Dilatational rheology of protein+non-ionic surfactant films at air–water and oil–water interfaces

Colloids and Surfaces A: Physicochemical and Engineering Aspects 143 (1998) 211–219

Dilatational rheology of protein+non-ionic surfactant films at air–water and oil–water interfaces Brent S. Murray *, Anne Ventura, Cendrine Lallemant Food Colloids Group, Procter Department of Food Science, University of Leeds, Leeds, LS2 9JT, UK Received 10 January 1997; accepted 30 September 1997

Abstract The dilatational rheology of adsorbed films of b-lactoglobulin has been studied at the air–aqueous solution interface and the n-tetradecane–aqueous interface using a Langmuir trough apparatus employing a pulse change in interfacial area. The effect of the addition of the non-ionic surfactant C E to films adsorbed from 10−3 wt% b-lactoglobulin at 12 6 pH 7 and 30°C was also examined. The surfactant appears to bind to b-lactoglobulin with a low molar ratio (one surfactant molecule per protein molecule), but this and/or co-adsorption has significant effects on the dilatational moduli. Films exhibit higher dilatational moduli at the oil–water interface compared to the air–water interface and show a stronger dependence on the surfactant concentration at the oil–water interface. At around equimolar ratios of protein and surfactant there is a slight maximum in the dilatational moduli, more noticeable at the oil–water interface, though separate measurements also indicated a slight enhancement of foam stability in this region. At higher surfactant concentrations there is a more marked decrease in the dilatational moduli to similar values for both types of interface. Moduli measured at strains up to at least 5% appear to be considerably non-linear. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Proteins; Surfactants; Dilatational; Rheology; Foams

1. Introduction It is often stated in the technical literature that a mixture of surfactants may result in maximum stability characteristics for emulsions and foams [1,2]. For the case of mixtures of low molecular weight surfactants and proteins (high molecular weight surfactants), this may be explained [3] as being due to a ‘‘lubricating’’ effect of the surfactants on the adsorbed protein. By this it is meant that the rather strongly, or rigidly, adsorbed proteins are made to be a little more flexible and mobile at the interface, either due to their partial * Corresponding author. Fax: +44 113 2332982; e-mail: [email protected]

displacement [4] and/or complex formation [5,6 ] with the surfactant at the interface. The combination of protein and surfactant may be able to respond more readily to varying rates and extents of deformation, more quickly recovering a surface film capable of preventing film rupture, with positive implications for foam or emulsion stability. The deformation of the adsorbed film may be characterised in terms of its dilatational and shear rheology. Several studies have found a correlation between the dilatational moduli of protein films and foam and emulsion stability, for example Sarker et al. [7] and Kim and Wasan [8]. Jiang and Chew [9] have demonstrated this for lysozyme at both the air–water (A–W ) and oil–water (O–W ) interface. Keen and Blake [10] stressed the impor-

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tance of high dilatational viscosity values in controlling bubble formation, growth and detachment. However, still relatively few interfacial rheological measurements have been made on mixed protein+surfactant systems, even though such mixtures are more common than pure surfactant or pure protein systems in real food colloids [11,12]. Where measurements have been made they have been restricted almost exclusively to the A–W interface. Changes in dilatational elasticity and viscosity are expected with increasing surfactant–protein molar ratio, R, due to conformational changes [13] and desorption of interfacial protein. Miyamoto et al. [14] studied the anti-foaming behaviour of sucrose esters in casein systems — in relation to certain drinks dispensers. Kokelaar et al. [15] and Kokelaar and Prins [16 ] have examined the surface dilatational rheology of surfactants and wheat proteins and found that an increase in the dilatational moduli correlates with retardation of foam collapse, with apparent consequences for bread improving properties. Clark et al. [17] examined mixtures of Tween 20+blactoglobulin at R values from 0 to 10 and found a reduction in the value of the dilatational storage modulus, e∞, from 32 to 15 mN m−1 and an increase in the loss modulus, e◊, from 1.6 to 4.4 mN m−1 with increasing R, whilst a slight increase in the corresponding foam stability was also observed [18]. Although the changes in dilatational moduli on mixing protein with surfactant may be slight, this can still indicate substantial changes in film stability. This is because the conditions of measurement are generally far removed from the highly turbulent conditions of foam or emulsion processing. If bulk concentrations of proteins and surfactants are used which are typical of those employed in commercial emulsions and foams, laboratory measurements generally cannot be made at high enough frequencies/shear rates to reveal the changes in interfacial concentration and tension because adsorption from the bulk is so fast. Yet because of the very high shear rates which occur during processing emulsions and foams, substantial interfacial tension gradients almost certainly arise because the rate of interfacial expansion is too fast to be compensated for by adsorption

and/or rearrangement in the surface. For this reason dilatational measurements at lower frequencies on protein/surfactant monolayers [19,20] or films adsorbed from low bulk concentrations may reveal information on mechanisms leading to stability or instability, because there is little or no adsorption which takes place from the bulk. Moduli describing film rheology are also unlikely to be independent of the large strains and rates of strain important technologically, particularly for protein or protein+surfactant films [1,2], where complex intramolecular and intermolecular interactions occur within the film. Thus small changes in apparent dilatational moduli measured under one set of conditions may indicate much more significant variations under different conditions — more important to real foam and emulsion formation and stability. This study is an investigation of the dilatational rheology of mixed protein+surfactant films over a range of frequencies and over a range of moderate deformations (though still at considerably lower deformations than occur on emulsification and foaming), including a comparison of behaviour at an A–W and O–W interface.

2. Materials and methods 2.1. Materials Bovine b-lactoglobulin (three times crystallised, lyophilised, desiccated, lot no. 91H7005), containing variants A and B, was purchased from SigmaAldrich (Poole, UK ). It was stored under refrigeration. Research grade hexaoxyethylene glycol n-dodecylether (C E ) was purchased from 12 6 Sigma-Aldrich (Poole, UK ) and was stored in the freezer. Trimethyl chlorosilane was from Aldrich. Granulated, activated charcoal (Prod. no. 33034), AnalR concentrated hydrochloric acid and AnalR concentrated nitric acid were from BDH-Merck (Poole, UK ). Imidazole (99%) and n-tetradecane (99%) were from Sigma-Aldrich (Poole, UK ). For every experiment, the water used was doubledistilled with a surface tension of 71.2 mN m−1 at 30°C.

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2.2. Preparation of protein solutions All protein solutions were buffered at pH 7.0 using 20 mM imidazole buffer. The pH of the buffer was adjusted by addition of hydrochloric acid. The buffer was stored under refrigeration. Unpurified and purified b-lactoglobulin solutions have been studied in this work. b-lactoglobulin solutions were purified with charcoal as follows. The charcoal was added to an imidazole buffered b-lactoglobulin solution at a ratio of 6 mg charcoal/mg protein and the sample was stirred for 30 min at room temperature. The charcoal was then removed by centrifugation (13 000 rpm, 20 min, 20°C ), in some cases followed by filtration, if suspended charcoal remained. 2.3. Equilibrium interfacial tension measurements Measurements of equilibrium interfacial tension as a function of surfactant concentration were made via a Kru¨ss K10 ST (Hamburg, Germany) tensiometer using a roughened platinum Wilhelmy plate. The solution was contained within a cleaned glass dish (cleaned by soaking in concentrated nitric acid overnight). All measurements were made at 30°C, unless otherwise stated. 2.4. Dilatational rheometry The apparatus for performing dilatational rheological measurements has been described in detail previously [19,20a,b]. Briefly, it consists of a rhombus shaped PTFE frame, or barrier, sitting in a rectangular PTFE trough. The design is similar to the ‘‘diamond trough’’ of Miller et al. [21], except that the barrier consists of one continuous piece, which has a number of advantages. The barrier contains the film of interest, which is expanded or compressed by flexing the corners, so changing the shape of the barrier. The interfacial tension, c, was measured via a Wilhelmy plate dipping into the interface, at the centre of the film. For measurements at the A–W interface a cleaned, roughened mica plate was used. For measurements at an O–W interface a 6–7 mm layer of oil was gently layered over the top of the aqueous phase and a hydrophobic mica plate was used, completely submerged

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below the surface of the oil layer. The plate was made hydrophobic by immersing it in a 5 wt% solution of trimethyl chlorosilane in heptane for 12 h. After this treatment, a convenient technique to maintain good hydrophobicity and keep the plate clean was to suspend the plate in a smoky Bunsen flame followed by heating in a clean flame to completely carbonise the black deposit on the plate and burn off any potential surface active material on the surface. All other surfaces coming into contact with the protein/surfactant solutions were cleaned with nitric acid and thoroughly rinsed with double-distilled water. After equilibrium interfacial tensions had been reached, films were expanded by a predetermined amount in 1.00 s and the resultant interfacial tension–time (t) decay curve recorded. Dilatational moduli were obtained by Fourier transformation of the decay curves according to Loglio et al. [22] — see below. All measurements were made at 30°C, unless otherwise stated. 2.5. Accelerated foam life-time test Foam stability was assessed using a simple shaking apparatus to generate the foam, whilst the foam was subjected to a low power, broad-band ultrasonic field to accelerate foam collapse. Samples were sealed in clean 100 ml glass bottles which were then attached to a shaker motor whilst immersed in a water bath to maintain constant temperature (30°C ). The bottles were shaken vertically for 30 s at 5 Hz. The bottles were then placed in a sonicating water bath and sonicated on low power until the foam had disappeared. The temperature of the water in the ultrasonic bath (model T9B, L&R, NJ, USA) was maintained at 30°C by circulating water from a thermostatted water bath though a series of copper coils immersed in the sonicating bath.

3. Results and discussion 3.1. Equilibrium surface tension values Fig. 1 shows the change in c versus time for 10−3 wt% b-lactoglobulin solutions, both purified

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Fig. 1. Interfacial tension, c, versus time, t, for 10−3 wt% b-lactoglobulin solutions, both purified (&) and unpurified ($).

Fig. 2. Final equilibrium c as a function of surfactant concentration, C , in the absence of b-lactoglobulin ($) and in the s presence of 10−1 wt% (+) and 10−3 wt% (&) b-lactoglobulin. The corresponding R scale is also indicated for 10−3 wt% protein.

and unpurified. Clearly the purification procedure, which was suggested by Clark and coworkers [7], may have a significant effect on the kinetics of c, particularly in the short time region. At longer times the effect of the purification is limited and the final equilibrium tension values for the purified and unpurified protein are almost the same. However, in all the following measurements reported, only the purified protein was used. The impurities in the b-lactoglobulin are probably fatty acids [7]. Fig. 2 shows the behaviour of the final equilibrium c as a function of surfactant concentration, C , in the absence of b-lactoglobulin and in the s

presence of two different concentrations of b-lactoglobulin, 10−1 and 10−3 wt%. In the absence of protein the break in the gradient suggests a critical micelle concentration (CMC ) of 2.82×10−3 wt%, or 6.5×10−5 mol dm−3, which is in good agreement with literature values [23]. The gradient of the plot at the CMC (assuming the Gibbs adsorption isotherm holds) gives an area ˚ 2. Plotted along the top of per molecule of 65 A the graph is the ratio, R, of the molar concentration of surfactant to the molar concentration of b-lactoglobulin corresponding to 10−3 wt% protein, assuming the monomeric form, although under these bulk solution conditions the protein exists as a dimer [24]. In the presence of 10−3 wt% b-lactoglobulin, there is no significant shift of the CMC, though c at low C values, below s the CMC, is considerably lower than in the absence of protein, suggesting co-adsorption of protein and surfactant. At 10−1 wt% b-lactoglobulin, the values of c are lower still and there is also a significant shift in the apparent CMC to 5.4× 10−3 wt% surfactant. This is probably the result of the binding of surfactant to the protein, reducing the concentration of free surfactant. If it is assumed that all the shift in the CMC is due to binding of surfactant to the protein to form a complex, then at 10−1 wt% the molar ratio of surfactant to protein monomer in the complex is calculated as 1.08:1. This is reasonable, since a number of studies have suggested that b-lactoglobulin binds nonionic surfactants at low binding ratios — one and sometimes two strong binding sites per monomer are usually indicated [4–6 ]. 3.2. Dilatational rheology of adsorbed protein films Films were aged for approximately 3–4 h before expansion, by which time which the interfacial tension, c, appeared to be constant. Fig. 3 shows the typical behaviour when 10−3 wt% blactoglobulin adsorbed films (no surfactant present) of area A were subjected to an area increase, DA, of 10%, i.e. DA/A=0.1. The change in interfacial tension, Dc, has been plotted against time. The curves shown are the average of at least four experiments. Within experimental error, a larger Dc was clearly observed for the film adsorbed at

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Fig. 3. Average interfacial tension, c, versus time, t, for 10−3 wt% b-lactoglobulin adsorbed films (no surfactant present) for DA/A=0.1 at A–W interface ($) and O–W interface (%).

the O–W interface compared with the A–W interface. Such Dc–t decay curves contain much useful information and can be analysed in a number of ways, many developed by the comprehensive studies of the late Paul Joos, to whom this volume is dedicated. Interfacial stress versus time, i.e. the Dc–t curve, may be Fourier transformed to obtain the dilatational elasticity, e, and viscosity, k, of the film as a function of frequency, f, of deformation. For a DA which proceeds faster than the time-scale (frequency) of the recovery considered [22,25]: e∞=e=

v (DA/A)

P

2

Dc(t) sin(vt) dt

(1)

0

and e◊=vk=

v

P

2

Dc(t) cos(vt) dt (2) 0 where e∞ and e◊ are the real (storage) and imaginary ( loss) parts of the complex modulus, v=2pf, and p=3.141…. Fig. 4 shows the results of applying Eqs. (1) and (2) to the data in Fig. 3. It is that seen that e∞ and to a lesser extent e◊ are greater at the O–W interface than at the A–W interface, reflecting the larger increase in Dc on expansion. This may be the result of greater unfolding of the protein at the O–W interface, due to better solvation by the oil of the hydrophobic side chains of the polypep(DA/A)

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Fig. 4. Fourier transform of data in Fig. 3 showing e∞ and e◊ at the A–W interface (# and %, respectively) and e∞ and e◊ at the O–W interface ($ and &, respectively) versus log f. 10

Fig. 5. Data from Fig. 4 showing log e and log k at the A–W 10 10 interface (# and %, respectively) and log e and log k at the 10 10 O–W interface ($ and &, respectively) versus log f. 10

tide. At high frequencies, e∞ levels off, approaching the Gibbs elasticity, e , given by [26 ]: 0 dc e =− (3) 0 d ln C where C is the surface excess concentration. Fig. 5 plots the dilatational elasticity, e, and viscosity, k, on a logarithmic scale. It is seen that k decreases markedly with increasing frequency. Fig. 6 takes the same data and plots them according to Van Hunsel and Joos [27] in the form of e∞/e versus 0 e◊/e , a Cole–Cole plot. The dashed line indicates 0 the theoretical curve expected for a diffusion controlled relaxation, which has a maximum of (앀2−1)/2=0.207 [28]. The dotted curve indicates the theoretical behaviour for a reorientation pro-

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holds then [27]: RTC2 e C 앀t = 0 0 D 앀D

Fig. 6. Cole–Cole plot of e◊/e versus e∞/e for the A–W ($) 0 0 and O–W (+) interface. The dashed line indicates the theoretical curve for a diffusion controlled relaxation, the dotted line indicates the theoretical behaviour for a reorientation process.

cess, which has a maximum of 0.5 [27,28]. The results indicate part conformance with a diffusion controlled process (at short times) but also part conformance with a process involving reorientation (at long times). Separate measurements of the relaxation of spread b-lactoglobulin monolayers (under slightly different conditions) have indicated that relaxation of molecules already adsorbed is not necessarily any slower than that due to adsorption of molecules from solution and their subsequent unfolding [19,20a,20b]. Thus a slower, reorientation process is not necessarily due to the molecules already adsorbed, but could be due to molecules which have just adsorbed through diffusion to the interface. Another method of analysing the data is to express the Dc decay in terms of a reduced tension, S, defined as: S=Dc/Dc

(4)

max

where Dc is the maximum value of Dc observed: max the difference between the maximum value of c measured just after the expansion and the equilibrium value. For relaxation which is dominated by a diffusion process it is appropriate to try and fit the S–t curve to the equation

A B A B t

erfc

t

1/2

(5) t t D D to obtain the diffusional relaxation time, t [27]. D Fitting Eq. (5) to the data in Fig. 3 gives t values D of 30.3 and 31.3 s for the A–W and O–W films, respectively. If the Gibbs adsorption equation

S=exp

(6)

where C is bulk concentration, R is the gas 0 constant, T the absolute temperature and D is the bulk diffusion coefficient of the surface active species. Although the surfactant in the system studied here is a protein and therefore unlikely to follow the Gibbs adsorption equation, it is still instructive to perform this calculation. The bulk diffusion coefficient of b-lactoglobulin (in the dimeric form at this pH ), as determined by centrifugation [29], is 7.45×10−11 m2 s−1 at 20°C. (Recent measurements in this laboratory have confirmed that this is the operative value for adsorption at the A–W interface under these conditions [30].) Using this value of D and the values of e of 45.3 and 68 mN m−1 from the Fourier 0 transformed curves above, Eq. (6) gives C values of 17.7 and 21.9 mg m−2 for the A–W and O–W interface, respectively, suggesting approximately equal coverages at the two interfaces. However, both these C values are unrealistically high, since they far exceed typical saturation monolayer coverages of 2–3 mg m−2 for adsorbed proteins. Using the diffusion coefficient for the whole molecule is inappropriate, since adsorption and lowering of c after expansion involves adsorption of segments of the newly arriving protein molecules and molecules already at the interface, as indicated above in the discussion of the Cole–Cole plot analysis. The difference in the moduli at the A–W and O–W interfaces qualitatively agrees with a more detailed study at a paraffin oil–water interface by Williams and Prins [31], though in a different range of bulk concentrations. Williams and Prins found that the difference between the moduli at the O–W and A–W interfaces decreases markedly as the concentration of protein is increased. Jiang and Chew [9] have also recently studied lysozyme at the corn O–W and A–W interfaces and Benjamins et al. [32] have also recently compared dilatational measurements on sodium caseinate, ovalbumin and bovine serum albumin films adsorbed at O–W and A–W interfaces. Benjamins et al. [32] found that e and k were lower at the

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O–W interface, though the oil used here was chemically quite different — a triacylglycerol oil obtained by purification of sunflower oil. The choice of oil type for such model studies may therefore be quite important in trying to determine the behaviour of real food oil systems. 3.3. Dilatational rheology of mixed protein+surfactant films The results for experiments on films at the A–W and O–W interfaces adsorbed from mixtures of b-lactoglobulin+C E are shown in Figs. 7 and 12 6 8. The b-lactoglobulin concentration was kept

Fig. 7. e∞ versus log C at f=6.31×10−3 Hz for films adsorbed 10 s from 10−3 wt% b-lactoglobulin+C E at the A–W ($) and 12 6 O–W (&) interface. The corresponding R scale is also indicated.

Fig. 8. e◊ versus log C at f=2.51×10−4 Hz for films adsorbed 10 s from 10−3 wt% b-lactoglobulin+C E at the A–W ($) and 12 6 O–W (&) interface. The corresponding R scale is also indicated.

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fixed at 10−3 wt%. At least three experiments were performed on each mixture, the decay curves averaged and Fourier transformed, as above. In order to compare the mixtures sensibly, values of e∞ and e◊ at representative frequencies of f=6.31×10−3 Hz ( log f=−2.2) and f=2.51× 10 10−4 Hz ( log f=−3.6) have been plotted as a 10 function of C . The corresponding R scale is also s indicated. Fig. 7 shows the behaviour of e∞ and Fig. 8 the corresponding behaviour of e◊, with error bars shown. The decrease in the magnitude of the dilatational moduli at higher non-ionic surfactant concentrations is not nearly as marked as with the interfacial shear moduli measured elsewhere (e.g. see Chen and Dickinson [33]), because the difference between the moduli for different proteins or between proteins and low-molecular-weight surfactants is not so large in the first place [1,2]. Within experimental error, the A–W moduli at these frequencies seem independent of C until s approximately 3×10−5 wt% (i.e. R#2, or approximately equal molar concentrations of surfactant and dimer). Above this concentration there is a fall in the moduli. For the O–W interface, however, it appears that there is a modest maximum in the moduli, somewhere between about 10−6 wt% and 10−4 wt%. Beyond about 10−4 wt% there is a sharp decrease in the moduli, as for the A–W interface. Part of the reason for some of the large errors indicated is given below, but the results suggest differences in the dynamics of response of the films at the A–W and O–W interfaces in the presence of relatively low concentrations of surfactant. This could be due to different states of unfolding of the proteins at the two interfaces (see above) and interactions between the protein molecules in the film induced by co-adsorption of the surfactant and/or due to differences in the surface active behaviour of the protein–surfactant complexes adsorbing from the bulk solution. The results in Figs. 7 and 8 suggest little effect of low surfactant concentrations on the dilatational moduli of the A–W films at these frequencies. Fig. 9 also shows the corresponding Gibbs elasticity, e , for these same films as a function of 0 C and the two lines indicate results from the s accelerated foam life-time test (a) for the

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Fig. 9. For A–W interface, e∞ (&) and e◊ (+) from Figs. 7 and 8, with the Gibbs elasticity, e ($), also shown. The lines drawn 0 indicate the accelerated foam life-times (a) for the protein+surfactant mixtures and (b) for the surfactant on its own.

protein+surfactant mixtures and (b) for the surfactant on its own. Despite the scatter in the e 0 data there is a suggestion of a slight enhancement in e at the A–W interface, which appears to match 0 a small but clear increase in the foam stability between C =10−5 and 10−4 wt%. ( These results s are similar to those reported elsewhere, obtained under slightly different conditions [1,2].) Part of the reason for the relatively large errors in the results for the mixtures, which perhaps obscures the true trend in the data, is the fact that some of the measurements were made at slightly different (but exactly known) DA/A, ranging from 8% to 12%. Although it is often claimed or assumed that the dilatational moduli are independent of the strain at a few per cent, it was decided to investigate this further. Figs. 10 and 11 show the behaviour of e∞ and e◊, respectively, as a function of the percentage DA/A. The results indicate a strong dependence of the moduli on the strain, i.e. the rheology is non-linear, down to at least 5%. It is planned to extend these measurements down to lower strains in the future, using a more sensitive strain gauge for monitoring the change in the pull on the Wilhelmy plate. (Below around 5% even the maximum values of Dc were too low to be measured reproducibly.) Another explanation of the strain-dependence and the scatter of the results is that there are c gradients set

Fig. 10. e∞ at f=6.31×10−3 Hz versus DA/A for films adsorbed from 10−3 wt% b-lactoglobulin at the A–W ($) and O–W (&) interface.

Fig. 11. e◊ at f=2.51×10−4 Hz versus DA/A for films adsorbed from 10−3 wt% b-lactoglobulin at the A–W ($) and O–W (&) interface.

up within the interface on expansion, due to the high viscosity typical of such protein films. The usual, above type of analysis assumes a homogeneous expansion of the film molecules, so that the macroscopic, measured value of c is assumed to rise and fall with exactly the same value at every point in the film, due to microscopic c gradients which dissipate through protein rearrangement and adsorption. With non-homogeneous expansion of the interface the Dc–t decay curve will vary depending on the exact location and orientation of the Wilhelmy plate relative to the expanding barrier [34,35]. It is obvious that during the processing of real emulsions and foams, interfaces must be subjected to percentage expansions even larger and less homogeneous than those studied here. Thus such experiments might be of more

B.S. Murray et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 143 (1998) 211–219

value than measurements at lower deformations in understanding the relationship between the rheological properties of the adsorbed films and their stabilising properties.

4. Conclusions Adsorbed films of b-lactoglobulin exhibit higher dilatational moduli at the O–W interface compared to the A–W interface. Only one molecule of the non-ionic surfactant C E binds to each molecule 12 6 of b-lactoglobulin, but this and/or surfactant co-adsorption has significant effects on the dilatational moduli, these effects being different at the A–W and O–W interfaces. Thus the effects of lowmolecular-weight surfactants on the interfacial dilatational rheology of protein films can be subtle — relatively low concentrations of surfactants may shift the magnitude of the dilatational moduli and the corresponding foam stability in different directions. Moduli measured at strains up to at least 5% may be considerably non-linear, which has implications for the many different methods used for measuring interfacial rheology and how these measurements are related to film formation and stability in systems containing protein+lipid.

Acknowledgment Financial support from the Nuffield Foundation is gratefully acknowledged.

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