biopolymers have been already reported.34-36 The salient features are reported in the following. β-Lactoglobulin (BLG) is a compact globular protein of the lipocalin family whose secondary structure contains approximately 50% β-sheet, 9-12% R-helix, 8-10% turn, and 30-35% random coil.37,38 The BLG powder used was composed of equal amounts of variants A and B (Lactalis, Retiers, France). The hydrodynamic radius (Rh) of the protein is 2.5 nm, as determined by dynamic light scattering (DLS), which is indicative of an equilibrium between the monomeric and dimeric forms of the protein.39 As dispersions were filtered through 0.22 µm microfilters before DLS measurements, a small amount of soluble aggregated species could also be present. This is important to notice, since aggregated proteins markedly changed the structure and stability of mixed dispersions.35 The electrophoretic mobility of BLG dispersions was in the range +0.05-0.1 µm‚cm‚V-1‚s-1 at pH 4.2, as determined using Zetaphoremeter II equipment (Sephy Technology). Acacia gum, also called gum arabic, is a complex arabinogalactan-type polysaccharide exuded by Acacia trees. The acacia gum is defined as an heteropolysaccharide, since it contains about (33) Schmitt, C.; Sanchez, C.; Lamprecht, A.; Renard, D.; Lehr, C.M.; de Kruif, C. G.; Hardy, J. Colloids Surf., B: Biointerfaces 2001, 20, 267. (34) Schmitt, C.; Sanchez, C.; Thomas, F.; Hardy, J. Food Hydrocolloids 1999, 13, 483. (35) Schmitt, C.; Sanchez, C.; Despond, S.; Renard, D.; Thomas, F.; Hardy, J. Food Hydrocolloids 2000, 14, 403. (36) Sanchez, C.; Renard, D.; Robert, P.; Schmitt, C.; Lefebvre, J. Food Hydrocolloids 2002, 16, 257. (37) Qi, X. L.; Holt, C.; McNulty, D.; Clarke, D. T.; Brownlow, S.; Jones, G. R. Biochem. J. 1997, 324, 341. (38) Sawyer, L.; Kontopidis, G. Biochim. Biophys. Acta 2000, 1482, 136. (39) Schmitt, C. Etude de la coacervation complexe entre la β-lactoglobuline et la gomme d’acacia en solution aqueuse. Ph.D. Thesis, I. N. P. L., Vandoeuvre-le`s-Nancy, France, 2000.

Self-Assembly of β-Lactoglobulin and Acacia Gum 2% of a polypeptide.40 Three major molecular species have been isolated, an arabinogalactan (AraG), an arabinogalactanpolypeptide complex (AraGP), and a glycoprotein (GP).41 HPSECMALLS measurements revealed that our gum sample was mainly characterized by the presence of the AraGP and AraG molecular fractions.36 The weight average molecular weight (Mw) of the first fraction was 2.34 × 106 g‚mol-1, and that of the second fraction was 2.68 × 105 g‚mol-1. The Mw of the whole sample (fraction 1 + 2) was 5.34 × 105 g‚mol-1. The polydispersity of each fraction was 1.2-1.3 whereas the polydispersity of the whole sample was 2.3. The radius of gyration (Rg) and the hydrodynamic radius (Rh) of acacia gum whole fractions were 14.2 and 11.9 nm, respectively, indicating a random coil shape (Rg/Rh ∼ 1.2). Small amounts of acacia gum aggregated structures have also been observed by DLS (Rh ∼ 200 nm). These aggregates are known to be always present in acacia gum samples42,43 or more generally in plant secretions containing AraGP.44 The electrophoretic mobility of acacia gum dispersions was -1.3 µm‚cm‚V-1‚s-1 at pH 4.2. Preparation of β-Lactoglobulin/Acacia Gum/Water Mixed Dispersions. Acacia gum (AG) and β-lactoglobulin (BLG) aqueous stock dispersions at 0.1 wt % (for SALS) or 1 wt % (for CSLM) total biopolymer concentration were prepared in deionized or bidistilled water, respectively. Stock dispersions were gently stirred for at least 2 h at 20 ( 1 °C and then stored for 18 h at 4 ( 1 °C to enable good hydration of biopolymers. The pH of BLG dispersions was adjusted to 4.75 (pH corresponding to the minimum solubility of the protein), and dispersions were centrifuged for 1 h at 10000g to remove insoluble aggregated structures. We have previously shown that BLG aggregates strongly accelerate coacervation with AG.35 The concentration of BLG in dispersions was checked by optical density at 278 nm (corrected for turbidity) using a molar absorption coefficient 1%1 cm ) 9.6.45 AG dispersions were centrifuged using the same conditions. The pH of the resulting dispersions was adjusted to 4.2. BLG/AG mixed dispersions at protein-to-polysaccharide weight ratios (Pr/Ps) of 1:1 or 2:1 were obtained by gently mixing stock dispersions. In the following, these two dispersions are referred to as BLG(1)/AG(1) and BLG(2)/AG(1), respectively. Experiments were duplicated. Confocal Scanning Laser Microscopy (CSLM). Biopolymers were labeled by covalently linking fluorescent markers. BLG was labeled with fluoresceine isothiocyanate (FTIC) with excitation/emission wavelengths of 485/530 nm, and AG was labeled with rhodamine B isothiocyanate (RITC) with excitation/ emission wavelengths of 530/590 nm.46 To achieve labeling, the pH values of stock BLG and AG dispersions were adjusted to 8.5 and 10.5, respectively, to dissociate charged groups on biopolymers. 25 µL of a 2 wt % FITC or RITC dispersion in DMSO was then added to 100 mL of a BLG or AG dispersion, respectively. The cross-linking reaction occurred under gentle stirring at room temperature during 1 h 30 min. The marker concentration used gave a biopolymer/marker ratio of about 4, which is sufficient to provide efficient labeling while minimizing the presence of free markers.47 The procedure avoids the need for any filtration or precipitation/drying post-treatment that could modify the molecular structure of biopolymers. Small volumes (200 µL) of BLG/ AG dispersions were placed onto a glass cell with a central cavity and sealed with a glass cover to avoid dehydration. The development of microstructure in BLG/AG dispersions at 1 wt % total biopolymer concentration was followed during 2 h at 20 °C using a Bio-Rad MRC 1024 laser scanning confocal imaging system equipped with an argon ion laser emitting at 488 and 514 (40) Anderson, D. M. W.; Bridgeman, M. M. E.; Farquhar, J. G. K.; McNab, C. G. A. Int. Tree Crops J. 1983, 2, 245. (41) Randall, R. C.; Phillips, G. O.; Williams, P. A. Food Hydrocolloids 1989, 3, 65. (42) Mukherjee, S. N.; Ghosh, K. B. J. Ind. Chem. Soc. 1949, 26, 81. (43) Ray, K.; Bird, P. B.; Iacobucci, G. A.; Clark, B. C., Jr. Food Hydrocolloids 1995, 9, 123. (44) Baldwin, T. C.; McCann, M. C.; Roberts, K. Plant Physiol. 1993, 103, 115. (45) Townend, R.; Winterbottom, R. J.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82, 3161. (46) de Belder, A. N.; Granath, K. Carbohydr. Res. 1973, 30, 375. (47) Lamprecht, A.; Scha¨fer, U. F.; Lehr, C.-M. Eur. J. Pharm. Biopharm. 2000, 49, 1.

Langmuir, Vol. 18, No. 26, 2002 10325 nm.47 The apparatus was coupled with an inverted Axiovert 1000 microscope (Carl Zeiss, Germany), allowing a magnification of 40-400×. The focal plane of observation was about 30 µm from the inverted objective of the microscope. The double illumination of the fluorescent probes was used. Assays were also performed with blends containing only one labeled biopolymer to detect any effect of labeling on the temporal evolution of microstructures. No effect was observed. Pictures were taken approximately every minute during the first hour and every 15 min thereafter to reduce the photobleaching of markers with time. Pictures were processed using the Laser Sharp MRC-1024 software version 3.2 from Bio-Rad. Structural details of coacervates were obtained by capturing areas of pictures using the CaptureImage freeware version 1.4.7 (Netsoft Tech., http://www.netsoft.com). Colors of pictures were then modified using the Scion Image freeware version Beta 4.0.2 (Scion Corporation). Small Angle Static Light Scattering (SALS). Before mixing, stock BLG and AG dispersions at 0.1 wt % total biopolymer concentration and pH 4.2 were filtered through 0.22 µm microfilters. Phase-ordering kinetics of BLG/AG mixtures were then followed during 10 h using SALS. Light scattering experiments were carried out using a Mastersizer S long bench (Malvern Ltd., U.K.). A He-Ne laser light (λ ) 0.6334 µm) was passed through a 0.5-mm-width measurement cell (Malvern Ltd) in which the BLG/AG mixtures were injected using a syringe. The beam was converged by a reverse Fourier 300-mm focusing lens. A series of 37 detectors covering the scattering wave vector Q range 2.1 × 10-2 to 10 µm-1 collected the scattered light. The turbidity τ of mixed dispersions was also recorded at low scattering angle according to τ ) d-1 ln(I0/It) with d the thickness of the measuring cell, I0 the incident intensity, and It the transmitted intensity. The background intensity was recorded once on filtered MilliQ water (recording time: 5 s) before starting the experiment on mixed dispersions. Experimental scattering intensity was obtained every 200 s (sample time: 10 s) and corresponded to the raw scattered intensity of the sample minus the background intensity. In addition, corrections were applied to take into account the scattering angle-dependent geometry of the detectors. The resulting scattered intensity was further corrected for turbidity according to the relationship48,49

Ic(θ) ) Im(θ) exp(τd/cos θ)[τd(cos-1 θ - 1)] × {exp[τd(cos-1 θ - 1)] - 1 } - 1 (1) with τd ) -ln T(t) where Ic and Im are respectively the corrected and measured scattered intensity distributions, d is the sample thickness, τ is the turbidity, and T(t) is the transmittance of the specimen at a given time t after the onset of phase separation. The corrected scattered intensity Ic will be described as I(q) hereafter. Scattered intensity functions I(q) versus q obtained for BLG(1)/AG(1) and BLG(2)/AG(1) dispersions were fitted by, respectively, weighted curve fit and cubic spline curve fit functions from the KaleidaGraph software version 3.5 (Synergy Software, Reading, MA). Maximum scattered intensities Imax and the corresponding wave vectors qmax were obtained from the fit results. Time-Resolved Small Angle Static Light Scattering. The separation of a homogeneous mixture into two phases may be described in theory by two different mechanisms, spinodal decomposition (SD) or nucleation and growth (NG), corresponding to two different types of time-dependent statistical fluctuations, respectively homophase and heterophase fluctuations.50-52 The main difference between SD and NG is that composition fluctuations are formed spontaneously and grow in amplitude (48) Stein, R. S.; Keane, J. J. J. Polym. Sci. 1955, 17, 21. (49) Hashimoto, T.; Itakura, M.; Hasegawa, H. J. Chem. Phys. 1986, 85, 6118. (50) Gunton, J. G.; San Miguel, M.; Sahni, P. S. In Phase Transition and Critical Phenomena; Domb, C., Lebowitz, J. L., Eds.; Academic Press: New York, 1983; Vol. 8, p 276. (51) Binder, K. In Materials Science and TechnologysA Comprehensive Treatment; Cahn, R. W., Haasen, P., Kramer, E. J., Eds.; VCH: Weinhem, 1991; Vol. 5, Chapter 7, p 405. (52) Bray, A. J. Adv. Phys. 1994, 43, 357.

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for SD (unstable state), whereas for NG an activation barrier has to be overcome before large amplitude localized concentration fluctuations appear (metastable state). The dynamics of phase separation are generally divided into early, intermediate, and late stages. The different stages of phase separation can be described by means of the temporal evolution of the (small angle) scattered intensity function.53,54 A priori, it is also possible to decide on a SD or NG mechanism from the scattering pattern. As we will see below, such a prediction may be misleading. When phase separation is induced by SD, the structure factor S(q) where q is the wave vector [q ) 4πn/λ sin (θ/2) with n the medium refraction index, λ the wavelength, and θ the scattering angle] exhibits a maximum scattering intensity located at qmax. The maximum is due to the instantaneous buildup of periodic concentration fluctuations throughout the whole sample. qmax is the wave vector referring to the dominant mode of fluctuation concentration. In the early stages of phase separation, the maximum scattering intensity increases exponentially with time but qmax remains steady. In the intermediate stage, the amplitude and the wavelength of concentration fluctuations grow. Here, the maximum scattering intensity increases and qmax is shifted toward smaller q values. The same evolution of the maximum scattering intensity is observed in the final stage of phase separation, but now structural domains grow in size in a selfsimilar way. Intermediate and late stages also differ in the relative thickness of the interface, which is larger and more diffuse in the intermediate regime and becomes smooth and narrower in the late stages.55,56 The growth of structural domains or droplets induced by phase separation generally follows power-law scaling in the late stages, with qmax ∼ t-R and Imax ∼ tβ. The hyperscaling relation β ) 3R is expected to hold during this stage.49,50,57 R parameter values are characteristic of coarsening mechanisms. It has been demonstrated both experimentally and numerically that diffusion-controlled coarsening through coalescence58 or Ostwald ripening59 gives R ) 1/3. Hydrodynamic interactions60 or droplet sedimentation61 increases the value of the exponent R. The existence of scaling laws indicates self-similarity of domain growth. In other words, there is only one length scale (or domain size) and time scale in the system.49,62,63 The scaled structure factor then collapses into a single master curve. This can be ascertained, assuming previously that S(q) ∼ I(q) when the form factor of particles is close to unity by plotting I(q)/Imax as a function of q/qmax. Experimental data can be compared to the empirical scaling structure function proposed by Furukawa62 for late stage phase separation and valid at q/qmax > 1:

3(q/qmax)2 I(q) ) Imax 2 + (q/qmax)2+γ where γ is an exponent equal to d + 1 for off-critical conditions, d being the Euclidian dimension. In three dimensions, interfaces between different phases are sharp when the exponent γ ) 4 (Porod’s law). Sharp interfaces mean that the width produced by the electron distribution of the basic structural elements of the phases is small compared to the average extent of the regions of constant density.64 An exponent between 3 and 4 reveals a rough or fractal surface structure.65,66 When the NG mechanism predominates, a monotonic decrease of scattered light intensity with angle is often observed.67-71 This (53) Mallamace, F.; Micali, N.; Trusso, S. J. Phys.: Condens. Matter 1996, 8, A81. (54) Dhont, J. K. G. J. Chem. Phys. 1996, 105, 5112. (55) Furukawa, H. Adv. Phys. 1985, 34, 703. (56) Lal, J.; Bansil, R. Macromolecules 1991, 24, 290. (57) Bates, F. S.; Wiltzius, P. J. Chem. Phys. 1989, 91, 3258. (58) Binder, K.; Stauffer, D. Phys. Rev. Lett. 1974, 33, 1006. (59) Lifshitz, I. M.; Slyozov, V. V. J. Phys. Chem. Solids 1961, 19, 35. (60) Siggia, E. D. Phys. Rev. A 1979, 20, 595. (61) Kumaran, V. J. Chem. Phys. 1998, 109, 2437. (62) Furukawa, H. Phys. A 1984, 123, 497. (63) Tanaka, H. J. Phys.: Condens. Matter 2000, 12, 207. (64) Ruland, W. J. Appl. Crystallogr. 1970, 4, 70. (65) Bale, H. D.; Schmidt, P. W. Phys. Rev. Lett. 1984, 53, 596. (66) Keefer, K. D.; Schaefer, D. W. Phys. Rev. Lett. 1986, 56, 2376.

Sanchez et al. scattering pattern is still considered as the signature of a NG mechanism. Although recent model simulations based on the Mie theory have shown that a zero-angle peak appears at the earliest time in the growth of polydispersed spheres, the increase in the sphere volume fraction coupled to non-independent and multiple scattering promotes the appearance of a nonzero angle peak.72 The peak position then shifts toward larger wave vectors. A typical length scale can also be observed in nucleating systems, apparently arising from nuclei which are surrounded by a depletion zone with a local density which is lower than the bulk density.51,73 Model calculations have shown that a maximum at a wave vector q * 0 can be theoretically obtained either at low concentrations of dispersed-phase particles when a depletion layer surrounded the particles or at high concentrations of particles due to the correlation produced by the location of individual scatterers in a constrained space.74,75 The appearance of a peak in the scattering pattern has been reported experimentally in aggregation of proteins,76 colloidal spheres,77-80 or rods,81 in crystallization of colloidal spheres,82 in phase separation of a polymer blend in the metastable region of the phase diagram,69 and in phase separation and gelation of biopolymer mixtures.83,84 For early times of nucleation, eventually, after an induction time, the maximum scattered light intensity Imax evolves as Imax ∼ tβ, with β values between 3 and 4.68,82 The maximum dimension of scatterers R, equal to 2π/qmax, follows the power law R(t) ∼ t1/2, indicative of a diffusion-limited growth of isolated phase particles surrounded by a depletion layer.82,83,85 After longer times, the depletion layers start to touch those of neighboring particles and the growth of particles slows down, entering a transition regime where power-law exponents are predicted to be between 1/6 and 1/ .83,85 In the late stage of the nucleation, coarsening of particles 3 occurs through coalescence or Ostwald ripening. The temporal evolution of Imax and qmax is the same as that in the late stage of spinodal decomposition, as described above. Experimental data can then be compared to the scaling structure function proposed by Furukawa62 for late stage SD where droplets with sharp interfaces are present.

Results CSLM. The temporal evolution of the microstructure of BLG(1)/AG(1) dispersions at pH 4.2 from double illumination of fluorescent probes is shown in Figure 1. About 60 s after mixing stock dispersions, a great number of coacervates were visible, induced by phase separation. The apparent diameters (da) of the coacervates ranged (67) Hashimoto, T. Current Topics in Polymer Science; Hansen: Munich, 1987; p 199. (68) Nunes, S. P.; Inoue, T. J. Membr. Sci. 1996, 111, 93. (69) Balsara, N. P.; Lin, C.; Hammouda, B. Phys. Rev. Lett. 1996, 77, 3847. (70) Girard-Reydet, E.; Sautereau, H.; Pascault, J. P.; Keates, P.; Navard, P.; Thollet, G.; Vigier, G. Polymer 1998, 11, 2269. (71) Liu, K.; Kiran, E. J. Supercrit. Fluids 1999, 16, 59. (72) Maugey, J.; van Nuland, T.; Navard, P. Polymer 2001, 42, 4353. (73) Sur, A.; Lebowitz, J. L.; Marro, J.; Kalos, M. H. Phys. Rev. B 1977, 15, 3014. (74) Eliç abe, G. E.; Larrondo, H. A.; Williams, R. J. J. Macromolecules 1997, 30, 6550. (75) Eliç abe, G. E.; Larrondo, H. A.; Williams, R. J. J. Macromolecules 1998, 31, 8173. (76) Georgalis, Y.; Umbach, P.; Soumpasis, D. M.; Saenger, W. J. Am. Chem. Soc. 1998, 120, 5539. (77) Carpineti, M.; Giglio, M. Phys. Rev. Lett. 1992, 68, 3327. (78) Sintes, T.; Toral, R.; Chakrabarti, A. Phys. Rev. E 1994, 50, 3330. (79) Butler, B. D.; Muzny, C. D.; Hanley, H. J. M. J. Phys.: Condens. Matter 1996, 8, 9457. (80) Ramirez-Santiago, G.; Gonzalez, A. E. Phys. A 1997, 236, 75. (81) van Bruggen, M. P. B.; Dhont, J. K. G.; Lekkerkerker, H. N. W. Macromolecules 1999, 32, 2256. (82) Scha¨tzel, K.; Ackerson, B. J. Phys. Rev. E 1993, 48, 3766. (83) Tromp, R. H.; Jones, R. A. Macromolecules 1996, 29, 8109. (84) Butler, M. F.; Heppentall-Butler, M. Biomacromolecules 2001, 2, 812. (85) Wagner, R.; Kampmann, R. In Materials Science and TechnologysA Comprehensive Treatment; Cahn, R. W., Haasen, P., Kramer, E. J., Eds.; VCH: Weinhem, 1991; Vol. 5, Chapter 4, p 213.

Self-Assembly of β-Lactoglobulin and Acacia Gum

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Figure 1. CSLM micrographs as a function of time (s) after mixing β-lactoglobulin (BLG) and acacia gum (AG) at a proteinto-polysaccharide weight ratio of 1:1, pH 4.2, and 1 wt % total biopolymer concentration. The scale bar represents 50 µm. Continuous and dotted white circles enclose the coacervates shown in Figure 3a and b, respectively.

Figure 2. CSLM micrographs as a function of time (s) after mixing BLG and AG at a protein-to-polysaccharide weight ratio of 2:1, pH 4.2, and 1 wt % total biopolymer concentration. The scale bar represents 50 µm. Continuous and dotted white circles enclose the coacervates shown in Figure 3c and d, respectively.

approximatively from 1 to 15 µm. The smallest and largely more numerous coacervates appeared in red. The largest coacervates had mainly the form of single vesicles. Large vesicles probably resulted from swelling by solvent of smaller ones. A multivesicular coacervate was also observed in the bottom right corner of the picture. The yellow color indicated the simultaneous presence of BLG and AG, that is, the assembly of macromolecular complexes. The red color at the surface of coacervates indicated the predominant presence of AG. From 39 to 910 s, the number of vesicular coacervates considerably increased. This could be caused both by the formation of new coacervates and by sedimentation of previously formed particles appearing later in the focal plane. The major structural characteristic of the system from 910 to 7200 s was the dominant presence of small coacervates (da < 5 µm) and the almost complete disappearance of large vesicles. The whole observation that large vesicles appeared before smaller coacervates in the focal plane suggested that different coarsening rates existed in the system; that is, either all coacervates were formed initially at the same time but some ones coarsened faster or alternatively some coacervates were formed and coarsened before a secondary demixing occurred. No significant changes in the structure of dispersions were noticed after 1900 s. Changing the BLG to AG weight ratio (2:1) altered the coarsening kinetic of the system as well as the structure and morphology of coacervates (Figure 2). A few seconds after mixing stock dispersions, some coacervates with da ranging from 5 to 10 µm were visible. However, the most

striking structural feature of the system was the presence of a myriad of red small coacervates (da ∼ 1-3 µm). No vesicular particles were observed at that time. The visible heterogeneous green shapes were thought to be slideadsorbed insoluble coacervates. After 160 s, small coacervates were still visible, but the number of larger coacervates (da > 5 µm) increased. Large coacervates were characterized by a nonuniform distribution of yellow, red, and green fluorescence. The green fluorescence indicated that not all proteins have interacted with polysaccharides, an observation in agreement with the weight ratio which was in favor of BLG. Vesicular coacervates were clearly observed. A great number among them were coalescing or partially coalescing, promoting the formation of multivesicular structures with a much higher frequency than that for the previous dispersion. From 160 to 7200 s, BLG(2)/AG(1) dispersions coarsened gradually, leading to a great number of coacervates in the focal plane. Large coacervates with heterogeneous shapes and apparent diameters above 10-20 µm were observed beyond 310 s. The heterogeneous shape of coacervates indicated that they were adsorbed onto the glass surface of the slide. Vesicular coacervates were still present, but larger magnification of pictures was needed to see them. After ∼2000 s, an almost continuous layer of coacervates was the hallmark of the pictures, and it became very difficult to distinguish structural details of particles. The heterogeneity of the whole structure increased as a function of time, and coacervates surfaces appeared “rough”. Details of the temporal evolution of specific coacervates whose positions did not change during 15 min were

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Figure 3. Details of coacervates extracted from Figure 1 on BLG(1)/AG(1) dispersions (pictures a and b) and from Figure 2 on BLG(2)/AG(1) dispersions (pictures c and d).

obtained after processing pictures. This may clarify some interesting features of the stability of coacervates. The first picture in Figure 3a was the multivesicular coacervate observed through the sagital plane in BLG(1)/AG(1) dispersions after 162 s and located in the top right corner of Figure 1. In the picture, white/blue and green colors represented, respectively, yellow/orange and red fluorescence. The presence of vacuoles as well as the outer/inner layer of AG can be clearly observed in the picture. Multivesicular coacervates showed internal rearrangements, since only one large vacuole surrounded by the coacervated phase and an outer layer of AG was present at 458 s. The tendency of such giant multivesicular structures was then to collapse. Figure 3b was included to show the apparent stability of coacervates against coalescence. Two vesicular coacervates interacted after 313 s, showing partial coalescence. However, the two particles moved away as time elapsed, suggesting the existence of repulsive forces. Structural rearrangements of multivesicular coacervates were also observed in BLG(2)/AG(1) dispersions, suggesting a weight-ratio-independent mechanism (Figure 3c). However, the phenomenon was minor as compared to the numerous examples of coacervates’ coalescence. Figure 3d shows such an example where at least four coacervates formed after 450 s a single particle. Hence, regarding CSLM pictures as a function of time, a clear difference between the two studied mixed dispersions was the stability against coalescence of coacervates formed at a 1:1 weight ratio. SALS. A classical way to study phase separation kinetics in unstable binary mixtures is by monitoring the turbidity of the dispersions during phase separation. The initial turbidity τ of BLG(1)/AG(1) dispersions increased slightly (∼10%) until 400 s, indicating a moderate development of density inhomogeneities (Figure 4). From 400 s to the end of the experiment, τ decreased slowly to reach 65% of its initial value. BLG(1)/AG(1) dispersions were quite stable as a function of time and displayed

Figure 4. Turbidity τ (m-1) as a function of time (s) after mixing at pH 4.2 BLG and AG at a protein-to-polysaccharide weight ratio of 1:1 (b) and 2:1 (O). Each point is the average of two experiments.

moderate structural changes. The evolution of the turbidity was in agreement with the previous CSLM observations. On the other hand, BLG(2)/AG(1) dispersions displayed a totally different turbidity profile, characterized by marked structural transitions (Figure 4). The initial turbidity of dispersions was high, ∼15 times larger than that for BLG(1)/AG(1) dispersions, and increased further rapidly up to a maximum turbidity value τmax, showing strong development of density inhomogeneities. The second phase began at around 1210 s and was characterized by a drastic reduction of the number of scatterers, since more than 30% of the turbidity value reached at τmax was lost. A transient and reliable increase of τ then


Figure 5. Time-resolved scattered light intensity I(q) as a function of the wave vector q recorded after mixing at pH 4.2 BLG and AG at a protein-to-polysaccharide weight ratio of 1:1: 60 s (b); 1010 s (O); 2020 s (9); 4040 s (0); 10100 s (2); 15150 s (4); 20200 s ([); 25250 s (]); 30300 s (1); 36360 s (3). Continuous lines are weighted curve fits. Inset: Maximum scattered light intensity Imax as a function of time. Each point is the average of two experiments.

appeared after around 3000 s, suggesting a secondary phase separation mechanism. Finally, the turbidity gradually decreased, reaching 16% of the τmax value. Marked structural transitions and a strong instability characterized the temporal evolution of BLG(2)/AG(1) dispersions. The scattered intensity function for BLG(1)/AG(1) dispersions displayed initially a correlation peak located at a wave vector qmax of about 0.6 µm-1 (Figure 5). The length scale R ()2π/qmax) obtained at the beginning of the phase separation was then around 10 µm. A value of 10 µm is in the range of coacervates’ apparent diameters estimated from CSLM micrographs. This value did not change significantly with time. On the other hand, Imax decreased from 0 to 4000 s and then increased significantly from 4000 until 12000 s and decreased again (see inset in Figure 5). A transient increase of density inhomogeneities appeared at long coarsening time for this system. However, changes in scattered light intensity were within an 0.75-1.35 au interval. Whereas subtle structural changes occurred with time in BLG(1)/AG(1) dispersions, the system was quite stable. The morphology of the phaseseparated interfaces was examined through the high q tail of the scattering curves. The decrease of the scattering functions followed a power law with an exponent in the range 0.9-1.1 (results not shown). In this case, sharp interfaces did not develop in the time window selected. Regarding BLG(2)/AG(1) dispersions, the scattered intensity function displayed strong changes as a function of time (Figure 6). As a general trend, a correlation peak appeared initially and then the maximum shifted toward smaller q values, indicating the growth of coacervates. At longer coarsening times, the main features of scattering functions were the appearance of a strong correlation peak at a qmax value of 0.2 µm-1 and the appearance of a shoulder at q > 1 µm-1 (inset in Figure 6). Figure 7 shows with more detail the shift of the correlation peak until 1200 s. Interestingly, both an increase of I(q) at small scattering angles and a decrease of the scattered intensity at qmax occurred between 1200 and 1400 s (inset in Figure 7), suggesting a clustering of droplets in larger structural

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Figure 6. Time-resolved scattered light intensity I(q) as a function of the wave vector q recorded after mixing at pH 4.2 BLG and AG at a protein-to-polysaccharide weight ratio of 2:1: 60 s (b); 1010 s (O); 2020 s (9); 4040 s (0); 6060 s (2); 8080 s (4); 10100 s ([); 15150 s (]); 20200 s (1); 25250 s (3); 30300 s (×); 36360 s (+). Continuous lines are cubic spline curve fits. Inset: same data in log-log coordinates showing the presence of a shoulder at long coarsening times.

Figure 7. Time-resolved initial evolution of the scattered light intensity I(q) as a function of the wave vector q recorded after mixing at pH 4.2 BLG and AG at a protein-to-polysaccharide weight ratio of 2:1: 60 s (b); 202 s (O); 404 s (9); 606 s (0); 808 s (2); 1010 s (4); 1212 s ([). Inset: 1212 s (b) and 1414 s (O). Continuous lines are weighted curve fits.

domains. The increase of I(q) at small scattering angles was a transient phenomenon, as shown in Figure 8. I(q) decreased after 1400 s and then increased again until the end of the experiment, indicating a continuous coarsening of the system. The evolution with time of the length scale R also revealed that at least two distinct growth phases characterized phase separation in BLG(2)/AG(1) blends. During the first growth phase, which corresponded to the first shift of the scattering functions, the R parameter increased from ∼15 to 45-50 µm after ∼3000 s (Figure 9). During the second coarsening phase, beginning, or discernible, with the development of the strong correlation peak observed at a qmax value of 0.2 µm-1, R increased slightly from 25 to 35 µm. The increase of R during the two growth phases followed the power law R(t) ∼ tR with

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Figure 8. Temporal evolution of the scattered light intensity I(q) at small scattering angles averaged over the q range 0.01240.0417 µm-1. Each point is the average of two experiments.

Figure 9. Temporal evolution of the length scale R (µm-1) obtained from the scattering profiles after mixing at pH 4.2 BLG and AG at a protein-to-polysaccharide weight ratio of 2:1. The lines represent the power law R ∼ tR with the R exponent values indicated on the graph. Each point is the average of two experiments.

an exponent R equal to 0.45 (first phase) and 0.2 (second phase) (Figure 9). The maximum scattered intensity Imax obtained for the two growth phases followed the power law Imax(t) ∼ tβ with approximately the same exponent β value (0.5) (Figure 10). The first and second growth phases were then characterized by the relationships β ∼ 1.3R and β ∼ 2.5R, respectively. The morphology of phaseseparated interfaces was analyzed by determining the high q tail of the scattering curves through a log-log plot of I(q) versus q. The slope values obtained increased from 2.7 after the beginning of phase separation to 3.7 at around 5000 s, remained steady from 5000 to 10000 s, and then decreased to 3.3 at the end of the experiment (Figure 11). It is important to notice that the decrease of the slope began at the time where a shoulder developed at q > 1 µm-1 in the scattering functions. At the end of the experiment, a slope of 4.1 could be found in the region between the peak and the shoulder. Dynamic scaling of

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Figure 10. Temporal evolution of the maximum scattered light intensity Imax obtained from the scattering profiles after mixing at pH 4.2 BLG and AG at a protein-to-polysaccharide weight ratio of 2:1. The line represents the power law R ∼ t0.5. Each point is the average of two experiments.

Figure 11. Temporal evolution of the slope value of log(I) vs log(t) at 3.546 e q e 10.414 µm-1 obtained from the scattering profiles after mixing at pH 4.2 BLG and AG at a protein-topolysaccharide weight ratio of 2:1. Each point is the average of two experiments.

the scattering function was checked by plotting I(q)/Imax versus q/qmax in log-log coordinates (Figure 12). Data scaling did not lead to a satisfactory master curve. As time elapsed, the high q tail of the scattering curves first shifted to larger q values until about 1200 s; then it shifted to lower q values, indicating a sharpening of length scale. Discussion The level of charge neutralization between BLG and AG clearly controls the kinetics of phase separation and the structure of BLG/AG dispersions. When mixing BLG and AG at a weight ratio of 2:1 or 1:1, dispersions become immediately turbid, indicating quasi-instantaneous phase separation. BLG(2)/AG(1) dispersions are much more turbid than BLG(1)/AG(1) dispersions, which is the proof that more efficient charge neutralization occurs in the former. However, a common structural characteristic of


Figure 12. Normalized scattered intensity I/Imax as a function of the normalized wave vector q/qmax from the results given in Figure 8: 60 s (b); 2020 s (O); 4040 s (9); 6060 s (0); 10100 s (2); 20200 s (4); 30300 s ([); 36360 s (]). The continuous curve (s) is the theoretical Furukawa scaling curve for spherical particles in the later stages of spinodal decomposition.

both dispersions is the formation of vesicular or multivesicular coacervates. To our best knowledge, the BLG/ AG pair is the first reported biopolymer system able to spontaneously form giant vesicles. Generally, discrete liquid or gelled coacervates are obtained.86-91 Vesicles have also been observed through electrostatic interactions between water-soluble polyanionic polymers and diblock copolymers containing two water-soluble blocks, one cationic and one neutral.92 However, such systems generally form core-shell micellar structures instead of vesicles.93-98 The formation of giant vesicles induced by electrostatic interactions between macromolecules is then not a common event and could be limited to specific biopolymer pairs and to a narrow range of experimental conditions. For the BLG/AG system, optimum conditions for the formation of vesicles are a BLG/AG weight ratio close to unity. With a ratio of 8:1, previous observations revealed no formation of vesicular droplets.39 The different results suggest that efficient charge neutralization of AG molecules by BLG favors the condensation of macromolecular complexes in discrete droplets while insufficiently neutralized macromolecular complexes, that is, with an excess of negative charges, favor the formation of vesicles. It is worth noting that Bungenberg de Jong soon demonstrated that only negatively charged gelatin/acacia (86) Newton, D. W.; McMullen, J. N.; Becker, C. H. J. Pharm. Sci. 1977, 66, 1327. (87) Je´gat, C.; Taverdet, J. L. Polym. Bull. 2000, 44, 345. (88) McMullen, J. N.; Newton, D. W.; Becker, C. H. J. Pharm. Sci. 1982, 71, 628. (89) Chilvers, G. R.; Morris, V. J. Carbohydr. Polym. 1987, 7, 111. (90) Le´vy, M.-C.; Andry, M.-C. Therapie 1989, 44, 365. (91) Remunãn-Lo´pez, C.; Bodmeier, R. Int. J. Pharm. 1996, 135, 63. (92) Gohy, J.-F.; Varshney, S. K.; Je´roˆme, R. Macromolecules 2001, 34, 2745. (93) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Macromolecules 1996, 29, 6797. (94) Kataoka, K.; Togawa, H.; Harada, A.; Yasugi, K.; Matsumoto, T.; Katayose, S. Macromolecules 1996, 29, 8556. (95) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288. (96) Harada, A.; Kataoka, K. J. Controlled Release 2001, 72, 85. (97) Cohen Stuart, M. A.; Besseling, N. A. M.; Fokkink, R. G. Langmuir 1998, 14, 6846. (98) Gohy, J.-F.; Varshney, S. K.; Antoun, S.; Je´roˆme, R. Macromolecules 2000, 33, 9298.

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gum coacervates were able to vacuolize under external stress.2 Our hypothesis is supported by a recent study on DNA/cationic polymer interactions where it was shown that the higher charge density polymer forms compact particles with DNA while the lower charge density polymer forms supramolecular structures resembling vesicles, as shown by AFM.99 The mechanism of formation of vesicles remains unclear. A working hypothesis, based on the micelle-vesicle transition observed by mixing micellar solutions of cationic and anionic surfactants,100-102 would be that microphase separation induced by complexation between two amphiphilic biopolymers results in aggregates resembling micelles that spontaneously rearrange to form vesicles. The formation of core-shell micellar structures from electrostatic interactions between polyelectrolytes is often observed, as mentioned above. A central question is to understand how the hypothetic micellar aggregates transform into vesicles. As suggested by one reviewer of this paper, it has been shown that the formation of vesicles is preceded by the formation of disklike aggregates103 and that the initial formation of vesicles is based on a balance between the unfavorable edge energy of disklike aggregates and the bending energy required to form spherical structures.104 Another possibility would be that porous or micellar aggregates rearranged in order to reduce the volume of the more viscoelastic coacervated phase.63 The driving force would be the minimization of the elastic energy associated with the formation of an heterogeneous structure in a more elastic medium.105 We are currently investigating the mechanism of formation and the detailed structure of these vesicles. In particular, it seems crucial to characterize kinetically and structurally the different entities preceding the formation of vesicles, for instance to determine whether aggregates of biopolymer complexes are formed and what is their nature (porous, like micelles, fractal), whether the formation of disklike aggregates is possible, and so forth. Coacervates in BLG(1)/AG(1) dispersions induce initially the formation of a shallow maximum in scattered light intensity functions, indicating a preferential length scale in the system. All derived parameters from scattering functions such as Imax, length scale R, and the asymptotic high q tail of scattering functions (called slope hereafter) do not change markedly as a function of time. Dispersions are almost stable during the entire duration of the experiment. The stability is due to the presence of the continuous layer of AG around coacervates, as revealed by CSLM, which promotes a steric and electrostatic stabilization of the particles. The same mechanism is involved in the stabilization of pectin-gelatin coacervates.88 The size of coacervates remains consequently small and marginally increases as a function of time. Both the presence of an outer layer of AG and the small size of coacervates could explain the low slope value (∼1) calculated from the scattering functions at high q. Such a value indicates either that sharp interfaces between phase-separated structures are not developed, that is, the ratio of the thickness of interfaces to the characteristic (99) Wolfert, M. A.; Dash, P. R.; Nazarova, O.; Oupicky, D.; Seymour, L. W.; Smart, S.; Strohalm, J.; Ulbrich, K. Bioconjugate Chem. 1999, 10, 993. (100) O’Connor, A. J.; Hatton, T. A.; Bose, A. Langmuir 1997, 13, 6931. (101) Campbell, S. E.; Zhang, Z.; Friberg, S. E.; Patel, R. Langmuir 1998, 14, 590. (102) Tondre, C.; Caillet, C. Adv. Colloid Interface Sci. 2001, 93, 115. (103) Xia, Y.; Goldmints, I.; Johnson, P. W.; Hatton, T. A.; Bose, A. Langmuir 2002, 18, 3822. (104) Shioi, A.; Hatton, T. A. Langmuir 2002, 18, 7431. (105) Tanaka, H. Phys. Rev. Lett. 1996, 76, 787.

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length of domains is not small enough,106 or that the length scale for which there are inhomogeneities in refractive index is in the same range as 1/q. In fact, the only structural changes observed by CSLM and SALS encompass a slow sedimentation of coacervates, leading gradually to the disappearance of single vesicles, the collapse of the few multivesicular coacervates formed, and a transient new increase in the number of coacervates induced by delay coarsening of the system. The latter two points are also observed in BLG(2)/AG(1) dispersions and merit specific comments. The collapse of multivesicular coacervates into single vesicles results necessarily from coalescence of flocculated coacervates. This implies a liquidlike character of the coacervated phase, allowing its drainage from the vesicle membranes. The second point refers to the delayed appearance of new coacervates in both dispersions; that is, some droplets are formed first or coarsen faster than the other ones. In other words, different rates of coarsening exist during complex coacervation of BLG and AG. The observed delay may simply be caused by the mixing of dispersions, since it is obvious that perfect homogenization cannot be achieved instantaneously. In this case, part of the biopolymers in the fluid contact area may coacervate first. An additional possibility would reside in the polydispersity of the stock BLG and AG dispersions. As mentioned in the Experimental Section, AG contains mainly two molecular fractions with different molecular masses. Additionally, some few AG aggregates are also present. Concerning BLG, process-induced aggregates were discarded; however, it is technically impossible to totally remove small soluble aggregates. Knowing that larger molecular masses favor complex coacervation, the delay observed in the appearance of coacervates may well reside in the size (and surface charge) distribution of biopolymers. In BLG(2)/AG(1) dispersions where more BLG is available to neutralize AG, the polysaccharide does not provide efficient steric and electrostatic stabilization of coacervates. Coarsening of the system is then observed by CSLM and SALS. Regarding light scattering results, the initial appareance of a maximum in the scattering function that moves toward smaller wave vectors can be induced both by spinodal decomposition (SD) and nucleation and growth (NG), as indicated in the section Time-Resolved Small Angle Static Light Scattering. It is doubtful that the scattering patterns obtained could be due to initial stages of SD, since an initial exponential growth of Imax is not observed, and that qmax moves from the beginning to smaller wave vectors. Another interesting observation is that the slopes obtained from the scattering functions as a function of time tend to 4, which demonstrates the emergence of a nucleating regime.76,81 Considering further that coacervates with apparent diameters in the micrometer range are initially present in mixed dispersions, either SD occurs but the initial stages are too rapid to be detected or, alternatively, NG occurs. The value of 0.5 obtained for R, the power-law growth of the length scale R, is compatible with the early stages of NG and late stages of both NG and SD. The value of 0.5 obtained for β, the power-law growth of Imax, is not compatible with the early stages of NG or with the theoretical β ) 3R relationships expected in the late stages of SD and NG. The conclusion is that the mechanism of phase separation during complex coacervation cannot be identified unambiguously as classical SD or NG on the basis of the temporal evolution of scattering patterns. Polydispersity of biopolymers,

existence of different coarsening rates, correlations between coarsening mechanisms, and strong attractive interactions can modify phase-ordering kinetics, resulting in a system-dependent light scattering signature. The only certitude is that, at the beginning of the experiments, a nucleating regime has been reached. The coarsening of the droplets obtained is faster than could be expected from a purely diffusive or coalescence controlled mechanism (R ) 1/3), since the exponent R was around 0.5. It is first important to note that an exponent of 1/3 does not provide a priori definite information on the precise coarsening mechanism. CSLM micrographs on 1 wt % BLG/AG dispersions show the presence of single vesicles that grow in size and a great number of multivesicular coacervates. Then both mass transport by diffusion and flocculation/coalescence must occur. In a system of spherical particles, both the quench depth and hydrodynamic interactions can enhance the growth exponent.54 Considering that hydrodynamic interactions mainly operate in concentrated dispersions, and if we assume that mixing two oppositely charged biopolymers can be assimilated to a deep pH quench, then rapid charge neutralization of coacervates can accelerate particles’ coarsening in BLG(2)/AG(1) dispersions. In addition, convective flow induced by sedimentation of some insolubilized particles may also contribute to enhance the growth exponent. After the first growth phase (from 0 to 1212 s), an important structural transition phase occurs (from 1212 to 4000 s). This transition is characterized by two major consecutive events. The first event is the decrease in the number of scatterers with a concomitant transient appearance of large structural domains, that contribute to the sharp reduction in turbidity. The second event is a secondary phase separation that transiently increases the turbidity. The possible origin of the secondary phase separation has been commented on above. The decrease in the number of scatterers can be caused both by a flocculation/coalescence process and by sedimentation of particles. These two phenomena have been observed by CSLM and may explain the transient increase of the scattered light intensity at small scattering angles. The high q tail of scattering functions recorded during the two first coarsening phases displays a power-law exponent increasing from 2.7 to 3.7 as a function of time. An exponent of 2.7 indicates the presence of compact particles with fractal structures. From exponent values higher than 3, the structure of the particle surface is probed. Considering further that the exponent γ ) 6 - ds, where ds is the surface fractal dimension,107 the surface fractal dimension of particles decreases from 2.9 (at 400 s) to 2.3 at 4000 s. A considerable change in the structure of the interface occurs, that becomes less diffuse as time elapses. The value of the exponent did not change significantly at longer times (until 10 000 s), indicating that the interfacial structure is equilibrated after the two first coarsening phases. Following the structural transition, a new growth regime is observed (from 4000 to 36 000 s). This growth is characterized by the appearance of a marked correlation peak and a shoulder in the scattering function. The intensity of the peak Imax strongly increases as a function of time. The power-law exponent R of the length scale R (0.2) is indicative of a diffusion-controlled coarsening mechanism. The relationship β ) 2.5R is obtained, which may indicate that a late stage SD or NG is reached. The early events of the second growth process then occur

(106) Koga, T.; Kawasaki, K.; Takenaka, M.; Hashimoto, T. Phys. A 1993, 198, 473.

(107) Zhao, Y.-P.; Cheng, C.-F.; Wang, G.-C.; Lu, T.-M. Surf. Sci. 1998, 409, 703.


simultaneously to the late stages of the first one, resulting in a correlation of coarsening mechanisms, as suggested above. In parallel, scattered light intensity at the smaller scattering angles also increases. The presence of a shoulder in the scattering function can be interpreted as a manifestation of a degree of local ordering.57,108 The results suggest that an increasing number of clustered particles are being organized in a typical global structure with secondary local ordering. The exponent of the power law calculated in the high q tail region of scattering functions at long coarsening times is 3.3. The morphology of the interfaces within the domains becomes less sharp and more rough or fractal as time elapses. Such a characteristic resembles that observed by CSLM in the focal plane after coarsening of settled coacervates. The rough characteristic of the formed structures may be caused by aggregation or precipitation of interacting coacervates. A similar phenomenon has been observed with rod colloids strongly interacting through polymer-induced depletion.109 The thin optical cell used (500 µm) in our study may confine the coacervates, contributing to their interaction, and allows the observation in a vertical plane of a similar structure to that observed in a horizontal plane by CSLM. The last growth phase observed by SALS could correspond to the final stages of coacervation, that is, the structuring of the concentrated coacervated phase. Finally, the absence of a master curve after dynamic scaling of scattering functions even at long coarsening times is discussed now. For dynamic scaling to apply in late stages of phase separation, the system must have reached the equilibrium composition and the phase separation must be described by only one length and time scale.84 From the above description of the different growth phases, it seems obvious that more than one length and time scale exist in BLG(2)/AG(1) dispersions during coacervation. In particular, the presence of an interfacial length scale due to the fractal character of interfaces can be one cause of the absence of scaling,84 as well as the different coarsening rates detected. The resulting polydispersity of dispersions can induce a spread out of the scattering intensity over a wider q range.110 Settling of particles may also enhance the inherent polydispersity of biological systems by separating large structures from small ones.57,111 For all these reasons, it is not surprising that dynamic scaling does not apply to our system and that the Furukawa function differs from experimental data in a wide time window. The Furukawa function fits better our results only at the longer times where equilibrium composition is reached. (108) Kawakatsu, T.; Kawasaki, K.; Furusaka, M.; Kanaya, T. J. Chem. Phys. 1995, 102, 2247. (109) van Bruggen, M. P. B.; Lekkerkerker, H. N. W. Macromolecules 2000, 33, 5532. (110) Tuinier, R.; Dhont, J. K. G.; de Kruif, C. G. Langmuir 2000, 16, 1497. (111) Rouw, P. W.; Woutersen, A. T. J. M.; Ackerson, B. J.; de Kruif, C. G. Phys. A 1989, 156, 876.

Langmuir, Vol. 18, No. 26, 2002 10333

Summary Electrostatic interactions between oppositely charged β-lactoglobulin (BLG) and acacia gum (AG) induce an attractive phase separation, called complex coacervation. This self-assembly process leads to the formation of supramolecular spherical structures called coacervates. Confocal scanning laser microscopy (CSLM) and small angle static light scattering (SALS) are adequate methods to study the structuring and phase-ordering kinetics of complex coacervation. In well defined conditions, like in the present study, coacervates appear as giant vesicles. Interactions between these vesicles contribute to the formation of transient multivesicular structures. When there is not enough BLG to efficiently neutralize the negative charges of AG, a surface layer of AG stabilizes coacervates, inhibiting their interactions. In these conditions, once coacervates are formed, they settle very slowly. When sufficient BLG is present, charge neutralization of AG is sufficient to render the system highly unstable. Coacervates rapidly increase in size through diffusioninduced mass transport, solvent swelling, and interactions between coacervates. Interfaces of separated phases sharpen with time but remain fractal. Neutralized coacervates and multivesicular structures settle and form a concentrated coacervated phase. Aggregation and precipitation of coacervates in a confined environment lead to the formation of an equilibrated heterogeneous structure whose interfaces are fractal. The inherent polydispersity of biological macromolecules results in different rates of coarsening in mixed dispersions. After mixing BLG and AG, one fraction of biopolymers strongly interacts, resulting in rapid demixing and coarsening of particles, whereas another fraction more weakly interacts, resulting in slower demixing and coarsening rates. The two mechanisms appear to occur simultaneously. Polydispersity of dispersions, different length and time scales, and gravity effects preclude dynamic scaling of scattering data. Regarding phase-ordering kinetics, the level of charge neutralization and possible correlations between different coarsening mechanisms result in a systemdependent light scattering signature. Thus, the temporal evolution of the parameters determined from light scattering patterns (Imax, R) does not allow us to unambiguously describe the mechanism of phase separation on the basis of the classical spinodal decomposition or nucleation and growth theories. Acknowledgment. The authors thank the “Ministe`re Franç ais de l’Education Nationale, de la Recherche et de la Technologie”, for its Ph.D. fellowship (C.S.). The German office for universitary exchanges (DAAD) financially supported CSLM experiments at the Department of Biopharmaceutics and Pharmaceutical Technology (University of Saarland, Germany). LA0262405

Self-Assembly of Î²-Lactoglobulin and Acacia Gum in Aqueous Solvent

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