In Situ Measurement of the Displacement of Protein Films from the Air

Jul 14, 2001 - Interfacial Characterization of β-Lactoglobulin Networks: Displacement by Bile Salts. Julia Maldonado-Valderrama , Nicola C. Woodward ...
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Biomacromolecules 2001, 2, 1001-1006

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In Situ Measurement of the Displacement of Protein Films from the Air/Water Interface by Surfactant Alan R. Mackie,*,† A. Patrick Gunning,† Mike J. Ridout,† Peter J. Wilde, and J. Rodriguez Patino‡ Institute of Food Research, Norwich Research Park, Colney, NR4 7UA, United Kingdom, and Departamento de Ingenieria Quimica, Facultad de Quimica, Universidad de Sevilla, 41012 Sevilla, Spain Received April 2, 2001; Revised Manuscript Received June 13, 2001

The displacement of spread protein films from the air/water interface by surfactant was followed using Brewster angle microscopy (BAM) and interfacial rheology. The displacement of β-lactoglobulin and β-casein by a nonionic surfactant was monitored as a function of both surface pressure and time. In both cases, protein displacement occurred over the same surface pressure range that had been observed previously by atomic force microscopy (AFM). In the case of the β-lactoglobulin, surfactant domains grew large enough in the protein film to be visible in the BAM images. The shapes of the domains were very similar to those seen previously by AFM in the late stages of displacement. The results from both proteins confirm the results published previously while highlighting some implications for the application of the “orogenic” model of displacement for large protein films. The surface rheological data showed that the β-lactoglobulin/surfactant mixed film retained much of its elasticity until the latter stages of displacement. This indicates that at least in the early stages of displacement, the mixed film was dominated by the behavior of the protein in the film. Introduction The majority of manufactured, processed foods are multiphase systems. That is, they contain two or more immiscible phases (aqueous, oil, or gas phases) in the form of foams and emulsions. Stability of these systems is generally achieved through a protective interfacial layer around emulsion droplets or foam bubbles. In food systems, the interfacial layer often comprises both proteins and surfactants. Considerable progress has been made in the understanding of the way in which proteins and surfactants stabilize multiphase systems.1-3 Proteins form an immobile viscoelastic interface forming a mechanical barrier, whereas lipids and surfactants rely on a high degree of mobility to stabilize interfaces by the Gibbs-Marangoni mechanism. Thus, interest lies in describing, and if possible, predicting, the interfacial behavior of mixed systems. Several methods have been used to study competitive adsorption between proteins and surfactants.4,5 Recent work has allowed the indirect study of competitive adsorption at a molecular level.6-8 This has led to the proposal that protein is displaced by a three-stage “orogenic” mechanism.6 In the first stage, the surfactant adsorbs into small defects in the protein film and this gradually forms small pools of surfactant, which increase the surface pressure of the film. As adsorption continues, the surfactant domains grow but the thickness of the protein layer is unaffected. This is the compression phase. In stage two, the surfactant domains continue to grow but the change in area of the * To whom correspondence should be addressed. Tel: 44 1603 255261. Fax: 44 1603 507723. E-mail. [email protected]. † Institute of Food Research. ‡ Universidad de Sevilla.

protein film is compensated for by a corresponding change in film thickness. This is the collapse phase. Finally, in stage three, when the surface pressure is sufficiently high the protein network breaks down and the protein is displaced from the interface probably in the form of aggregates. However, until now it has not been possible to visualize this orogenic displacement of protein by surfactant directly at the liquid/gas interface. This would definitively rule out any idea that some of the features seen by atomic force microscopy (AFM) were due to the sample preparation. The advent of high-resolution Brewster angle microscopy (BAM) has made it possible to directly visualize interfacial films with a reasonable spatial resolution (ca. 1 µm). Over the past few years, much work has been done using BAM to study mixed lipid/protein systems. However, this work has generally looked at lipid monolayers perturbed by the insertion of proteins.9-11 In particular, the work of Rodriguez Patino et al. in Seville has studied β-casein and its interaction with monoglycerides.12-14 This work led to the conclusions that the thickness of a β-casein layer at the air/water interface was a function of the surface pressure and that monoolein was unable to completely displace β-casein from the interface. Previous measurements of β-lactoglobulin displacement6,7 have used AFM to characterize the orogenic mechanism by which the displacement occurs. These studies showed that in the later stages of displacement by the nonionic surfactant Tween 20, many surfactant domains perforated the protein film. A proportion of these domains were found to be greater than 10 µm in diameter. Because of their size, these domains should be visible optically.

10.1021/bm015540i CCC: $20.00 © 2001 American Chemical Society Published on Web 07/14/2001

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Interest has not been confined to surface composition. Interfacial rheology has been seen as key to understanding the interfacial behavior of protein systems.15,16 In particular, the relationship between dilatational rheology and functional properties such as foam stability has been studied.17 Thus, we have sought to use it here to provide information about the strength of the interfacial protein film as an adsorbing surfactant disrupts it. This then provides information about the strength of interactions within the film and how these change when the protein film is challenged by surfactant. Materials and Methods The milk proteins used in this study were β-lactoglobulin (L-0130, lot 91H7005) and β-casein (C-6905, lot 12H9550) from Sigma Chemicals (Poole, U.K.). Samples were initially prepared at 2 mg mL-1 in water. The water used in this study was surface pure (γ0 ) 72.6 mN m-1 at 20 °C) cleaned using a water purification system. The Tween 20 (polyoxyethylene sorbitan monolaurate) was obtained as a 10% solution (Surfact-Amps 20) from Pierce (Rockford, IL). The other surfactant used in the study was sodium lauryl sulfate (SDS), obtained as a 10% solution (L-4522, lot 97H8505) from Sigma Chemicals (Poole, U.K.). The Brewster angle microscope used in this study was a BAM2 from NFT (Gottingen, Germany). Details of both the technique and the instrument are available in the literature.18 In brief, the relative reflectivity (I) at the subphase Brewster angle is given by the following equation. I ) Cd2

(1)

where C is a constant dependent upon the optical properties of the interfacial film and d is the thickness of the film. In the experiments presented here, the reflectivity for the protein films was relatively high and that for surfactant films relatively low. This is consistent with measurements made previously by others on mixed protein surfactant/lipid systems.19 Thus, in all BAM images the light regions are protein rich and the dark regions are surfactant rich. The imaging conditions were adjusted to optimize image quality and not to enable quantitative measurement of reflectance. Thus, generally as the surface pressure increased the shutter speed was also increased. Surface pressure measurements were made during the BAM experiments using a platinum Wilhelmy plate and a Langmuir trough with one mobile barrier and a maximum useable area of 562 cm2. All experiments were performed at 20 °C with distilled water as the subphase. Protein was spread at the interface in the trough by dropwise deposition of a 2 mg/mL solution from a pipet. The surface rheological measurements were made using a ring-trough, the details of which have been described elsewhere.20 The apparatus consists of a ground glass ring with an internal area of 75 cm2, which is sinusoidally oscillated vertically while the surface tension within the ring is measured. The frequency of oscillation was kept at 0.13 Hz, and the change in area was kept to approximately 5% of the total area. In these experiments, the protein was spread drop by drop from a 2 mg/mL solution within the ring and allowed to equilibrate.

Figure 1. A series of BAM images showing (a) β-lactoglobulin, Π ) 10 mN/m; (b) β-lactoglobulin/Tween 20, Π ) 20.4 mN/m; (c,d) β-lactoglobulin/Tween 20, Π ) 22.3 mN/m; (e) β-lactoglobulin/Tween 20, Π ) 23.8 mN/m; and (f) Tween 20, Π ) 25.2 mN/m. All images are 429 µm × 322 µm.

Tween 20 was then added to the subphase. Both the surface dilatational rheology and the surface pressure were measured as a function of time over the duration of the experiment. Results An interfacial film of β-lactoglobulin was formed by spreading 64 µg drop by drop from a solution containing 2 mg/mL protein in water. This represented coverage of 1.14 mg/m2 and was chosen so that the surface pressure could be raised to 10 mN/m without too much compression. This value of surface pressure was chosen to match previous experiments.6 The surface tension was measured for 15 min while the monolayer equilibrated. The final surface pressure was 4 mN/m. The surface was then compressed until the surface pressure reached 10 mN/m (approximately 1.5 mg/m2), at which point the surfactant Tween 20 was added to the subphase to give a concentration of 2 µM. BAM images were taken periodically. Further injections of Tween 20 were made as necessary to further increase the surface pressure and displace the protein. Figure 1 shows a sequence of BAM images taken as the surface pressure increased. The images shown are selected from over 50 taken of each system and are intended to be representative of what was seen. Figure 1a shows the protein film at a surface pressure of 10 mN/m before any surfactant had been added. The protein film is clearly homogeneous with dust causing the only visible features. As the surfactant adsorption progressed and the surface pressure increased, domains of surfactant appeared. Figure 1b shows an image of a protein film that was taken at a surface pressure of 20.4 mN/m. There are distinct regions in the image. These regions are a bright, proteinrich region on the left, a dark region on the right where the protein has been displaced by the surfactant, and a region in the center, displaying intermediate brightness. The region on the left of the image is very bright suggesting that this

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Figure 2. AFM images of (a) β-lactoglobulin/Tween 20 mixed film transferred at Π ) 22.5 mN/m (11 µm × 11 µm) and (b) β-casein/ Tween 20 mixed film transferred at Π ) 19.2 mN/m (6.4 µm × 6.4 µm).

area is purely protein and contains little or no Tween 20. However, the central region contains some holes that are large enough to be visible, and the gray level of the surrounding film is low enough to suggest that it contains a significant amount of Tween 20. The majority of the holes (if any) are too small to resolve in this central region of the image. The next image, Figure 1c, was taken at a surface pressure of 22.3 mN/m and illustrates that the displacement had progressed further. The image shows many holes in the protein film extending across the whole field of view. At this point in the displacement, moving the BAM to different areas of the trough showed the same patterns. Figure 1d also shows that the surfactant domains permeate the majority of the remaining protein film, although the network is still protein continuous. The last two BAM images of this system show the final stages of displacement in which the protein network breaks down leaving islands of protein (Figure 1e) which themselves finally desorb, leaving an interface occupied entirely by Tween 20 (Figure 1f). In previous work on protein displacement, AFM was used to measure both film thickness and the proportion of the interfacial film occupied by protein as a function of the surface pressure of the mixed protein/surfactant film.6 However, it is not possible to use BAM to quantify the displacement in terms of either protein film thickness or percent area occupied by protein because of the relatively limited resolution. Despite this, it is still useful to make a qualitative comparison with data collected previously by AFM. Figure 2 shows AFM images of Langmuir-Blodgett (LB) films of the same systems. The first image (Figure 2a) is of a β-lactoglobulin film transferred at a surface pressure of 22.5 mN/m and shows domains with a mean diameter of 0.5 µm. The BAM image (Figure 1d), taken at a surface pressure of 22.3 mN/m, contains domains with a mean diameter of 7.5 µm. The difference is likely to be due primarily to the difference in length scales probed by the two techniques. The BAM cannot resolve the small holes, and the AFM does not have a wide enough field of view to measure the large ones. Despite the fact that the domains are larger in the BAM images (Figure 1c,d), a comparison with Figure 2 suggests that they represent the same phenomenon. In work previously published,6 it was shown that the area occupied by the protein changed as a function of surface pressure (Figure 4, symbols). It is evident from this figure that the β-lactoglobulin was largely displaced from

Figure 3. Surface tension plotted as a function of time for 19 µg of spread β-lactoglobulin + Tween 20 (curve 1) and 10 µg of spread β-casein + Tween 20 (curve 3). Also shown is the surface elastic modulus for the two samples, curves 2 and 4, respectively. The arrows mark the times at which additions of Tween 20 were made to increase the subphase concentration by 1 µM.

Figure 4. Surface dilatational elastic modulus plotted as a function of surface pressure for 1 µM β-lactoglobulin (curve 1), 1-3 µM Tween 20 (curve 2), and 19 µg of spread β-lactoglobulin + 1-2 µM Tween 20 (curve 3). Also shown are data for area occupied by protein as a function of surface pressure (symbols); data are from from Mackie et al. (ref 6).

the interface between 18 and 25 mN/m. The BAM images also show displacement over this surface pressure range. The previous work showed that displacement was via an orogenic mechanism, but it was unclear how this was affected by the rheology of the interface. Initial measurements suggested that the surface pressure had to be high enough to, at least partially, collapse the protein film before displacement could take place. To investigate this further, rheological measurements were made on the same systems as were measured in the BAM. Protein (19 µg, 2.5 mg/m2) was spread in the ring of a ring-trough and allowed to equilibrate for 40 min before Tween 20 was added to the subphase to give an initial concentration of 1 µM (Figure 3, curves 1 and 2). Further additions of Tween 20 were made to increase the surface pressure as required. The results (Figure 4, curve 3) show that as the surfactant adsorbed, the surface pressure increased from 10.4 to 14.5 mN/m. This coincided with an initial decrease in the dilatational elastic modulus. Subsequently, the elastic modulus increased slightly to a peak at a surface pressure of 21 mN/m before decreasing again toward the curve for Tween 20 alone. Thus, there appears to be two regions where the Tween 20 affects the mixed film, first in the initial stages of surfactant adsorption and second and most significantly in the later stages where the surfactant begins to dominate the behavior.

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Figure 5. A series of BAM images showing (a) β-casein/Tween 20, Π ) 18.6 mN/m; (b) β-casein/Tween 20, Π ) 21.4 mN/m; (c) β-casein/ Tween 20, Π ) 21.4 mN/m; and (d) β-casein/Tween 20, Π ) 23.8 mN/m. All images are 429 µm × 322 µm.

For comparison, the surface dilatational elasticity values for the two pure adsorbing components are also included (curves 1 and 2). The data for the protein alone show the same peak in elastic modulus at 21 mN/m as the mixed system although the values for the early stages of adsorption appear much lower than those for the spread protein film. The reason for this is that in setting up the mixed film a protein layer was initially spread. This film reached an equilibrium surface pressure of 10.4 mN/m after about 10 min. However, for the following 30 min the elastic modulus continued to rise while the surface pressure remained essentially static (Figure 3, curve 2). This rise is responsible for the difference in elasticity between the two curves in Figure 4 (curves 1 and 3) at 10.4 mN/m and is an indication of the amount of rearrangement of the protein that occurred on the surface. With regard to the Tween 20 curve, the apparent measured elasticity is actually a function of the exchange of adsorbed surfactant with the bulk and is thus very dependent on the conditions under which the measurements were made. However, the frequency and amplitude used throughout the experiment were kept constant. Thus, the values should at least be representative of the final state of the mixed film where the exchange rate of the Tween 20 between the surface and the bulk should not be significantly affected. To cover the required dynamic range of surface pressures, two concentrations of Tween 20 were used, 1 and 3 µM. The data for both concentrations are included in the figure and indicate that the elasticity seemed to be not a function of concentration but more a function of surface pressure. The other protein used in this study, β-casein, behaved slightly differently. The interfacial film was formed by spreading 50 µg (0.89 mg/m2) of β-casein from a 2 mg/mL solution in water. After 15 min, the surface pressure was 1.1 mN/m. The film was then compressed to 10 mN/m. As with the β-lactoglobulin, the initial β-casein film was homogeneous except for the occasional dust particle. Successive injections of Tween 20 were then made into the subphase. Figure 5 shows several BAM images obtained at increasing surface pressures. The first features started to

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appear in the film at about 17 mN/m with a subphase concentration of 3 µM Tween 20. However, it was not until the surface pressure had reached 21.3 mN/m that large, clearly defined features became visible. Figure 5a shows the type of feature that appeared early on in the displacement. The feature shows up merely as a region of lower reflectance, implying that the mean protein density was lower. The most likely explanation is that the Tween 20 had penetrated the protein layer but any surfactant domains were too small to resolve by BAM. Figure 5b shows the displacement front, which was the boundary between the protein continuous and surfactant continuous regions. The left side of the image is protein continuous, whereas the right side of the image is surfactant continuous. The surfactant region clearly contains islands of protein, and the protein region also shows some evidence of structure. In fact, the reflectance of the protein film increased further away from the displacement front. This suggests that the Tween 20 concentration in the protein film decreased with increasing distance from the front. The other key feature that is amenable to BAM measurements was the difference in mobility between the protein film on the left of the image, which was immobile, and the surfactant domain on the right, which was highly mobile. Displacement did not occur solely at a “front” but also occurred in regions surrounded by continuous protein. One such region is shown in Figure 5c. In this image, surfactant domains are clearly visible and were of many different sizes ranging from those that were too small to resolve up to about 40 µm for the largest domain in the center. Figure 5d demonstrates how fluid flow in the surfactant domain caused sections of the protein film to be “peeled off”. In this case, the flow was from the bottom of the image toward the top. The figure shows a linear section of the protein film, which had been coiled back on itself by the flow. The second AFM image in Figure 2 (image b) shows a β-casein interfacial film transferred at a surface pressure of 19.2 mN/m. The image clearly shows the typical circular domains of β-casein films at the air/water interface,6 which in this case had a mean diameter of 0.3 µm. The BAM images (Figure 5) showed few resolvable domains except for the single large domain adjoining the displacement front. The main problem would appear to be that the BAM cannot resolve the small holes and the protein film breaks up before the holes get large enough to be visible. The domains that were of a size that could be measured showed no specific size characteristic of a given surface pressure but covered a very broad range of sizes. The symbols in Figure 6 show that β-casein was largely displaced from the interface over the surface pressure range from 17 to 22 mN/m when previously measured by AFM. The BAM images also show displacement over this range of surface pressure. In surface rheological measurements made on the same system, 10 µg (1.3 mg/m2) of protein was spread on the surface inside the ring-trough and allowed to equilibrate for 30 min. Tween 20 was then added to the subphase to give an initial concentration of 1 µM. As in the case of the β-lactoglobulin film, further additions of Tween 20 were made as required to increase the surface pressure

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Figure 6. Surface dilatational elastic modulus plotted as a function of surface pressure for 1 µM β-casein (curve 1), 1-3 µM Tween 20 (curve 2), and 10 µg of spread β-casein + 1-2 µM Tween 20 (curve 3). Also shown are data for area occupied by protein as a function of surface pressure (symbols); data are from Mackie et al. (ref 6).

(Figure 3, curves 3 and 4). The results (Figure 6, curve 3) show that as the surface pressure increased from 16 to 19 mN/m due to surfactant adsorption the dilatational elastic modulus followed the curve for the protein alone. However, after 19 mN/m the elastic modulus rose toward the curve for Tween 20 alone. The data suggest that there was no protein left at the interface by the time the surface pressure reached 23 mN/m. For comparison, the data for the two pure adsorbing components are also included. The elastic modulus for the pure protein film also shows an increase from 19 mN/m but rises much more steeply than that for the mixed film. Thus, at all surface pressures higher than 19 mN/m the mixed film shows a lower surface elasticity than the pure protein film. It is important to highlight the fact that although β-casein forms a weak surface film it is still an immobile 2-D gel. In previous work,8 displacement of β-lactoglobulin by the charged surfactant SDS was studied by AFM. In that work, surfactant domains were formed in the protein film but remained small until close to the point at which the interface became surfactant continuous (the 2-D percolation threshold). In an attempt to visualize this displacement directly by BAM, a β-lactoglobulin film was spread to a surface pressure of 7.7 mN/m and after equilibration for 15 min was compressed to 10.2 mN/m. SDS was then introduced into the subphase to gradually increase the concentration to 400 µM after 45 min. One hour after the first addition, the surface pressure was 21.8 mN/m. The reflectivity remained essentially unchanged. This confirmed the results collected previously by AFM that showed that the charged surfactant did not begin to displace the protein from the interface until much higher surface pressure (36 mN/m) compared with the nonionic surfactant.6 The size of the SDS domains seen previously by AFM suggested that they would not be resolvable by BAM. Discussion In this paper, we have sought to demonstrate that the features seen in partially displaced protein films, when observed indirectly by AFM, may also be observed directly, in situ at the air-water interface using BAM. In the case of

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the β-lactoglobulin, the BAM images in Figure 1 appear to match those measured by AFM in the later stages of displacement shown in Figure 2a, at least in terms of shape. It has been postulated that the shape of the surfactant domains in the protein films is governed by the intermolecular interactions of the protein.6 The comparatively weak film formed by β-casein gives rise to the circular holes due to isotropic expansion of the surfactant domains, whereas the stronger β-lactoglobulin film only allows anisotropic expansion along lines of weakness in the film. The images from both measurement techniques appear to show surfactant domains that have a self-similar geometry. That is, the relative size and shape of the surfactant domains at the interface is the same regardless of the magnification of the technique used for their characterization. This lends credence to the idea that the orogenic displacement mechanism plays a role at the scale of emulsions (as detected with the magnification of AFM) and of foams (as detected with the magnification of BAM). Despite these similarities, there were some distinct differences including the presence of a displacement front. Another feature of the displacement of both proteins that is not apparent from the BAM images was the flow of the film. In all cases, the protein film was observed to flow away from the areas where displacement was occurring. One possible explanation for this is that in the areas where displacement was taking place surfactant was adsorbing into the film. The surface pressure within the surfactant domain increased, leading to expansion of the domain compressing the protein film, causing it to collapse. Thus, where the surfactant was adsorbing into the film there was expansion and where there was no displacement occurring there was collapse (contraction). This would cause the protein film to flow away from areas where displacement was taking place. This behavior is predicted by the orogenic displacement model but has not been observed previously. The main requirement for the observation of this flow is that adsorption of surfactant into the protein layer must be confined initially to a small region of the film. This could be the case if there were local concentration gradients due to poor mixing in the bulk phase. This may have been the case in the BAM experiments as the trough had a large surface area (562 cm2) but was only 5 mm deep and the subphase was not stirred. Once the local displacement has started, the protein film not involved in the displacement becomes compressed making it more difficult for the surfactant to adsorb into it. The protein displacement studies have shown that some of the key features controlling the behavior are the strength and compressibility of the film. With this in mind, we have looked at the interfacial rheology of the two main systems described above. The results show that both proteins exhibit a transition in the surface dilatational elastic modulus over the surface pressure range where displacement takes place. In the case of β-lactoglobulin, the transition goes from the highly elastic protein film to the essentially inelastic surfactant film. However, the protein film appears to retain a great deal of the elastic properties even at comparatively low coverage. The data for the β-casein are complicated by the fact that the elastic modulus for the protein film is lower

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than the “apparent” elastic modulus of the Tween 20. In the case of both proteins, the values for elasticity of the mixed films were essentially indistinguishable from the values for the proteins alone down to surface area coverage of about 35%. It was only as the surface pressure rose from that point that the film was then weakened by the adsorption of Tween 20 into the protein layer. Comparison with data collected previously on the thickness of the protein layer as a function of surface pressure suggests that in both cases the protein layer would have increased in thickness possibly adding to the strength of the protein film. The rheological data on these two systems highlight the dominant role the protein plays at the interface in mixed systems. In this study, we have measured in situ the displacement of protein by a nonionic surfactant. The BAM images appear to support the idea that displacement takes place via an orogenic mechanism. Although the BAM does not have the same resolution as AFM, it still serves to validate the Langmuir-Blodgett data measured previously. The surface rheological behavior also serves to support the idea of an orogenic displacement. Acknowledgment. This work was funded by the BBSRC as part of the core grant to the Institute. References and Notes (1) Wilde, P. J. J. Colloid Interface Sci. 1996, 178, 733. (2) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (3) Dickinson, E. Colloids Surf., B 1999, 15, 161.

Mackie et al. (4) Petkov, J. T.; Gurkov, T. D.; Campbell, B. E.; Borwankar, R. P. Langmuir 2000, 16, 3703. (5) Green, R. J.; Su, T. J.; Joy, H.; Lu, J. R. Langmuir 2000, 16, 5797. (6) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (7) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. (8) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 8176. (9) Zhao, J.; Vollhardt, D.; Brezesinski, G.; Siegel, S.; Wu, J.; Li, J. B.; Miller, R. Colloids Surf., A 2000, 168, 287. (10) Zhang, H. J.; Wang, X. L.; Cui, G. C.; Li, J. B. Colloids Surf., A 2000, 175, 77. (11) Vollhardt, D.; Fainerman, V. B. AdV. Colloid Interface Sci. 2000, 86, 103. (12) Rodriguez Patino, J. M.; Carrera Sanchez, C.; Rodriguez Nino, Ma. R. Langmuir 1999, 15, 4777. (13) Rodriguez Patino, J. M.; Carrera Sanchez, C.; Rodriguez Nino, Ma. R. J. Agric. Food Chem. 1999, 47, 4998. (14) Rodriguez Patino, J. M.; Carrera Sanchez, C.; Rodriguez Nino, Ma. R. Food Hydrocolloids 1999, 13, 401. (15) Izmailova, V. N.; Yampolskaya, G. P.; Tulovskaya, Z. D. Colloids Surf., A 1999, 160, 89. (16) Pezennec, S.; Gauthier, F.; Alonso, C.; Graner, F.; Croguennec, T.; Brule, G.; Renault, A. Food Hydrocolloids 2000, 14, 463. (17) Fruhner, A.; Wantke, K.-D.; Lunkenheimer, K. Colloids Surf., A 1999, 162, 193. (18) Rodriguez Patino, J. M.; Carrera Sanchez, C.; Rodriguez Nino, Ma. R. Langmuir 1999, 15, 2484. (19) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Kragel, J.; Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko, O. V. AdV. Colloid Interface Sci. 2000, 86, 39. (20) Kokelaar, J. J.; Prins, A.; De Gee, M. J. Colloid Interface Sci. 1991, 146, 507.

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