Orogenic Displacement in Mixed â-Lactoglobulin/â-CaseinFilms at the Air/Water Interface

Alan R. Mackie,* A. Patrick Gunning, Michael J. Ridout, Peter J. Wilde, andVictor 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 â-casein/â-lactoglobulin films to the air/water interface and the subsequentdisplacement by the nonionic surfactant Tween 20 was studied. A combination of fluorescent labeling ofthe protein and Langmuir-Blodgett deposition was used to study the mixed protein layer. The adsorptionwas also monitored using two surface rheological techniques, shear and dilatation. Fluorescent labelingwas able to show that to within the limits of optical resolution the two proteins were well mixed at theinterface. We also show that the film remained well mixed after 3 days. Surface rheological data from thetwo techniques used was self-consistent and showed that during the initial stages of development, the filmswere dominated by the adsorption of the â-casein. Both fluorescence microscopy and atomic force microscopywere used to follow the displacement of the mixed film by surfactant. Results on films displaced by thenonionic surfactant Tween 20 showed that â-casein was preferentially displaced from the mixed film beforeâ-lactoglobulin.

Introduction

Proteins are widely used in the food industry asemulsifiers and foaming agents. Consequently there hasbeen much interest in understanding the way in whichprotein structure and interfacial function are related.1-4

Furthermore, because of the complex nature of mostfood systems, there has been substantial interest inunderstanding the interactions between proteins andsurfactants5-7 and between different proteins.8,9 This isof importance because proteins and emulsifiers stabilizeinterfacial films by different and conflicting mechanisms.In particular, two groups of food proteins have beenstudied, caseinates and whey proteins. The predominantwhey protein is â-lactoglobulin while a major componentof caseinate is â-casein. These two proteins show consid-erable differences in both structure and behavior. Sometime ago it was observed that â-casein could displace Rs1-casein from an oil/water interface but that â-lactoglobulincould not displace â-casein from such an interface.However, work done by others on â-casein10,11 suggeststhat it is much more difficult to displace from the oil/water interface than from the air/water interface. Dis-placement of â-casein required approximately 10 mN/m

higher surface pressure at the oil/water interface. Thus,it is not clear that the same behavior would be observedat the air/water interface.

Recently we have used atomic force microscopy (AFM)to look at the way in which proteins are displaced froman interface by surfactants. This has led to the develop-ment of an “orogenic” displacement model.10-12 This modeldescribes a mechanism that works in the following way:Heterogeneity in the protein film allows the displacingsurfactant to adsorb into localized defects, and thesenucleated sites then grow. The expansion of the pools ofsurfactant compresses the protein network, which initiallyincreases in density without increasing in thickness. Oncea certain critical density is reached, the protein layerthickness increases such that the protein film volume ismaintained as the surfactant domains expand. At suf-ficiently high surface pressures the network fails, releasingprotein, which then desorbs from the interface. Morerecently this model has been confirmed in situ at the air/water interface by Brewster angle microscope experi-ments.13

Here we attempt to extend this model by looking at thedisplacement of a mixed protein film by a nonionicsurfactant (Tween 20). More importantly we attempt tosee whether one protein can displace another from aninterface by a similar mechanism. Thus, we have usedfluorescent labeling to distinguish between proteins andto allow the observation of any phase separation that mightoccur at the interface. Although this is not the first timethat such techniques have been used,14 this is the firsttime that such a high degree of spatial resolution hasbeen obtained. The high spatial resolution is importantbecause of the range over which one might expect to seeprotein phase separation. In the case of mixed proteinand charged surfactant films (probably the closest com-

* To whom correspondence should be addressed. Tel: 44 1603255261. Fax: 44 1603 507723. E-mail: alan.mackie@bbsrc.ac.uk.

(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.

(12) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir2000, 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.

6593Langmuir 2001, 17, 6593-6598

10.1021/la010687g CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 09/14/2001

parison to protein-protein), the surfactant domains werein the order of tens of nanometers in size.12 In fact thelargest surfactant domains seen in a mixed proteinsurfactant film before the final rupture of the proteinnetwork were some only 100 µm in diameter.

Materials and Methods

The milk proteins used in this study were â-lactoglobulin (L-0130, lot 91H7005) and â-casein (C-6905, lot 12H9550) fromSigma Chemicals (Poole, U.K.). Samples were initially preparedat 2 mg‚mL-1 in water. The water used in this study was surfacepure (γ0 ) 72.6 mN‚m-1 at 20 °C) cleaned using an Elga ElgastatUHQ water purification system. The Tween 20 (polyoxyethylenesorbitan monolaurate) was obtained as a 10% solution (Surfact-Amps 20) from Pierce (Rockford, IL). The proteins were labeledusing amine reactive dyes, fluorescein isothyocyanate (FITC)from Sigma (Poole, U.K.) in the case of â-casein and 6-carboxy-rhodamine 6G succinimidyl ester (Molecular Probes Inc., Eugene,OR) in the case of â-lactoglobulin. The reaction was performedusing the recommended protocols (Molecular Probes), and theconjugate was purified by gel filtration to remove unbound dyeusing a Sephadex G-25 column.

Surface tension measurements were made using a wettedground glass Wilhelmy plate and a Langmuir trough (LabconLtd, Durham, U.K.). The PTFE trough was 255 × 112 × 16 mm,a volume of 450 mL, with one fixed and one mobile barrier. Allexperiments were performed at room temperature (20 °C) withdistilled water as the subphase. For the AFM measurements,the adsorbed films were produced from solutions containing 0.5µM â-casein, 0.5 µM â-lactoglobulin, and 0.5 µM Tween 20.Surface tension measurements were made as a function of timefor 30 min while the monolayer equilibrated. After this periodof equilibration, the subphase was perfused with 2 L of 0.5 µMTween 20 at a rate of 1.1 mL‚s-1. Further additions of surfactantto the subphase were then made in order to increase the surfacepressure as required. The Langmuir-Blodgett (LB) films wereformed on hydrophilic freshly cleaved mica substrates. LB filmswere produced by lowering a freshly cleaved piece of mica sheet,mounted perpendicular to the interface, down through theinterface and then pulling it back out again. The mounted pieceof mica was driven at a constant rate of 0.2 mm‚s-1. Surfacetension was monitored during the dip and showed that the filmwas only transferred onto the mica on the upward stroke. TheAFM images were measured under butanol. The precise detailsof the AFM and the imaging technique are given elsewhere.15

Fluorescence measurements were made using an OlympusBX60 microscope. Excitation was from a mercury arc lamp viaa 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 takenwere of LB films deposited onto washed glass coverslips in themanner described above.

Rheological measurements were made using both surface shearand surface dilation techniques. The surface dilation measure-ments were made using the method of Kokelaar et al.16 Thetechnique involves periodically raising and lowering a 10-cmground-glass ring into a vessel containing 250 mL of sample.The resulting change in surface area was kept to 5%, and thefrequency of dilation/compression was kept at 0.8 rads‚s-1. Thesurface shear measurements were made using a Camtel CIR100 surface shear rheometer (Camtel, Royston, U.K.). Thisinstrument uses a Du Nouy ring oscillating in normalizedresonance mode.17 The ring is oscillated in air at its resonantfrequency of approximately 3 Hz. When the ring is placed at aninterface, the resonant frequency and amplitude will changedepending on the elasticity and viscosity of the interface.Feedback currents restore the frequency and amplitude of theoscillations back to resonance. The values of the feedback signalrequired to restore the frequency are used to calculate the surface

shear elastic modulus (G′), and the signal required to restore theamplitude enables calculation of the viscous component (G′′).

Results and Discussion

In this paper we attempt to improve our understandingof mixed protein interfaces by investigating the structureand physical properties of a model system comprising twoproteins: â-lactoglobulin and â-casein. In previous paperswe have shown that surfactants disrupt and displaceproteins films by an orogenic mechanism, which involvesthe expansion of phase-separated surfactant domains. Themain aim of this work is to discover whether structuresand phase separation processes are likely to occur in amixed protein interface, similar to those observed in theprotein/surfactant systems studied previously. The resultsare discussed in terms of the various stages in theformation and stabilization of the interface. More specif-ically, the stages are (i) adsorption, (ii) phase separation,and (iii) displacement.

Adsorption. Protein adsorption is mainly determinedby surface hydrophobicity, molecular weight, solubility,and flexibility.18 â-Casein is a much more flexible moleculethan â-lactoglobulin and is therefore capable of unfoldingat the interface more rapidly. It has been found thatrandom coil proteins such as â-casein adsorb and lowerinterfacial tension more rapidly than globular proteins.19

â-Casein also has very different surface elasticity toâ-lactoglobulin, so the surface elasticity was used todynamically probe the adsorption of the mixed proteinsystem.

Two types of interfacial rheology were employed,dilatational and shear. Figure 1 shows the dilatationalelastic modulus for the two individual protein films andfor a 1:1 mixture. All three had the same total proteinconcentration (1 µM). â-Lactoglobulin and â-casein showmarkedly different behavior, which has been shown beforeby others.20 â-Casein shows a typical peak at earlyadsorption times which is caused by the collapse of thehighly charged N-terminal “tail” region into the sub-phase.21 The elasticity reaches a plateau of approximately10 mN/m after less than 10 min. It should be mentionedthat the â-casein film had virtually reached its equilibriumsurface pressure in about 5 min (data not shown). Theâ-lactoglobulin simply showed a steady increase in elasticmodulus to a pseudoplateau of approximately 78 mN/mafter 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 functionof time for adsorbing 1 µM â-casein (line 1), 1 µM â-lactoglobulin(line 2), and a 1:1 mixture of the two proteins at a total proteinconcentration of 1 µM (line 3).

6594 Langmuir, Vol. 17, No. 21, 2001 Mackie et al.

surface pressure of the film was still steadily increasingafter 105 min (data not shown). The mixed system showedfeatures of both proteins. The peak from the partialcollapse of the â-casein is present although shifted toslightly longer times. The much broader peak fromâ-lactoglobulin was also visible but reduced by nearly afactor of 4 to 22 mN/m. Subsequently the dilatationalelasticity climbed very slowly to 25.4 mN/m after nearly4 h. The surface pressure of the mixed film took about 30min to reach equilibrium (data not shown). The datasuggest that both proteins were observed to adsorb,resulting in a mixed â-lactoglobulin/â-casein interface.However, the surface elasticity of the mixed systemsuggests that â-casein was the dominant protein at thisstage in the adsorption. This agrees with previous workshowing the more rapid adsorption of â-casein. If, rathersimplistically, one assumes the surface dilatational elasticmodulus of the separate components to be merely additive,then the curve for an interface containing 85% casein fitsthe data reasonably well, especially in the latter stages.Further investigation will be required in order to fullyascertain the true nature of the rheological behavior ofthe mixed interfacial film. Especially in terms of the degreeto which such immobile systems may become phaseseparated.

The data from the measurement of surface shearelasticity are shown in Figure 2. Again, the figure showsdata for the individual proteins and the mixed system.The film formed by â-casein was so weak as to be almostimmeasurable while the film formed by â-lactoglobulingave quite high values. The values for â-lactoglobulin roseto a peak of 4.9 mN/m after 50 min and then decreasedin elasticity to about 2 mN/m after 3 h. The mixed filmshows a gradual slow rise to 1 mN/m after 3 h. The resultsshow a similar trend to the surface dilatational elasticity,in that â-casein was clearly the dominant protein at theseconcentrations. However, the surface shear elasticitywould suggest that there was almost no adsorbed â-lac-toglobulin. The difference between the results from thesurface shear and the surface dilatational rheology canbe put down to the different methodologies. Surfacedilatational rheology measures the changes in surfacepressure during compression and expansion of the inter-face whereas surface shear rheology measures directlythe mechanical properties of the interface under shear.Both methods are sensitive to protein structure and thenumber and strength of intermolecular interactions.However, surface shear tends to be more sensitive toprotein interactions and surface dilatation more sensitiveto adsorbed structure and composition. Hence, the surfacedilatation reveals that under these conditions â-casein

was the predominant protein at the surface but a smallamount of â-lactoglobulin was present. Despite thispresence, the mechanical strength of the adsorbed film(as measured by the surface shear elasticity) was almosttotally dominated by the â-casein component.

Therefore both surface rheological methods suggest thatat these equal concentrations â-casein adsorbed morerapidly than â-lactoglobulin and dominated the surfacerheological properties. To investigate further, fluorescencemicroscopy was used to see if there was a surfaceconcentration excess of â-casein and to observe any phaseseparation which may occur.

Phase Separation. The aim here was to determinethe spatial distribution of the two proteins at the interfaceand thus to ascertain if the proteins were evenly mixedor if phase separation occurred and how the proteindistribution could help us understand the surface rheo-logical behavior. Previous work on protein/surfactantmixed interfaces has shown, using AFM, how the sur-factant phase separates into domains to displace theprotein. AFM was ideally suited to these investigationsbecause it could easily distinguish between the separateprotein and surfactant domains. AFM cannot howeverdistinguish between two different proteins. To determinethe spatial distribution of the two proteins in the mixedfilm, fluorescence microscopy was employed.

In these experiments, the two proteins of interest werelabeled with fluorescent probes, â-casein with FITC andâ-lactoglobulin with rhodamine 6G. In both cases theextent of labeling was approximately 1:1 protein to dye.The total protein concentration in the Langmuir troughwas 0.5 µM, and adsorption was allowed to continue for3 days. Periodically, sections of the interface weretransferred onto glass coverslips by the Langmuir-Blodgett method. Figure 3a is from an interface afteradsorption for 2 days (surface pressure ) 21.5 mN/m) andshows a rather homogeneous deposition of protein. Thedominant protein was the FITC-labeled â-casein. Therewas a low level of very localized heterogeneity observedin all the images throughout the duration of the experi-ments. To check whether the heterogeneity seen in thisimage was representative of a small amount of phaseseparation of the proteins on the microscopic scale, asimilar experiment was performed using only the fluo-

Figure 2. Shear elastic modulus plotted as a function of timefor adsorbing 1 µM â-casein (line 1), 1 µM â-lactoglobulin (line2), and a 1:1 mixture of the two proteins at a total proteinconcentration of 1 µM (line 3).

Figure 3. A series of fluorescence images following thedisplacement of a mixed, dual stained protein film. â-Caseinwas labeled green and lactoglobulin was labeled red. All imagesare 63 µm by 48 µm. Image a shows a mixed protein filmtransferred at a surface pressure of 21.5 mN/m. Also shown isthe mixed film at various stages of displacement transferredat 27 (b), 28.5 (c), and 29.5 mN/m (d).

Mixed Protein Displacement Langmuir, Vol. 17, No. 21, 2001 6595

rescent â-lactoglobulin. When the autocorrelation func-tions of the two image sets were compared (data notshown), they were found to be very similar. This suggeststhat the small-scale heterogeneities were due simply torandom adsorption of the protein. There were no changesin the spatial distribution of the two proteins and no phaseseparation processes were observed over the experimentalperiod of 3 days. This behavior is very different from thephase separation observed with the surfactant/proteinsystems studied10 that could show phase separation inless than 1 h. The main differences between adsorbedproteins and surfactants are that (a) adsorbed surfactantis able to exchange with nonadsorbed surfactant and (b)adsorbed surfactant can freely diffuse at the interface. Incontrast, proteins, once adsorbed and interacting withneighboring proteins, can neither exchange with the bulknor diffuse at the interface. The mobility of a surfactantallows molecular segregation to occur and the resultantdomains to expand and deform. The distinct lack ofmobility of the adsorbed protein molecule arrests anysegregation process, and consequently there is no drivingforce for phase separation. Hence the results of thefluorescence experiments shown in Figure 3a demonstratethat no phase separation was observed, even after 3 days.Any local heterogeneity in the surface distribution ofprotein is due to the initial adsorption process, after whichno measurable redistribution was observed. The lack ofrearrangement and phase separation observed here is incontrast to the work published recently by Sengupta andDamodaran.14 We had observed previously that excessivefluorescent labeling could affect the surface properties ofthe protein. Measurements made on the surface rheologyof the fluorescent-labeled mixed protein film in the presentstudy showed it to be consistent with the unlabeled system.This indicates that the labeling had not significantlyaffected the strength of the film.

In summary, fluorescent microscopy demonstrated thatâ-casein was indeed the dominant adsorbed protein underthese conditions and that even after 3 days, no segregationor phase separation of the adsorbed proteins was observed.

Displacement. The displacement of the mixed â-lac-toglobulin/â-casein interface by the nonionic surfactantTween 20 was studied. The aim was to see if the structureof the mixed protein interface influenced how the indi-vidual proteins were displaced. This is important forunderstanding how real protein mixtures interact withlipids and surfactants in food foams and emulsions.

The displacement was measured first on unlabeledprotein using AFM. Figure 4 shows AFM images of a mixedâ-lactoglobulin/â-casein film at various stages of displace-ment by Tween 20. It can be observed that the appearanceof the protein films changed as displacement progressed.The light areas are where the protein is located, and thedark areas are where surfactant occupied the surface.Figure 4a shows a mixed film transferred at a surfacepressure of 21.6 mN/m where protein covered 69% of thesurface area. The second image (Figure 4b) shows themixed film after further displacement. This image is of afilm transferred at a surface pressure of 25.0 mN/m andshows protein film occupying an area of 30.5%. Forcomparison panels c and d of Figure 4 show pure â-caseinand pure â-lactoglobulin films, respectively. The adsorbedâ-casein film was transferred at a surface pressure of 20.7mN/m, and the film had a mean protein area of 51%. Theadsorbed â-lactoglobulin film was transferred at a surfacepressure of 26.1 mN/m, and the protein occupied an areaof 49%. It is clear that the holes visible in the mixed proteinfilm show characteristics of both single-component proteinfilms. The shapes of the holes were neither circular as

with the isotropic compression of the â-casein nor asirregular as those seen in the â-lactoglobulin film. Theshape of the displacement patterns gives a clear indicationthat there was indeed a mixed film at the interface.However, these images alone do not provide enoughinformation to draw firm conclusions about the spatialdistribution of the individual proteins within the film.

All AFM images were analyzed to provide informationabout both the surface area occupied by the protein andthe thickness of the protein layer. Figures 5 and 6 showthe protein area and thickness, respectively, as a functionof the surface pressure at which the film was transferred.For comparison both figures show data on films of pureâ-casein and of pure â-lactoglobulin. The displacement ofthe mixed film occurred over a range of some 7 mN/m andfalls between the displacement curves for the individualproteins (Figure 5). The fact that the displacement fallslinearly between the two curves for the individual proteins

Figure 4. AFM images showing mixed â-casein and â-lacto-globulin films being displaced by Tween 20. The films weretransferred 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 shownare AFM images of a â-casein film being displaced by Tween20 at a surface pressure of 20.7 mN/m (c) and a â-lactoglobulinfilm being displaced by Tween 20 at a surface pressure of 26.1mN/m (d). The image sizes are 9.0 and 4.0 µm, respectively.

Figure 5. The surface area (%) occupied by protein is plottedas a function of the surface pressure at which the LB film wastransferred. Data are shown for films of â-casein alone ([),â-lactoglobulin alone (9), and the 1:1 mixed film (2). Also shownare data from fluorescent-labeled films displaced after agingfor 3 days (b).

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suggests that there has been no phase separation betweenthe proteins in the mixed film. This mixed film requireda higher surface pressure to displace than a â-casein filmbut a lower surface pressure than a â-lactoglobulin film.If there had been a high degree of phase separation, onemight have expected to see the displacement for the mixedsystem follow the curves for the single proteins at leastover part of the range. In the early stages the â-caseindomains would have been displaced; thus the mixed filmwould have followed the displacement curve for theâ-casein alone. In the latter stages only the â-lactoglobulinwould have remained at the interface, and thus the mixedfilm displacement curve would have resembled the â-lac-toglobulin displacement curve. The other possibility isthat there was very little â-casein on the surface becauseit had already been displaced by the â-lactoglobulin. Thiswould mean that at the lowest surface pressure at whichthe mixed surfactant protein film was measured (21.2mN/m), any domains of purely â-casein would have alreadycollapsed and been replaced by either Tween 20 orâ-lactoglobulin.

The film thickness data showed a similar trend to thearea data. However, Figure 6 is not quite as simple tointerpret because the thickening of the mixed film occursat surface pressures where a pure â-casein film would notexist. Despite this the mixed film data seem to show atransition from the â-casein data to the â-lactoglobulindata with an apparent decrease in thickness during thistransition. Above about 24 mN/m the mixed film showsessentially the same thickness as the equivalent â-lac-toglobulin film.

To gain more information about the displacementprocess of the mixed proteins, the interface formed usingthe fluorescent-labeled protein was displaced by theaddition of Tween 20. The interface had been aged for 3days, and the displacement data are shown in Figure 5.The aging clearly makes the protein more difficult todisplace due to the development of interprotein interac-tions over long adsorption times. This aging phenomenonis 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 ofFigure 3 show the progressive displacement of the proteinfilm by the adsorbing Tween 20. Figure 3b is an image ofa film transferred at a surface pressure of 27 mN/m andshows the protein film still occupying 66% of the totalarea. The image showed slightly less green and slightlymore red than the previous image making it appear more

yellow. This would seem to suggest that the film wascompressed at the expense of the FITC-labeled â-casein.Figure 3c shows an image of a film transferred at a surfacepressure of 28.5 mN/m. The protein film in this imageoccupies only 48% of the total area, and again the imageappeared more yellow than the previous indicating thatmore FITC-labeled â-casein has been lost from theinterface. The final image in the series, Figure 3d, wastransferred at a surface pressure of 29.5 mN/m and showsthe protein film occupying only 42% of the total surfacearea. The protein film in this image appeared to be verysimilar in color to the previous image. To quantify thesecolor changes, each image was separated into hue (color),saturation (color density), and intensity (brightness). Thehue, or color scale, was then adjusted to cover only red togreen, and histograms of the images were calculated.These histograms are plotted in Figure 7. This shows thatthere was a significant change in the color of thefluorescent protein film as the displacement progressed.The color change indicated that the ratio of FITC â-caseinto rhodamine 6G â-lactoglobulin decreased. This in turnsuggests that the Tween 20 preferentially displaced theâ-casein from the interface. Thus, either the displacementwas not entirely “orogenic” as protein appears to havebeen lost from the interface at all stages of displacementor there were local heterogeneities in the surface proteindistribution resulting in very small domains of almostpure â-casein in a continuum of mixed â-casein/â-lacto-globulin.

ConclusionsIn this work we have used various methods to probe the

interactions between â-casein and â-lactoglobulin. Surfacerheological measurements demonstrated that â-caseinadsorbs to the interface more rapdly than â-lactoglobulin.Thus, the interface is dominated by the â-casein in theearly stage of protein film development. It is evident fromthe microscopy that the â-lactoglobulin that subsequentlyadsorbs does not cause any form of large-scale phaseseparation. This is almost certainly due, at least in part,to the lack of diffusion of the protein molecules within themixed film. The rheological data suggest that at theconcentrations used in this study, a 1:1 molar solutionleads to a film containing about 80% â-casein. The evidencefrom both the AFM and the fluorescence microscopysuggests that the two proteins form a homogeneouslymixed film which because of the high surface concentrationratio of â-casein to â-lactglobulin, contained small (<200nm) domains of almost pure â-casein.

Figure 6. The film thickness of the protein layer is plotted asa function of the surface pressure at which the LB film wastransferred. Data are shown for films of â-casein alone ([),â-lactoglobulin alone (9), and the 1:1 mixed film (2).

Figure 7. Histograms of the proportion of the total measurefluorescence in the images in Figure 3 that comes from theFITC-labeled â-casein. Line 1 is for Figure 3a, line 2 is forFigure 3b, line 3 is for Figure 3c and line 4 is for Figure 3d.

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From the displacement studies we have seen that theâ-casein is displaced from the interface in preference tothe â-lactoglobulin. In the final stages of displacement,the shapes of the holes in the film resemble those in aâ-lactoglobulin film and the surface pressure required toreach such a position correlates well with those requiredby a pure â-lactoglobulin film. All of the informationcombined leads to a coherent picture of the mixed proteinfilm. The early stages of formation were dominated by thehighly flexible â-casein, but the compressed film wasdominated by stronger interactions of the â-lactoglobulinallowing the â-casein to be removed from the interface.

The use of different surface rheological techniquestogether with imaging methods allows a more completeunderstanding of the influence of protein composition andinteractions on the mechanical properties of interfacialfilms formed by complex mixtures of food proteins.

Acknowledgment. This work was funded by theBBSRC as part of the core grant to the institute.

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