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Orogenic Displacement in Mixed β-Lactoglobulin/β-Casein Films at the Air/Water Interface Alan R. Mackie,* A. Patrick Gunning, Michael J. Ridout, Peter J. Wilde, and Victor J. Morris Department of Materials Science, Institute of Food Research, Norwich Research Park, Colney NR4 7UA, U.K. Received May 8, 2001. In Final Form: July 17, 2001 The adsorption of mixed β-casein/β-lactoglobulin films to the air/water interface and the subsequent displacement by the nonionic surfactant Tween 20 was studied. A combination of fluorescent labeling of the protein and Langmuir-Blodgett deposition was used to study the mixed protein layer. The adsorption was also monitored using two surface rheological techniques, shear and dilatation. Fluorescent labeling was able to show that to within the limits of optical resolution the two proteins were well mixed at the interface. We also show that the film remained well mixed after 3 days. Surface rheological data from the two techniques used was self-consistent and showed that during the initial stages of development, the films were dominated by the adsorption of the β-casein. Both fluorescence microscopy and atomic force microscopy were used to follow the displacement of the mixed film by surfactant. Results on films displaced by the nonionic surfactant Tween 20 showed that β-casein was preferentially displaced from the mixed film before β-lactoglobulin.
Introduction Proteins are widely used in the food industry as emulsifiers and foaming agents. Consequently there has been much interest in understanding the way in which protein structure and interfacial function are related.1-4 Furthermore, because of the complex nature of most food systems, there has been substantial interest in understanding the interactions between proteins and surfactants5-7 and between different proteins.8,9 This is of importance because proteins and emulsifiers stabilize interfacial films by different and conflicting mechanisms. In particular, two groups of food proteins have been studied, caseinates and whey proteins. The predominant whey protein is β-lactoglobulin while a major component of caseinate is β-casein. These two proteins show considerable differences in both structure and behavior. Some time ago it was observed that β-casein could displace Rs1casein from an oil/water interface but that β-lactoglobulin could not displace β-casein from such an interface. However, work done by others on β-casein10,11 suggests that it is much more difficult to displace from the oil/ water interface than from the air/water interface. Displacement of β-casein required approximately 10 mN/m * To whom correspondence should be addressed. Tel: 44 1603 255261. Fax: 44 1603 507723. E-mail:
[email protected]. (1) Dickinson, E. Colloid Surf., B 1999, 15, 161. (2) Mackie, A. R.; Husband, F. A.; Holt, C.; et al. Int. J. Food Sci. Technol. 1999, 34, 509. (3) Izmailova, V. N.; Yampolskaya, G. P. Adv. Colloid Interface Sci. 2000, 88, 99. (4) McGuire, J.; Bower, C. K.; Bothwell, M. K. Aust. J. Dairy Technol. 2000, 55, 65. (5) Fruhner, H.; Wantke, K. D.; Lunkenheimer, K. Colloid Surf., A 2000, 162, 193. (6) Nino, M. R. R.; Wilde, P. J.; Clark, D. C.; Patino, J. M. R. Langmuir 1998, 14, 2160. (7) Nasir, A.; McGuire, J. Food Hydrocolloids 1998, 12, 95. (8) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (9) Husband, F. A.; Wilde, P. J.; Mackie, A. R.; Garrood, M. J. J. Colloid Interface Sci. 1997, 195, 77. (10) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (11) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242.
higher surface pressure at the oil/water interface. Thus, it is not clear that the same behavior would be observed at the air/water interface. Recently we have used atomic force microscopy (AFM) to look at the way in which proteins are displaced from an interface by surfactants. This has led to the development of an “orogenic” displacement model.10-12 This model describes a mechanism that works in the following way: Heterogeneity in the protein film allows the displacing surfactant to adsorb into localized defects, and these nucleated sites then grow. The expansion of the pools of surfactant compresses the protein network, which initially increases in density without increasing in thickness. Once a certain critical density is reached, the protein layer thickness increases such that the protein film volume is maintained as the surfactant domains expand. At sufficiently high surface pressures the network fails, releasing protein, which then desorbs from the interface. More recently this model has been confirmed in situ at the air/ water interface by Brewster angle microscope experiments.13 Here we attempt to extend this model by looking at the displacement of a mixed protein film by a nonionic surfactant (Tween 20). More importantly we attempt to see whether one protein can displace another from an interface by a similar mechanism. Thus, we have used fluorescent labeling to distinguish between proteins and to allow the observation of any phase separation that might occur at the interface. Although this is not the first time that such techniques have been used,14 this is the first time that such a high degree of spatial resolution has been obtained. The high spatial resolution is important because of the range over which one might expect to see protein phase separation. In the case of mixed protein and charged surfactant films (probably the closest com(12) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 8176. (13) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Biomacromolecules, in press. (14) Sengupta, T.; Damodaran, S. J. Colloid Interface Sci. 2000, 229, 21.
10.1021/la010687g CCC: $20.00 © 2001 American Chemical Society Published on Web 09/14/2001
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parison to protein-protein), the surfactant domains were in the order of tens of nanometers in size.12 In fact the largest surfactant domains seen in a mixed protein surfactant film before the final rupture of the protein network were some only 100 µm in diameter. Materials and Methods The milk proteins used in this study were β-lactoglobulin (L0130, 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 an Elga Elgastat UHQ water purification system. The Tween 20 (polyoxyethylene sorbitan monolaurate) was obtained as a 10% solution (SurfactAmps 20) from Pierce (Rockford, IL). The proteins were labeled using amine reactive dyes, fluorescein isothyocyanate (FITC) from Sigma (Poole, U.K.) in the case of β-casein and 6-carboxyrhodamine 6G succinimidyl ester (Molecular Probes Inc., Eugene, OR) in the case of β-lactoglobulin. The reaction was performed using the recommended protocols (Molecular Probes), and the conjugate was purified by gel filtration to remove unbound dye using a Sephadex G-25 column. Surface tension measurements were made using a wetted ground glass Wilhelmy plate and a Langmuir trough (Labcon Ltd, Durham, U.K.). The PTFE trough was 255 × 112 × 16 mm, a volume of 450 mL, with one fixed and one mobile barrier. All experiments were performed at room temperature (20 °C) with distilled water as the subphase. For the AFM measurements, the adsorbed films were produced from solutions containing 0.5 µM β-casein, 0.5 µM β-lactoglobulin, and 0.5 µM Tween 20. Surface tension measurements were made as a function of time for 30 min while the monolayer equilibrated. After this period of equilibration, the subphase was perfused with 2 L of 0.5 µM Tween 20 at a rate of 1.1 mL‚s-1. Further additions of surfactant to the subphase were then made in order to increase the surface pressure as required. The Langmuir-Blodgett (LB) films were formed on hydrophilic freshly cleaved mica substrates. LB films were produced by lowering a freshly cleaved piece of mica sheet, mounted perpendicular to the interface, down through the interface and then pulling it back out again. The mounted piece of mica was driven at a constant rate of 0.2 mm‚s-1. Surface tension was monitored during the dip and showed that the film was only transferred onto the mica on the upward stroke. The AFM images were measured under butanol. The precise details of the AFM and the imaging technique are given elsewhere.15 Fluorescence measurements were made using an Olympus BX60 microscope. Excitation was from a mercury arc lamp via a blue excitation (FITC) filter set or a green excitation (rhodamine) filter set. Images were taken using a cooled chip CCD camera (SP-eye, Photonic Sciences, Cambridge, U.K.). The images taken were of LB films deposited onto washed glass coverslips in the manner described above. Rheological measurements were made using both surface shear and surface dilation techniques. The surface dilation measurements were made using the method of Kokelaar et al.16 The technique involves periodically raising and lowering a 10-cm ground-glass ring into a vessel containing 250 mL of sample. The resulting change in surface area was kept to 5%, and the frequency of dilation/compression was kept at 0.8 rads‚s-1. The surface shear measurements were made using a Camtel CIR 100 surface shear rheometer (Camtel, Royston, U.K.). This instrument uses a Du Nouy ring oscillating in normalized resonance mode.17 The ring is oscillated in air at its resonant frequency of approximately 3 Hz. When the ring is placed at an interface, the resonant frequency and amplitude will change depending on the elasticity and viscosity of the interface. Feedback currents restore the frequency and amplitude of the oscillations back to resonance. The values of the feedback signal required to restore the frequency are used to calculate the surface
In this paper we attempt to improve our understanding of mixed protein interfaces by investigating the structure and physical properties of a model system comprising two proteins: β-lactoglobulin and β-casein. In previous papers we have shown that surfactants disrupt and displace proteins films by an orogenic mechanism, which involves the expansion of phase-separated surfactant domains. The main aim of this work is to discover whether structures and phase separation processes are likely to occur in a mixed protein interface, similar to those observed in the protein/surfactant systems studied previously. The results are discussed in terms of the various stages in the formation and stabilization of the interface. More specifically, the stages are (i) adsorption, (ii) phase separation, and (iii) displacement. Adsorption. Protein adsorption is mainly determined by surface hydrophobicity, molecular weight, solubility, and flexibility.18 β-Casein is a much more flexible molecule than β-lactoglobulin and is therefore capable of unfolding at the interface more rapidly. It has been found that random coil proteins such as β-casein adsorb and lower interfacial tension more rapidly than globular proteins.19 β-Casein also has very different surface elasticity to β-lactoglobulin, so the surface elasticity was used to dynamically probe the adsorption of the mixed protein system. Two types of interfacial rheology were employed, dilatational and shear. Figure 1 shows the dilatational elastic modulus for the two individual protein films and for a 1:1 mixture. All three had the same total protein concentration (1 µM). β-Lactoglobulin and β-casein show markedly different behavior, which has been shown before by others.20 β-Casein shows a typical peak at early adsorption times which is caused by the collapse of the highly charged N-terminal “tail” region into the subphase.21 The elasticity reaches a plateau of approximately 10 mN/m after less than 10 min. It should be mentioned that the β-casein film had virtually reached its equilibrium surface pressure in about 5 min (data not shown). The β-lactoglobulin simply showed a steady increase in elastic modulus to a pseudoplateau of approximately 78 mN/m after 30 min followed by a slight decrease thereafter. The
(15) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Morris, V. J. Biopolymers 1998, 46, 245. (16) Kokelaar, J. J.; Prins, A.; De Gee, M. J. Colloid Interface Sci. 1991, 46, 507. (17) Sherrif, M.; Warburton, B. Polymer 1974, 15, 253.
(18) Garofalakis, G.; Murray, B. S. Colloids Surf., B 1999, 12, 231. (19) Mitchell, J. R. Dev. Food Proteins 1986, 4, 291. (20) Williams, A.; Prins, A. Colloids Surf., A 1996, 4, 267. (21) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans 1995, 91, 2847.
Figure 1. Dilatational elastic modulus plotted as a function of time for adsorbing 1 µM β-casein (line 1), 1 µM β-lactoglobulin (line 2), and a 1:1 mixture of the two proteins at a total protein concentration of 1 µM (line 3). shear elastic modulus (G′), and the signal required to restore the amplitude enables calculation of the viscous component (G′′).
Results and Discussion
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Figure 2. Shear elastic modulus plotted as a function of time for adsorbing 1 µM β-casein (line 1), 1 µM β-lactoglobulin (line 2), and a 1:1 mixture of the two proteins at a total protein concentration of 1 µM (line 3).
surface pressure of the film was still steadily increasing after 105 min (data not shown). The mixed system showed features of both proteins. The peak from the partial collapse of the β-casein is present although shifted to slightly longer times. The much broader peak from β-lactoglobulin was also visible but reduced by nearly a factor of 4 to 22 mN/m. Subsequently the dilatational elasticity climbed very slowly to 25.4 mN/m after nearly 4 h. The surface pressure of the mixed film took about 30 min to reach equilibrium (data not shown). The data suggest that both proteins were observed to adsorb, resulting in a mixed β-lactoglobulin/β-casein interface. However, the surface elasticity of the mixed system suggests that β-casein was the dominant protein at this stage in the adsorption. This agrees with previous work showing the more rapid adsorption of β-casein. If, rather simplistically, one assumes the surface dilatational elastic modulus of the separate components to be merely additive, then the curve for an interface containing 85% casein fits the data reasonably well, especially in the latter stages. Further investigation will be required in order to fully ascertain the true nature of the rheological behavior of the mixed interfacial film. Especially in terms of the degree to which such immobile systems may become phase separated. The data from the measurement of surface shear elasticity are shown in Figure 2. Again, the figure shows data for the individual proteins and the mixed system. The film formed by β-casein was so weak as to be almost immeasurable while the film formed by β-lactoglobulin gave quite high values. The values for β-lactoglobulin rose to a peak of 4.9 mN/m after 50 min and then decreased in elasticity to about 2 mN/m after 3 h. The mixed film shows a gradual slow rise to 1 mN/m after 3 h. The results show a similar trend to the surface dilatational elasticity, in that β-casein was clearly the dominant protein at these concentrations. However, the surface shear elasticity would suggest that there was almost no adsorbed β-lactoglobulin. The difference between the results from the surface shear and the surface dilatational rheology can be put down to the different methodologies. Surface dilatational rheology measures the changes in surface pressure during compression and expansion of the interface whereas surface shear rheology measures directly the mechanical properties of the interface under shear. Both methods are sensitive to protein structure and the number and strength of intermolecular interactions. However, surface shear tends to be more sensitive to protein interactions and surface dilatation more sensitive to adsorbed structure and composition. Hence, the surface dilatation reveals that under these conditions β-casein
Figure 3. A series of fluorescence images following the displacement of a mixed, dual stained protein film. β-Casein was labeled green and lactoglobulin was labeled red. All images are 63 µm by 48 µm. Image a shows a mixed protein film transferred at a surface pressure of 21.5 mN/m. Also shown is the mixed film at various stages of displacement transferred at 27 (b), 28.5 (c), and 29.5 mN/m (d).
was the predominant protein at the surface but a small amount of β-lactoglobulin was present. Despite this presence, the mechanical strength of the adsorbed film (as measured by the surface shear elasticity) was almost totally dominated by the β-casein component. Therefore both surface rheological methods suggest that at these equal concentrations β-casein adsorbed more rapidly than β-lactoglobulin and dominated the surface rheological properties. To investigate further, fluorescence microscopy was used to see if there was a surface concentration excess of β-casein and to observe any phase separation which may occur. Phase Separation. The aim here was to determine the spatial distribution of the two proteins at the interface and thus to ascertain if the proteins were evenly mixed or if phase separation occurred and how the protein distribution could help us understand the surface rheological behavior. Previous work on protein/surfactant mixed interfaces has shown, using AFM, how the surfactant phase separates into domains to displace the protein. AFM was ideally suited to these investigations because it could easily distinguish between the separate protein and surfactant domains. AFM cannot however distinguish between two different proteins. To determine the spatial distribution of the two proteins in the mixed film, fluorescence microscopy was employed. In these experiments, the two proteins of interest were labeled with fluorescent probes, β-casein with FITC and β-lactoglobulin with rhodamine 6G. In both cases the extent of labeling was approximately 1:1 protein to dye. The total protein concentration in the Langmuir trough was 0.5 µM, and adsorption was allowed to continue for 3 days. Periodically, sections of the interface were transferred onto glass coverslips by the LangmuirBlodgett method. Figure 3a is from an interface after adsorption for 2 days (surface pressure ) 21.5 mN/m) and shows a rather homogeneous deposition of protein. The dominant protein was the FITC-labeled β-casein. There was a low level of very localized heterogeneity observed in all the images throughout the duration of the experiments. To check whether the heterogeneity seen in this image was representative of a small amount of phase separation of the proteins on the microscopic scale, a similar experiment was performed using only the fluo-
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rescent β-lactoglobulin. When the autocorrelation functions of the two image sets were compared (data not shown), they were found to be very similar. This suggests that the small-scale heterogeneities were due simply to random adsorption of the protein. There were no changes in the spatial distribution of the two proteins and no phase separation processes were observed over the experimental period of 3 days. This behavior is very different from the phase separation observed with the surfactant/protein systems studied10 that could show phase separation in less than 1 h. The main differences between adsorbed proteins and surfactants are that (a) adsorbed surfactant is able to exchange with nonadsorbed surfactant and (b) adsorbed surfactant can freely diffuse at the interface. In contrast, proteins, once adsorbed and interacting with neighboring proteins, can neither exchange with the bulk nor diffuse at the interface. The mobility of a surfactant allows molecular segregation to occur and the resultant domains to expand and deform. The distinct lack of mobility of the adsorbed protein molecule arrests any segregation process, and consequently there is no driving force for phase separation. Hence the results of the fluorescence experiments shown in Figure 3a demonstrate that no phase separation was observed, even after 3 days. Any local heterogeneity in the surface distribution of protein is due to the initial adsorption process, after which no measurable redistribution was observed. The lack of rearrangement and phase separation observed here is in contrast to the work published recently by Sengupta and Damodaran.14 We had observed previously that excessive fluorescent labeling could affect the surface properties of the protein. Measurements made on the surface rheology of the fluorescent-labeled mixed protein film in the present study showed it to be consistent with the unlabeled system. This indicates that the labeling had not significantly affected the strength of the film. In summary, fluorescent microscopy demonstrated that β-casein was indeed the dominant adsorbed protein under these conditions and that even after 3 days, no segregation or phase separation of the adsorbed proteins was observed. Displacement. The displacement of the mixed β-lactoglobulin/β-casein interface by the nonionic surfactant Tween 20 was studied. The aim was to see if the structure of the mixed protein interface influenced how the individual proteins were displaced. This is important for understanding how real protein mixtures interact with lipids and surfactants in food foams and emulsions. The displacement was measured first on unlabeled protein using AFM. Figure 4 shows AFM images of a mixed β-lactoglobulin/β-casein film at various stages of displacement by Tween 20. It can be observed that the appearance of the protein films changed as displacement progressed. The light areas are where the protein is located, and the dark areas are where surfactant occupied the surface. Figure 4a shows a mixed film transferred at a surface pressure of 21.6 mN/m where protein covered 69% of the surface area. The second image (Figure 4b) shows the mixed film after further displacement. This image is of a film transferred at a surface pressure of 25.0 mN/m and shows protein film occupying an area of 30.5%. For comparison panels c and d of Figure 4 show pure β-casein and pure β-lactoglobulin films, respectively. The adsorbed β-casein film was transferred at a surface pressure of 20.7 mN/m, and the film had a mean protein area of 51%. The adsorbed β-lactoglobulin film was transferred at a surface pressure of 26.1 mN/m, and the protein occupied an area of 49%. It is clear that the holes visible in the mixed protein film show characteristics of both single-component protein films. The shapes of the holes were neither circular as
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Figure 4. AFM images showing mixed β-casein and β-lactoglobulin films being displaced by Tween 20. The films were transferred at surface pressures of 21.6 (a) and 25.0 mN/m (b). The image sizes are 4.0 and 5.0 µm, respectively. Also shown are AFM images of a β-casein film being displaced by Tween 20 at a surface pressure of 20.7 mN/m (c) and a β-lactoglobulin film being displaced by Tween 20 at a surface pressure of 26.1 mN/m (d). The image sizes are 9.0 and 4.0 µm, respectively.
Figure 5. The surface area (%) occupied by protein is plotted as a function of the surface pressure at which the LB film was transferred. Data are shown for films of β-casein alone ([), β-lactoglobulin alone (9), and the 1:1 mixed film (2). Also shown are data from fluorescent-labeled films displaced after aging for 3 days (b).
with the isotropic compression of the β-casein nor as irregular as those seen in the β-lactoglobulin film. The shape of the displacement patterns gives a clear indication that there was indeed a mixed film at the interface. However, these images alone do not provide enough information to draw firm conclusions about the spatial distribution of the individual proteins within the film. All AFM images were analyzed to provide information about both the surface area occupied by the protein and the thickness of the protein layer. Figures 5 and 6 show the protein area and thickness, respectively, as a function of the surface pressure at which the film was transferred. For comparison both figures show data on films of pure β-casein and of pure β-lactoglobulin. The displacement of the mixed film occurred over a range of some 7 mN/m and falls between the displacement curves for the individual proteins (Figure 5). The fact that the displacement falls linearly between the two curves for the individual proteins
Mixed Protein Displacement
Figure 6. The film thickness of the protein layer is plotted as a function of the surface pressure at which the LB film was transferred. Data are shown for films of β-casein alone ([), β-lactoglobulin alone (9), and the 1:1 mixed film (2).
suggests that there has been no phase separation between the proteins in the mixed film. This mixed film required a higher surface pressure to displace than a β-casein film but a lower surface pressure than a β-lactoglobulin film. If there had been a high degree of phase separation, one might have expected to see the displacement for the mixed system follow the curves for the single proteins at least over part of the range. In the early stages the β-casein domains would have been displaced; thus the mixed film would have followed the displacement curve for the β-casein alone. In the latter stages only the β-lactoglobulin would have remained at the interface, and thus the mixed film displacement curve would have resembled the β-lactoglobulin displacement curve. The other possibility is that there was very little β-casein on the surface because it had already been displaced by the β-lactoglobulin. This would mean that at the lowest surface pressure at which the mixed surfactant protein film was measured (21.2 mN/m), any domains of purely β-casein would have already collapsed and been replaced by either Tween 20 or β-lactoglobulin. The film thickness data showed a similar trend to the area data. However, Figure 6 is not quite as simple to interpret because the thickening of the mixed film occurs at surface pressures where a pure β-casein film would not exist. Despite this the mixed film data seem to show a transition from the β-casein data to the β-lactoglobulin data with an apparent decrease in thickness during this transition. Above about 24 mN/m the mixed film shows essentially the same thickness as the equivalent β-lactoglobulin film. To gain more information about the displacement process of the mixed proteins, the interface formed using the fluorescent-labeled protein was displaced by the addition of Tween 20. The interface had been aged for 3 days, and the displacement data are shown in Figure 5. The aging clearly makes the protein more difficult to displace due to the development of interprotein interactions over long adsorption times. This aging phenomenon is a feature of protein films and has been noted by others.18 Initially the subphase concentration was 1 µM Tween 20, but this was increased to 2 µM after 1 h. Panels b-d of Figure 3 show the progressive displacement of the protein film by the adsorbing Tween 20. Figure 3b is an image of a film transferred at a surface pressure of 27 mN/m and shows the protein film still occupying 66% of the total area. The image showed slightly less green and slightly more red than the previous image making it appear more
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Figure 7. Histograms of the proportion of the total measure fluorescence in the images in Figure 3 that comes from the FITC-labeled β-casein. Line 1 is for Figure 3a, line 2 is for Figure 3b, line 3 is for Figure 3c and line 4 is for Figure 3d.
yellow. This would seem to suggest that the film was compressed at the expense of the FITC-labeled β-casein. Figure 3c shows an image of a film transferred at a surface pressure of 28.5 mN/m. The protein film in this image occupies only 48% of the total area, and again the image appeared more yellow than the previous indicating that more FITC-labeled β-casein has been lost from the interface. The final image in the series, Figure 3d, was transferred at a surface pressure of 29.5 mN/m and shows the protein film occupying only 42% of the total surface area. The protein film in this image appeared to be very similar in color to the previous image. To quantify these color changes, each image was separated into hue (color), saturation (color density), and intensity (brightness). The hue, or color scale, was then adjusted to cover only red to green, and histograms of the images were calculated. These histograms are plotted in Figure 7. This shows that there was a significant change in the color of the fluorescent protein film as the displacement progressed. The color change indicated that the ratio of FITC β-casein to rhodamine 6G β-lactoglobulin decreased. This in turn suggests that the Tween 20 preferentially displaced the β-casein from the interface. Thus, either the displacement was not entirely “orogenic” as protein appears to have been lost from the interface at all stages of displacement or there were local heterogeneities in the surface protein distribution resulting in very small domains of almost pure β-casein in a continuum of mixed β-casein/β-lactoglobulin. Conclusions In this work we have used various methods to probe the interactions between β-casein and β-lactoglobulin. Surface rheological measurements demonstrated that β-casein adsorbs to the interface more rapdly than β-lactoglobulin. Thus, the interface is dominated by the β-casein in the early stage of protein film development. It is evident from the microscopy that the β-lactoglobulin that subsequently adsorbs does not cause any form of large-scale phase separation. This is almost certainly due, at least in part, to the lack of diffusion of the protein molecules within the mixed film. The rheological data suggest that at the concentrations used in this study, a 1:1 molar solution leads to a film containing about 80% β-casein. The evidence from both the AFM and the fluorescence microscopy suggests that the two proteins form a homogeneously mixed film which because of the high surface concentration ratio of β-casein to β-lactglobulin, contained small (
