Morphological Changes in Adsorbed Protein Films at the Oil−Water

Langmuir 2008, 24, 1979-1988

1979

Morphological Changes in Adsorbed Protein Films at the Oil-Water Interface Subjected to Compression, Expansion, and Heat Processing Rong Xu, Eric Dickinson, and Brent S. Murray* Food Colloids Group, Procter Department of Food Science, UniVersity of Leeds, Leeds LS2 9JT, United Kingdom ReceiVed September 10, 2007. In Final Form: October 31, 2007 Adsorbed films of milk proteins at the oil-water (O-W) interface have been imaged using a Brewster angle microscope (BAM). Special adaptations were made to the BAM to allow imaging of the O-W interface and to enable in situ heating and cooling of the adsorbed films. The proteins β-lactoglobulin (β-L) and Rs1-, β-, and κ-casein were studied over a range of bulk protein concentrations (Cb) and surface ages at pH 7 and for β-L at pH 5 also. The adsorbed films were subjected to incremental compression and expansion cycles, such that the film area was typically varied between 125% and 50% of the original film area, and the resulting film structure was recorded via the BAM at 25.0 °C. Structuring of β-L films (the formation of ridges and cracks) was more pronounced at pH 5 (closer to the protein’s isoelectric point) than at pH 7 and for longer adsorption times and/or higher Cb. Structuring was also much more apparent at the O-W interface than at the A-W interface on compression/expansion/aging, especially at pH 7. After heating β-L films adsorbed at low Cb (0.005 wt %) to 80 or 90 °C, an even greater degree of film structuring was evident, but β-L films adsorbed at higher Cb (g0.05 wt %) showed fewer but larger fractures. The adsorbed caseins showed little evidence of such features, either before or after heating, apart from slight structuring for the heated films of Rs1- and κ-casein films after 1 day. Changes in the dilatational elastic modulus of the β-L films (Cb ) 0.005 wt %) were correlated with the variations in the structural integrity of the films as observed via the BAM technique. In particular, there was a marked increase in the elastic modulus on heating, while the cycle of compression and expansion appeared to result in a net film weakening overall. The β-L films adsorbed at higher Cb (g0.05 wt %) behaved as if an even stronger elastic skin completely covered the interface. The overall conclusion is that interfacial protein films subjected to these types of thermal and mechanical perturbations, which are typical of those that occur in food colloid processing, can become highly inhomogeneous, depending on the type of protein and the bulk solution conditions. This undoubtedly has implications for the stability of the corresponding emulsions and foams.

Introduction In previous work,1 we demonstrated the structuring that can develop in adsorbed protein films at the A-W interface when the films are subjected to incremental compression and expansion. This was particularly noticeable after aging of the films and/or when the pH approached the isoelectric pH of the protein. Two proteins that are particularly important in the stabilization of food foams were studiedsβ-lactoglobulin (β-L) and ovalbumin (OA). Brewster angle microscopy (BAM) was used to image the film structure. The BAM technique is extremely sensitive to any changes in film thickness or optical density, and it has the advantage of being completely noninvasive, i.e., none of the film components have to be labeled with chromophores (as in fluorescence microscopy) and the films can be examined in situ. The structuring observed is relevant to the stability of the corresponding foams stabilized by such proteins, since processing of these foams frequently involves the application of high shear forces during whipping, stirring, and pumping, as well as pressure cycling during the formation and dispensing of the foamed products. These processes usually result in considerable compression and expansion of the adsorbed protein film stabilizing the system, which may disrupt the structure of the adsorbed films. Recent work has shown2-4 that common food proteins, * Author to whom correspondence should be addressed. Tel. 44 (0)113 3432962; Fax. 44 (0)113 3432982; E-mail [email protected]. (1) Xu, R.; Dickinson, E.; Murray, B. S. Langmuir 2007, 23, 5005. (2) Murray, B. S.; Dickinson, E.; Lau, C. K.; Schmidt, E. Langmuir 2005, 21, 4622. (3) Murray B. S.; Cox, A.; Dickinson, E.; Nelson, P. V.; Wang, Y. In Food Colloids: Self-Assembly and Material Science; Dickinson, E., Leser, M., Eds.; Royal Society of Chemistry: Cambridge, 2007; p 369.

even at quite high protein concentrations, do not have the capacity to prevent the coalescence of air bubbles when the interfaces are expanded at relatively modest rates and extents of expansion. In particular, it has been found that proteins that show the most pronounced structuring, most notably ovalbumin,1 provide the least resistance to coalescence on bubble expansion.2 The effects of expansion and compression of adsorbed proteins are probably equally important at the oil-water (O-W) interface. These effects have relevance to the formation and processing of emulsions. The present study extends our earlier work at the A-W interface to the O-W interface. Certain differences might be expected between the behavior of the same proteins at the two types of interface, even in the absence of compression and expansion, because the configuration of proteins at O-W and A-W interfaces is not necessarily the same. Air is not a good solvent for the hydrophobic regions of a protein, whereas an oil phase will effectively solvate the hydrophobic amino acid residues. On the basis of experiments on spread and adsorbed monolayers of β-L and caseins, Murray et al.5-7 suggested that, overall, proteins appear to be more unfolded and possibly more flexible at the O-W interface, and this results in higher dilatational moduli for globular proteins. This effect is most likely due to the better solvency of the oil for the side chains of the hydrophobic amino acid residues of the proteins. Thus, proteins tend to form more fluidlike films at the O-W interface, whereas at the A-W (4) Heuer, A.; Cox, A. R.; Singleton, S.; Barigou, M.; van Ginkel, M. Colloids Surf., A 2007, 311, 112. (5) Murray, B. S.; Færgemand, M.; Trotereau, M.; Ventura, A. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, 1998; p 223. (6) Murray, B. S. Prog. Colloid Polym. Sci. 1997, 103, 41. (7) Færgemand, M.; Murray, B. S. J. Agric. Food Chem. 1998, 46, 885.

10.1021/la702806t CCC: $40.75 © 2008 American Chemical Society Published on Web 01/23/2008

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interface, they may behave more like insoluble aggregates, the protein molecules being strongly adsorbed but also more likely to stick together. Certainly, ovalbumin is well-known for coagulation on adsorption at the A-W interface.9 It is interesting, therefore, that most previous BAM work1,10,11 suggests that β-L at the A-W interface does not readily form folds or ridges at the microscopic level on compression or expansion, indicating that a strong, coherent film capable of supporting such features is not formed. However, recently it was shown1 that the inclusion of a low area fraction (less than 0.01%) of 20 µm polystyrene latex spheres at the A-W interface highlights some structuring that is not readily observable in the absence of such particles. Greater unfolding of proteins at the O-W interface may lead to more extensive intermolecular cross-linking that strengthens the films. Indeed, this may be the reason that the values of interfacial shear and dilatational elasticity and viscosity are higher at the O-W interface compared the A-W interface.5-7 Thus, films at the O-W interface may be more resistant to deformation on expansion/compression compared to films at the A-W interface. On the other hand, this greater resistance may make them more brittle and susceptible to fracture. Up to now, there have been very few studies of adsorbed film structure at the O-W interface,12,13 and so it is important to investigate these issues further. Apart from processing conditions that may result in expansion and compression of adsorbed films, another key processing variable is temperature, since many emulsified products (and to a lesser extent foamed products) receive a pasteurization treatment after the proteins have adsorbed. Typical pasteurization temperatures (around 90 °C) are above the thermal denaturation temperatures of most food proteins, and, although the adsorbed proteins are expected already to be unfolded to some extent after adsorption to the interface, such temperatures will induce considerable further changes in the adsorbed configuration. Indeed, Dalgleish et al.14 have shown that one effect of temperature can be to change the relative surface activities of β-L and the caseins. Thermally unfolded β-L is able to compete much more effectively with the caseins for adsorption at the interface. Roth et al.15 showed that heating can increase the surface shear viscosity of β-L films and make them much less susceptible to desorption by the non-ionic surfactant Tween 20, but the effects of compression and expansion of the films were not tested. This paper reports the technologically important case of the measurement of the structural changes of adsorbed films in response to compression and expansion at the O-W interface for films that have been heat-processed. In addition, as previously reported, we have made measurements of the interfacial stress (tension) response of the films to try to relate the structural changes (as observed via BAM) to the mechanical properties of the films. Materials and Methods Materials. The β-lactoglobulin from bovine milk (∼90% PAGE, crystallized and lyophilized; lot no. 033K7003) and κ-casein (∼80% PAGE, crystallized and lyophilized) were from Sigma-Aldrich Chemical Co. (Poole, UK). The β-casein and Rs1-casein were freeze(8) Murray, B. S.; Lallemant, C.; Ventura, A. Colloids Surf., A 1998, 143, 211. (9) MacRitchie, F. AdV. Colloid Interface Sci. 1986 254, 341. (10) Murray, B. S.; Garofalakis, G. Colloids Surf., B 1999, 12, 231. (11) Rodrı´guez Patino, J. M.; Sa´nchez, C. C.; Rodrı´guez Nin˜o, M. R.; Fernandez, M. C. J. Colloid Interface Sci. 2001, 242, 141. (12) Benjamins, J.-W., Ph.D. Thesis, Lund University, 2004. (13) Benjamins, J.-W.; Jo¨nsson, B.; Thuresson, K.; Nylander, T. Langmuir 2002, 18, 6437. (14) Dalgleish, D. G.; Goff, H. D.; Luan, B. B. Food Hydrocolloids 2002, 16, 295. (15) Roth, S.; Murray, B. S.; Dickinson, E. J. Agric. Food Chem. 2000, 48, 1491.

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Figure 1. (A) Schematic diagram of the Brewster angle microscope setup and side view of the specialist Langmuir trough apparatus: laser (a), CCD camera and objective lens (b), guide tube for laser to O-W interface (c), guide tube for beam reflected from the O-W interface (d), point of incidence at O-W interface (e), upper oil phase (f), lower aqueous phase (g), elastic rubber barrier containing O-W interface for compression and expansion (h), stainless steel pins for change the area of the elastic barrier (i), and wall of trough (j). (B) View of the trough arrangement from above with some of the same labels as in (A). The position of the Wilhelmy plate (k) is indicated for separate experiments when the interfacial tension is measured. Arrows show the direction of movement of the pins (i) for changing the area enclosed by the elastic barrier (h). dried samples (98% purity) from the Hannah Research Institute (Ayr, Scotland). All other reagents, including imidazole buffer, sodium chloride, hydrochloric acid, acetic acid, and acetone were AnalR grade from VWR International Ltd. (Poole, UK). Dow Corning Silicone oil “200/10cS” was also purchased from VWR International Ltd. Millipore water (Millipore Ltd, Watford, UK), free from surfaceactive impurities and with a conductivity of less than 10-7 S cm-1, was used throughout. Proteins were dissolved in pH 7.0 buffer solutions formed from 0.05 mol dm-3 imidazole + 0.05 mol dm-3 NaCl, adjusted to pH 7.0 by addition of HCl. A pH 5.0 buffer was also used in some experiments, formed from 0.01 mol dm-3 sodium acetate (+0.05 mol dm-3 NaCl) and adjusted to pH 5 by addition of acetic acid. Protein concentrations varying between 0.005 and 0.5 wt % were studied. Methods. Images of adsorbed protein films at the O-W interface were recorded using a BAM2plus Brewster angle microscope (NFT, Go¨ttingen, Germany), modified (see Figure 1A) and combined with a specialist Langmuir trough (see Figure 1B). The basic principles of BAM16 and the specific instrument used here have been explained elsewhere1,10 and are not discussed in detail here. Modification of the BAM was required because the BAM2plus instrument is designed for imaging at the A-W interface. If an oil layer is placed on top of the aqueous phase, there are several effects that have to be addressed, as follows. (a) The Brewster angle at the O-W interface is lower than at the A-W interface for typical oils, and so the angle of incidence has to be adjusted from the normal settings for the A-W interface (the Brewster angle, θB, is defined by θB ) tan-1(n2/n1), where n1 ) the refractive index of medium 1 (air for the A-W interface and oil for the O-W interface) and n2 is the refractive index of medium 2 (the aqueous phase)). More importantly, the smaller difference in refractive index between oil and water, compared to that between air and water, means that at the O-W interface the intensity of the reflected beam due to the presence of an adsorbed film is lower. (b) Some imaging light intensity is lost (16) He´non, S.; Meunier, J. ReV. Sci. Instrum. 1991, 62, 936.

Morphological Changes in Adsorbed Protein Films due to the additional incidence of the laser beam on the surface of the oil and particularly due to the beam being reflected from the O-W interface as it exits the oil phase to the air. (c) The effect of the oil layer is to displace the optical path of the beam in the vertical plane compared to the situation where the oil phase is absent, so that the positions of the microscope objective lens and CCD camera collecting the reflected beam (see Figure 1A) have to be adjusted. Points (a) and (c) above cause problems with the commercial instrument, because without serious mechanical modifications, the goniometers do not allow angles of incidence less than approximately 43°, which happens to be the Brewster angle of incidence at the O-W interface for a typical straight-chain hydrocarbon like n-tetradecane or n-hexadecane (refractive index ≈ 1.43). (For the A-W interface, the Brewster angle is 53.13°.) Also, it is not easy to adjust the vertical position of the lens and camera without compromising the alignment and adjustment of the whole instrument. The solution adopted to get around these problems was to mount light guides on the instrument, as illustrated in Figure 1A. Thus, the laser light is not incident on the surface of the oil phase at all, but is transmitted to the O-W interface, at the O-W Brewster angle, by a steel tube with a small optical flat sealed to its end, dipping in the bulk oil phase, just above the O-W interface. Similarly, the beam reflected from the O-W interface is transmitted to the objective lens via a second similar tube. The optic flats were 10 mm thick and 72 mm in diameter, made of quartz manufactured by Scientific Optics Ltd (Hastings, UK). Both faces of the flats were highly polished and exactly parallel to each other. They were carefully mounted in the tubes so that their faces were exactly at right angles to the beam, to avoid refraction and reflection on their faces. Experiments were also conducted with solid glass rods as an alternative. However, these proved too difficult to align reliably. Further technical details on the apparatus used here are available under the Supporting Information. Interestingly, Benjamins et al.12,13 independently came up with a similar solution to that used here, also after experimenting with solid glass rods. The oil phase was polydimethysiloxane (PDMS), i.e., silicone oil. There were two main reasons for choosing this oil here rather than the hydrocarbon n-tetradecane or n-hexadecane, which is frequently used in model studies. First, the refractive index of silicone oils is somewhat lower than that of hydrocarbons, and thus the Brewster angle is a little higher (43.6° for a refractive index of 1.40). This meant that further adjustment to the instrument’s goniometers was not necessary to allow this angle of incidence (see point (a) above). Second, and more importantly, the higher chain length silicone oils are not nearly as volatile as the liquid hydrocarbons, which was important for this study because the protein films adsorbed at the O-W interface were to be heated to up to 90 °C. On testing with n-tetradecane, for example, excessive evaporation occurred, reducing the height of the oil layer significantly during the experiment, causing the end of the light-guide tubes to be only partially submerged in the oil phase. This interfered with the path of the beam. Also, the oil tended to condense elsewhere in the instrument, causing contamination problems. A range of PDMS oils are available, up to very high molecular weights, high viscosities, and of low volatility. Some of the higher-viscosity silicone oils were tested, but the high viscosity sometimes made spreading of the oil over the interface difficult. It was found that the 200/10cS oil was adequate for the experiments conducted here. As the product code implies, it has an average molecular weight of approximately 200 Da and a viscosity of approximately 10 centiStokes (i.e., approximately 10 times that of water at 25 °C). The interfacial tension at the O-W interface, γow, was measured as 33 mN m-1. This γow value is lower than that for n-tetradecane (53 mN m-1) and other pure hydrocarbons, due to the slightly higher polarity of silicone oil. However, measurements made previously17,18 have shown that the adsorption of proteins (β-L and sodium caseinate, for example) at the silicone O-W (17) Murray, B. S. Unpublished results presented at 10th International Conference on Colloid and Interface Science, 23-28 July, Bristol, 2000. (18) Akhtar, M.; Dickinson, E. Colloids Surf., B 2003, 31, 125.

Langmuir, Vol. 24, No. 5, 2008 1981 interface is very similar to adsorption at the hydrocarbon O-W interface, for low-viscosity silicone oils. The square trough containing the aqueous phase was made from anodized aluminum alloy, with a hollowed-out base for circulation of water from a temperature-controlled bath. This arrangement gave accurate ((0.1 °C) and rapid temperature control and reduced the problem of thermal convection, which can prevent clear imaging of the interface. The area of the trough was 90 cm2, which was smaller than that used previously,1 in order to reduce the required volume of the aqueous phase and oil. The barrier used to compress the interface, illustrated in Figure 1B, was also different in design from that used previously, partly to accommodate the reduced volume of the trough. It consisted of a thin rubber diaphragm, stretched around four pins to form a square-shaped barrier. The pins were connected to gears and tracking that allowed their synchronized movement along the diagonal of the square. In this way, the film contained within the barrier could be subjected to purely areal compression and expansion. This type of flexible barrier idea is like that originally developed by Benjamins et al.19 The height of the diaphragm was approximately half the depth (3 cm) of the trough, so that roughly 2/3 of the volume of the trough could be filled with aqueous phase and the remaining 1/3 with oil, to allow the barrier to compress and expand films at the O-W interface, as illustrated in Figure 1B. Leaving clean water in contact with the rubber diaphragm for 3-4 h, and then compressing the A-W interface, did not result in any significant decrease in the surface tension. This suggested that the diaphragm did not contain surface-active materials that could leach out of the barrier and significantly affect the properties of the adsorbed films studied. The O-W interface was formed by adding approximately 200 mL of the buffered protein solution to the trough, taking care to avoid the formation of bubbles. The starting position of the barrier was usually such that the surface area enclosed by the barrier was 31.1 cm2. This is referred to throughout as the reference, starting area, A0. In all experiments, the trough temperature was initially set at 25.0 ( 0.1 °C, unless stated otherwise. Any bubbles that were adventitiously trapped within the barrier were aspirated away using a Pasteur pipet and vacuum pump; this usually took 10-30 s. Immediately after this, a volume of 50-60 mL of silicone oil was carefully layered over the aqueous phase. This point was defined as the zero adsorption time. After waiting for adsorption times of either 4 h or 1 day, the interface was compressed and expanded to different areas, A, in several stages. The maximum area that the film was expanded to was usually 54.8 cm2, or A/A0 ) 1.78, and the minimum area was 13.7 cm2, or A/A0 ) 0.48, for comparison with the measurements made previously at the A-W interface.1 Images were collected from the center of the barrier, as indicated in Figure 1A. The minimum area was also dictated by the requirement to avoid the barrier touching the optics conveying the laser light to and from the interface. Between each compression/expansion stage, the motion of the barrier was stopped for 5 min to allow collection of an image. Although the actual snapshot takes less than 0.04 s to acquire, this 5 min waiting time was necessary to allow for any mechanical disturbances to subside and to facilitate any adjustment to focusing, and so forth, that might be necessary to improve the quality of the image. It was not possible to obtain good quality images while the barrier was actually moving at the speeds employed, because the barrier movement caused disturbance of the interfacial features and consequent blurring of the images. The laser output power was always kept at 50-60% of its maximum and the camera shutter speed fixed at 50 s-1. The actual angle of incidence employed at the silicone oil-water interface was 45 ( 0.2°, adjusted within these limits to give the best contrast. The effective resolution of the microscope was approximately 2 µm, and the image dimensions were 768 × 572 pixels. The analyzer and polarizer angles were set at zero. As explained previously,1 although slightly improved resolution could sometimes be obtained by varying these settings, there was then the additional difficulty of reliably (19) Benjamins, J.; de Feijter, J. A.; Evans, M. T. A.; Graham, D. E.; Phillips, M. C. Faraday Discuss. 1976, 59, 218.

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Figure 2. Typical BAM images and changes in interfacial pressure (∆π) for a β-L film, Cb ) 0.005 wt %, at pH 7, after 4 h adsorption when subjected to the typical compression (a) and expansion (b) cycles employed. The area ratios (A/A0) at which the images are taken are as follows: (A) 1.00, (B) 0.68, (C) 0.44, (D) 0.68, (E) 1.00, and (F) 1.78. The white arrow on (A) indicates the concentric lines due to optical interference. The scale bar ) 150 µm. comparing systems under different settings. Minor changes in image brightness or contrast were made using the BAM2plus software. In separate experiments where γow was monitored during compression/expansion cycles, a Wilhelmy plate of roughened mica (width 1.5 cm) was positioned in the interface at the center of the barrier. The Wilhelmy plate was suspended from a force transducer and a digital recording device (Maywood Instruments, Basingstoke, UK) connected to a PC. Force data were recorded at a rate of 5 or 10 readings per second.

Results and Discussion Morphological Characteristics of Adsorbed Films of β-Lactoglobulin without Heating. Figure 2 illustrates typical BAM images and changes in interfacial pressure (∆π) for a β-L film adsorbed from a bulk protein concentration Cb ) 0.005 wt %, at pH 7, after 4 h adsorption when subjected to the typical compression (Figure 2a) and expansion (Figure 2b) cycles employed. The overall compression and expansion took place in 3 and 5 stages, respectively, and images were collected at the end of each stage. The compression stages were from A/A0 ) 1 to 0.85, from 0.85 to 0.68, and from 0.68 to 0.44. These stages took 48, 55, and 94 s, respectively. The expansion stages were

Xu et al.

from 0.44 to 0.55, from 0.55 to 0.68, from 0.68 to 1.00, from 1.00 to 1.36, and from 1.36 to 1.78. These expansion stages took 94, 55, 48, 98, and 98 s, respectively. Each experiment was repeated at least 3 times. The exact details of the images were different every time, but the images shown in Figure 2 (and in all subsequent figures) are representative of the changes that were always observed under the given conditions. It should be remembered that the interfacial tension γOW was measured in separate experiments but under identical conditions as in the collection of the BAM images. This procedure was necessary because it was not possible to measure γOW and record an image at the center of the barrier simultaneously, due to the Wilhelmy plate interfering with the optical path of the laser. Also, if the laser was incident on the interface close to the Wilhelmy plate, even very slight movements of the plate introduced interfacial convection that blurred the images. The scatter in the ∆π data in Figure 2 is typical of the variation between experiments. It is rather large in proportion to the overall changes in ∆π, partly due to the relatively small changes in ∆π and the inherent noise in the data for these fairly large compression ratios, as noted previously at the A-W interface.1 At the A-W interface, the data were always more noisy on compression than expansion, which was attributed to film collapse and interfacial aggregation, but at the O-W interface, it was observed that there was little difference between compression and expansion in terms of the noise in the data. Part of the scatter is also due to the inherent error in the measurement, estimated as around (0.2 mN m-1, due to the smaller Wilhelmy plate employed (due to the smaller trough), so that smaller overall forces were measured. The ∆π data were generally evenly scattered about a smooth curve, the gradient of which could be used to characterize the viscoelastic response of the film (see later). Figure 2 shows that, in fact, no film structure was observable in the 4 h old β-L film at pH 7. The concentric lines seen in Figure 2 are not due to surface structure. They are present on all raw BAM images, to different extents; they arise from optical interference, as discussed previously.1 For static images, these artifacts can be removed by subtraction of a background image, for example. However, in all images that will be presented later, where true surface structures evolve, this subtraction is difficult because the structures generally change their size and shape slightly and/or drift in and out of view in the time it takes to acquire a single image. Therefore, in Figure 2 and all of the following BAM images, the raw images are presented, except for the occasional adjustment in brightness or contrast of the whole image. The lack of structure at the O-W interface at pH 7 is exactly what was observed at the A-W interface under the same conditions.1,11 At the A-W interface, a small amount of surface structuring was observed only on compressing the films to the minimum area at a lower pH (pH 5), particularly for films that had been aged for longer (e.g., ca. 1 day). This surface structuring took the form of the occasional brighter band within the film. An example is shown in Figure 3A, taken from the earlier publication,1 for a 20 h old film formed by adsorption at the same Cb (0.005 wt %) at pH 5, after compression to A/A0 ) 0.48 (without added latex particles). Such bright bands, or striations, have been interpreted to be ridges or folds of thicker regions of protein. Such structures are more likely to form after longer adsorption time due greater unfolding and more extensive crosslinking of the adsorbed molecules. Similarly, since pH 5 is closer to the isoelectric pH of β-L (pI ) 5.6) than pH 7, the reduced electrostatic repulsion between adjacent molecules will tend to enhance intermolecular cross-linking at pH 5. The interfacial

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Figure 3. Typical BAM images for various compressed films of β-L, Cb ) 0.005 wt %. (A) 20 h old film at the A-W interface at pH 5. (B) 1 day old film at the O-W interface, pH 5, with inset showing the corresponding image for the 1 day old film interface at pH 7. The white arrows indicate a few faint striations that appeared. (C) 1 day old film at pH 5 (not heated), then cooled and imaged at 15 °C. (D) 6 h old film at pH 5 after the 80 °C heat treatment. The inset shows the corresponding image at pH 7. (E) 1 day old film at pH 5 after the 80 °C heat treatment. (F) 1 day old film at pH 7 after the 80 °C heat treatment. The white arrow indicates an example of the rare occurrence of striations that appeared. (G) 1 day old film at pH 7 after the 80 °C heat treatment, then cooled and imaged at 15 °C. (H) 6 h old film at pH 7 after the 90 °C heat treatment. (I) 1 day old film at pH 7 after the 90 °C heat treatment. All images were at A/A0 ) 0.44, apart from (A), which was at A/A0 ) 0.48 (without added latex particles), taken from an earlier publication,1 and imaged at 25 °C, unless stated otherwise. The scale bar ) 150 µm.

viscoelastic character of most globular proteins increases with increased adsorption time and reduced net molecular charge.20 Morphological Characteristics of β-Lactoglobulin Adsorbed Films after Heat Treatment. Because of the differences at pH 5 mentioned above, β-L was also studied at the O-W interface at pH 5, for 4 h and 1 day old films, for comparison with the results at the A-W interface. In the work described in this paper, however, the emphasis was to see how heat processing would affect film structure. So, heating and cooling at both pH 7 and pH 5 were also studied. The results are summarized in the rest of Figure 3, which shows the typical images obtained at the O-W interface after compression to the minimum area, A/A0 ) 0.44, for comparison with the 1 day old film at the A-W interface compressed to A/A0 ) 0.48 (Figure 3A). If film structures were formed at all, they were usually noticeable at this high degree of compression. Two heating procedures were used: (i) heating the adsorbed film at 80 °C for 30 min, and (ii) heating at 90 °C for 60 min. Heating began after allowing adsorption for 4 h. After heating, the films were cooled and imaged at either 25 or 15 °C, at a film age of either 6 h or 1 day. Occasionally, films were cooled from 25 to 15 °C and imaged at 15 °C without prior heat treatment, since previous work had suggested that cooling below ambient temperature may also induce film structure.21 At pH 5, very little was observed at A/A0 ) 0.44 for 4 h adsorption (images not shown), but Figure 3B shows that for 1

day old films at pH 5 a few faint striations appeared. These structures are similar to those observed at the A-W interface at pH 5 (not heated). The inset of Figure 3B shows the corresponding image for the 1 day old film at the O-W interface at pH 7, where no structure was visible, as at the A-W interface. Both at pH 5 and at pH 7, the 4 h and 1 day old films (not heated) were cooled to 15 °C and imaged. For the 4 h old films at pH 7 and pH 5, there was little difference between the images obtained at either temperature, but for the 1 day old film at pH 5, there was much more noticeable structure at pH 5, as can be seen in Figure 3C. This is interesting because the type of bonding that will universally strengthen protein-protein interactions on cooling is hydrogen bonding. A marked increase in the surface shear viscosity of gelatin films formed on adsorption from Cb ) 0.001 wt % was noted by Castle et al.22 Apparently, the reduction in electrostatic intermolecular repulsion at pH 5 plus the slight lowering of the temperature from 25 to 15 °C results in a much stronger film capable of supporting folds and corrugations. Figure 3D shows that the 80 °C heat treatment at pH 5 starts to induce a different type of film structure in the 6 h old film. In contrast, structuring was still not visible at pH 7 under the same conditions (inset of Figure 3D). Even more pronounced structuring was observed at pH 5 for the film that was heatprocessed at 80 °C and then imaged after 1 day (Figure 3E).

(20) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2002, 7, 426. (21) Murray, B. S.; Cattin, B.; Schu¨ler, E.; Sonmez, Z. O. Langmuir 2002, 18, 9476.

(22) Castle, J.; Dickinson, E.; Murray, B. S.; Stainsby, G. In Proteins at Interfaces; Brash, J. L., Horbett, T. A., Eds; ACS Symposium Series No. 343; American Chemical Society: Washington, DC, 1987; Chapter 8.

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Large globule-like structures arranged in lines across the interface were visible. At pH 7, after the same heat-processing at 80 °C, only for the aged films did some structuring start to become visiblesas illustrated by the example of the single striation across the image in Figure 3F. Cooling the films to 15 °C after the 80 °C heat treatment at pH 7 increased the frequency of occurrence of these types of structure considerably, as shown in Figure 3G, but again no structuring was observed for the 4 h old films at pH 7, even after cooling to 15 °C after the 80 °C heat treatment (images not shown). In separate experiments, some films were aged for 4 days at pH 7 without heat treatment and then cooled to 15 °C, but still no film structure was observed so that, at pH 7, lowering the temperature alone was not sufficient to induce film structuring. However, the combination of increased film age, 80 °C heat treatment, plus subsequent lowering of the temperature to 15 °C apparently does induce some the structuring of the films at pH 7, though not as much as at pH 5. Heating will undoubtedly aid unfolding and cross-linking of the film, while cooling will aid the formation of intermolecular hydrogen bonds, as noted above. Although the increased tendency for structuring at pH 5 is of interest, many food emulsion products have a pH which is closer to 7; otherwise, the major milk proteins (the caseins) tend to precipitate. Also, we note that the 80 °C heat treatment is a little mild compared to many pasteurization temperatures. For these reasons, it was decided to concentrate on the behavior at pH 7, but using the more severe heat treatments90 °C for 60 min. Figure 3H,I shows the much more pronounced structuring induced by such treatments at pH 7 for the 6 h and 1 day old films, respectively, imaged at 25 °C. Thus, it is clear that a higher temperature and longer treatment time induces the formation of a more coherent film that can support the formation of ridges and folds on compression. Roth et al.15 have studied the interfacial shear rheology of β-L films subjected to very similar heat processing at 90 °C. On cooling to 25 °C, the heat-treated films showed much higher interfacial shear viscosity (and rigidity) and a much higher concentration of surfactant (Tween 20) was required to displace the β-L from the O-W interface, compared to non-heat-treated adsorbed films of β-L. This is in agreement with the formation of a much more coherent gel-like film, that is more resistant to orogenic displacement by non-ionic surfactant.23 Figures 4 and 5 give more detail on the full compression/ expansion cycles of the 6 h and 1 day old β-L films, respectively, at pH 7 after the 90 °C heat treatment. The surface pressure changes and the typical film structures are shown at the end of the three compression stages and at the end of three of the five expansion stages, exactly as in Figure 2, for comparison with the unheated films. It is worth noting that, on compression, although quite pronounced creases and folds appeared, on re-expansion these seemed to disappear and no obvious fractures in the films were observed. This is contrast to the behavior previously observed at the A-W interface,1 where the sequence of images suggested that cracks appeared in the films, although such features were really only made visible anyway by the addition of PS particles, which seemed to accumulate at the cracks in the film. There are reasonable arguments to suggest that the particles did not induce the fractures, again suggesting that the films at the A-W interface were inherently more brittle than at the O-W interface. In addition, many of the types of structure observed at the O-W interface, where particles were not added, were similar to those observed at the A-W interface with particles present, at least on compression. Thus, it still seems unlikely that (23) Mackie, A.; Wilde, P. AdV. Colloid Interface Sci. 2005, 117, 3.

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Figure 4. Typical BAM images and changes in interfacial pressure (∆π) for a 6 h old β-L film at pH 7, after the 90 °C heat treatment, Cb ) 0.005 wt %, when subjected to the typical compression (a) and expansion (b) cycles employed, i.e., (A)-(F) correspond to the same area ratios (A/A0) as in Figure 2. The scale bar ) 150 µm.

the added particles were solely responsible for the fractures in the A-W films. It remains to be seen though if similar particles affect the structure of heat-processed films at the O-W interface. By comparing Figures 4 and 5, it is seen that the surface pressure behavior of the 6 h and 1 day old films is remarkably similar, but the film morphology is slightly different. There seem to be fewer striations overall in the 1 day old films, but those striations which did occur were found to be darker and more pronouncedsand therefore more likely to be cracks in the film, deficient in protein. This same trend for less frequent but more pronounced features was observed on adsorption from a 10× higher concentration of β-L (Cb ) 0.05 wt %) as shown in Figure 6. These films are even more uniform overall compared to those at 0.005 wt % β-L, with the occasional very marked feature, like that highlighted in Figure 6B at the end of the film expansion, which looks like a large slit or tear opening out in the film. The same trend continues in the images presented in Figure 7, which shows the results for films adsorbed from an even higher Cb value (0.5 wt %). Although some very thick banding appeared on compression (Figure 7a), on expansion (Figure 7b) the films were remarkably uniform. This was despite the fact that large protein aggregates clearly formed in the bulk aqueous phase at

Morphological Changes in Adsorbed Protein Films

Figure 5. Typical BAM images and changes in interfacial pressure (∆π) for a 1 day old β-L film at pH 7, after the 90 °C heat treatment, Cb ) 0.005 wt %, when subjected to the typical compression (a) and expansion (b) cycles employed, as in Figure 2.

this concentration after the heat treatment, since the bulk phase went cloudy. (At Cb ) 0.005 wt %, the aqueous phase remained clear, while at 0.05 wt %, the aqueous phase was very slightly cloudy after heating.) Apparently, these aggregates could not adsorb to the interface, however, or they would have been visible in the BAM images. The different behavior at different Cb values merits further discussion. After 4 h of adsorption at pH 7 from Cb ) 0.005 wt %, the interface is essentially saturated and adsorption from 0.05 or 0.5 wt % gives essentially the same interfacial tension.1 However, faster adsorption at a higher bulk concentration can kinetically trap many adsorbed molecules in an initially less unfolded state than for their adsorption from a lower value of Cb.24 Thus, the state of protein unfolding, and therefore the mechanical properties of the films adsorbed at different Cb but which exhibit the same γOW, can be quite different. There is also the possibility of the formation of protein multilayers at higher Cb that might enhance the overall film strength. Although incontrovertible proof of protein multilayer adsorption is hard to find,25 it is expected that heat treatment enhances the chances of further bulk protein becoming incorporated into the first protein (24) Mitchell, J. R. In DeVelopments in Food Proteins; Hudson, B. J. E., Ed.; Elsevier: London, 1986; Vol 4, p 291. (25) Dickinson, E.; Murray, B. S.; Stainsby, G. In AdVances in Food Emulsions and Foams; Dickinson, E., Stainsby, G., Eds.; Elsevier Applied Science: London, 1988; Chapter 4.

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Figure 6. Typical BAM images and changes in interfacial pressure (∆π) for a 6 h old β-L film at pH 7, after the 90 °C heat treatment, Cb ) 0.05 wt %, when subjected to the typical compression (a) and expansion (b) cycles employed, as in Figure 2. The white arrow on (b) highlights what looks like a large slit opening out in the film. The scale bar ) 150 µm.

layer adsorbed, due to thermally induced unfolding of the protein in the bulk. This bulk unfolded protein might then be expected to cross-link more easily with the unfolded protein at the interface. Mechanical Properties of Adsorbed Films of β-Lactoglobulin. Whatever the reasons for the apparently greater structural coherence visible in the BAM images at 0.05 wt % β-L (Figure 6) and 0.5 wt % β-L (Figure 7), the ∆π versus A/A0 behavior was certainly different than at Cb ) 0.005 wt %. At 0.05 and 0.5 wt % β-L, ∆π decreased on compression and increased on expansion. Normally, the surface tension should decrease (and therefore ∆π increase) as an adsorbed film is compressed, due to the greater packing and surface coverage at the interface, and vice versa on expansion. The results were quite reproducible and similar at Cb ) 0.05 and 0.5 wt %. This might be due to a purely mechanical effect of a thick interfacial film on the Wilhelmy plate. If the whole protein film forms a completely coherent skin at the interface, attached to the barrier and the Wilhelmy plate, then compressing and buckling of the whole film could have the effect of pushing the plate down into the subphase, increasing the force measured on the force transducer, which is calculated as a rise in interfacial tension (decrease in ∆π). The opposite

1986 Langmuir, Vol. 24, No. 5, 2008

Xu et al.

Figure 8. Dilatational elasticities (av) calculated at the start of each compression and expansion cycle for β-L, pH 7, Cb ) 0.005 wt %: 4 h old film (filled circle); 6 h old after the 90 °C heat treatment (open circle); 1 day old after the 90 °C heat treatment (open square). The arrows on the lines connecting the points indicate the sequence of changes in A/A0.

Figure 7. Typical BAM images and changes in interfacial pressure (∆π) for a 1 day old β-L film at pH 7, after the 90 °C heat treatment, Cb ) 0.5 wt %, when subjected to the typical compression (a) and expansion (b) cycles employed, as in Figure 2. The scale bar ) 150 µm.

would serve to decrease the pull on the plate on expansion. Thus, the ∆π measurements shown in Figures 6 and 7 are meaningless in terms of interfacial tension in the normal sense and really reflect the mechanical behavior of a thick, gel-like skin at the interface. It is interesting that on expansion beyond A/A0 ) 1 there was essentially no further change in the force. This would occur if the barrier broke away from the gel layer, as the barrier expanded to an area greater than that of the initially formed film. Although the above is a plausible explanation of the apparent changes in ∆π, the breaking away from the barrier of such a film was not observed by the naked eye. Possibly, such effects were masked by the cloudiness that developed in the aqueous phase on heating, or because the films were still thin compared to the wavelength of visible light. The changes in ∆π depicted in Figures 2, 4, and 5 have been analyzed in order to provide some quantitative assessment of the changes in the mechanical properties of the films due to compression, expansion, and heat processing. The plots of ∆π versus A/A0 were converted to plots of ∆π versus ln(A) and the gradient taken as a measure of the complex dilatational elasticity . As used previously,1 the initial value of  measured at the start of each compression or expansion stage was taken in order to compare the different systems. In this case, it involved fitting the first 100 data points (i.e., over 5-10 s) of the ∆π versus ln(A) plot to a straight line to give av. The values of av obtained are

plotted in Figure 8 against the corresponding values of A/A0 at the start of the compression or expansion stage. The considerable scatter in the raw data means that there is no point in fitting a more complex function to the trend to obtain av, and the estimated error is of the order of (0.5 mN m-1. In Figure 8, the arrows on the lines connecting the points indicate the sequence of change in area from A/A0 ) 1 initially, to A/A0 ) 0.44 (i.e., the compression), back to A/A0 ) 1, and then to A/A0 ) 1.78 (i.e., the subsequent expansion). The main conclusion that can be drawn from Figure 8 is that av is significantly higher after the 90 °C heat treatment of the films adsorbed at Cb ) 0.005 wt % β-L at pH 7. Strengthening of these films as a result of heat processing was similarly suggested by the BAM images. Within the experimental error, it also appears that, as a net result of the combined compression and expansion cycle, the heated films end up being weaker (i.e., av lower) than before the cycle. This is most notable for the heated and aged (1 day old) film. There is some initial increase in av on compression, which might be expected if the film components are being forced together, whereas a net decrease, particularly for the aged and heated film, would be expected if these films begin to fracture. For the unheated, 4 h old film, the net result of the compression/expansion cycle seems to be the opposites but the absolute values of av are all much lower and the changes are similar to the estimated error in av ((0.5 mN m-1), so perhaps the result is inconclusive. One other feature worth pointing out is that all the values of av at the O-W interface, including those for the heated films, are at least an order of magnitude lower than the corresponding values reported earlier1 under the same conditions at the A-W interface. Such a large difference cannot be accounted for by the noise in the data. It was noted in the Introduction that dilatational storage and loss moduli for spread and adsorbed films of β-L (under similar conditions) have been reported as being larger at the O-W interface than at the A-W interface.5-8 However, this was on the basis of a different measurement technique, where a small (5% or 10%) increase in film area was applied and the resulting ∆π versus time plot was Fourier transformed. In fact, these earlier results suggested that the difference between the O-W and A-W interfaces was quite small at lower frequencies (longer times) or was even reversed. Similarly, other work has suggested that the interfacial moduli may be higher or lower

Morphological Changes in Adsorbed Protein Films

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Figure 9. Film elasticity (E) calculated at the start of each compression and expansion cycles for β-L, pH 7: Cb ) 0.05 wt %, 6 h old after the 90 °C heat treatment (filled circle); Cb ) 0.5 wt %, 1 day old after the 90 °C heat treatment (open square). The arrows on the lines connecting the points indicate the sequence of changes in A/A0.

depending upon the type of protein, the protein concentration,26-28 or even the type of oil type used.29,30 With regard to the ∆π data in Figures 6 and 7, for the heated films at the higher Cb value of 0.05 and 0.005 wt %, respectively, it was pointed out earlier that there is a problem in interpreting these data as interfacial tensions in the usual sense. However, if the films are treated as coherent skins connected to the barrier and Wilhelmy plate, as suggested above, an elastic modulus can be calculated by taking the strain in the film as the ratio of the change in width of the film to the overall width of the film, and the ∆π values as the change in the stress normal to the direction of compression/expansion. The ratio of the stress to the strain gives a film elasticity E. Middelberg et al.31 have performed similar calculations using a novel type of interfacial rheometer. Here, however, the stress measurement in the film was performed by measuring the lateral force on two parallel pieces of optic fiber that floated at the interface, connected to a force transducer. The movement of the fibers toward or away from each other was controlled via an “inchworm motor” linear piezoelectric positioning device. In the same way as in Figure 8, only the initial part of the ∆π data was used to characterize the value of E at the start of each compression/expansion stage. Again, a linear fit to the first 100 data points was used. It is seen that this is a good approximation for each of the changes shown in Figures 6 and 7, and the lower noise in the data (compared to that in Figures 2, 4, and 5) means that the estimated error in E is much smaller as a percentage of the typical values in E, compared to the estimated percentage error in av. The calculated values of E are shown in Figure 9 and the maximum error in E is (7.5 mN m-1. It is not meaningful to compare the values of av with E, but the same sort of trend in E as function of the change in A/A0 is seen in that there is a net reduction in the film strength as a result of the full compression-expansion cycle, particularly for the heated film at the higher Cb ) 0.5 wt %, despite this having the highest initial value of E. This is consistent with the film at this higher concentration exhibiting fewer but more catastrophic breakages as seen in the BAM images. The values of E obtained are (26) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403, 415, 427. (27) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227, 240. (28) Adams, D. J.; Evans, M. T. A.; Mitchell, J. R.; Phillips, M. C.; Rees, P. M. J. Polym. Sci., Part C 1971, 34, 167. (29) Williams, A.; Prins, A. Colloids Surf., A 1996, 114, 267. (30) Benjamins, J.; Cagna, A.; Lucassen-Reynders, E. H. Colloids Surf., A 1996, 114, 245. (31) Jones, D. B.; Middelberg, A. P. J. Chem. Eng. Sci. 2002, 57, 1711.

Figure 10. Typical BAM images for various compressed films of the caseins, pH 7, Cb ) 0.005 wt % (A) 1 day old β-casein film after the 90 °C heat treatment, A/A0 ) 0.44. The black arrows indicate examples of the numerous very small dark pits that were just discernible. (B) 6 h old Rs1-casein film after the 90 °C heat treatment, A/A0 ) 0.44. The white arrow indicates the occasional streakiness that was observed. (C) 1 day old Rs1-casein film after the 90 °C heat treatment, after compression to A/A0 ) 0.44 and reexpansion A/A0 ) 1. (D) 6 h old κ-casein film after the 90 °C heat treatment, A/A0 ) 0.44. The black arrows indicate examples of the numerous very faint striations that were observable. (E) 1 day old κ-casein film after the 90 °C heat treatment, A/A0 ) 0.44. (F) 1 day old κ-casein film after the 90 °C heat treatment, after compression to A/A0 ) 0.44 and reexpansion A/A0 ) 1. The black arrows indicate examples of possible fracturing of the films. The scale bar ) 150 µm.

similar to the range of values (ca. 100 mN m-1) measured by Middelberg et al. using their similar technique, but for unheated β-L films. Morphological Characteristics of Adsorbed Films of the Caseins. Finally, we present some results for the disordered caseins to compare with those of the globular β-L. Figure 10 shows selected images taken from adsorbed films of the β-, Rs1-, and κ-casein (Cb ) 0.005 wt %) at pH 7 after the 90 °C heat treatment. Without heat treatment, none of these proteins exhibited any kind of film structuring when subjected to the same compression-expansion cycle as above for β-L. Figure 10A shows an example of an image for the 1 day old β-casein film at the minimum area employed, A/A0 ) 0.44. On close observation, the appearance of numerous very small dark pits is just discernible, but no ridges or fissures were observed. Nothing was observable at all for the 6 h old films. Figure 10B shows the corresponding image for the 6 h old Rs1-casein film. Quite a large amount of structuring is visible in terms of numerous dark pits (that are much larger and more noticeable than with β-casein) and also the occasional streakiness. These types of structure were observable at most values of A/A0. The only other type of structure that was observed with Rs1-casein was the appearance of what might have been the onset of film fracturing after re-expansion of the 1 day old film to A/A0 ) 1. An example

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is shown in Figure 10C. This was not observed for the 6 h old films. Finally, κ-casein revealed a different kind of structuring again. For the 6 h old film, after compression, there appeared to be numerous very faint striations throughout the film, that persisted on subsequent expansion. An example is given in Figure 10D. For the 1 day old films, curious looplike structures were also observed (Figure 10E) and possibly some evidence of fracturing (Figure 10F) on re-expansion of the films after compression. However, it should be mentioned that the behavior with κ-casein was not as reproducible as with the other proteins, in that sometimes the features illustrated were not always present when the experiments were repeated. Origin of the Structural Changes Due to Heating. The origin of the strengthening of the films on heating and the exact nature of the changes in intermolecular interactions under the specific conditions employed awaits further investigation. The maximum temperatures of the heat treatments used were well above the accepted thermal denaturation temperature of β-L in bulk solution. Above this temperature, β-L is well-known to undergo a series of aggregation steps leading to the formation of disulfide crosslinked protein networks, catalyzed by the increased exposure of the one free SH group of β-L due to its unfolding.32 Of course, β-L is already expected to be unfolded to some extent, particularly at the O-W interface, as discussed in the Introduction. The enhanced unfolding and flexibility of β-L at the O-W interface may aid the formation of intermolecular disulfide cross-links. Indeed, Dickinson and Matsumura33 have provided evidence that intermolecular disulfide cross-links can slowly form at the O-W interface even in the absence of heating. Similarly, κ-casein is known to form disulfide cross-links with other proteins on heating. Thus, there is the capacity for disulfide cross-linking to explain the change in properties of the films as a result of the heat treatment. On the other hand, Croguennec et al.34 have found that intermolecular disulfide bonds are not necessary for the formation of adsorbed β-L films that possess high interfacial viscoelasticity. Thus, close packing of molecules and/or the reformation of multiple, cooperative intermolecular hydrogen bonds on cooling may be responsible for the strengthening of the films.22,35 Previously, the sort of BAM instrument that has been used here has been calibrated so that the brightness (gray level) in the images can be used to measure the reflectivity of the interface and therefore provide further information on the thickness and density of adsorbed protein films as a function of compression and time of adsorption.36,37 This was not attempted here for the heated films at the O-W interface for a number of reasons. First, as the films become more inhomogeneous and fragmented, it becomes more difficult to find an average gray level that represents the average film thickness or density. In contrast, nonheated films at the A-W interface (without included particles) are relatively homogeneous, even after compression and expansion, as noted in the Introduction. Second, the reflectivity at the O-W interface is always lower than at the A-W interface, reducing the sensitivity. Third, during the time it takes to perform the heating and cooling cycles, there is a drift in the response of the CCD camera that makes it difficult to relate the intensities measured before heating to those measured after heating. (32) Verheul, M.; Roefs, S. P. F. M.; de Kruif, K. G. J. Agric. Food Chem. 1998, 46, 896-903. (33) Dickinson, E.; Matsumura, Y. Int. J. Biol. Macromol. 1991, 13, 26-30. (34) Croguennec, T.; Renault, A.; Bouhallab, S.; Pezennec, S. J. Colloid Interface Sci. 2006, 302, 32-39. (35) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2007, 12, 232-241. (36) Garofalakis, G.; Murray, B. S. Colloids Surf., B 2001, 21, 3. (37) Rodriguez Patino, J. M.; Carrera Sanchez, C.; Rodriguez Nin˜o, M. R. Food Hydrocolloids 1999, 13, 401.

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However, it would certainly be useful to perform such measurements with a more suitable instrument, such as a scanning ellipsometer.38

Conclusions Taken all together, the BAM images and the analysis of the interfacial tension data lead to the inescapable conclusion that the typical heat processing of emulsions stabilized by β-L leads to the formation of much stronger, more coherent films when the bulk protein concentration is higher. The behavior at the highest Cb value tested (0.5 wt %) is probably more relevant to real emulsions, which only tend to be stabilized at and above this sort of protein concentration. The images obtained at this concentration of β-L show that strengthening of the films can lead to fewer but more pronounced inhomogeneities on compression and expansion. In this respect, it is again relevant to draw attention to the fact that fracturing seems much more prevalent for the strongest A-W films, i.e., at lower pH and longer adsorption times, though these were not heated. This supports the idea that A-W films tend to behave more as close-packed aggregates rather than as coherent, unfolded, and cross-linked films. Such films cannot prevent coalescence on rapid expansion. (It was not possible to carry out heating experiments on adsorbed films at the A-W interface in the same way as at the O-W interface because this caused excessive evaporation of the aqueous phase, which changed the concentration of the subphase.) The experimental observations of wrinkling and fracturing mirror previous Brownian dynamic computer simulations39 of compression and expansion of adsorbed films and reinforce the view that the gellike model of protein films is especially valid for films at the O-W interface, and even more so following heat treatment. In contrast, the caseins seem to exhibit less structuring on adsorption at the O-W interface, either before or after heat processing. The β-casein films showed no structuring at all. The Rs1-casein and κ-casein films exhibited some structuring, possibly even fracturing, but only after aging and heat processing. The film structure differences between the caseins and β-L correlate with the well-known capacity of the globular proteins to form films with more intermolecular cross-links and higher interfacial viscoelasticities than the disordered caseins.19 Nevertheless, even though the film structuring for the caseins overall was not so marked, it is interesting to recall that both Rs1-casein and κ-casein exhibit higher surface shear viscosities at the O-W interface than β-casein.40 One technologically important issue still to be pursued is how adsorbed films formed from mixtures of the caseins and β-L will behave when subjected to similar conditions of heating, compression, and expansion. Acknowledgment. The authors would like to thank BBSRC (BBS/B/0501X) for funding this research and Mr. Phillip V. Nelson for designing and constructing the Langmuir trough apparatus and the adaptations to the Brewster angle microscope. Supporting Information Available: Additional graphics/figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. LA702806T (38) Harke, M.; Teppner, R.; Schulz, O.; Motschmann, H.; Orendi, H. ReV. Sci. Instrum. 1997, 68, 3130. (39) Pugnaloni, L. A.; Ettelaie, R.; Dickinson, E. J. Colloid Interface Sci. 2005, 287, 401. (40) Murray, B. S. In Proteins at Liquid Interfaces; Miller, R., Mo¨bius, D., Eds.; Elsevier Science: Amsterdam, 1998; p 179.