Disruption of Viscoelastic β-Lactoglobulin Surface Layers at the Air

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Disruption of Viscoelastic β-Lactoglobulin Surface Layers at the Air-Water Interface by Nonionic Polymeric Surfactants B. Rippner Blomqvist,*,†,‡ M. J. Ridout,§ A. R. Mackie,§ T. Wa¨rnheim,† P. M. Claesson,‡ and P. Wilde§ YKI, Institute for Surface Chemistry, P.O. Box 5607, 114 86 Stockholm, Sweden, Department of Chemistry, Surface Chemistry, Royal Institute of Technology, Drottning Kristinas va¨ g 51, 100 44 Stockholm, Sweden, and Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, United Kingdom Received June 11, 2004. In Final Form: August 17, 2004 Nonequilibrium interfacial layers formed by competitive adsorption of β-lactoglobulin and the nonionic triblock copolymer PEO99-PPO65-PEO99 (F127) to the air-water interface were investigated in order to explain the influence of polymeric surfactants on protein film surface rheology and foam stability. Surface dilatational and shear rheological methods, surface tension measurements, dynamic thin-film measurements, diffusion measurements (from fluorescence recovery after photo bleaching), and determinations of foam stability were used as methods. The high surface viscoelasticity, both the shear and dilatational, of the protein films was significantly reduced by coadsorption of polymeric surfactant. The drainage rate of single thin films, in the presence of β-lactoglobulin, increased with the amount of added F127, but equilibrium F127 films were found to be thicker than β-lactoglobulin films, even at low concentration of the polymeric surfactant. It is concluded that the effect of the nonionic triblock copolymer on the interfacial rheology of β-lactoglobulin layers is similar to that of low molecular weight surfactants. They differ however in that F127 increases the thickness of thin liquid films. In addition, the significant destabilizing effect of low molecular weight surfactants on protein foams is not found in the investigated system. This is explained as due to long-range steric forces starting to stabilize the foam films at low concentrations of F127.

Introduction Several properties of surface active molecules have been recognized as important factors for the stabilization of fluid dispersions, such as foams and emulsions, e.g., their adsorption behavior (rate of adsorption, ability to lower the surface tension, adsorbed layer thickness, amount adsorbed, ability to desorb), their surface lateral mobility, and their ability to change conformation and to interact with other molecules at the interface and across thin films. Many of these properties can be traced in the surface rheological behavior, i.e., the time-dependent response to deformations of surface layers. Therefore surface rheology can be essential for foaming properties, and high values of the surface shear and dilatational moduli are found for protein-stabilized foam films.1,2 There are several recent reviews considering the rheological properties of fluid interfaces occupied by mixtures of proteins and surfactants,1,2 by nonionic homopolymers,3 and by surfactants.4 Following adsorption to the air-water interface, globular proteins such as β-lactoglobulin, bovine serum albumin, or R-lactalbumin form strong viscoelastic layers, in which * To whom correspondence should be addressed: Brita Rippner Blomqvist, YKI, Institute for Surface Chemistry, P.O. Box 5607, 114 86 Stockholm, Sweden. E-mail: [email protected]. Telephone: +46-8-5010 6079. † YKI, Institute for Surface Chemistry. ‡ Department of Chemistry, Surface Chemistry, Royal Institute of Technology. § Institute of Food Research. (1) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176-181. Murray, B. S. Curr. Opin. Colloid Interface Sci. 2002, 7, 426-431. (2) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437-471. (3) Noskov, B. A.; Akentiev, A. V.; Bilibin, A. Y.; Zorin, I. M.; Miller, R. Adv. Colloid Interface Sci. 2003, 104, 245-271. (4) Langevin, D. Adv. Colloid Interface Sci. 2000, 88, 209-222.

the molecules are essentially immobile.5,6 The protein molecules are interacting through a combination of van der Waals and dipolar forces, ionic, hydrophobic, and hydrogen bonds, and possibly also by covalent bonds (disulfide bridges).7 This interfacial network of highly entangled and cross-linked molecules can stabilize liquid films against rupture by opposing film stretching and by slowing down film thinning and drainage.5,6 Conventional surfactants can also stabilize liquid films by intermolecular forces, but to a significantly smaller extent. One of the ways that low molecular weight (LMW) surfactants stabilize foam films is by the Gibbs-Marangoni mechanism,8,9 which requires lateral mobility in the foam lamellae. When a film is subjected to a local stretching due to some external disturbance, the increase in surface area will be accompanied by a local decrease in the surface excess of adsorbed surfactant, which results in the generation of a surface tension gradient (the Gibbs effect). In this situation the stabilization depends on the local change of the surface tension with time (the Marangoni effect). The surface tension can be returned to a uniform level by two mechanisms: the adsorption of surfactant from the bulk or the lateral diffusion of molecules already adsorbed at the surface. It is the latter mechanism that causes a stabilization by also leading to a transport of (5) Clark, D. C.; Coke, M.; Mackie, A. R.; Pinder, A. C.; Wilson, D. R. J. Colloid Interface Sci. 1990, 138, 207-219. (6) Coke, M.; Wilde, P. J.; Russell, E. J.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489-504. (7) Dickinson, E. J. Chem. Soc., Faraday Trans. 1998, 94, 16571669. (8) Schick, M. J.; Schmolka, I. R. Foaming/Nonionic surfactants; Schick, M. J., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1987; Vol. 23. (9) Ewers, W. E.; Sutherland, K. L. Aust. J. Sci. Res. 1952, A5, 697710.

10.1021/la0485475 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/12/2004

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underlying solution into the thinner part of the film, thus restoring the film thickness.9 Finally, an interlayer repulsion due to electrostatic double-layer forces and steric interactions may also provide film stabilization to a varying degree depending on surface charge density and the structure of the interfacial layer. The complexity of the interplay of the different mechanisms of foam stabilization is illustrated by the fact that foams generated in the presence of both proteins and LMW surfactants are often less stable than those stabilized by each component alone.1,2,6 The reason is that the surfactants break down the viscoelastic protein layer, while at the same time the presence of the protein at the surface impedes the lateral mobility of surfactant molecules. Consequentially, none of the mentioned stabilizing mechanisms can work efficiently, except the stabilization due to repulsive interactions across the foam lamellae. The rheology at the air-water interface of mixed adsorbed layers and the displacement of proteins by conventional surfactants have often been studied with the focus on milk proteins together with various surfactants, such as β-lactoglobulin-Tween 20 (polyoxyethylene[20]sorbitan monolaurate),10-13 β-lactoglobulin-SDS (sodium dodecyl sulfate),14 β-lactoglobulin-C12E6,15 β-lactoglobulin-sucrose monoesters,16 β-casein-Tween 20,11,12 or R-lactalbumin-Tween 20.12 These studies have shown that the coadsorption of LMW surfactants into protein layers at the interface reduces the surface viscoelasticity and brings about a fluidization of the layers. The intermolecular cross-links in the protein layer are disrupted, which may be caused by displacement (nonionic surfactants) and/or by solubilization due to the formation of a highly water-soluble complex between the protein and the surfactant (mainly ionic surfactants).7 It has been demonstrated that LMW surfactants displace the proteins by first adsorbing at defects in the protein film. Next, phase-separated surfactant domains are formed, which grow with increasing adsorption of the surfactant, compressing the protein network, and finally displacing the protein from the interface.12,17 In the present work we have investigated the effect of relatively high molecular weight polymeric surfactants on the surface rheology of adsorbed β-lactoglobulin layers. The polymeric surfactants are nonionic block copolymers of ethylene oxide and propylene oxide, PEO-PPO-PEO, which on their own can be effective stabilizers of foams and emulsions, an effect usually discussed in terms of steric repulsion. In addition to their ability to form monolayers at the air-solution interface, some general properties of nonionic poly(ethylene oxide)-based block copolymers, which are relevant for foam stability, are their ability to self-aggregate in solution above the critical micelle concentration (cmc) and the reversed temperature (10) Kragel, J.; Wustneck, R.; Clark, D.; Wilde, P.; Miller, R. Colloid Surf., A 1995, 98, 127-135. Roth, S.; Murray, B. S.; Dickinson, E. J. Agric. Food Chem. 2000, 48, 1491-1497. (11) Kragel, J.; Wustneck, R.; Husband, F.; Wilde, P. J.; Makievski, A. V.; Grigoriev, D. O.; Li, J. B. Colloids Surf., B 1999, 12, 399-407. (12) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157-166. (13) Petkov, J. T.; Gurkov, T. D.; Campbell, B. E.; Borwankar, R. P. Langmuir 2000, 16, 3703-3711. (14) Kragel, J.; O’Neill, M.; Makievski, A. V.; Michel, M.; Leser, M. E.; Miller, R. Colloids Surf., B 2003, 31, 107-114. Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 81768181. (15) Murray, B. S.; Ventura, A.; Lallemant, C. Colloids Surf., A 1998, 143, 211-219. (16) Garofalakis, G.; Murray, B. S. Colloids Surf., B 2001, 21, 3-17. (17) Mackie, A. R.; Gunning, A. P.; Pugnaloni, L. A.; Dickinson, E.; Wilde, P. J.; Morris, V. J. Langmuir 2003, 19, 6032-6038.

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dependence of solubility.18 In this paper the investigated polymer (F127) is referred to as “block copolymer” or “polymeric surfactant”. The block copolymers have properties in common with both LMW surfactants and certain proteins. First, the diffusion rate, which initially controls the adsorption to nonpolar interfaces, is comparatively low. However, at equilibrium the block copolymers, like many LMW surfactants, lower the surface tension more effectively than proteins in general.2 Second, like proteins, they are able to change their conformation upon adsorption. At low surface coverage, both the PPO and the PEO part can adsorb in an almost flat two-dimensional conformation. At high coverage loops and tails of the more hydrophilic PEO chains will extend toward the solution and eventually form a brush structure19 of densely packed PEO chains. Because of their molecular size, both proteins and PEOPPO-PEO polymeric surfactants may form thick adsorbed layers and thus interact with its surroundings by steric repulsion. In addition, for globular proteins, the adsorption process is effectively irreversible with respect to dilution of the bulk phase.7 For the block copolymers, the exchange rate between the surface and the bulk phase is extremely slow, which means that the adsorption of large polymers can be considered as irreversible within normal experimental periods of time. In this sense the polymeric surfactants are likely to show similarities with flexible random coil proteins such as β-casein. Regarding the viscoelastic behavior, dilatational relaxation processes for LMW surfactants are usually determined by the diffusional exchange between the surface layer and the bulk solution and by rapid lateral motions within the layer, whereas the relaxation in polymer systems also proceed within the adsorbed film, by an exchange of polymer segments between different regions of the surface layer.3,4 The aim of the present study was to elucidate how the block copolymer, upon coadsorption with a protein (βlactoglobulin), affected the protein network in terms of lateral interactions and mobility in surface layers and thin foam films. Layers adsorbed at the air-water interface have been characterized by surface shear and dilatational techniques, film thickness and drainage measurements, and surface diffusion experiments. We relate the results to the foam stability of mixed systems and discuss briefly mechanisms for foam stabilization. Experimental Section Materials. β-Lactoglobulin (Mw ) 18 281 g/mol), containing genetic variants A and B, from bovine milk was obtained from Sigma-Aldrich Co., U.K. (L-0130, lot no. 91H7005). The block copolymer Synperonic PE/F127 (PEO99-PPO65-PEO99, Mw(mean) ) 12 500 g/mol according to the manufacturer, batch no. 2804BK0518) was kindly provided by ICI Surfactants, Uniqema, The Netherlands. The F127 sample was used without further purification. The notation F127 refers to F for flakes. The first two numbers and the last number are indicative of the molecular weight of the PPO block (≈3780 g/mol) and the weight fraction of the PEO block (≈70%), respectively. The subscripts in the structural formula are the numbers of monomers. All solutions studied were below the phase separation temperature (cloud point at 800 µM ) >100 °C)20 and the cmc21,22 for the main component of F127 (reported values of the cmc are 600 µM (25 °C),21 3200 (18) Malmsten, M. Adsorption of Polymers at the Solid/Liquid Interface. Ph.D. Thesis, University of Lund, 1992. (19) Milner, S. T. Science 1991, 251, 905-914. (20) Desai, P. R.; Jain, N. J.; Sharma, R. K.; Bahadur, P. Colloids Surf., A 2001, 178, 57-69. (21) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414-2425. (22) Diakova, B.; Kaisheva, M.; Platikanov, D. Colloids Surf., A 2001, 190, 61-70.

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(20 °C),21 and 2000 µM22). Ultrapure deionized water (Elga Elgastat LabWater system, 18.2 MΩ cm resistivity) was used. Sodium phosphate buffer (10 mM, pH 7.0, γ0 ) 72.7 mN/m) was prepared by dissolving NaH2PO4 and Na2HPO4 (AnalaR grade from British Drug Houses) in water. NaCl (AnalaR grade) was purchased from Sigma-Aldrich Co., U.K. All glassware and the Wilhelmy plates were washed with Micro-90 (#9050, International Products Corp., Burlington, NJ) and rinsed thoroughly with water, followed by 2-propanol (AnalaR grade), and then water. Stock solutions of β-lactoglobulin were purified according to the method of Clark et al.,23 by using granular active charcoal (product no. 33034, batch 452619OG) from BDH Chemicals Ltd., U.K. Surface active contaminants (e.g., fatty acids) bound to the protein have been shown to reduce the dilatation elastic modulus of adsorbed films, but these compounds are possible to remove by treatment with charcoal.23 Protein concentrations were determined by UV spectroscopy (Lambda 9 UV/VIS/NIR, PerkinElmer), using a molar absorption coefficient of E279 ) 0.93 mg mL-1 cm-1.24 Stock solutions of F127 were prepared by dissolving the polymer in water by gentle stirring overnight at least 1 day before the measurement. Solutions were stored under nitrogen at room temperature until use. All measurements were made at constant β-lactoglobulin concentration (10 µM ) 0.18 g/L) and varying F127 concentration. Solutions were obtained by dilution with sodium phosphate buffer of freshly made protein stock solutions, and addition of appropriate amounts of F127 solution, and were allowed to equilibrate for 30 min before any measurement. The fluorescent probe for FRAP measurements was 5-(N-octadecanoyl)aminofluorescein (ODAF, Mw ) 613.79 g/mol) from Molecular Probes Europe BV, the Netherlands (O-332, lot no. 2051-1). A stock solution of ODAF (3 mM in EtOH) was prepared, which was added to aqueous solutions of the polymeric surfactant. Surface Dilatational Rheology. A low-frequency ring trough method as described by Kokelaar et al.25 was used. The advantage with this technique is that the change in area is almost completely dilatational and the shear effects are minimized. The apparatus consisted of a ground glass cylinder (diameter 10 cm) that was dipped and raised in the vertical direction by a sinusoidal drive, in a glass trough containing the solution. The surface tension was measured using a ground glass Wilhelmy plate placed inside of the cylinder. The periodic (ω ) 0.82 rad/s ) 0.13 Hz) expansion and compression caused an area change (dA/A) of 5%. The adsorbed films were investigated at room temperature (20-21°C) for 1 h. The surface tension γ, the complex dilatational modulus E (E ) dγ/d ln A ) dγ/(dA/A), E ) Ed + iηdω)26), the real part of the modulus Ed, and the imaginary part of the modulus ηdω were measured as a function of time. Ed is the storage modulus connected with the elasticity, whereas the loss past, ηdω, represents the dissipation processes in the layer (including the friction between molecules in the layer and diffusional exchange of molecules between the surface and the underlying bulk solution).26 The elasticity Ed and viscosity ηd are related to the phase angle θ, which is the phase shift between the sinusoidal oscillations of surface tension and surface area, via the expression tan(θ) ) ηdω/Ed. ω is the angular frequency of the sinusoidal deformations.26 The sample solution volume 250 mL was introduced in the trough, and the adsorption began at t ) 0. The first data point was recorded after 30 s. The adsorbed films were investigated at room temperature (20-21 °C) for 1 h. The range of F127 concentrations was 0.02-200 µM, i.e., a 500:0.05 molar ratio of β-lactoglobulin/F127. Protein-free aqueous solutions of F127 (0.02-1600 µM) were also investigated. It was confirmed that the measured dilatation parameters of the F127 samples (23) Clark, D. C.; Husband, F.; Wilde, P. J.; Cornec, M.; Miller, R.; Kragel, J.; Wustneck, R. J. Chem. Soc., Faraday Trans. 1995, 91, 19911996. (24) Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 319326. The absorbance was calculated from the extinction coefficient as estimated with the method of Gill et al according to the Swiss-Prot/ TrEMBL database (PO2754, 05/02/2003) (25) Kokelaar, J. J.; Prins, A.; Degee, M. J. Colloid Interface Sci. 1991, 146, 507-511. (26) Lucassen, J., van den Tempel, M. J. Colloid Interface Sci. 1972, 41, 491-498.

Rippner Blomqvist et al. were unaffected by the presence/absence of 10 mM sodium phosphate buffer. Surface Shear Rheology. Measurements were carried out by using an automatic in-plane oscillatory ring apparatuss Camtel CIR 100 (Camtel Ltd., Royston, U.K.) The technique has been described previously.27 Briefly, a platinum Du Nou¨y ring (diameter 13 mm) was placed in the plane of the air-liquid interface. The oscillation resonance frequency was 3 Hz, and the strain amplitude was set to 5 × 10-3 rad. The shear elastic, G′, and shear viscous, G′′, moduli were calculated from the applied forces required to maintain resonance frequency and amplitude (normalized resonance mode). Water was used as the reference interface, and a reference measurement was taken once a day. Solutions were poured into a glass vessel (Φ ) 4.6 cm), and the surface shear rheology of the adsorbed layer was measured for 1 h. Each measurement was performed in duplicate at room temperature (20-21 °C). Adsorption from solutions of β-lactoglobulin and F127 (range 0.1-2 µM) and of F127 only for comparison was investigated. FRAP (Fluorescence Recovery after Photobleaching). The surface lateral diffusion coefficient of the surface-active fluorescent probe ODAF in block copolymer films at the airwater interface was determined by using a technique described previously.28 The method involves irreversible photobleaching of fluorescent molecules by means of a short-lived high-intensity laser flash, within a limited area at the surface of a thin foam film. The repopulation with (nonbleached) fluorescent molecules by lateral diffusion into the bleached area is monitored as a function of time. The rate of recovery of fluorescence is used to calculate the surface lateral diffusion coefficient of the fluorophore.29 The wavelength of the laser was 488 nm (Ar+ 10 W, Coherent Innova 100-10) and the illuminated spot diameter was 2.85 µm. Thin foam films were formed in a ground glass ring (3 mm internal diameter), which was mounted horizontally in a chamber, containing a small trough filled with the solution in order to achieve a vapor-saturated atmosphere. Films were formed from solutions containing F127 (2, 20, 200, and 1600 µM) and 0.014 mol of ODAF per mole of surfactant, except at the lowest concentration (2 µM), where the probe concentration had to be raised to 0.14 mol per mole of surfactant to achieve a detectable signal of the fluorescence. The concentration of ODAF in the solutions was 0.3-3 µM. At least 10-15 fluorescence recovery curves for each film were collected. All FRAP measurements were performed at 24 °C on films that had drained to its equilibrium thickness. The data were analyzed by nonlinear leastsquares fittings to an expression30 defining the time dependence of fluorescence recovery, as described in the article by Clark et al.28 Thin-Film Measurements. The thickness of single suspended foam films stabilized by β-lactoglobulin and F127 was measured interferometrically using the setup described previously,28 based on the dynamic thin-film drainage method.31 A laser beam (λ ) 632.8 nm) from a 10 mW HeNe source (model 1125P, Uniphase, C. A.), was focused on the film using a microscope objective (×40/040 Nikon). The laser beam could be cut off by a shutter, while the drainage pattern and appearance of the film were observed visually, by means of reflected light from an inverted light microscope (white light, Nikon Optiphot) using the same objective (×40/040). The thin films were formed in the same glass capillary ring as used for the FRAP experiments. The diameter of the films was adjusted to 350-400 µm. At equilibrium the thickness was checked to be equal at several positions of the film. The time scale of the experimental procedure was standardized. A 30 µL droplet was introduced in the glass ring and immediately placed in the chamber, followed by an (27) Warburton, B. Surface rheology. In Technologies in rheological measurement; Collyer, A. A., Ed.; Chapman & Hall: London, 1993; pp 55-97. (28) Clark, D. C.; Dann, R.; Mackie, A. R.; Mingins, J.; Pinder, A. C.; Purdy, P. W.; Russell, E. J.; Smith, L. J.; Wilson, D. R. J. Colloid Interface Sci. 1990, 138, 195-206. (29) Beck, K.; Peters, R. Spectroscopy and the Dynamics of Molecular Biological Systems; Bayley, P. M., Dale, R. E., Eds.; Academic Press: New York/London, 1985; p 177. (30) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055. (31) Scheludko, A. Adv. Colloid Interface Sci. 1967, 1, 391-464.

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adsorption period of 5 min before formation of a film. All films were observed for at least 1 h at 24 °C. At least duplicate measurements (more often three to four repeats) were performed. The equivalent water film thickness hw was calculated using the refractive index of water (nw ) 1.33), according to Scheludko’s expression:31

hw )

( )

(

λ arcsin 2πnw

1+

)

∆ 4R(1 - ∆) (1 - R)2

1/2

(1)

where ∆ ) (I - Imin )/(Imax - Imin ), λ is the wavelength of the laser, and R ) (nw - 1)2/(nw + 1)2. Imax and Imin were taken as the intensity level at the last interference maximum and the intensity when no film was present in the chamber, respectively. The solutions contained 10 mM sodium phosphate buffer, pH 7.0, and NaCl (100 mM) was added to further suppress electrostatic double-layer interactions. Foam Stability. Foam was created in a glass column by sparging gas (pure compressed air, AGA, Sweden, filtered through a 0.2 µm particle filter) through the solution via a porous glass frit (34 mm frit diameter, 16-40 µm pore size), by using automatic equipment (Foamscan, I. T. Concept, Longessaigne, France). Mixed solutions of 10 µM β-lactoglobulin and 0.02-200 µM F127 were investigated. The solutions were made up in 10 mM sodium phosphate buffer, pH 7.0. The foam volume was calculated from an image of the column that was recorded by a CCD camera and registered continuously with time. Twenty milliliters of solution was introduced into the bottom of the column. The gas flow was set to 50 mL/min and was turned off when the foam volume had reached 100 mL. The time to 100 mL was 92-94 s for pure β-lactoglobulin decreasing to 87-88 s at the highest F127 concentration (200 µM). The decay of the foam was monitored at room temperature (20-22 °C) for 1 h. All measurements were performed in triplicate. Presented values of the foam stability represent the foam volume relative to the volume of foam formed by β-lactoglobulin alone (relative foam stability (%) ) foam volume/foam volumeβ-lactoglobulin × 100) at t ) 1 h. Foaming data for Tween 20 was redrawn from ref 53.

Results and Discussion Adsorption Properties. The effect of adsorption of β-lactoglobulin and the triblock copolymer F127 to the air-water interface was determined by measuring the surface tension decrease with time for 60 min (Figure 1a), by means of the Wilhelmy plate method. The protein concentration was kept constant at 10 µM (0.18 g/L) while the content of F127 was varied between 0 and 200 µM. The adsorption equilibrium state was not attained within the hour of adsorption for any of the compositions studied, since the surface tension continued to decrease slowly throughout the experiments. However, after ≈20-30 min an almost constant surface tension state was observed for β-lactoglobulin alone and for mixed solutions containing 20 and 200 µM F127. At the intermediate copolymer concentrations (0.5-2 µM), the rate of surface tension decrease was somewhat more significant toward the end of the experiment. This indicates that the time course of the dynamic processes in the surface layer changed with the bulk concentration of F127. The time to reach the true surface tension equilibrium for block copolymers is often found to be very long (days), e.g.,32,33 due to the slow diffusion, the polydisperse nature of the block copolymers, and the slow exchange rate between the adsorbed layer and the bulk phase. Regarding proteins, Graham and Phillips34 measured the time dependence of the surface (32) Rippner, B.; Boschkova, K.; Claesson, P. M.; Arnebrant, T. Langmuir 2002, 18, 5213-5221. (33) Jiang, Q., Chiew, Y. C., Valentini, J. E. Colloids Surf., A 1996, 113, 127-134. (34) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403-414.

Figure 1. (a) Surface tension as a function of time for mixed solutions of 10 µM β-lactoglobulin and various amounts of the block copolymer F127 (0-200 µM, as indicated to the right). Data were obtained by the Wilhelmy plate method. (b) Surface tension versus the concentration of F127 for solutions in the presence (b) and absence (2) of 10 µM β-lactoglobulin. The values represent the surface tension after 60 min of adsorption.

tension and the surface excess (Γ) for several proteins and concluded that the surface tension may change with time at constant Γ due to conformational rearrangements of the protein molecule. The time scale for these conformation changes could be 1-8 h. Therefore it is not surprising that we see a slow continuous change of the surface tension with time (Figure 1a, 10 µM protein/0, 20, and 200 µM F127) even when the surface, which will be shown below, is dominated presumably by only one species, either the β-lactoglobulin or the block copolymer. Reported values of solution diffusion coefficients (D) of the β-lactoglobulin are 9.7 × 10-11 and 7.7 × 10-11 m2/s35 for the monomer and dimer,36 respectively, and 6 × 10-11 m2/s as recently determined by NMR at pH 7.37 The bulk diffusion coefficient of F127 was 6 × 10-11 m2/s,38 as measured by NMR at 27 °C.39 Thus the block copolymer and the protein were expected to diffuse at a similar rate from the bulk solution to the interface and the initial surface composition would scale with the solution composition. (35) Gilbert, L. M.; Gilbert, G. A. Nature 1961, 192, 1181. Cecil, R.; Ogston, A. G. Biochemistry 1949, 44, 33-35. (36) At pH 7.0 and 10 mM salt buffer added β-lactoglobulin monomers (Mw ) 18 kDa) self-associate into dimers (Mw ) 37 kDa) held together by noncovalent interactions, and there is an equilibrium between dimers and monomers in solution, with predominance for the dimer. McKenzie, H. A.; Sawyer, W. H. Nature 1967, 214, 1101. (37) Le Bon, C.; Nicolai, T.; Kuil, M. E.; Hollander, J. G. J. Phys. Chem. B 1999, 103, 10294-10299. (38) To be viewed as an average between monomers and micelles since the measured concentration was close to the cmc.39 D for the monomer would be expected to be slightly lower. (39) Malmsten, M.; Lindman, B. Macromolecules 1992, 25, 54405445.

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Figure 3. The surface dilatational modulus (E) plotted as a function of surface pressure, showing the transition from a highly viscoelastic protein layer to a weaker layer dominated by the block copolymer. The various symbols represent experiments with 10 µM β-lactoglobulin (b) and added F127 in the concentration range 0-200 µM (], 0.02; 4, 0.2; +, 0.5; O, 1; [, 2; 2, 20; 9, 200 µM). The dilatational modulus for layers formed by adsorption from solutions of F127 alone (solid lines, 0-200 µM) is shown for comparison. The surface pressure, π, is given by the difference (π ) γ0 - γ) between the surface tension of water, γ0, and the solution surface tension, γ.

Figure 2. Surface dilatational rheology for adsorbed layers formed from mixed solutions of 10 µM β-lactoglobulin and the block copolymer F127 (concentration range 0-200 µM) as measured in a ring trough at the frequency 0.13 Hz, with a surface area change of 5%. (a) The evolution of the dilatational modulus (E) with time. The F127 concentrations varied as indicated in the figure. (b) The complex dilatational modulus (E, O), the elastic component (Ed, 2), and the viscous component (ηdω, b) at t ) 60 min as a function of the F127 concentration.

The surface tension after 60 min of adsorption from mixed β-lactoglobulin and F127 solutions is compared to the corresponding values for pure F127 solutions in Figure 1b. The surface tension for 10 µM β-lactoglobulin without added F127 was 52.4 mN/m after 1 h of adsorption, in agreement with previously published values.12,13 The surface tension of F127 also agrees with previously reported results.22 The surface layer is dominated by protein at low surfactant concentration, although the effect of coadsorption of F127 is already evident at 0.02 µM. The two curves converge at a F127 concentration close to 2 µM, which indicates that up to a F127 concentration of about 2 µM, a mixed layer is formed. Above this concentration of F127, the surface tension of the mixed solutions coincide with that of pure F127, which suggests that the interface is completely dominated by the polymeric surfactant. Surface Dilatation Rheology of Adsorbed Layers. The evolution of the dilatational modulus was followed at the frequency of 0.13 Hz at constant β-lactoglobulin concentration and a range of F127 concentrations (Figure 2a). This frequency was tuned to give a good response for β-lactoglobulin, as it allows the collection of enough data points within the chosen adsorption time. Under the experimental conditions used, the protein on its own is effectively irreversibly adsorbed.5,6 Any exchange with the bulk of the protein which may occur would be at time scales longer than 0.1 Hz.40 From this it follows that a

change of the dilatational modulus compared to that of the protein is caused by the coadsorption of the polymeric surfactant. For the pure protein film, the modulus increased gradually with time to reach 130 mN/m after 60 min (end of experiment), showing that a mechanically strong intermolecular network is formed at the air-water interface.The time-dependent increase of the modulus is a combined effect of adsorption and development of molecular interactions and conformation changes at the surface. With increasing amounts of the polymeric surfactant the modulus clearly decreased, as seen for example for 10 µM protein/20 µM of F127, where the modulus was only 5 mN/m after 1 h. For the solutions containing 0.2 and 0.5 µM F127 a viscoelastic protein layer initially (e5 min) began to build up, but as the coadsorption of block copolymer continued, the modulus passed through a maximum and then decreased with time, suggesting that F127 displaced the protein from the interface. The elastic part, Ed, of the complex modulus is larger than the viscous part, ηdω, for all interfacial compositions studied (Figure 2b). Surface layers formed from the solutions of F127 only are also predominantly elastic (data not shown). Thus, on this point adsorption layers of proteins, block copolymers, and LMW surfactants41,42 qualitatively show similar properties, i.e., being mainly elastic upon dilatation in the low-frequency range. The effect of the polymeric surfactant F127 on the surface dilatational rheology of β-lactoglobulin layers at the interface may be concluded by displaying the dilatational modulus as a function of the surface pressure (Figure 3), where an increased surface pressure corresponds to an increased adsorption time. Note that the data presented in Figure 3 are based on the same (40) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227-239. (41) Rodriguez Nino, M. R.; Wilde, P. J.; Clark, D. C.; Rodriguez Patino, J. M. J. Agric. Food Chem. 1998, 46, 2177-2184. (42) Stubenrauch, C.; Miller, R. J. Phys. Chem. B 2004, 108, 64126421.

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experiments as shown in Figure 2. Thus, each curve (marked with the various symbols) in Figure 3 represents the surface pressure and the dilatational modulus during 1 h of adsorption from the mixed protein-F127 solutions. The dilatational modulus at different surface pressures of F127 without the presence of any protein, represented by the solid lines, is included for comparison. There are several overlapping lines because, as for the mixed system, the modulus was measured for 1 h in separate experiments, using different bulk solution concentrations, to collect data for the whole range of surface pressures. The different experiments for the pure F127 overlap which shows that the measured surface dilatational modulus in this case is a unique function of the surface pressure, independently of the adsorption time and the bulk concentration used to reach this surface pressure. The variations of E between experiments at a certain surface pressure was small (less than 1 mN/m), which implies that variations of the bulk solution composition due to polydispersity of F127 may have a small effect, but do not affect the interpretation of data. The evolution of the viscoelasticity of the β-lactoglobulin layer was completely different compared to that of F127. It continued to increase throughout the experiment, due to protein unfolding that expose more hydrophobic parts toward air and formation of intermolecular cross-links. The strong, mainly elastic layer, of β-lactoglobulin is weakened by coadsorption of F127. As the F127 concentration increases, the behavior gradually changes from the protein-like behavior to triblock copolymer-like behavior. An effect of F127 is found even at the lowest surfactant concentration (0.02 µM), although the surface was still covered mainly by the protein. The surface pressure of 0.02 µM F127 on its own reached only 5 mN/m at the end of the experiment (at 60 min). It is clear that β-lactoglobulin and F127 coadsorb and form mixed surface layers up to at least 2 µM of F127. Thus (which is not obvious from the surface tension data), the protein is not fully displaced at 2 µM. The viscoelasticity for the block copolymer decreases at higher surface pressure (Figure 3, solid line, 20-35 mN/ m). The latter could be a result of increasing exchange of molecules between the interface and bulk phase. Frequency variations of the dilatational viscoelastic properties and the coupling with the exchange of soluble surfactant between the surface and the bulk are well-known.3,4 Upon compression some molecules can dissolve into the underlying water, to restore the equilibrium surface concentration. At low frequency, when there is time to reach “equilibrium” in each cycle, this process occurs fully and there is no resistance to compression-expansion. On the other hand, at high frequency, when the exchange rate is much slower than the frequency of the disturbance, the layer behaves as an insoluble monolayer.4 Apart from the exchange with the bulk solution, there are frequency variations of the dilatational parameters related to relaxation processes within the adsorbed layer that are induced upon deformation. For many LMW surfactants the modulus increases with frequency at a given concentration.42,43 An important matter is whether the observed effect of F127 on the protein film would be qualitatively different if measured at a different frequency. If the dilatational modulus of F127-dominated layers would be higher than that for the protein layer at a higher frequency, this could, on the contrary to what we conclude above, give the impression that the lateral strength of the block

copolymer film is stronger than that of the protein. We measured the dilatational modulus for the block copolymer at the concentration 20 µM over the frequency range 0.010.3 Hz. The modulus increased 3 mN/m in this interval. Others have measured the frequency dependence of the dilatational elasticity for adsorption layers of F127 and similar PEO-PPO-PEO block copolymers by capillary wave techniques over broad frequency and concentration ranges.33,44 The data presented by these authors show that the elastic modulus increases with frequency, reaching a plateau or a maximum that does not exceed 30 mN/m at any concentration. This is far below the dilatational elasticity of the β-lactoglobulin film. Therefore we can conclude that the observed weakening of the protein layer is a real effect due to the presence of F127. Surface Shear Rheology of Adsorbed Layers. Surface shear rheology measurements tend to be more sensitive than dilatational rheology to interactions between molecules in the surface layer. The reason is that the resistance to the movement of the oscillatory ring placed in the plane of the interface is measured directly, whereas the dilatation rheology measures the changes in surface tension upon compression and expansion of the surface. The surface shear storage modulus (G′) for layers adsorbed from 10 µM β-lactoglobulin solutions increased rapidly with time and exceeded the maximum measurable value of the instrument within a few minutes (Figure 4a). A weakening of the protein layer was observed when F127

(43) Wantke, K.-D.; Fruhner, H.; Fang, J.; Lunkenheimer, K. J. Colloid Interface Sci. 1998, 208, 34-48.

(44) Mun˜oz, M. G.; Monroy, F.; Herna`ndez, P.; Ortega, F.; Rubio, R. B.; Langevin, D. Langmuir 2003, 19, 2147-214.

Figure 4. Surface shear rheology as measured with the oscillating ring method (frequency 3 Hz, strain amplitude 5 × 10-3 rad) for adsorbed layers of 10 µM β-lactoglobulin and the polymeric surfactant F127. The concentrations of added F127 are indicated in the figures. (a) G′ (shear storage modulus) as a function of time. (b) G′′ (shear loss modulus) as a function of time.

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Table 1. Drainage Rate and Equilibrium Film Thickness of Thin Liquid Films Stabilized by β-Lactoglobulin and the Polymeric Surfactant F127a solution 10 µM β-lactoglobulin 10 µM β-lactoglobulin/0.2 µM F127 10 µM β-lactoglobulin/2 µM F127 10 µM β-lactoglobulin/20 µM F127 20 µM F127 c

drainage time (n) ( std dev (min)b

film thickness (hw) ( std dev (nm)c

>180 29 (3) ( 1.4 7.7 (3) ( 1.4 4.7 (2) ( 0.1 3.9 (4) ( 0.7

29.9 28.0 ( 1.06 38.4 ( 1.98 46.7 ( 0.14 45.5 ( 1.48

a The adsorption time was 5 min before formation of the thin film. Film diameter was 350-400 µM. b To reach equilibrium film thickness. At equilibrium. n is the number of determinations and std dev is the standard deviation.

Figure 5. Surface lateral diffusion coefficient of the fluorophore ODAF, as a function of F127 concentration, in the surface of model thin films stabilized by F127.

was added to the solutions, and the viscoelastic properties of mixed layers showed essentially similar features to when investigated with the dilatational rheometer. As suggested by the dilatation results, the initial adsorption and behavior are dominated by the protein at the intermediate molar ratios investigated. During the initial 10-15 min, interactions between protein molecules started to build up and reached a maximum, but as increasing amounts of F127 adsorbed, G′ decreased with time (10 µM protein/0.2 µM F127 and 10 µM protein/0.4 µM F127). The storage modulus at 10 µM protein/2 µM F127 coincides with that for a pure surfactant film, i.e., a minimal response to the applied shear compared to the protein is observed, indicating weaker interactions at the interface. This could be an indication that no β-lactoglobulin at all adsorbed in the presence of 2 µM F127. However, this is not consistent with the dilatational modulus which clearly showed that some protein was present at the surface for the mixed solution of 10 µM β-lactoglobulin/2 µM F127. The interpretation of the shear experiments must be that the low number of interactions in the protein network in the presence of 2 µM F127 was not detectable by this technique. The shear loss modulus G′′ was lower than the storage modulus G′ as seen in Figure 4b. Thus, also under shear the adsorbed layers were primarily elastic. Surface Lateral Diffusion. The aim of including FRAP measurements was to measure the ability of the fluorophore to move laterally at the interface stabilized by the block copolymer. The diffusion coefficient of the fluorescent probe, ODAF, in adsorbed layers of F127 was determined, which gives an estimate of the lateral movement at the surface and an indirect measure of the viscosity in the layer. The diffusion coefficient is shown in Figure 5, and it decreases with increasing bulk concentration and consequently with the surface coverage and surface packing density. Due to the high molecular weight of these polymeric surfactants, it is likely that molecular rearrangements can occur after adsorption. This

will allow the surfactant to adopt different conformations depending on packing density. Therefore it would appear that at higher surface pressures, the F127 surfactant forms a thicker, denser adsorbed layer, as evidenced by the decrease in surface lateral diffusion of the probe molecule. For β-lactoglobulin on its own, the ODAF probe is completely immobilized.45 Thus, the result indicates that F127 is mobile at the interface, which would enable a Gibbs-Marangoni stabilization mechanism of foam films, in contrast to what is found for (globular) proteins. However, compared to low molecular weight surfactants such as Tween 20 (Mw ≈ 1228 g/mol) and SDS (Mw ) 288.4 g/mol) for which DODAF ≈ 2 × 10-7 12 and 6 × 10-7 cm2/s,28 respectively, the lateral mobility of the probe in a F127 layer is at least 1 order of magnitude lower. This is an indication that the surface diffusion of F127 in adsorbed layers at the air-water interface is significantly smaller than the surface diffusion of a LMW surfactant, which may compromise its ability to stabilize thin films by the Gibbs-Marangoni mechanism. Thin-Film Measurements. The drainage time and equilibrium thickness of thin liquid films stabilized by β-lactoglobulin or F127, as well as mixed films, are shown in Table 1. The appearance of the films was observed through an inverted light microscope, and the film characteristics during drainage are summarized in Table 2. The protein concentration was kept at 10 µM, the mixed solutions contained F127 in the range 0.2-20 µM, and films of F127 on its own at 20 µM were also examined. Since the competitive adsorption process is time dependent, the adsorption time before the formation of a thin film was set to 5 min in all experiments. The main driving force for film thinning is the difference between the capillary pressure in the lamellae and the bulk liquid surrounding the film, which model the foam lamellae and the adjacent Plateau borders of a foam. Since the film was suspended horizontally in a closed vaporsaturated chamber, the influences of gravity and evaporation were minimized in the model system. In the metastable equilibrium state, the sum of forces (if net repulsive) acting between the interfacial layers across the thin film will balance the capillary pressure. If the sum of these forces is attractive, the thinning will continue until the film ruptures. The stability of nonionic block copolymer films is governed by electrostatic and steric forces. Several explanations to the origin of the electrostatic force have been proposed, of which a specific adsorption of OH- ions is the most favored.46 In our experiments, 100 mM NaCl was added in order to screen repulsive electrostatic double-layer forces. The repulsive forces observed will in this case thus be mainly of steric origin. Draining foam films are generally of nonuniform (45) Sarker, D. K.; Wilde, P. J.; Clark, D. C. Colloids Surf., B 1995, 3, 349-356. (46) Karraker, K. A.; Radke, C. J. Adv. Colloid Interface Sci. 2002, 96, 231-264. Stubenrauch, C.; von Klitzing, R. J. Phys., Condens. Matter 2003, 15, R1197-R1232.

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Table 2. Characteristics of Thin Liquid Films Stabilized by β-Lactoglobulin and F127 during Drainage solution 10 µM β-lactoglobulin

10 µM β-lactoglobulin/0.2 µM F127

10 µM β-lactoglobulin/2 µM F127

10 µM β-lactoglobulin/20 µM F127 20 µM F127

drainage behavior Formation of a centrally “dimpled” film with uniform concentric Newton’s rings (film thickest in the center). Drained uniformly and very slowly. Last interference ring in the center disappeared after 1.5 h. Film then evenly gray, but needed >3 h to become flat. Uniform concentric rings formed, but faster drainage than the film stabilized only by protein. Fast “bulk drainage” during the first minutes, then drainage rate slowed and eventually the form of the last rings became a little irregular. A first plateau thickness (hw ) 45.7 nm) at t ) 15-16 min, which was stable for ≈12-15 min, was then transformed to the equilibrium thickness (gray) in a one-step, instantaneous manner. Uniform concentric rings formed initially, but these became irregular within t ) 2-4 min. Fast “bulk drainage” during the first minutes, then somewhat slower drainage rate to equilibrium film thickness (grey), which remained the same to t ) 1 h. Irregular appearance of the film with varying interference colors and rapid drainage. Behaved as if only polymeric surfactant present. Irregular appearance of the film with varying interference colors and rapid drainage. Film looked gray at equilibrium. The thickness (eq) remained the same throughout the experiment (1 h).

thickness. A thicker region (a dimple) forms in the central part of a circular film, and it is separated from the Plateau border by a ring of thinner thickness. Dimples are especially noticeable in protein films. The drainage rate is limited by the viscosity of the bulk liquid that has to flow through the thinnest region close the edge of the film and by the viscoelastic properties of the interfacial layers. Rigid films, typically a viscoelastic protein film formed by globular proteins, are less mobile and offer resistance to deformation during thinning, whereas a typical surfactant film is mobile and flexible, which result in faster drainage. The results from our experiments are consistent with this picture. Thus, the time to reach the equilibrium thickness for the protein film (>3 h) was largely reduced after addition of only 0.2 µM F127, and the drainage time was gradually further decreased as more surfactant was added. The time to reach equilibrium for a mixed film with 20 µM F127 was 4.7 min, which is very close to the drainage time for a pure surfactant film at 20 µM. The bulk viscosities did not differ significantly between the (dilute) solutions, even though the local effective viscosity in the confined environment of the film may have an effect. This behavior is similar to that observed for LMW surfactant systems,6 and it is also consistent with the surface dilatational data already presented, i.e., a significant compositional transition in the surface layer takes place at F127 concentrations between ≈2 and 20 µM. The time to reach the equilibrium thickness for the different compositions of the films indicates a transition from a rigid, viscoelastic film dominated by the protein to a mobile fast-draining surfactant-dominated film. The film formed from a F127 solution alone at 20 µM drained slightly faster than when it also contained 10 µM β-lactoglobulin, which could be due to some complex formation that slowed the drainage by increasing the local bulk viscosity. Another indication of binding between F127 and β-lactoglobulin was observed at 10 µM protein/0.2 µM F127, but in none of the other films. In this case, the film stayed at an intermediate plateau thickness that was stable for 12-15 min, before it suddenly drained to the equilibrium thickness. The difference between the intermediate and equilibrium thickness was 17.7 nm. However, the visual characteristics of films at 20 µM F127 with and without β-lactoglobulin appeared identical (Table 2). Nonionic surfactants generally interact weakly with proteins in solution, but since β-lactoglobulin possesses a hydrophobic ligand binding site, this protein has been shown to specifically bind molecules such as retinol and fatty acids23 and to form 1:1 complexes with Tween 206

and C12E6.15 The size of F127 is considerably larger than the mentioned ligands and would probably not fit into the hydrophobic pocket of β-lactoglobulin. This study does not provide enough information to draw conclusions about any binding of F127 to β-lactoglobulin, but any potential protein-block copolymer aggregate formation does not seem to affect the drainage rate to any larger extent. All equilibrium films were relatively thick common black films that were gray in color. The protein film was thicker than expected (29.9 nm) for a monolayer comparing with the molecular dimensions of the β-lactoglobulin dimers (3.6 × 3.6 × 6.9 nm47) and the measured monolayer thickness obtained using neutron reflectivity48 or atomic force microscopy.49 However, it is in agreement with the thickness of foam films of β-lactoglobulin11 at the low disjoining pressures employed by the dynamic thin film method and close to the thickness as indicated by the onset of the steric repulsion of disjoining pressure isotherms.50 The films became thicker than that of the pure protein film with increasing amounts of the polymeric surfactant. It is reasonable that the film thickness increases with the surface coverage of F127, since the ethylene oxide chains are forced to stretch out from the interface as the space becomes more limited. All F127 concentrations were below the cmc for the main component, to avoid contributions from micelles to the dynamics and thickness. The thickness of films stabilized solely by the polymeric surfactant was 45.5 nm for the bulk solution concentration of 20 µM, which is considerably larger than the about 15 nm observed for Tween 20.6 We note that several observations have been made that the equilibrium foam film thickness even at high electrolyte concentrations is larger than twice the adsorption layer thickness of block copolymers, as estimated by ellipsometry on a hydrophobized silica surface32 and by disjoining pressure measurements at the air-water interface51 or by theoretical scaling predictions.52 The explanation given is that, since the copolymers are (47) McKenzie, H. A. Milk Proteins Chemistry and Microbiology; Academic Press: London, 1971; Vol. II. (48) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847-2854. (49) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Morris, V. J. Langmuir 2001, 17, 6593-6598. (50) Dimitrova, T. D.; Leal-Calderon, F.; Gurkov, T. D.; Campbell, B. Langmuir 2001, 17, 8069-8077. (51) Khristov, K.; Jachimska, B.; Kazimierz, M.; Exerowa, D. Colloids Surf., A 2001, 186, 93-101. (52) Exerowa, D.; Sedev, R.; Ivanova, R.; Kolarov, T. Colloids Surf., A 1997, 123, 277-282.

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Figure 6. The effect of adding F127 (solid line) on the foam stability (relative foam stability (%) ) foam volume/foam volumeβ-lactoglobulin × 100 at t ) 1 h) of 10 µM β-lactoglobulin. The effect of added Tween 20 (dashed line) is plotted as a comparison.

polydisperse and the polymer segment density varies normal to the surface, the steric repulsion at large separations arises from a fraction of some long polymer tails protruding into the solution. Foam Stability. On the basis of the characterization of the mixed surface layers and single thin films, an effort was made to relate these findings to the stability properties of macroscopic foam formed from solutions with the same compositions as used earlier. A predetermined volume of foam was formed and, as a measure of the foam stability, the foam volume 60 min after formation was determined and is presented relative to the foam volume of pure β-lactoglobulin. The effect of added block copolymer on the stability of β-lactoglobulin foams is shown in Figure 6. It should be recognized that the time to reach the predetermined foam volume (100 mL) was very similar, suggesting a similar foamability of the solutions. There was no evidence of the foam being destabilized by F127 at any concentration. This is in sharp contrast to the behavior commonly encountered with LMW surfactants, where dramatic destabilization is often observed.1,2,6 For comparison, the effect of the LMW surfactant Tween 20 on foam stability is shown.53 In this case, once a critical concentration of surfactant has been reached, the foam stability begins to fall. This is because the surfactant begins to disrupt the surface layer of protein and the adsorbed protein layer is no longer able to stabilize the foam by the viscoelastic mechanism. Equally, there is not enough surfactant present to stabilize the foam by the Gibbs-Marangoni mechanism. Hence the integrity of the mixed interface is poor, and the foam stability falls. Once (53) Wilde, P. J. J. Colloid Interface Sci. 1996, 178, 733-739.

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the surfactant displaces the protein from the surface, it is able to stabilize the foam in its own right, and the foam stability increases once more, to values associated with the surfactant alone.53 The fact that no effect on the β-lactoglobulin foam stability was observed, although a weakening of the interfacial protein film in the lateral direction was evident from both the dilatational and the shear rheology of adsorbed layers and the drainage rates of thin films, suggests that the stability in this time scale (1 h) of the mixed foams is mainly determined by other processes than the surface rheological. Instead, the long-range steric forces, which were shown to increase the thickness of mixed films, may be responsible for the stabilization. The lack of effect on the protein foam upon addition of the block copolymer, despite the clear effects on the surface layer in terms of composition and surface rheology, clearly shows the complexity of the mechanisms for foam stabilization. This will be further addressed in a subsequent paper. Conclusion The triblock copolymer F127 is able to disrupt the viscoelastic adsorbed layer of β-lactoglobulin and displace the protein from the surface in a manner similar to that previously reported for low molecular weight surfactants. This was evidenced by a gradual decrease in surface dilatational and shear elasticity and viscosity and by increased rates of thin film drainage in the presence of increasing amounts of the polymeric surfactant. The response to dilatation and shear of F127 surface layers was mainly elastic, like for the β-lactoglobulin and many LMW surfactants. In contrast to the protein, the block copolymer is mobile at the surface, though the surface mobility of F127 is lower than that of conventional LMW surfactants. This may compromise its ability to stabilize foam lamellae against rupture by the Gibbs-Marangoni effect. Despite the disruption of the highly viscoelastic interfacial layer of protein, the stability of foams formed from mixtures of F127 and β-lactoglobulin was not affected, in contrast to what is found for LMW surfactants. This was explained by the thick films formed by the block copolymer and the accompanying long-range steric interaction across foam lamellae. Acknowledgment. This work was in part supported by funds from the European Union within the Marie Curie Training Site fellowship program (Contract No. QLK1CT-2000-60030) at the Institute for Food Research in Norwich. LA0485475