Langmuir 1997, 13, 7151-7157
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Molecular Diffusion and Drainage of Thin Liquid Films Stabilized by Bovine Serum Albumin-Tween 20 Mixtures in Aqueous Solutions of Ethanol and Sucrose Peter J. Wilde,† Ma. Rosario Rodrı´guez Nin˜o,‡ David C. Clark,†,§ and Juan M. Rodrı´guez Patino*,‡ Institute of Food Research, Food Biophysics Department, Norwich Laboratory, Norwich Research Park, Norwich NR4 7UA, United Kingdom, and Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/Prof. Garcı´a Gonza´ lez, s/num. 41012-Sevilla, Spain Received June 23, 1997. In Final Form: September 29, 1997X
The drainage characteristics and surface lateral diffusion properties of thin liquid films (foam lamellae) formed by BSA (bovine serum albumin) and Tween 20 were studied using optical microscopy including epi-illumination and fluorescence recovery after photobleaching. The concentrations of the surfactant and protein at the aqueous phase interface, the composition of the interface (BSA, Tween 20, and BSA-Tween 20 mixtures), and the aqueous phase composition (1 M ethanol and 0.5 M sucrose) were the variables studied. This study has revealed many complicated effects arising from film-film and film-subphase interactions as a function of the variables studied. The interactions between Tween 20 and BSA at the interface were found to dependent not only on the interfacial composition but also on the subphase composition.
Introduction Thin liquid films have developed large scientific and industrial applications. Aqueous films suspended in air are considered to be a sound model for foam lamellae1,2 and aqueous films in oil as a model for emulsions.3,4 Studying the drainage of thin liquid films is important for understanding the processes involved in emulsion and foam stabilization.5-8 The extent and rate of drainage of liquid from the interior of the lamellae are two of the most important factors determining the stability of foam films. Thin films can be stabilized by two distinct mechanisms, the one which dominates is dependent upon the molecular composition of the interface.5,6 Low molecular weight surfactants such as polar lipids congregate at the interface and form a fluid adsorbed layer at temperatures above their transition temperature. The free lateral diffusion at the interfacial layer confers stability in surfactantstabilized thin films. This process is often referred to as the Marangoni mechanism.9 In contrast, the adsorbed * Author to whom the correspondence should be addressed: tel., +34 5 4557183; fax, +34 5 4557134; e-mail,
[email protected]. † Institute of Food Research. ‡ Universidad de Sevilla. § Current address: DMV International, NCB-laan 80, PO Box 13, 5460 BA Veghel, The Netherlands. X Abstract published in Advance ACS Abstracts, November 15, 1997. (1) Coke, M.; Wilde, P. J.; Russel, E. J.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489. (2) Clark, D. C.; Coke, M.; Wilde, P. J.; Wilson, D. R. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; Royal Society of Chemistry: Cambridge, 1991; p 272. (3) Clark, D. C.; Mackie, A. R.; Wilde, P. J.; Wilson, D. R. Faraday Discuss. 1994, 98, 253. (4) Wilde, P. J.; Clark, D. C. J. Colloid Interface Sci, 1993, 155, 48. (5) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsifiers and Their Applications; Hartel, R., Hasenhuette, Eds.; Chapman & Hall: New York, 1977; Chapter 5. (6) Clark, D. C. In Characterization of Food: Emerging Methods; Gaonkar, A., Ed.; Elsevier: Amsterdam, 1995; p 23. (7) Kruglyakov, P. M. Surf. Sci. Ser. (Thin Solid Films) 1988, 59, 767. (8) Rao, A. A.; Wasan, D. T.; Manev E. D. Chem. Eng. Commun. 1982, 15, 63. (9) Ewers, W. E.; Sutherland, K. L. Aust. J. Sci. Res. Ser. 1952, A5, 697.
S0743-7463(97)00664-1 CCC: $14.00
layer in protein-stabilized thin films is much stiffer and typically has viscoelastic properties.10,11 These derive from the extensive protein-protein interactions that form in the adsorbed layer,5,6,12 in which lateral diffusion of molecules in the adsorbed layer is inhibited.1,13,14 Thin film instability can result in systems that contain mixtures of proteins and low molecular weight surfactants,1,2,11,15 as is the case in many foods. The origin of this instability derives from the incompatibility of the two stabilization mechanisms. The changes in adsorbed layer structure at the airwater and oil-water interfaces caused by competitive adsorption between proteins and lipids have been studied in detail by different methods.11,13,14,16,17 It has been shown that the presence of protein-lipid interactions can have a pronounced effect on the interfacial layer that in turn can influence the stability or instability of the dispersed emulsion or foam.13,14,18-20 These studies demonstrate the great sensitivity of emulsion and foam stability to the nature of protein-lipid interactions at fluid-fluid interfaces. Although the formation and properties of the interfacial layer on the emulsion droplet or the foam bubble are strongly linked to the properties of the aqueous environ(10) Castle, J.; Dickinson, E.; Murray, B. S. ACS Symp. Ser. 1987, No. 343, 118. (11) Sarker, D. K.; Wilde, P. J.; Clark, D. C. Colloids Surf. B 1995, 3, 349. (12) Prins, A.; Jochems, A. M. P.; Van Kalshbeek, H. K. A. I.; Boerboom, J. F. G.; Wijnen, M. E., Williams, A. Prog. Colloid Polym. Sci. 1996, 100, 321. (13) Clark, D. C.; Coke, M.; Mackie, A. R.; Pinder., A. C.; Wilson, D. R. J. Colloid Interface Sci. 1990, 138, 207. (14) 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. (15) Wilde, P. J. J. Colloid Interface Sci. 1996, 178, 733. (16) Clark, D. C.; Wilde, P. J.; Bergink-Martens, D.; Prins, A. In Food Colloids and Polymers. Structure and Dynamics; Dickinson, E., Walstra, P., Eds.; RSC Special Publication 113; Royal Society of Chemistry: Cambridge, 1993; p 354. (17) Courthaudon, J.-L.; Dickinson, E.; Dalgleish, D. C. J. Colloid Interface Sci. 1991, 145, 390. (18) Chen, J.; Dickinson, E. Colloids Surf., A 1995, 100, 255. (19) Chen, J.; Dickinson, E. Colloids Surf., A 1995, 100, 267. (20) Chen, J. Dickinson, E. Food Hydrocolloids 1995, 9, 35.
© 1997 American Chemical Society
7152 Langmuir, Vol. 13, No. 26, 1997
Wilde et al. to the equation 113,14,25,26
ment, little data have been published concerning the effect of the aqueous phase composition on the equilibrium properties of thin liquid films.21 In this paper, we describe a series of experiments that investigate the subphase composition (ethanol and sucrose aqueous solutions) and the interfacial composition (bovine serum albumin (BSA) and Tween 20) and their influence upon thin film drainage and surface diffusion. By incorporating solutes into the aqueous phase, we hope to approach the behavior of a simple well-defined model of a complex food formulationssuch as ice creams, desserts, soft drinks, cream liqueurs, cereal-based foods, etc.22,23
where F(t) is the fluorescence signal at time t after bleaching, F0 and F∞ are the calculated fluorescence intensities at t ) 0 and infinity, respectively, τD is the recovery time, and β is a value dependent on the degree of bleaching. The diffusion coefficient D is then calculated by
Materials and Methods
D ) ω2/4τD
Materials. BSA (bovine serum albumin, more than 96% pure, Fluka), Tween 20 (polyoxyethylene sorbitan monolaurate Fluka, Product No. 326896/1693), ethanol (99.8% pure, Merck), sucrose (99.5% pure, Fluka), potassium dihydrogen phosphate (99,5% pure, Merck), dipotassium hydrogen phosphate (99% pure, Merck), and ODAF (5-N-(octadecanoyl)aminofluorescein, Molecular Probes Inc. No. 0-322) were used as supplied. All the samples were prepared in 50 mM sodium phosphate buffer using double-distilled surface chemically pure water and adjusted to pH 7.0. Aqueous solutions of 1 M ethanol and 0.5 M sucrose were studied as variables. The measurements were made as a function of Tween 20 concentration. Tween 20 was added to the protein solution in the concentration range of 0-30 µM. All the experiments were carried out at 20 °C. Thin film drainage. The apparatus used in the formation and observation of thin liquid films is essentially similar to that described in detail previously.13,14 Briefly, a single foam lamella was formed in a small glass annulus (3.5 mm internal diameter) by withdrawing the solution down a capillary side arm. Withdrawal of liquid from the film initially created a very thick film which was then allowed to drain to its equilibrium thickness. This assembly was contained within a temperature-controlled chamber, fitted with optically flat glass windows which permitted observation of the film using epi-illumination on an inverted microscope (Nikon Diaphot) connected to a video camera (Nikon FE2), attached to a TV monitor. This device allowed direct study of thin film drainage behavior, such as the drainage time and the appearance of the thin films as a function of drainage time as well as at equilibrium. Photographs of the thin films as a function of drainage time were taken from the video film using a SONY CVP-M3E video-printer. An equilibrium period of about 30 min was necessary before experimental measurements were started to allow for protein-lipid adsorption, temperature adjustment, and saturation of the vapor pressure. However, more time was necessary to study the drainage behavior in ethanol aqueous solutions, due to the lower rate of protein film formation.24 In these systems the time dependence on drainage behavior was studied as variable. Different Tween 20 concentrations were added to a constant BSA concentration of 0.1% (wt/wt) in water and aqueous solutions of 1 M ethanol and 0.5 M sucrose. Surface Lateral Diffusion. Surface lateral diffusion of BSA and BSA-Tween 20 mixtures on water and aqueous solutions of ethanol and sucrose were determined by the fluorescence recovery after photobleaching (FRAP) technique. The approach used was similar to that reported previously.13,14 A low molecular weight surface active fluorescent probe (ODAF) at 0.5 µM was included in the BSA and BSA-Tween 20 solutions. The use of ODAF at low concentrations ensured that it did not itself induce changes in thin film characteristics.11 The fluorescence recovery curve after bleaching was collected by microcomputer and fitted
were ω is the diameter of the laser spot on the thin film. Different spot sizes were used to determine whether the ODAF mobility was due to lateral diffusion or linear flow in the thin film. These phenomena can be distinguished since the characteristic recovery time is proportional to the spot diameter under conditions of flow and to the spot diameter squared when diffusion is the dominant process.6,27 The films were allowed to drain to equilibrium thickness before measurements were initiated. Five to ten data curves were collected and averaged prior to analysis.
(21) Brierley, E. R.; Wilde, P. J.; Onishi, A.; Hughes, P. S.; Simpson, W. J. Clark, D. C. J. Sci. Food Agric. 1996, 70, 531. (22) Krog, N. J.; Riison, T. H.; Larsson, K. In Encyclopaedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1985; Vol. 2, p 321. (23) Leadbetter, S. L. Food Focus No. 9, Leathered Food R. A., Brit. Food Mfg. Ind. Res. Assoc., 1990. (24) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R. Colloids Surf., A 1995, 103, 91.
F∞t βτD F(t) ) t 1+ βτD F0 +
(1)
(2)
Results Thin Film Drainage. Differences in the drainage properties of thin films as a function of interfacial and aqueous bulk phase compositions are shown in Figure 1. The drainage of thin films stabilized by Tween 20 demonstrated the typical features of a noninteracting surfactant,14 exhibiting behavior commonly referred to as mobile or “chaotic” drainage (Figure 1a-c). Figure 1a shows the film just after formation, the colors of the film are interference patterns and denote the film thickness. The interference patterns are very fluid and rapid, demonstrating the mobile nature of the adsorbed layer allowing free drainage of the film. After approximately 0.5 min drainage (Figure 1b) black spots appear in the film, representing the final transition to the films equilibrium thickness (approximately 12 nm). After 2.5-3 min, the film is completely black and essentially at equilibrium; that is, no further drainage takes place. The main drainage characteristics of Tween 20-stabilized thin films as a function of lipid concentration and aqueous phase composition are presented in Table 1. In contrast, thin films stabilized by BSA showed drainage characteristics typical of a protein-stabilized film.13 Figure 1d shows a BSA-stabilized thin film 1 min after formation. The concentric interference rings within the film, Newton’s rings (denoting points of equal thickness, thickest at the center), remain virtually immobile. The low surface mobility and high surface viscoelasticity drastically reduce drainage from the film; thus the concentric rings slowly disappear over a period in excess of 3 h. However, differences existed as the drainage progressed as a function of BSA concentration and aqueous phase composition (Table 2). The existence of surface aggregates was observed in the presence of 1 M ethanol (Figure 1e,f). The aggregates are the small, local, thicker regions observed in the films (Figure 1e, t ) 7.5 min; Figure 1f, t ) 23 min). In the presence of 0.5 M sucrose a very slow drainage rate was observed with wide interference fringes (Figure 1g, t ) 12 min; Figure 1h, t ) 8.5 min). (25) Axeldord, D. E.; Koppel, D. E.; Schellinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055. (26) Axelrod, D. In Spectroscopy and Dynamic of Molecular Biological Systems; Nayley, P. M., Dale, R. E., Eds.; Academic Press: London, 1985; p 163. (27) Lalchev, Z. I.; Wilde, P. J.; Clark, D. C. J. Colloid Interface Sci. 1994, 167, 80.
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Figure 1. Photographs of the drainage of Tween 20 (a-c), BSA (d-h), and BSA-Tween 20 (i-l) mixed films as a function of Tween 20 and BSA concentrations, BSA-Tween 20 ratio, and aqueous phase composition (1 M ethanol and 0.5 M sucrose). For an explanation see text and Tables 1-3.
The drainage behavior of mixed BSA-Tween 20 films was intermediate between the chaotic Tween 20 drainage (Figure 1a) and the uniform BSA drainage (Figure 1d). The films showed distortion of the uniform, concentric Newton’s rings and more rapid drainage as the concentration of Tween 20 increased. Ultimately, when the Tween 20 concentration was high enough, the film drainage behavior was identical to that of Tween 20 alone. In the presence of 1 M ethanol (Figure 1i,j and Table 3), a more rapid drainage together with interfacial aggregates was observed. Aggregates were also observed in the presence of 0.5 M sucrose (Figure 1k, l and Table 3). Surface Diffusion in the Adsorbed Layer. Typical FRAP data for mixed BSA-Tween 20 thin films on water and aqueous solutions of ethanol and sucrose are shown
in Figure 2. The prebleach level, the reduction in fluorescence due to the bleaching pulse, and the postbleaching recovery are displayed in Figure 2a. The experimental data, computed fit, and the residuals (experimental data minus the computed fit) for BSATween 20 mixed films on water (Figure 2b), 1 M ethanol (Figure 2c) and 0.5 M sucrose (Figure 2d) solutions are also shown. A summary of the measured surface lateral diffusion coefficient (D) of BSA and BSA-Tween 20 thin films as a function of Tween 20 concentration is shown in Figure 3. Each subphase studied showed either limited or time dependent mobility below a certain critical Tween 20 concentration. The results show some interesting features: (a) on water or 1 M ethanol, the transition between a protein-like film (immobile, D ≈ 0 cm2‚s-1) and a lipid-
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Table 1. Characteristics of Drainage of Thin Films Stabilized by Tween 20 on the Air-Aqueous Phase Interface at 20 °C subphase
Tween 20 (µM)
water water
1 10
water
50
water ethanol (1 M) ethanol (1 M)
100 1 10
ethanol (1 M) ethanol (1 M) sucrose (0.5 M)
50 100 1
sucrose (0.5 M) sucrose (0.5 M)
10 50
sucrose (0.5 M)
100
a
drainage characteristics fluid and very mobile film; unstable, ruptures a few seconds after formation typical lipid-like mobile film drainage; unstable, ruptures after equilibrium thickness (2-3 min) typical lipid-like mobile film drainage; black spots denoted final transition to black film; stable; see Figure 1a (ta ) 0.1 min), Figure 1b (t ) 0.5 min), and Figure 1c (t ) 2.5 min) typical lipid film; very stable typical lipid film; unstable film; rapid drainage typical lipid film; the film turns to white and then to black; black spots which merged quickly to form a black film at equilibrium similar behavior to that of Tween 20 at 10 µM homogeneous film; white film which turns to gray with time; stable and fluid film typical lipid film; white film that changed to dark film with time; unstable, rupture occurred before equilibrium thickness similar to that of Tween 20 at 1 µM typical lipid film; fluid film with black spots at the periphery; white “star” at the center of the film; black spots merged with time giving a black film at equilibrium similar to that of Tween 20 at 50 µM, but more stable
t, time after formation of the thin film.
Table 2. Characteristics of Drainage of Thin Films Stabilized by BSA on the Air-Aqueous Phase Interface at 20 °C subphase
BSA (%, wt/wt)
water water water water ethanol (1 M) ethanol (1 M)
1 × 10-4 1 × 10-3 1 × 10-2 1 × 10-1 1 × 10-3 1 × 10-2
ethanol (1 M)
1 × 10-1
sucrose (0.5 M) sucrose (0.5 M) sucrose (0.5 M)
1 × 10-4 1 × 10-3 1 × 10-2
sucrose (0.5 M)
1 × 10-1
a
drainage characteristics typical colored protein film; unstable, ruptured about 30 s after formation typical colored fringes; unstable, ruptured about 2 min after formation typical protein film, slow drainage Newton’s fringes (drainage time greater than 3 h) typical of protein drainage, Newton’s fringes, stable film, slow drainage, see Figure 1d (t ) 1 min)a unstable and fluid film; the film is unstable even after allowing 3 h for adsorption typical of protein films; aggregates observed; film stability is time dependent; the fresh film is unstable after formation; after 5 min of adsorption the film lifetime is around 5 min typical of protein films; black film at the periphery (presence of aggregates); slow drainage; see Figure 1e (t ) 7.5 min) and Figure 1f (t ) 23 min) existence of fringes at the beginning of formation; fluid film; unstable, the film burst within 1 min typical protein film; wide rings; unstable, the film burst within 3 min typical protein film; aggregated; black ring at the periphery which grows with time; very stable film (the life time was greater than 4 h); see Figure 1h (t ) 8.5 min) typical of protein film; stable and slow drainage; wide rings; the drainage takes more than 4 h, and continues to be stable; see Figure 1g (t ) 12 min)
t, time after formation of the thin film.
Table 3. Characteristics of Drainage of Thin Films Stabilized by BSA-Tween 20 Mixtures on the Air-Aqueous Phase Interface at 20 °Ca subphase water water water water ethanol (1 M)
ethanol (1 M) ethanol (1 M) sucrose (0.5 M) sucrose (0.5 M) sucrose (0.5 M) sucrose (0.5 M) a
Tween 20 (µM) 1 5 7 10 1
2 5 2 5 7 10
drainage characteristics typical protein-film drainage: concentric colored fringes close to transition; unstable film; the film burst frequently similar to that at 7 µM Tween 20 typical behavior of lipid film drainage; existence of many black spots; spot collapse; central black disk at steady state unstable and very fluid film; presence of aggregates (white spots on the interface); the films burst frequently; after an elapse time necessary to allow protein adsorption, the film was stable; black ring at the periphery which grows as a function of time; black ring at steady state; see Figure 1i (t ) 8.5 min)b similar to that at 1 µM Tween 20; see Figure 1j (t ) 0.3 min) similar to that at 1 µM Tween 20 protein-like film; very slow drainage; existence of many aggregates; see Figure 1k (t ) 11 min) protein-like film; very slow drainage; existence of aggregates (bright points) colored fringes; existence of aggregates a interfacial segregation; see Figure 1l (t ) 4.75 min) typical of lipid film; fluid and stable film; dark spot at the periphery which grows toward the center of the film
BSA concentration in the aqueous bulk phase: 1 × 10-1 %, wt/wt. b t: time after formation of the thin film.
like film (D > 0 cm2‚s-1) was observed at a Tween 20 concentration of around 5 µM. (b) In the presence of 1 M ethanol, at Tween 20 concentrations around the transition point, D was time dependent. Just after film formation, the film fluidity was high and the films were unstable. At longer times, the films were more stable, and D was reduced, often to zero. ( c) With a 0.5 M sucrose solution the transition point was observed at a Tween 20 concentration of 10 µM. (d) The Tween 20 concentration dependence of D on ethanol aqueous solutions was
different for thin films on water or the sucrose solution. The surface diffusion coefficient on the ethanol solution showed a more complex dependence on Tween 20 concentration. The surface diffusion coefficient described a maximum at a Tween 20 concentration of 10 µM. (e) At the highest Tween 20 concentration studied (20 µM), the superficial diffusion coefficient did not depend on the aqueous phase composition. That is, the value of D was practically the same on either water, 1 M ethanol, or 0.5 M sucrose.
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Figure 2. Typical FRAP data obtained from a film stabilized by a mixture of BSA (0.1%, wt/wt) and Tween 20 (15 µM) on water (A). The recovery portion of a FRAP curve obtained with a mixture of BSA (0.1%, wt/wt) and Tween 20 (15 µM) on water (B), and aqueous solutions of 1 M ethanol ( C) and 0.5 M sucrose (D).
Figure 3. The Tween 20 concentration dependence of the surface diffusion coefficient of surface-adsorbed 5-N-(octadecanoyl)aminolfuorescein in a thin film stabilized by the mixtures BSA (0.1%, wt/wt)-Tween and adsorbed on water (4), and aqueous solutions of 1 M ethanol (b) and 0.5 M sucrose (×).
Discussion According to the DLVO theory, the factors mainly responsible for the drainage and stability of thin films are the capillary suction at the plateau borders, the van der Waals attractive forces, and the electrostatic repulsion. The capillary pressure and the van der Waals forces create a difference in pressure between the interlamellar fluid within the film, and the bulk phase, causing the liquid to drain out. Thin liquid films often rupture on reaching a certain critical thickness.7,28,29 Rupture is not seen either when the film is thermodynamically stable at a thickness greater than that of the critical thickness or when the film can exist in a stable form below the critical thickness. It is known that the critical thickness decreases with increasing surfactant concentration8 and reaches an (28) Scheludko, A. Adv. Colloid Interface Sci. 1967, 1, 391. (29) Vrij, A. Discuss. Faraday Soc. 1966, 42, 23.
almost constant value near the critical micelle concentration (cmc). Low surfactant concentrations cause the films to rupture at a large critical thickness. As the surfactant concentration increases, the film drains to lower thickness before rupture and black spot formation is seen.30 These data are in agreement with our results on Tween 20 thin film stability as a function of surfactant concentration (Table 1). On water, below the cmc of Tween 2031 the drainage rate was high and the films were unstable (i.e., the films burst shortly after formation). Above the cmc, as both micelles and monomers are able to diffuse to the surface, the monolayer is saturated by Tween 20 and then the drainage rate decreased and the film stability increased. The characteristics of thin films (drainage and surface diffusion) formed from BSA and BSA-Tween 20 mixtures depended on the compositions of both the interface (i.e., the BSA/Tween 20 ratio) and the aqueous phase (i.e., the presence of sucrose or ethanol). This dependence is of practical importance because most food formulations contain proteins and surfactants as emulsifiers as well as solutes in the aqueous phase, such as ethanol (alcoholic beverages, bakery or cake products, etc.) or sucrose (ice cream, cream liqueurs, bakery or cake products, etc.). Effect of Interfacial Composition. Thin film drainage and FRAP techniques provide complementary data relating the behavior of thin liquid films. Under conditions where BSA is dominant at the interface, no diffusion is observed (Figure 3). These films display extremely slow drainage in the form of a series of concentric fringes (Newton’s rings) which eventually become black at extended time periods (>3 h) (Figure 1d, Table 2). As the (30) Manev, E.; Scheludko, A.; Exerowa, D. Colloid Polym. Sci. 1974, 252, 586. (31) Rodrı´guez Patino, J. M. Rodrı´guez Nin˜o, Ma. R.; Alva´rez, J. M. G. Food Hydrocolloids 1997, 11, 49.
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Tween 20 concentration in the system increased (Table 3), changes in the drainage behavior were observed, the drainage became more rapid, and the films were less stable. This was followed by the onset of surface mobility (Figure 3) and the rapid drainage properties and surface diffusion observed with Tween 20 alone. It has long been recognized that proteins and lipids can interact at interfaces as well as in the bulk,32-36 which can affect the properties of the adsorbed layer at the airwater and oil-water interfaces and, in turn, the stability of the foam or emulsion.18-20 These interactions are difficult to quantify reliably in the laboratory because of their complexity:5 (a) proteins and lipids can interact in an attractive process associated with coadsorption or competitive adsorption at the interface; (b) proteins can interact with lipids in solution either as individual molecules or as micelles at lipid concentrations above the cmc. In general, lipids may interact with proteinstabilized interfaces through solubilization by complex formation or replacement mechanisms, depending on lipid surface interactions and lipid-protein bonding. The transition in adsorbed layer structure at the airwater interface11,16 and the oil-water interface17-20 by competitive adsorption between protein and emulsifiers is well documented in the literature. Analysis of the data obtained in this work and comparison with surface tension data31,37 and surface viscoelastic properties38 of BSA and BSA-Tween 20 mixtures adsorbed on the same aqueous solutions allows us to conclude that the Tween 20 concentration at which a transition occurred in the mobility of the adsorbed films, correlated with the change in the surface dilational modulus vs Tween 20 concentration plot. This transition occurred at Tween 20 concentrations higher than but of the same order of magnitude as the cmc. Effect of Solutes. The BSA concentration dependence of the drainage behavior of thin films (mobility and stability) is also evident in the data presented (Table 2). In fact, on water, 1 M ethanol, and 0.5 M sucrose it was necessary to have a BSA concentration above monolayer coverage to obtain stable protein films. Above this critical BSA concentrationsrange between 10-3 and 10-2 % (wt/ wt)24sthe protein-protein interactions in the adsorbed layer are maximal. These interactions result in the formation of a gel-like adsorbed layer12 and/or multilayer formation can also occur, which correlate with a marked increased in film viscoelasticity.39,40 The behavior of Tween 20 thin films in the presence of ethanol and sucrose is quite different. The higher observed film stability and mobility could be associated with lipidethanol or lipid-sucrose interactions both at the interface and in the bulk phase. Increasing the lipid-solute interactions could decrease both lipid-lipid interactions (and hence the van der Waals forces) and electrostatic repulsion between lipid head groups, as was observed with (32) Cornell, D. G.; Carroll, R. J. J. Colloid Interface Sci. 1985, 108, 226. (33) Erickson, B.; Hegg, P. O. Prog. Colloid Polym. Sci. 1985, 70, 92. (34) MacRitchie, F. Adv. Protein Chem. 1978, 32, 283. (35) Tanford, C. J. Am. Chem. Soc. 1962, 84, 4240. (36) Wu¨stneck, R.; Wetzel, R.; Buder, E.; Hermel, H. Colloid Polym, Sci. 1988, 266, 1061. (37) Rodrı´guez Nin˜o, Ma. R. Doctoral Dissertation, University of Seville, Spain, 1997. (38) Rodrı´guez Nin˜o, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Submitted for publication in AIChE J. (39) Rodrı´guez Nin˜o, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 1997, 45, 3010. (40) Rodrı´guez Nin˜o, Ma. R., Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 1997, 45, 3016.
Wilde et al.
polar lipids at the air-aqueous phase interface.41-44 The film then thins smoothly down to its critical thickness (Table 1), where it either ruptures or forms thinner stable structures by a jump transition according to previous data.8 The addition of ethanol to water decreased the surface tension value, which indicated that ethanol may be concentrated at the interface. Therefore, ethanol would be able to interact with lipids and proteins both at the interface and in the bulk phase. If ethanol acts as a protein denaturing agent,35 then protein-protein interactions at the interface diminish, which agrees with the higher film fluidity observed from film drainage (Figure 1e,f, Table 2) and the high surface diffusion coefficient (Figure 3). The surface dilational rheology38 provides further data which also supports this hypothesis. In fact, the film fluidity and mobility from drainage and FRAP techniques correlated well with a reduction in the film viscoelasticity upon the addition of ethanol to the subphase. Moreover, denatured protein could facilitate lipid-protein interactions, which agrees with the observed film segregation (Figure 1i,j). The denaturing effect of ethanol could also produce a decrease in protein solubility which could have a negative effect on the film stability. On the other hand, if ethanol acts as a stabilizing agent for proteins,45 the native state could be present at the interface which agrees with thin film appearance by drainage experiments (Figure 1j) at higher Tween 20 concentrations than that corresponding to transition. Finally, the time dependence of the film behaviorsobserved by film drainage (Tables 2 and 3) and FRAP experiments (Figure 3)scould be explained by the competitive adsorption of ethanol, protein, and lipid molecules at the interface. The addition of sucrose to water increases the surface tension value, which indicates its strong cohesive force on water. This also indicates that sucrose is excluded from the intermediate domain of the protein in aqueous solutions.46,47 That is, when sucrose is present in the aqueous solution, the protein is preferentially hydrated. Thus we tentatively suggest that if sucrose limits protein unfolding and/or protein-protein interactions, the reduction in aggregation allowed more protein to be involved in film formation. The consequences of these effects are as follows: Firstly, there is increased stability of thin films (Figure 1g,h, Table 2) which could be attributed to a higher viscosity in the bulk phase due to the sucrose. Secondly, there are decreased lipid-protein interactions at the interface, which agrees with the appearance of thin films (Figure 1k,l). It is possible that native protein and lipid could both be present at the interface but as immiscible, noninteracting species. The presence of native protein at the interface can be supported by the higher rate of adsorption of BSA in the presence of sucrose.24 Moreover, a decrease in surface viscoelasticity was observed with these systems,40 consistent with reduced protein-protein interactions in the adsorbed layer. Thirdly, a higher Tween 20 concentration was necessary to displace the protein from the interface, which correlates with the FRAP data (Figure 3). That is, in the presence of sucrose, BSA tends to be preferentially adsorbed at the interface. (41) Fuente, J. F.; Rodrı´guez Patino, J. M. Colloids Surf. A 1995, 104, 29. (42) Fuente, J. F.; Rodrı´guez Patino, J. M. AIChE J. 1996, 42, 1416. (43) Rodrı´guez Patino, J. M.; Ruı´z, D. M., Fuente, J. F. J. Colloid Interface Sci. 1992, 154, 146. (44) Rodrı´guez Patino, Ruı´z, M. D.; Fuente, J. F. J. Colloid Interface Sci. 1993, 157, 343. (45) Brands, J. F.; Hunt, L. J. Am. Chem. Soc. 1967, 89, 4826. (46) Crowe, J. H.; Crowe, L. M.; Crowe, J. F.; Wistrom, C. A. Biochem. J. 1987, 242, 1. (47) Lee, J. C.; Timasheff, S. N. J. Biol. Chem. 1981, 256, 7193.
Properties of Thin Liquid Films
Clearly, interfacial composition is not the only parameter that can influence the properties of thin liquid films. This study has revealed complicated effects arising from film-film and film-subphase interactions. The existence of these interactions at the interface also appears to be important for the surface rheological characteristics38-40 and the structure of adsorbed protein-lipid films (data not published), which are of tremendous importance for the formation and stability of food emulsions18-20 and foams.5,6,31
Langmuir, Vol. 13, No. 26, 1997 7157
Acknowledgment. This research was supported in part by DGICYT through Grants PB93-0923 and PB941459. J.M.R.P. is also grateful to DGICYT for providing Grant PR95-175. J.M.R.P. and M.R.R.N. thank IFR for providing excellent facilities and support for this work. P.J.W. and D.C.C. acknowledge the support of the Biotechnology and Biological Sciences Research Council and the EC Network, Project ERBCHRXT930322. LA970664V
