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The adsorption of mixed β-casein/β-lactoglobulin films to the air/water interface and the subsequent
displacement by the nonionic surfactant Tween 20 was studied. A combination of fluorescent labeling of
the protein and Langmuir-Blodgett deposition was used to study the mixed protein layer. The adsorption
was also monitored using two surface rheological techniques, shear and dilatation. Fluorescent labeling
was able to show that to within the limits of optical resolution the two proteins were well mixed at the
interface. We also show that the film remained well mixed after 3 days. Surface rheological data from the
two techniques used was self-consistent and showed that during the initial stages of development, the films
were dominated by the adsorption of the β-casein. Both fluorescence microscopy and atomic force microscopy
were used to follow the displacement of the mixed film by surfactant. Results on films displaced by the
nonionic surfactant Tween 20 showed that β-casein was preferentially displaced from the mixed film before
β-lactoglobulin.
(15) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Morris, V. J. (18) Garofalakis, G.; Murray, B. S. Colloids Surf., B 1999, 12, 231.
Biopolymers 1998, 46, 245. (19) Mitchell, J. R. Dev. Food Proteins 1986, 4, 291.
(16) Kokelaar, J. J.; Prins, A.; De Gee, M. J. Colloid Interface Sci. (20) Williams, A.; Prins, A. Colloids Surf., A 1996, 4, 267.
1991, 46, 507. (21) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M.
(17) Sherrif, M.; Warburton, B. Polymer 1974, 15, 253. J. Chem. Soc., Faraday Trans 1995, 91, 2847.
Mixed Protein Displacement Langmuir, Vol. 17, No. 21, 2001 6595
surface pressure of the film was still steadily increasing Figure 3. A series of fluorescence images following the
after 105 min (data not shown). The mixed system showed displacement of a mixed, dual stained protein film. β-Casein
features of both proteins. The peak from the partial was labeled green and lactoglobulin was labeled red. All images
collapse of the β-casein is present although shifted to are 63 µm by 48 µm. Image a shows a mixed protein film
transferred at a surface pressure of 21.5 mN/m. Also shown is
slightly longer times. The much broader peak from the mixed film at various stages of displacement transferred
β-lactoglobulin was also visible but reduced by nearly a at 27 (b), 28.5 (c), and 29.5 mN/m (d).
factor of 4 to 22 mN/m. Subsequently the dilatational
elasticity climbed very slowly to 25.4 mN/m after nearly was the predominant protein at the surface but a small
4 h. The surface pressure of the mixed film took about 30 amount of β-lactoglobulin was present. Despite this
min to reach equilibrium (data not shown). The data presence, the mechanical strength of the adsorbed film
suggest that both proteins were observed to adsorb, (as measured by the surface shear elasticity) was almost
resulting in a mixed β-lactoglobulin/β-casein interface. totally dominated by the β-casein component.
However, the surface elasticity of the mixed system Therefore both surface rheological methods suggest that
suggests that β-casein was the dominant protein at this at these equal concentrations β-casein adsorbed more
stage in the adsorption. This agrees with previous work rapidly than β-lactoglobulin and dominated the surface
showing the more rapid adsorption of β-casein. If, rather rheological properties. To investigate further, fluorescence
simplistically, one assumes the surface dilatational elastic microscopy was used to see if there was a surface
modulus of the separate components to be merely additive, concentration excess of β-casein and to observe any phase
then the curve for an interface containing 85% casein fits separation which may occur.
the data reasonably well, especially in the latter stages. Phase Separation. The aim here was to determine
Further investigation will be required in order to fully the spatial distribution of the two proteins at the interface
ascertain the true nature of the rheological behavior of and thus to ascertain if the proteins were evenly mixed
the mixed interfacial film. Especially in terms of the degree or if phase separation occurred and how the protein
to which such immobile systems may become phase distribution could help us understand the surface rheo-
separated. logical behavior. Previous work on protein/surfactant
The data from the measurement of surface shear mixed interfaces has shown, using AFM, how the sur-
elasticity are shown in Figure 2. Again, the figure shows factant phase separates into domains to displace the
data for the individual proteins and the mixed system. protein. AFM was ideally suited to these investigations
The film formed by β-casein was so weak as to be almost because it could easily distinguish between the separate
immeasurable while the film formed by β-lactoglobulin protein and surfactant domains. AFM cannot however
gave quite high values. The values for β-lactoglobulin rose distinguish between two different proteins. To determine
to a peak of 4.9 mN/m after 50 min and then decreased the spatial distribution of the two proteins in the mixed
in elasticity to about 2 mN/m after 3 h. The mixed film film, fluorescence microscopy was employed.
shows a gradual slow rise to 1 mN/m after 3 h. The results In these experiments, the two proteins of interest were
show a similar trend to the surface dilatational elasticity, labeled with fluorescent probes, β-casein with FITC and
in that β-casein was clearly the dominant protein at these β-lactoglobulin with rhodamine 6G. In both cases the
concentrations. However, the surface shear elasticity extent of labeling was approximately 1:1 protein to dye.
would suggest that there was almost no adsorbed β-lac- The total protein concentration in the Langmuir trough
toglobulin. The difference between the results from the was 0.5 µM, and adsorption was allowed to continue for
surface shear and the surface dilatational rheology can 3 days. Periodically, sections of the interface were
be put down to the different methodologies. Surface transferred onto glass coverslips by the Langmuir-
dilatational rheology measures the changes in surface Blodgett method. Figure 3a is from an interface after
pressure during compression and expansion of the inter- adsorption for 2 days (surface pressure ) 21.5 mN/m) and
face whereas surface shear rheology measures directly shows a rather homogeneous deposition of protein. The
the mechanical properties of the interface under shear. dominant protein was the FITC-labeled β-casein. There
Both methods are sensitive to protein structure and the was a low level of very localized heterogeneity observed
number and strength of intermolecular interactions. in all the images throughout the duration of the experi-
However, surface shear tends to be more sensitive to ments. To check whether the heterogeneity seen in this
protein interactions and surface dilatation more sensitive image was representative of a small amount of phase
to adsorbed structure and composition. Hence, the surface separation of the proteins on the microscopic scale, a
dilatation reveals that under these conditions β-casein similar experiment was performed using only the fluo-
6596 Langmuir, Vol. 17, No. 21, 2001 Mackie et al.
From the displacement studies we have seen that the The use of different surface rheological techniques
β-casein is displaced from the interface in preference to together with imaging methods allows a more complete
the β-lactoglobulin. In the final stages of displacement, understanding of the influence of protein composition and
the shapes of the holes in the film resemble those in a interactions on the mechanical properties of interfacial
β-lactoglobulin film and the surface pressure required to films formed by complex mixtures of food proteins.
reach such a position correlates well with those required
by a pure β-lactoglobulin film. All of the information
combined leads to a coherent picture of the mixed protein Acknowledgment. This work was funded by the
film. The early stages of formation were dominated by the BBSRC as part of the core grant to the institute.
highly flexible β-casein, but the compressed film was
dominated by stronger interactions of the β-lactoglobulin
allowing the β-casein to be removed from the interface. LA010687G