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Phase Separation and Association of Globular Protein Aggregates in the Presence of Polysaccharides: 2. Heated Mixtures of Native β-Lactoglobulin and K-Carrageenan Philippe Croguennoc, Taco Nicolai,* and Dominique Durand Polyme` res, Colloı¨des, Interfaces, UMR CNRS, Universite´ du Maine, 72085 Le Mans Cedex 9, France
Allan Clark Unilever Research Colworth, Sharnbrook, Bedford MK441LQ, U.K. Received November 30, 2000. In Final Form: March 24, 2001 Aqueous solutions of globular proteins (β-lactoglobulin) at pH 7 and 0.1 M NaCl were heated in the presence of various concentrations of polysaccharide (κ-carrageenan). The fraction of unaggregated proteins was determined as a function of heating time with size exclusion chromatography. The rate at which the proteins aggregate is independent of the polysaccharide concentration at least up to 9 g/L κ-carrageenan. The protein aggregates were characterized using light scattering. At modest concentrations (up to 1 g/L) the presence of κ-carrageenan accelerates the growth of the aggregates and therefore the gel formation, but the structure of the aggregates is not modified. At higher concentrations κ-carrageenan induces phase separation of protein aggregates.
Introduction Heat-denatured globular proteins aggregate and form gels at sufficiently high concentrations.1 This phenomenon is important for applications in the food industry and has been intensively investigated in the past. More recently, mixed systems of globular proteins and polysaccharides have become the focus of attention, because addition of a polysaccharide can modify the texture of a protein gel. A broad range of phenomena may be observed for mixed gels of globular proteins and polysaccharides, because many polysaccharides form gels themselves under certain conditions. In addition, protein may form a complex with polysaccharides. In this paper we will consider the case where the polysaccharide may be considered as a semiflexible chain and does not show specific interactions either with the protein present or with other polysaccharide chains. For a few such systems the influence of a polysaccharide on the elastic modulus and the microscopic structure of a heat-set globular protein gel has been investigated.2-9 * To whom correspondence may be addressed. Email: Taco.
[email protected]. (1) Clark, A. H. Gelation of globular proteins. In Functional Properties of Food Macromolecules 1999, p 77-142. (2) Syrbe, A.; Fernandes, P. B.; Dannenberg, F.; Bauer, W.; Klostermeyer, H. Whey Protein + Polysaccharide Mixtures: Polymer Incompatibility and Its Application, in Food macromolecules and colloids; Dickinson, E., Walstra, P., Lorient, D., Eds.; The Royal Society of Chemistry: Cambridge; 1995; pp 328-339. (3) Ndi, E. E.; Swanson, B. G.; Barbosa-Canovas, G. V.; Luedecke, L. O. J. Agric. Food Chem. 1996, 44, 86. (4) Manoj, P.; Kasapis, S.; Hember, M. W. N. Carbohydr. Polym. 1997, 32, 141. (5) Mleko, S.; Li-Chan, E. C. Y.; Pikus, S. Food Res. Int. 1997, 30, 427. (6) Fernandes, P. B. Interactions in Whey Protein/Polysaccharide Mixtures at pH 7. In Polysaccharide Association Structures in Food; Walter, R. H., Ed.; Marcel Dekker, Inc.: New York, 1998. (7) Capron, I.; Nicolai, T.; Smith, C. Carbohydr. Polym. 1999, 40, 233. (8) Neiser, S.; Draget, K. I.; Smidsrod, O. Food Hydrocolloids 2000, 14, 95. (9) Ould Eleya, M. M.; Turgeon, S. L. Food Hydrocolloids 2000, 14, 29.
Pure globular protein gels vary in homogeneity depending on the pH10 and the ionic strength.11,12 If the pH is close to the isoelectric point of the protein, or if the ionic strength is high, so-called particulate gels are formed that contain protein-rich domains with a diameter of the order of micrometers. Away from the isoelectric point so-called finely stranded gels are formed that are homogeneous above the length scale of a few hundred nanometers. In the latter case the presence of a polysaccharide generally induces the formation of protein-rich microdomains,2,3,6,8 while in the former case it may actually inhibit their formation.8 The microdomains are formed by a process of microphase separation that occurs during the protein gelation, which is halted by the gel formation. The presence of polysaccharides may induce gelation of globular proteins in conditions where the pure proteins do not gel.3,9 In other cases the gel modulus may be increased or decreased by the presence of polysaccharides depending on the concentration of the latter.7,8 Apart from their industrial applications, one may argue that heated globular protein solutions are good model systems for irreversibly aggregating colloids. In contrast to other model systems such as silica or gold spheres, the aggregation of globular proteins is easily quenched by dropping the temperature. In addition, the aggregates are stable to dilution and may therefore be investigated by various techniques at different stages of the process. Heated mixtures of globular proteins and polysaccharides therefore represent good models to study the influence of flexible polymers on the aggregation and gelation of small colloids. In heated mixtures of globular proteins and polysaccharides the aggregation and phase separation events occur simultaneously, with very different kinetics. The advantage of using proteins is that the aggregation kinetics (10) Langton, M.; Hermansson, A.-M. Food Hydrocolloids 1992, 5, 523. (11) Bowland, E. L.; Foegeding, E. A. Food Hydrocolloids 1995, 9, 47. (12) Verheul, M.; Roefs, S. P. F. M. J. Agric. Food Chem. 1998, 46, 4909.
10.1021/la001675i CCC: $20.00 © 2001 American Chemical Society Published on Web 06/12/2001
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can be almost halted, by cooling so that the effect of phase separation can be studied for differently sized aggregates. In contrast to the heated mixtures, mixed systems of aggregated proteins and polysaccharides at room temperature have received very little attention so far.13,14 In part 1 of this series, however, we have reported a study of phase separation in mixtures of κ-carrageenan (κ-car) and aggregates of preheated β-lg. In the conditions used in these experiments (0.1 M NaCl and pH7) κ-car has the conformation of a flexible coil and is fully compatible with native β-lg. We showed that above a certain κ-car concentration protein-rich microdomains are formed by the β-lg aggregates. These microdomains then slowly precipitate, and a macroscopic phase separation is observed. The phase separation leads to fractionation of the size distribution of the aggregates, with the larger aggregates residing in the microdomains and the smaller aggregates, plus the κ-car, being in the supernatant. The lower size limit of the aggregates that phase separate decreases with increasing κ-car concentration but is only weakly dependent on the protein concentration. The microdomains are homogeneous down to length scales equal to about the size of the smallest phase-separated β-lg aggregates. We have also shown that the microdomains are initially liquid and disperse into the original aggregates after dilution. However, after some time, the aggregates in the microdomains associate irreversibly and form microgel particles. Here we investigate the influence of κ-car in situ during the heat-induced aggregation and gelation processes of β-lg. Earlier we reported an investigation of such gelled mixtures using mechanical measurements and transmission electron microscopy.7 The aim of the work presented here was to study the influence of κ-car on the aggregation process of β-lg that precedes the gel formation. Experimental Section Materials. The κ-carrageenan used for this study was an alkali-treated extract from Eucheuma cottonii supplied by SKW Biosystems, (Baupte, France). The solutions were prepared as follows: A freeze-dried sample of κ-carrageenan in the sodium salt form was dissolved while stirring for a few hours in hot Millipore water (70 °C). The pH was adjusted to 9 to eliminate the risk of hydrolysis during the preparation. The solution was first dialyzed against Millipore water at pH 7 to eliminate excess salt and subsequently against 0.1 M NaCl. A 200 ppm NaN3 (0.003 M) solution was added to avoid bacterial growth. The solutions always contained a small amount of large aggregates which perturbed the light scattering results, so these aggregates were removed by filtration through 0.2 µm pore size Anotop filters. The absence of aggregated material, and other spurious scatterers, was checked by dynamic light scattering. κ-car shows a reversible coil-helix transition on lowering the temperature below a critical value (Tc).15,16 The value of Tc depends on the concentration and type of ions in the systems. In the helix conformation, κ-car usually aggregates and forms a self-supporting gel if the concentration is sufficient. For the present study we used κ-car with sodium counterions, and we adjusted the ionic strength with NaCl. Under these conditions κ-car has the coil configuration at temperatures above 11 °C. The κ-car used in this study was characterized using size exclusion chromatography (SEC) and static and dynamic light scattering. The following characteristics were obtained: weight average (13) Syrbe, A. Polymer Incompatibility in Aqueous Whey Protein and Polysaccharide Solutions: phase separation phenomena and microgel particle formation. Munich Technical University: Munich, 1997; p 246. (14) Tuinier, R.; Dhont, J. K. G.; De Kruif, K. G. Langmuir 2000, 16, 1497. (15) Rochas, C.; Landry, S. Carbohydr. Polym. 1987, 7, 435. (16) Meunier, V.; Nicolai, T.; Durand, D. Macromolecules 2000, 33, 2497.
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Figure 1. Schematic representation of the aggregation process of β-lg at pH 7 and 0.1 M salt. molar mass Mw ) 4.3 × 102 kg/mol, polydispersity index Mw/Mn ≈ 2, z-average radius of gyration Rgz ) 72 nm, and z-average hydrodynamic radius Rhz ) 30 nm.16 The β-lg used in this study was a gift from Lactalis (batch no. 754). High-performance liquid chromatography shows that the sample consists of equal fractions of genetic variants A and B. Solutions were prepared by dialyzing against distilled and deionized water at pH 7. The solutions were filtered through 0.2 or 0.45 µm pore size Anatope filters depending on the concentration. The concentrations were determined after filtration by SEC with refractive index detection using a refractive index increment dn/dc ) 0.189 g/mL for β-lg17 and dn/dc ) 0.145 g/mL for κ-car. The protein concentration was also determined using UV absorption and an extinction coefficient 0.96 g/(L/cm).18 Both methods gave the same results within a few percent. Heat-induced aggregation was induced by placing the mixtures covered with a layer of paraffin oil in a thermostat bath set at the heating temperature within 0.2 °C. The samples were at the set heating temperature within a few minutes. Methods. Light scattering measurements were made using an ALV-5000 multibit multitau correlator and a solid-state laser (Spectra Physics, Millenium II) operating with vertically polarized light with wavelength λ ) 532 nm. The range of scattering wave vectors covered was 3.0 × 10-3 < q < 3.5 × 10-2 nm-1. The temperature was controlled by a thermostat bath set at 20 ( 0.1 °C. SEC experiments were carried out at room temperature with a TSK PW 5000 + PW 6000 column set (30 cm + 60 cm) in series. We used a combination of refractive index detection and UV detection at 278 nm. The columns were eluted with a 0.1 M NaNO3 solution at a flow rate of 1 mL/min; 200 ppm NaN3 was added as a bacteriostatic agent. The injected volume was 300 µL, and the injected concentration was approximately 0.1%. Confocal scanning laser microscopy measurements were made in collaboration with V. Normand (Unilever Research, Colworth, U.K.) with a MRC 600 CSLM (Bio-Rad Inc., Hemel Hempstead, U.K.) in combination with an Ortholux microscope (Leica, Milton Keynes, U.K.). Optical microscopy experiments were done in collaboration with D. Ausserre (PSPI, Universite´ du Maine, France) using a new visualization method.
Results and Discussion We have investigated the heat-induced aggregation and gelation of 20 g/L β-lg at 0.1 M NaCl and pH 7 in the presence of varying amounts of κ-car. But before we discuss the influence of κ-car, we will first summarize briefly what is known from earlier studies about the aggregation and gelation of heat-denatured β-lg without added polysaccharide.19-22 Figure 1 shows a schematic drawing of the aggregation process at pH 7. At room temperature native β-lg is present mainly in the form of dimers in equilibrium with a small (17) Perlmann, G. E.; Longsworth, L. G. J. Am. Chem. Soc. 1948, 70, 2719. (18) Townend, R.; Winterbottom, R. J.; Timasheff, S. N. J. Am. Chem. Soc. 1960, 82, 3161. (19) Gimel, J.-C., Durand, D.; Nicolai, T. Macromolecules 1994, 27, 583. (20) Aymard, P.; Gimel, J.-C.; Nicolai, T.; Durand, D. J. Chim. Phys. 1996, 93, 987. (21) Le Bon, C.; Nicolai, T.; Durand, D. Macromolecules 1999, 32, 6120. (22) Le Bon, C., Nicolai, T.; Durand, D. Int. J. Food Sci. Technol. 1999, 34, 451.
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Figure 2. Fraction of unaggregated β-lg as a function of heating time at 70 °C in a solution containing 20 g/L β-lg and various concentrations of κ-car indicated in the figure.
fraction of monomers. If we increase the temperature, the equilibrium shifts toward the monomer which has an increasing probability to denature. Denatured monomers cluster to form small well-defined particles that contain approximately 100 monomers. In a second step these primary aggregates cluster to form large polydisperse aggregates with self-similar structures characterized by a fractal dimension df ) 2. The structure and the size distribution of the aggregates are independent of the concentration of the proteins and the heating temperature. If the β-lg concentration is larger than a certain minimum (about 7 g/L in 0.1 M salt), the aggregates can grow sufficiently to fill the whole space and a gel is formed. Interestingly, the second step of the aggregation process is inhibited by electrostatic repulsion, but not the first step which is thought to involve exchange of disulfide bridges. The temperature dependence of the aggregation rate and also the gel time is controlled by the unfolding of the proteins and is an activated process with activation energy 300 kJ/mol. The first step of the aggregation process, which determines the rate at which monomers are used, increases with the square root of the protein concentration. On the other hand the time needed to form large aggregates and the gel increases strongly with decreasing the protein concentration and diverges at about 7 g/L. The reason is that the growth of the aggregates slows down as the reservoir of native proteins decreases. The growth stagnates if most native proteins have aggregated before the aggregates are sufficiently large to fill up the whole space and the system does not gel. Figure 2 shows the fraction of unaggregated proteins as a function of heating time at 70 °C for a solution containing 20 g/L β-lg and varying amounts of κ-car between 0 and 8 g/L. This fraction was determined using SEC by measuring the surface area corresponding to the native protein peak. Within the experimental error there is no influence of κ-car on the rate of protein consumption, i.e., on the rate of the first aggregation step. This observation is consistent with dynamic scanning calorimetry (DSC) measurements that showed that the denaturation temperature of β-lg is not modified by the presence of κ-car.9 We determined the structure of the β-lg aggregates by measuring the q-dependent scattered light intensity of highly diluted samples at room temperature. In all cases the contribution of κ-car and residual unaggregated β-lg to the total scattering intensity (I(q)) was negligible. We have calculated the scattering intensity of the aggregates relative to that of β-lg monomers at the same concentration (Imon): Ir(q) ) I(q)/Imon. Notice that in the range covered by light scattering Imon does not depend on q. We may
Figure 3. (a) Double logarithmic representation of the q dependence of the relative scattering intensity of higly diluted β-lg aggregates formed by heating a solution containing 20 g/L β-lg and 1 g/L κ-car at 70 °C for various times indicated in the figure. (b) Same data as in part a after rescaling the vertical axis with mw ) Ir(qf0) and the horizontal axis with Rgz.
write Ir(q) in terms of the weight averaged number of proteins per aggregate, mw, and the structure factor, S(q):
Ir(q) ) mwS(q)
(1)
The solutions were sufficiently dilute that interactions can be considered negligible and S(q) represents the z-averaged structure factor of the aggregates. The zaveraged radius of gyration, Rgz, may be calculated from the initial q dependence of S(q) using the Zimm approximation: S(q)-1 ) 1 + 1/3(qRgz)2. We have shown elsewhere for pure β-lg that S(q) is independent of heating time if plotted as a function of qRgz. In addition S(q) does not depend on the protein concentration or on the heating temperature. Figure 3a shows Ir(q) of a solution containing 20 g/L β-lg heated for different lengths of time at 70 °C in the presence of 1 g/L κ-car. The aggregation number mw ) Ir(qf0) increases with increasing heating time and the q dependence becomes more important. Figure 3b shows Ir normalized by mw, i.e., S(q), as a function of qRgz. In this representation all data superimpose as was observed
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Figure 4. Structure factor of highly diluted β-lg aggregates formed by heating a solution containing 20 g/L β-lg and various concentrations of κ-car indicated in the figure. The straight line has slope -2.0.
Figure 5. Double logarithmic representation of the weight average aggregation number as a function of the z-average radius of gyration for β-lg aggregates formed by heating a solution containing 20 g/L β-lg and various concentrations of κ-car indicated in Figure 4. The straight line has slope 2.0.
earlier for pure β-lg. This feature can be exploited to obtain values of mw and Rgz for large aggregates where the Zimm approximation is no longer valid. Figure 4 shows S(q) obtained in the same way for samples in the presence of different κ-car concentrations between 0 and 1 g/L. Clearly, S(q) is the same for all samples investigated, which implies that the structure of the aggregates is not influenced by κ-car in the range investigated. For large values of qRgz we find Ir ∝ q-2.0, which shows that the aggregates have a self-similar structure with fractal dimension df ) 2.0. Of course, Figure 4 only shows that the structure of the aggregates is the same on length scales probed by light scattering. That the local structure is also the same may be deduced from Figure 5 where we have plotted mw as a function of Rgz. Again with the data superimposed for all κ-car concentrations and for larger Rgz, we find mw ∝ Rgz2.0. The exponent is the fractal dimension and its value is consistent with that obtained from S(q). The fact that the data at different κ-car concentrations not only have the same slope but actually superimpose implies that the local structure of the aggregates is not modified by κ-car, because a different local structure would lead to a different prefactor of the power law relation. This means that the structure of the primary aggregates is not influenced by the presence of κ-car. It appears that neither the rate of the native protein consumption nor the structure of the aggregates are
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Figure 6. Double logarithmic representation of the time dependence of the weight average aggregation number and the z-average radius of gyration of β-lg aggregates formed in a solution containing 20 g/L β-lg and various concentrations of κ-car indicated in Figure 4 heated at 70 °C.
modified by the presence of κ-car. On the other hand, κ-car accelerates the growth of the aggregates as is shown in Figure 6. When we compare the growth at different κ-car concentrations, we find that initially the growth is the same as in the absence of κ-car. But once the β-lg aggregates have reached a certain size, the growth rate increases and a gel is formed rapidly. The influence of κ-car becomes significant when its concentration approaches the overlap concentration, which may be estimated as C* ) 3Mw/(Na4πRgz3) and is about 0.5 g/L.23 The size of the β-lg aggregates where κ-car influences the growth rate decreases rapidly with increasing κ-car concentration between 0.25 and 1.0 g/L, although the correlation length of κ-car varies little in this range (2616 nm).23 If we use higher κ-car concentrations, we observe very rapid formation of large particles together with small aggregates and the residual fraction of native proteins. These systems either develop into a gel or phase separate into a turbid bottom phase and a transparent top phase. Whether these systems gel or phase separate depends not only on the concentrations of β-lg and κ-car but also on the temperature. For example if a solution containing 20 g/L β-lg and 3 g/L κ-car is heated at 70 °C, we observe macroscopic phase separation, while if the same solution is heated at 76 °C, a self-supporting stable gel is formed. Optical microscopy shows the presence of spherical microdomains that have approximately the same size in the precipitate formed at 70 °C and the gel formed at 76 °C: see Figure 7. The κ-car concentration where we observe phase separation in the heated mixtures is similar to that where we observed the phase separation with preheated protein aggregates at room temperature. Notice that the rate of formation of the primary aggregates and thus the protein consumption is not influenced by κ-car at least up to 9 g/L where ξ ) 3.4 nm,23 even though the aggregates phase separate. We have investigated the structure of the gels on a larger scale using confocal microscopy. Figure 8 shows micrographs of heated solutions containing 20 g/L β-lg and different concentrations of κ-car. Within the resolution of the confocal microscope, the β-lg gels formed in the presence of no, or a small amount of, κ-car appear homogeneous. However, in the presence of more κ-car one observes a microphase separation leading to the formation of β-lg rich domains that appear to cluster. One also observes cavities on the length scale of tens of micrometers. Gels formed in the presence of larger amounts of κ-car are (23) Croguennoc, P.; Meunier, V.; Durand, D.; Nicolai, T. Macromolecules 2000, 33, 7471.
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Figure 7. Micrographs obtained from optical microscopy of solutions containing 20 g/L β-lg and 3 g/L κ-car heated during several hours at 70 °C (top) and 76 °C (bottom).
easily compressed by centrifugation and we find most of the κ-car in the supernatant. In ref 7 we presented micrographs at higher resolution using transmission electron microscopy on which clusters of protein-rich spherical microdomains are clearly visible. We also visualized the κ-car using antibodies which showed that it is situated in the cavities between the microdomains. The micrographs shown in Figure 8 were made on samples heated separately and transferred to the confocal microscope at room temperature. If we observe such a sample through the microscope during the heating process, we see behavior typical of spinodal decomposition, i.e., a sudden appearance of lighter and darker domains over the whole sample with a contrast that increases in time. We do not give a quantitative analysis of this phenomenon here, mainly because we believe that there is a strong influence from the proximate interface with air or the glass cover slip. The dynamics of the spinodal decomposition of small protein aggregates in the presence of an exopolysaccharide has been studied in detail by Tuinier et al.14 using small angle light scattering. As we showed in ref 7, the presence of κ-car has a profound influence on the mechanical properties of the gels. On one hand the gels form more quickly with increasing κ-car concentration, while on the other hand the coarsening of the gel structure caused by the microphase separation weakens the gel. The gel modulus as a function of κ-car concentration goes through a maximum, and, as mentioned above, in some cases we do not even observe gel formation, but rather macroscopic phase separation.
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Our findings on heated β-lg solutions in the presence of κ-car substantiate those of Syrbe13 on whey proteins in the presence of pectin which is also an anionic polysaccharide. Syrbe found using mainly optical microscopy that protein-rich spherical microdomains are formed that have a tendency to stick to each other. The size of the microdomains decreased with increasing pectin concentration but depended very little on the protein concentration. He also observed that if the temperature was slowly raised, the microdomains were larger than if they are quickly brought to the final temperature. All observations may be explained by the phase separation of protein aggregates induced by polysaccharide. As discussed in part 1 and ref 14, a possible driving force for the phase separation is depletion of polysaccharide chains at the surfaces of the aggregates. It is not possible to describe the effect of depletion quantitatively with currently available theories, because the aggregates have a fractal structure and are not fully rigid which reduces the effect compared to hard spheres with the same size. Nevertheless, it may be understood on a qualitative level that larger aggregates will feel the effect of depletion at lower polysaccharide concentrations. Notice, however, that depletion is not necessarily the only driving force for the phase separation. A slight incompatibility between proteins and polysaccharides may not be noticeable for individual low molar mass proteins because of the large contribution of entropy of mixing. But it could nevertheless induce phase separation for larger aggregates for which the entropy of mixing is much lower. For a given κ-car concentration the heat-induced aggregation of β-lg proceeds largely unperturbed by the κ-car until the aggregates reach a certain size. At that point the aggregates feel an effective attractive interaction possibly induced by depletion, which locally increases the aggregate concentration and thereby accelerates the growth. As the aggregates grow, the attractive interaction increases, which in turn increases the growth rate even more, until a gel is formed. There is a delicate interplay between the rate of aggregation and the strength of the interaction. The aggregation rate is very sensitive to the temperature, but not to the polysaccharide concentration as long as the aggregates are small enough so that the incompatibility is negligible. On the other hand the depletion interaction is not a strong function of the temperature, but it increases strongly with increasing polysaccharide concentration. At low polysaccharide concentrations the attractive interaction is not strong enough to drive phase separation, but favors the association of large aggregates, which leads to an increase of the growth rate of the aggregates without modifying their structure. Higher polysaccharide concentrations cause the formation of spherical microdomains that cluster and form a gel. The microdomains are denser with increasing polysaccharide concentration, which means that for a given protein concentration the volume fraction of the microdomains is smaller. This explains why the gel coarsens and becomes mechanically weaker with increasing polysaccharide concentration. Of course, the volume fraction of the microdomains also decreases with decreasing protein concentration with a similar effect on the structure and the strength of the gel. At high polysaccharide concentrations and low protein concentrations the volume fraction of the microdomains may become so small that no space-filling gel is formed, but instead the microdomains sediment. The final structure of the gel depends not only on the concentration of protein and polysaccharide but also on the heating temperature. At high temperatures the
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Figure 8. Micrographs obtained from confocal laser scanning microscopy of solutions containing 20 g/L β-lg and various concentrations of κ-car indicated in the figure heated during several hours at 70 °C.
aggregation may be faster than the formation of the microdomains and the system may gel before the microdomains are fully developed. Syrbe showed that the rate at which the sample is heated is very important.13 Even if the final temperature is high enough so that the aggregation is very fast, the phase separation still has a possibility to develop if this temperature is reached very slowly. Notice that, in the present work, the samples were quickly heated to the final temperature. Once the gel is formed, further growth and densification of the microdomains are inhibited. Therefore we would expect that more homogeneous and stronger gels form at higher temperatures. But a more systematic investigation is needed of the gel structure and the shear modulus as a function of the heating temperature and the concentrations of β-lg and κ-car in order to substantiate this point. For a given heat treatment the elastic modulus of globular protein gels increases at low polysaccharide concentrations and decreases at higher concentrations.7,8 It has not yet been established, however, whether a small amount of phase separation actually reinforces the gel. It could simply be a kinetic effect because the aggregation rate increases with increasing polysaccharide concentration. It may be argued that the interplay between phase separation and aggregation of heat-denatured globular proteins is important not only in the presence of polysaccharides but also for pure protein solutions. As mentioned
in the Introduction, similar protein-rich microdomains are observed in heat-set protein gels close to the isoelectric point or at high ionic strength, i.e., when electrostatic repulsion is reduced. This may explain why the structure of the gel and thus its mechanical properties are sensitive to the temperature and heating rate, while the structure of the aggregates themselves does not depend on the heating temperature. Conclusions The presence of κ-car in solutions of heat-denatured β-lg does not influence the rate of protein consumption nor the structure of the aggregates. However, it increases the aggregation rate and at higher κ-car concentrations it induces phase separation which leads to the formation of protein-rich spherical microdomains. The influence of κ-car is felt only for protein aggregates above a critical size which decreases with increasing κ-car concentration. The microdomains cluster and form either a gel or precipitate depending on the concentration and the temperature. The mesoscopic structure of the gel is determined by the balance between protein aggregation and phase separation. Acknowledgment. P.C. acknowledges financial support from Unilever Research. LA001675I
