Effect of Surfactant Type on SurfactantâProtein ... - ACS Publications

Biomacromolecules 2004, 5, 984-991

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Effect of Surfactant Type on Surfactant-Protein Interactions at the Air-Water Interface Paul A. Gunning,* Alan R. Mackie, A. Patrick Gunning, Nicola C. Woodward, Peter J. Wilde, and Victor J. Morris Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney NR4 7UA, United Kingdom Received November 26, 2003; Revised Manuscript Received February 25, 2004

The displacement of the proteins (β-lactoglobulin and β-casein) from an air-water interface by the nonionic (Tween 20 and Tween 60) and ionic (sodium dodecyl sulfate, cetyltrimethylammonium bromide, and lysophosphatidylcholine-lauroyl) surfactants has been visualized by atomic force microscopy (AFM). The surface structure has been sampled by the use of Langmuir-Blodgett deposition onto mica substrates to allow imaging in the AFM. In all cases, the displacement process was found to occur through the recently proposed orogenic mechanism (Mackie et al. J. Colloid Interface Sci. 1999, 210, 157-166). In the case of the nonionic surfactants, the displacement involved nucleation and growth of surfactant domains leading to failure of the protein network and subsequent loss of protein into the bulk phase. The surface pressure dependence of the growth of surfactant domains and the failure of the network were found to be the same for both Tween 20 and Tween 60, demonstrating that the breakdown of the protein film was dominated by the mechanical properties of the network. The displacement of protein by ionic surfactants was found to be characterized by nucleation of surfactant domains with little domain growth prior to failure of the network. The size of the domains formed by ionic surfactants was found to be limited by the strong intersurfactant repulsive forces between the charged headgroups. Screening of these charges led to an increase in the size of the domains. The surface pressure at which the network continuity was lost was found to be dependent on the type of surfactant and, in all cases, to occur at higher surface pressures than that required for nonionic surfactants. This has been attributed to surfactant-protein binding that initially strengthens the protein network at low surfactant concentrations. Evidence obtained from surface shear rheology supports this assertion. Introduction The control of foam stability has application across a wide range of industries. Foams are stabilized by surface-active species which largely fall into two main categories, namely, surfactants and proteins. These two species confer stability on foam films via different and incompatible mechanisms. Proteins form a viscoelastic network, sometimes referred to as a two-dimensional gel,1 which relies upon the strength of the intermolecular interactions to maintain its coherence. In contrast to proteins, surfactants rely on a high degree of surface mobility to counter deformation via the Marangoni effect. Over the past decade, improvements in various techniques have made interfacial films more amenable to detailed analysis. The application of various imaging techniques has led to an improved understanding of the structures formed by interfacial films.2-4 In particular, the combined use of Langmuir-Blodgett (LB) deposition and atomic force microscopy (AFM) techniques has revealed the detailed mechanism by which surfactants competitively displace proteins from interfaces. This has led to the development of a new “orogenic” displacement model.5-9 The use of LB methods * Author to whom correspondence should be addressed. Tel.: 44 1603 255261. Fax: 44 1603 507723. E-mail: [email protected].

is possible because the proteins assemble into an elastic network at the interface, providing sufficient mechanical resistance to deformation during the transfer process. Protein molecules adhere to the mica substrate immediately upon transfer, providing even further mechanical resistance to deformation of the L-B protein monolayer during and after transfer. The orogenic model of protein displacement describes the way in which a protein film is displaced from an interface by a surfactant. The model has been established for displacement of proteins by nonionic surfactants and works in the following manner. Heterogeneity in the protein film, due to packing limitations, initially allows the added surfactant to adsorb into localized defects in the protein film. For nonionic surfactants, the subsequent expansion of these surfactant domains compresses the protein network, which initially increases in density without increasing in thickness.5 Once a certain critical density is reached, the thickness of the protein layer increases such that the total protein film volume is maintained as the surfactant domains expand. At sufficiently high surface pressures (Π), the continuous protein network fails, releasing protein and leaving the interface surfactant-continuous with islands of protein, which can subsequently desorb from the interface.5,7 The density of the nucleation sites seems to be linked to the type of displacing

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Surfactant-Protein Interactions

surfactant,5,8 with preliminary studies suggesting that charged surfactants produce a high level of nucleation sites with only a small degree of domain expansion between nucleation and percolation (protein network failure, resulting in a surfactantcontinuous interface with islands of protein). A crucial aspect of the development of this model has been the establishment that the AFM studies on LB films represent the morphological changes occurring at the liquid interface and in real foams. This process has been observed directly through in situ dynamic AFM studies of protein displacement from graphite surfaces by surfactant solutions.6 The later stages of domain growth have been observed by Brewster angle microscopy directly at the air-water interface10 and in microscopic studies of liquid lamellae, where coexisting lipid and protein domains can be detected11 with morphological differences which match those imaged by AFM. The thickening of the protein domains prior to release of the protein into the bulk has also been seen in the studies on liquid lamellae.12 This model has highlighted the importance of breaking the protein network to allow the release of protein into the bulk phase. The surface pressure induced by the adsorbing surfactant within the growing domain is resisted by the elasticity of the protein film, which resists the expansion of the surfactant domains. Several studies have looked at the interfacial rheology of both protein films13-15 and mixed protein-surfactant films.15-17 The studies of large deformation interfacial rheology on protein films18-20 seem most likely to shed further light on the parameters that control the domain expansion. In particular, the yield stresses measured by Bos et al.20 highlight the differences between the protein films formed by β-casein and β-lactoglobulin. Although protein-surfactant interactions in bulk solution have been studied,21-23 it is still unclear what the precise role of these interactions is in protein displacement from interfaces. Therefore, this work aims to investigate, by studying different types of ionic surfactants, the effect of altered ionic interactions on the structure and morphology of mixed protein-surfactant interfacial films. Materials and Methods The milk proteins used in the present study were β-lactoglobulin (L-0130, lot 91H7005) and β-casein (C-6905, lot 12H9550), both obtained from Sigma Chemicals (Poole, U.K.). Protein solutions were initially prepared at a concentration of 2 mg mL-1 in water. The water used for this study was obtained from an Elga Elgastat UHQ water purification system and had a surface tension γ0 ) 72.6 mN m-1 at 20 °C. Sodium dodecyl sulfate (SDS) was obtained as a 10% solution (L4522, lot 97H8505) from Sigma Chemicals (Poole, U.K.). Lyso-phosphatidylcholine-lauroyl (LPC-L) was obtained as a powder (lot 80F83551) from Sigma Chemicals (Poole, U.K.). Cetyltrimethylammonium bromide (CTAB) was obtained from Fluka (Buchs, Switzerland). Tween 20 (polyoxyethylene sorbitan monolaurate) was obtained as a 10% solution (Surfact-Amps 20) from Pierce (Rockford, IL). Tween 60 (polyoxyethylene sorbitan monostearate) solutions were obtained from Quest International

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(Kent, U.K.). All other chemicals used were of analytical grade. Surface tension measurements were made using a wetted ground glass Willhelmy plate and a Langmuir trough. The polytetrafluoroethylene (PTFE) trough was built in-house with dimensions of 255 × 112 × 16 mm and a volume of 450 mL and was equipped with a mobile and a fixed PTFE barrier. The proteins β-lactoglobulin (70 µg) and β-casein (36 µg) were spread at the air-water interface as aqueous 2 mg/mL solutions. The spread protein films were allowed to equilibrate for 30 min. It has been shown5 from surface tension and surface rheological measurements that after this period of time the proteins have formed elastic networks and achieved a quasi-equilibrium state. Appropriate concentrations of surfactant (Tweens 0-1 µM, CTAB 0-220 µM, LPC-L 0-200 µM, SDS 0-1.1 mM) were added to the subphase with care being taken to prevent the surfactants from spreading at the interface by adding the surfactant to the trough behind the fixed barrier. All additions of surfactant and LB dips were made within 4 h of the protein film reaching quasi-equilibrium. For the LPC-L surfactant, additional studies were made to investigate possible effects of the aging of the films on protein displacement. This involved collecting data on LB dips made over longer periods (up to 24 h) of time and comparing the results obtained at different times. LB dips were performed at a dipping speed of 8.4 mm min-1 on a hydrophilic substrate of freshly cleaved mica. The surface pressure was closely monitored during each dip to confirm the efficient transfer of the adsorbed surface layer onto the mica. Transfer to the mica occurred only on the upstroke as the mica substrate was withdrawn from the interface. LB monolayers were rinsed in water, and LB monolayers containing LPC-L were further rinsed in 50:50 chloroform/methanol (both BDH chemicals, Poole U.K.). Initially, some images of LB dips of monolayers containing LPC-L were obtained prior to rinsing in the chloroform/ methanol solution. The images obtained were the same as those obtained from LB dips which had been rinsed in solvent, but the image contrast was lower and the image noise was higher. The rinsing steps were carried out to remove surfactant to aid visualization of the remaining protein film in the AFM. The excess solvent was evaporated by allowing the sample to stand in air for about 10 min. AFM images were produced using an East Coast Scientific atomic force microscope (ECS, Cambridge, U.K.). The probes used were Nanoprobe (Digital Instruments, Santa Barbara, CA) cantilevers with a quoted force constant of 0.38 N m-1. Samples were imaged in direct current contact mode under redistilled butanol. Images were subsequently analyzed using Image Pro 4.5 image analysis software (Media Cybernetics Corp., U.S.A.). Protein areas were calculated using a thresholding technique that determined the number of pixels above a threshold level as a percentage of the overall number of pixels in the image. This yields the percentage surface area covered by protein. Image analysis data were averaged over at least 16 images for each surface pressure, with standard deviations never greater than 5%. The AFM images use 256 gray levels to represent the heights between 0 and the maximum thickness. The thickness of the

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Figure 1. AFM images of LB films of spread β-lactoglobulin displaced by (a) nonionic Tween 20, Π ) 25.0 mN/m, 4 µm × 4 µm, and (b) nonionic Tween 60, Π ) 25.7 mN/m, 4 µm × 4 µm.

Figure 2. AFM images of LB films of spread β-lactoglobulin displaced by (a) anionic SDS, Π ) 36.0 mN/m, 1 µm × 1 µm, (b) cationic CTAB, Π ) 22.8 mN/m, 1 µm × 1 µm, and (c) zwitterionic LPC-L, Π ) 22.0 mN/m, 1 µm × 1 µm.

protein layers are determined by a histogram of gray levels within an image. Measured histograms are bimodal, with the lower peak corresponding to the substrate and the upper peak representing the adsorbed protein. Mean protein layer thicknesses were calculated from the difference in gray levels between the two peaks of the histogram. Surface shear rheological measurements of 1 µM solutions of β-lactoglobulin and of coadsorbed mixtures of the protein with either LPC-L or Tween 20 surfactants were made in 10 mM sodium phosphate buffer at pH 7. Data were obtained using a Bohlin CS10 controlled stress rheometer (Bohlin Instruments, Cirencester, U.K.) equipped with an IFG10 interfacial geometry bob at 25 °C. Results The effect of surfactant type (nonionic or ionic) on protein displacement was visualized by AFM imaging of mixed protein-surfactant structures formed at an air-water interface. The samples were produced using LB deposition on mica substrates, and representative results are shown in Figures 1 and 2. Brighter regions in each AFM image signify regions of greater height above the mica substrate and indicate the presence of protein. Dark areas correspond to regions originally occupied by surfactant. As mentioned in Materials and Methods, the LB dips were rinsed after recovery from the trough. This removed surfactant by

dissolving it out of the LB films, thus maximizing the height differences to optimize the image contrast for examination in AFM. Parts a and b of Figure 1 show AFM images of β-lactoglobulin films being displaced by the nonionic surfactants, Tween 20 and Tween 60, respectively. The scan size is the same for both images, and large dark areas representing surfactant-rich domains are clearly evident. Parts a-c of Figure 2 show AFM images of β-lactoglobulin films being displaced by the ionic surfactants SDS, CTAB, and LPC-L, respectively. The scan size is the same for all three images. These images show no evidence of the large surfactant domains evident in images obtained from the nonionic surfactant experiments shown in Figure 1. The fact that the scan size is four times greater for Figure 1 than for Figure 2 emphasizes the differences observed. The important conclusion is that ionic surfactants displace the protein by means of limited growth of many small domains as compared to the nucleation and growth of larger surfactant domains which is observed for displacement by the nonionic Tweens. To quantify the differences in behavior observed between nonionic surfactant and ionic surfactant displacement, image analysis data of the AFM results was undertaken and is presented in Figures 3 and 4. A graph of the proportion of the surface area occupied by β-lactoglobulin as a function of the surface pressure in the presence of Tween 20 or Tween 60 is given in Figure 3. Tween 20 and Tween 60 have the


Figure 3. Protein surface area coverage plotted as a function of the surface pressure for spread β-lactoglobulin films displaced by nonionic (2) Tween 20 and (9) Tween 60.

Figure 4. Protein surface area coverage plotted as a function of the surface pressure for spread β-lactoglobulin films displaced by ionic (b) SDS, (2) CTAB, and (9) LPC-L.

same headgroups but have different side chains (C12 for Tween 20, mostly C18 and some C16 for Tween 60). The displacement curves show very similar protein displacement behavior for both nonionic surfactants. For comparison, Figure 4 is a graph of the β-lactoglobulin surface area coverage as a function of the surface pressure in the presence of the ionic surfactants SDS, CTAB, and LPC-L. The surface pressure at which onset and completion of displacement occurs by each surfactant differs. For LPC-L, some studies were undertaken to assess the effects of aging on the displacement curves. Close to the percolation threshold (transition from a protein-continuous to a surfactant-continuous interface), any effects due to aging of the films were found to be negligible. At lower surface pressures, small differences in the displacement curves could be detected, but the changes were insignificant compared to the differences in the displacement curves observed for different surfactants. Protein displacement occurred over a wider range of surface pressures compared to the nonionic surfactants. Complete displacement occurred at higher surface pressures for the ionic surfactants, and the anionic surfactant SDS displaced protein at much higher surface pressures than any of the other surfactants studied. Further quantitative analysis of AFM data (not shown) confirmed that the average number of surfactantrich domains, for a given surface area and pressure, was 2.5-3 times greater for the ionic surfactants than for the


Figure 5. Protein surface area coverage (solid line) and height (dashed line) plotted as a function of the surface pressure for spread β-lactoglobulin films displaced by ionic (b) SDS and (2) CTAB.

nonionic surfactants. The thickness of adsorbed protein LB films as determined by quantitative measurement of AFM images is given in Figure 5. The surface area coverage and film thickness of β-lactoglobulin films are plotted as a function of the surface pressure in the presence of SDS or CTAB. The graph reveals smaller increases in the film thickness as the protein was displaced compared to the increases observed previously with Tween 20.5 In addition, the thicknesses observed in the presence of CTAB are lower. This is possibly due to the effect of positively charged CTAB on tip-substrate interactions. Similar effects of charged molecules on the heights measured by AFM tips have been reported previously.24 It has been shown by AFM25 that the surface rheology of protein films can affect the morphology of the surfactant domains formed during displacement by nonionic surfactants. β-Casein has a more random-coil molecular configuration than that of the globular β-lactoglobulin. The less elastic β-casein films formed at the air-water interface are less able to resist mechanical deformation than films produced by β-lactoglobulin.5 Figure 6a,b shows AFM images of β-casein films which have been partially disrupted by adsorption of Tween 20 (nonionic) and CTAB (cationic) surfactants at an air-water interface. It is apparent from these images that the ionic surfactant (CTAB) displaces the β-casein to form many small surfactant-rich domains, producing a very similar appearance to that observed when the globular protein, β-lactoglobulin, is displaced by the same surfactant (Figure 2). By contrast, large surfactant domains are produced by the action of nonionic Tween 20 on β-casein films (illustrated in Figure 6a), although as reported previously25 the domains are more circular than the domains observed in a β-lactoglobulin film (Figure 1). These studies demonstrate that it is not simply the elasticity of the protein film that inhibits growth of surfactant domains in the case of the ionic surfactants. To investigate the effect of charge screening on domain size, 0.2 M sodium phosphate buffer at pH 7 was used to screen electrostatic interactions. The effect of charge screening on the displacement of a β-lactoglobulin film by CTAB is illustrated by the AFM image shown in Figure 7. The scan size is 1µm × 1µm allowing direct comparison with Figure 2. Fewer, larger surfactant domains can be seen in the film

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Figure 6. AFM images of LB films of spread β-casein displaced by (a) nonionic Tween 20, Π ) 19.2 mN/m, 2 µm × 2 µm, and (b) ionic CTAB, Π ) 14.5 mN/m, 1 µm × 1 µm.

Figure 9. Surface elastic shear modulus of β-lactoglobulin/LPC-L coadsorbed films as a function of the LPC-L concentration. Figure 7. AFM image of an LB film of spread β-lactoglobulin displaced by cationic CTAB in 0.2 M sodium phosphate buffer at pH 7, Π ) 24 mN/m, 1 µm × 1 µm.

Figure 8. Surface shear rheology of β-lactoglobulin/LPC-L coadsorbed films as a function of time at different LPC-L concentrations: (2) 0.0 µM, (b) 3.1 µM, ([) 25.0 µM, and (9) 100.0 µM.

as compared to that observed for β-lactoglobulin displaced by CTAB in water (Figure 2). Surface rheological data for coadsorbed β-lactoglobulinLPC-L films at an air-water interface with increasing concentrations of the zwitterionic surfactant LPC-L are presented in Figure 8. For 1 µM β-lactoglobulin, the elastic shear modulus of the air-water interface is plotted as a function of time for each concentration of LPC-L. The

β-lactoglobulin films show an increase in the storage modulus, G′, with time, reaching a plateau value of about 0.015 Pa after about 10 min. For reasons of clarity, not all of the concentrations of LPC-L measured are reproduced in Figure 8. A complete data set is given in Figure 9, which shows the plateau values of the elastic shear modulus achieved by 1 µM β-lactoglobulin as a function of each added LPC-L concentration. It can be seen from Figure 9 that at low concentrations of added LPC-L a higher value of G′ was reached than for the pure protein film. The value of G′ then steadily decreased at higher concentrations of added surfactant, until at 100 µM LPC-L the elastic modulus was difficult to measure. Surface rheological data are presented in Figure 10 for 1 µM β-lactoglobulin with added nonionic surfactant Tween 20 for comparison with the zwitterionic LPC-L data. The plateau value of the elastic shear modulus is plotted as a function of Tween 20 concentration. It is apparent from Figure 10 that the addition of Tween 20 always resulted in a decrease in the value of G′, even at the lowest concentrations of surfactant studied, in contrast to the LPC-L experiments. Discussion It has been shown that the orogenic model of protein displacement is generic and applies for both spread and coadsorbed protein films, for air-water, oil-water, and solid-water interfaces, and for water-soluble and oil-soluble


Figure 10. Surface elastic shear modulus of β-lactoglobulin/Tween 20 coadsorbed films as a function of the Tween 20 concentration.

nonionic surfactants.5-9,25-29 The generic nature of the model is due to the fact that the proteins form networks that must be broken by the surfactant to allow displacement of protein by the surfactants. In the present studies, we have begun to investigate the nature of surfactant structure on the details of the displacement process. In the case of the Tweens it is possible to investigate the effects of changes in the length of the surfactant’s hydrophobic carbon chain (C12 for Tween 20, mostly C18 and some C16 for Tween 60) on the displacement process. Figure 1a,b illustrates the similarity of the displacement of β-lactoglobulin by Tween 20 and Tween 60 and demonstrates that the displacement of spread β-lactoglobulin films with Tween 60 occurs via the wellestablished orogenic mechanism.5 At similar surface pressures, the domain size and shape for Tween 20 and Tween 60 are very similar. Although higher bulk concentrations of Tween 60 are required to displace the protein, the data shown in Figure 3 illustrate that the growth of the domains and the percolation threshold of the network occur at the same surface pressures for the two surfactants. The domain growth and loss of continuity of the network depends on the mechanical properties of the network and are unaffected by the changes in the chain length of the surfactant. The irregular shape of the domain boundaries suggests that the surfactant domains cannot expand uniformly. This implies that the protein network is not uniform and that some regions yield more easily than others. The fact that failure of the network occurs at the same surface pressure for both Tween surfactants suggests that any binding between the protein and the surfactant does not alter the mechanical properties of the protein network. Figure 2 illustrates the salient features of the displacement of spread β-lactoglobulin films by the anionic surfactant SDS (Figure 2a), the cationic surfactant CTAB (Figure 2b), and the zwitterionic surfactant LPC-L (Figure 2c). These data confirm earlier, preliminary conclusions8 drawn about the displacement of protein from air-water interfaces by SDS. In all cases (Figure 2), the displacement is characterized by the presence of a large number (the average number of domains per unit area of the interface was 2.5-3 times higher than that observed for nonionic surfactants) of very small domains distributed throughout the protein network. The domain size, shape, and distribution appear to be largely independent of the polarity of the charged headgroup


of the surfactant. Displacement is characterized by nucleation of domains but with little subsequent domain growth. This is in contrast to the behavior observed for the nonionic surfactants and is illustrated in Figure 1. The displacement curves are different for each ionic surfactant (Figure 4), are also different to those seen for the nonionic surfactants (Figure 3), and cover a larger range of surface pressures, with an ultimately higher surface pressure necessary for complete failure of the protein network than for the nonionic Tweens. This suggests that the binding of the ionic surfactants with the proteins has modified the mechanical properties of the network. The changes in height observed for the ionic surfactants SDS (Figure 5a) and CTAB (Figure 5b) are relatively small, suggesting that the surfactants mainly colonize defects in the protein network and that the small subsequent expansion of these domains arises principally because of the compaction of the protein network through the refolding of individual protein molecules. A characteristic feature of orogenic displacement with ionic surfactants is the presence of a high density of small domains. It is interesting to consider whether the dominant factor controlling domain size is the strength of the protein network (which can be influenced by protein-surfactant binding) or is dominated by the surfactant-surfactant interactions. It is known from previous studies5 that β-casein is easier to displace from air-water interfaces than β-lactoglobulin. Figure 6a shows the displacement of spread β-casein films from an air-water interface with Tween 20. The displacement involves nucleation and growth of surfactant domains, but the domains are now circular in shape. This reflects the weaker network structure and the uniform growth of the Tween 20 domains. Figure 6b shows the typical displacement of a spread β-casein film at an air-water interface with the ionic surfactant CTAB. Even for the weaker β-casein network, the ionic surfactant still forms high densities of small-sized domains, similar to that seen with the β-lactoglobulin networks (Figure 2a). This would suggest that, in the case of ionic surfactants, it is the surfactantsurfactant interactions that determine the size and shape of the domains and not the strength of the protein film. A strong repulsive interaction between charged surfactants would favor dispersal of the surfactants across the interface rather than concentration within a few large domains. These strong repulsive interactions can be reduced by screening the charges by raising the ionic strength of the subphase in the Langmuir trough. Figure 7 shows displacement of a spread β-lactoglobulin film from an air-water interface by CTAB in the presence of 0.2 M sodium phosphate buffer at pH 7. It is clear from the AFM image that the addition of buffer has led to a substantial increase in the size of the surfactant domains. These experiments suggest that the most likely explanation for the small size of the domains is that the charge-charge repulsions between ionic surfactants favor uniform dispersal of the surfactant across the interface. Given that the presence of the protein network places some limitation on where the ionic surfactant can adsorb at the air-water interface, this dispersal is achieved by the formation of a large number of small domains.

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The interpretation of the displacement data for ionic surfactants suggests that these surfactants may have two distinct effects. The fact that the protein networks fail at higher surface pressures suggests that binding between the surfactant and the protein can enhance the strength of the network. However, at higher surfactant concentrations an orogenic effect ultimately weakens the network, eventually resulting in its failure. There are reports that ionic surfactants can form complexes with proteins16,30 and also that such binding may affect the rheology of the protein network.30 Interfacial rheology provides a method for probing the structure of the protein network. Surface shear measurements will probe the low strain distortion of the protein network and should be sensitive to the local degree of association of the proteins. The surface rheological experiments carried out here provided evidence for synergistic surfactant-protein interactions in the case of the zwitterionic surfactant LPCL. At low concentrations of added surfactant, an increase in G′ was observed, and then at higher LPC-L concentrations, a progressive decrease in G′ was observed (Figures 8 and 9). This is consistent with the observations on the displacement process obtained from the AFM data. It is proposed that at low LPC-L concentrations binding between the LPC-L and the protein strengthens the protein network leading to the observed increase in G′. At higher LPC-L concentrations the LPC-L starts to weaken the network by the orogenic mechanism and G′ starts to decrease. The presence of the peak in G′ seen in Figure 9 is the result of the competition between these two conflicting effects of the surfactant LPCL. At LPC-L concentrations above 100 µM there is no measurable value of G′, suggesting that orogenic displacement is the main factor determining the mechanical properties of the protein network at higher concentrations. The AFM displacement studies discussed earlier were carried out at LPC-L concentrations at, or above, 100 µM. Similar, interfacial shear rheological measurements were made for coadsorbed β-lactoglobulin-Tween 20 mixtures at air-water interfaces. In the case of added Tween 20 there was no evidence of an enhancement of the G′ values on addition of surfactant (Figure 10). Although there are reports31 that Tween 20 binds to β-lactoglobulin, the present studies suggest that any such binding does not enhance the strength of the protein network. Conclusions The displacement of β-lactoglobulin and β-casein from air-water interfaces by both nonionic and ionic surfactants has confirmed the generic nature of the orogenic model of protein displacement.14 The chain length of the nonionic surfactants Tween 20 and Tween 60 alter the efficacy of displacement. However, the displacement and breakdown of the protein network, as a function of surface pressure, is the same for both surfactants. The decrease in interfacial shear rheology of coadsorbed protein-surfactant films can be attributed to the progressive disruption of the protein network by the surfactant. Protein displacement by ionic surfactants is dominated by domain nucleation rather than domain growth. It has been

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suggested that this is due to electrostatic repulsions between surfactant molecules, and screening of these interactions has been shown to increase the domain size. For ionic surfactants, the surface pressure at which the protein network fails is dependent on the nature of the surfactant and, in all cases, is greater than the value at which failure occurs for the nonionic surfactants. These data suggest that interactions between the surfactants and protein alter the mechanical properties of the protein network. This conclusion is supported by interfacial shear rheology of coadsorbed mixtures of β-lactoglobulin-LPC-L films, which show an enhancement of film strength at low LPC-L concentrations. Acknowledgment. The research described in this article was supported by funding from the BBSRC strategic grant to the Institute and by funding from the BBSRC responsive mode grant D14067. References and Notes (1) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2002, 7, 426. (2) Patino, J. M. R.; Sanchez, C. C.; Nino, M. R. R. J. Agric. Food Chem. 1999, 47, 4998. (3) Sengupta, T.; Damodaran, S. J. Agric. Food Chem. 2001, 49, 3087. (4) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Morris, V. J. Biopolymers 1998, 46, 245. (5) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (6) Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Langmuir 1999, 15, 4636. (7) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. (8) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 8176. (9) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2001, 17, 6593. (10) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Patino, J. M. R. Biomacromolecules 2001, 2, 1001. (11) Clark, D. C.; Coke, M.; Wilde, P. J.; Wilson, D. R. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; Royal Society Special Publication No. 82; Royal Society of Chemistry: Cambridge, 1991; p 272. (12) Coke, M.; Wilde, P. J.; Russell, E. J.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489. (13) Williams, A.; Prins, A. Colloids Surf., A 1996, 114, 267. (14) Dickinson, E. Colloids Surf., B 1999, 15, 161. (15) Prins, A.; Van Kalsbeek, H. K. A. I. Colloids Surf., A 2001, 186, 55. (16) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Kragel, J.; Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko, O. V. AdV. Colloid Interface Sci. 2000, 86, 39. (17) Bos, M. A.; Van Vliet, T. AdV. Colloid Interface Sci. 2001, 91, 437. (18) Martin, A. H.; Bos, M. A.; van Vliet, T. Food Hydrocolloids 2002, 16, 63. (19) Martin, A. H.; Grolle, K.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. J. Colloid Interface Sci. 2002, 254, 175. (20) Bos, M. A.; Grolle, K.; Kloek, W.; van Vliet, T. Langmuir 2003, 19, 2181. (21) Sarker, D. P.; Wilde, P. J.; Clark, D. C. Colloids Surf., B 1995, 3, 349. (22) Waninge, R.; Paulsson, M.; Nylander, T.; Ninham, B.; Sellers, P. Int. Dairy J. 1998, 8, 141. (23) Kelly, D.; McClements, D. J. Food Hydrocolloids 2003, 17, 73. (24) Rossell, J. P.; Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Ultramicroscopy 2003, 96, 37. (25) Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Surf. Interface Anal. 1999, 27, 433. (26) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. In Food Colloids: Fundamentals of Formulation; Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: London, 2001; p 13.

Surfactant-Protein Interactions (27) Gunning, A. P.; Mackie, A. R.; Kirby, A. R.; Morris, V. J. Langmuir 2001, 17, 2013. (28) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Patino, J. R. Biomacromolecules 2001, 2, 1001. (29) Clark, D. C.; Mackie, A. R.; Wilde, P. J.; Wilson, D. R. Faraday Discuss. 1994, 98, 253.

Biomacromolecules, Vol. 5, No. 3, 2004 991 (30) Miller, R.; Fainerman, V. B.; Kragel, J.; Loglio, G. Curr. Opin. Colloid Interface Sci. 1997, 2, 578. (31) Coke, M.; Wilde, P. J.; Russel, E. J.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489.

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Effect of Surfactant Type on SurfactantâProtein ... - ACS Publications

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