Langmuir 2001, 17, 6593-6598 6593

Orogenic Displacement in Mixed β-Lactoglobulin/β-Casein

Films at the Air/Water Interface
Alan R. Mackie,* A. Patrick Gunning, Michael J. Ridout, Peter J. Wilde, and
Victor J. Morris
Department of Materials Science, Institute of Food Research, Norwich Research Park,
Colney NR4 7UA, U.K.

Received May 8, 2001. In Final Form: July 17, 2001

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.

Introduction higher surface pressure at the oil/water interface. Thus,

Proteins are widely used in the food industry as
emulsifiers and foaming agents. Consequently there has
been much interest in understanding the way in which
protein structure and interfacial function are related.1-4
Furthermore, because of the complex nature of most
food systems, there has been substantial interest in
understanding the interactions between proteins and
surfactants5-7 and between different proteins.8,9 This is
of importance because proteins and emulsifiers stabilize
interfacial films by different and conflicting mechanisms.
In particular, two groups of food proteins have been
studied, caseinates and whey proteins. The predominant
whey protein is β-lactoglobulin while a major component
of caseinate is β-casein. These two proteins show consid-
erable differences in both structure and behavior. Some
time ago it was observed that β-casein could displace Rs1-
casein from an oil/water interface but that β-lactoglobulin
could not displace β-casein from such an interface.
However, work done by others on β-casein10,11 suggests
that it is much more difficult to displace from the oil/
water interface than from the air/water interface. Dis-
placement of β-casein required approximately 10 mN/m
* To whom correspondence should be addressed. Tel: 44 1603
255261. Fax: 44 1603 507723. E-mail: alan.mackie@bbsrc.ac.uk.
(1) Dickinson, E. Colloid Surf., B 1999, 15, 161.
(2) Mackie, A. R.; Husband, F. A.; Holt, C.; et al. Int. J. Food Sci.
Technol. 1999, 34, 509.
(3) Izmailova, V. N.; Yampolskaya, G. P. Adv. Colloid Interface Sci.
2000, 88, 99.
(4) McGuire, J.; Bower, C. K.; Bothwell, M. K. Aust. J. Dairy Technol.
2000, 55, 65.
(5) Fruhner, H.; Wantke, K. D.; Lunkenheimer, K. Colloid Surf., A
2000, 162, 193.
(6) Nino, M. R. R.; Wilde, P. J.; Clark, D. C.; Patino, J. M. R. Langmuir
1998, 14, 2160.
(7) Nasir, A.; McGuire, J. Food Hydrocolloids 1998, 12, 95.
(8) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176.
(9) Husband, F. A.; Wilde, P. J.; Mackie, A. R.; Garrood, M. J. J.
Colloid Interface Sci. 1997, 195, 77.
(10) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid
Interface Sci. 1999, 210, 157.
(11) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir
2000, 16, 2242.
it is not clear that the same behavior would be observed
at the air/water interface.
Recently we have used atomic force microscopy (AFM)
to look at the way in which proteins are displaced from
an interface by surfactants. This has led to the develop-
ment of an “orogenic” displacement model.10-12 This model
describes a mechanism that works in the following way:
Heterogeneity in the protein film allows the displacing
surfactant to adsorb into localized defects, and these
nucleated sites then grow. The expansion of the pools of
surfactant compresses the protein network, which initially
increases in density without increasing in thickness. Once
a certain critical density is reached, the protein layer
thickness increases such that the protein film volume is
maintained as the surfactant domains expand. At suf-
ficiently high surface pressures the network fails, releasing
protein, which then desorbs from the interface. More
recently this model has been confirmed in situ at the air/
water interface by Brewster angle microscope experi-
ments.13
Here we attempt to extend this model by looking at the
displacement of a mixed protein film by a nonionic
surfactant (Tween 20). More importantly we attempt to
see whether one protein can displace another from an
interface by a similar mechanism. Thus, we have used
fluorescent labeling to distinguish between proteins and
to allow the observation of any phase separation that might
occur at the interface. Although this is not the first time
that such techniques have been used,14 this is the first
time that such a high degree of spatial resolution has
been obtained. The high spatial resolution is important
because of the range over which one might expect to see
protein phase separation. In the case of mixed protein
and charged surfactant films (probably the closest com-
(12) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir
2000, 16, 8176.
(13) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J.
Biomacromolecules, in press.
(14) Sengupta, T.; Damodaran, S. J. Colloid Interface Sci. 2000, 229,
21.

10.1021/la010687g CCC: $20.00 © 2001 American Chemical Society

Published on Web 09/14/2001
6594 Langmuir, Vol. 17, No. 21, 2001
parison to protein-protein), the surfactant domains were
in the order of tens of nanometers in size.12 In fact the
largest surfactant domains seen in a mixed protein
surfactant film before the final rupture of the protein
network were some only 100 µm in diameter.
Materials and Methods
The milk proteins used in this study were β-lactoglobulin (L-
0130, lot 91H7005) and β-casein (C-6905, lot 12H9550) from
Sigma Chemicals (Poole, U.K.). Samples were initially prepared
at 2 mg‚mL-1 in water. The water used in this study was surface
pure (γ0 ) 72.6 mN‚m-1 at 20 °C) cleaned using an Elga Elgastat
UHQ water purification system. The Tween 20 (polyoxyethylene
sorbitan monolaurate) was obtained as a 10% solution (Surfact-
Amps 20) from Pierce (Rockford, IL). The proteins were labeled
using amine reactive dyes, fluorescein isothyocyanate (FITC)
from Sigma (Poole, U.K.) in the case of β-casein and 6-carboxy-
rhodamine 6G succinimidyl ester (Molecular Probes Inc., Eugene,
OR) in the case of β-lactoglobulin. The reaction was performed
using the recommended protocols (Molecular Probes), and the
conjugate was purified by gel filtration to remove unbound dye
using a Sephadex G-25 column.
Surface tension measurements were made using a wetted
ground glass Wilhelmy plate and a Langmuir trough (Labcon
Ltd, Durham, U.K.). The PTFE trough was 255 × 112 × 16 mm,
a volume of 450 mL, with one fixed and one mobile barrier. All
experiments were performed at room temperature (20 °C) with
distilled water as the subphase. For the AFM measurements,
the adsorbed films were produced from solutions containing 0.5
µM β-casein, 0.5 µM β-lactoglobulin, and 0.5 µM Tween 20.
Surface tension measurements were made as a function of time
for 30 min while the monolayer equilibrated. After this period
of equilibration, the subphase was perfused with 2 L of 0.5 µM
Tween 20 at a rate of 1.1 mL‚s-1. Further additions of surfactant
to the subphase were then made in order to increase the surface
pressure as required. The Langmuir-Blodgett (LB) films were
formed on hydrophilic freshly cleaved mica substrates. LB films
were produced by lowering a freshly cleaved piece of mica sheet,
mounted perpendicular to the interface, down through the
interface and then pulling it back out again. The mounted piece
of mica was driven at a constant rate of 0.2 mm‚s-1. Surface
tension was monitored during the dip and showed that the film
was only transferred onto the mica on the upward stroke. The
AFM images were measured under butanol. The precise details
of the AFM and the imaging technique are given elsewhere.15
Fluorescence measurements were made using an Olympus
BX60 microscope. Excitation was from a mercury arc lamp via
a blue excitation (FITC) filter set or a green excitation (rhodamine)
filter set. Images were taken using a cooled chip CCD camera
(SP-eye, Photonic Sciences, Cambridge, U.K.). The images taken
were of LB films deposited onto washed glass coverslips in the
manner described above.
Rheological measurements were made using both surface shear
and surface dilation techniques. The surface dilation measure-
ments were made using the method of Kokelaar et al.16 The
technique involves periodically raising and lowering a 10-cm
ground-glass ring into a vessel containing 250 mL of sample.
The resulting change in surface area was kept to 5%, and the
frequency of dilation/compression was kept at 0.8 rads‚s-1. The
surface shear measurements were made using a Camtel CIR
100 surface shear rheometer (Camtel, Royston, U.K.). This
instrument uses a Du Nouy ring oscillating in normalized
resonance mode.17 The ring is oscillated in air at its resonant
frequency of approximately 3 Hz. When the ring is placed at an
interface, the resonant frequency and amplitude will change
depending on the elasticity and viscosity of the interface.
Feedback currents restore the frequency and amplitude of the
oscillations back to resonance. The values of the feedback signal
required to restore the frequency are used to calculate the surface
(15) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Morris, V. J.
Biopolymers 1998, 46, 245.
(16) Kokelaar, J. J.; Prins, A.; De Gee, M. J. Colloid Interface Sci.
1991, 46, 507.
(17) Sherrif, M.; Warburton, B. Polymer 1974, 15, 253.
Mackie et al.
Figure 1. Dilatational elastic modulus plotted as a function
of time for adsorbing 1 µM β-casein (line 1), 1 µM β-lactoglobulin
(line 2), and a 1:1 mixture of the two proteins at a total protein
concentration of 1 µM (line 3).
shear elastic modulus (G′), and the signal required to restore the
amplitude enables calculation of the viscous component (G′′).
Results and Discussion
In this paper we attempt to improve our understanding
of mixed protein interfaces by investigating the structure
and physical properties of a model system comprising two
proteins: β-lactoglobulin and β-casein. In previous papers
we have shown that surfactants disrupt and displace
proteins films by an orogenic mechanism, which involves
the expansion of phase-separated surfactant domains. The
main aim of this work is to discover whether structures
and phase separation processes are likely to occur in a
mixed protein interface, similar to those observed in the
protein/surfactant systems studied previously. The results
are discussed in terms of the various stages in the
formation and stabilization of the interface. More specif-
ically, the stages are (i) adsorption, (ii) phase separation,
and (iii) displacement.
Adsorption. Protein adsorption is mainly determined
by surface hydrophobicity, molecular weight, solubility,
and flexibility.18 β-Casein is a much more flexible molecule
than β-lactoglobulin and is therefore capable of unfolding
at the interface more rapidly. It has been found that
random coil proteins such as β-casein adsorb and lower
interfacial tension more rapidly than globular proteins.19
β-Casein also has very different surface elasticity to
β-lactoglobulin, so the surface elasticity was used to
dynamically probe the adsorption of the mixed protein
system.
Two types of interfacial rheology were employed,
dilatational and shear. Figure 1 shows the dilatational
elastic modulus for the two individual protein films and
for a 1:1 mixture. All three had the same total protein
concentration (1 µM). β-Lactoglobulin and β-casein show
markedly different behavior, which has been shown before
by others.20 β-Casein shows a typical peak at early
adsorption times which is caused by the collapse of the
highly charged N-terminal “tail” region into the sub-
phase.21 The elasticity reaches a plateau of approximately
10 mN/m after less than 10 min. It should be mentioned
that the β-casein film had virtually reached its equilibrium
surface pressure in about 5 min (data not shown). The
β-lactoglobulin simply showed a steady increase in elastic
modulus to a pseudoplateau of approximately 78 mN/m
after 30 min followed by a slight decrease thereafter. The
(18) Garofalakis, G.; Murray, B. S. Colloids Surf., B 1999, 12, 231.
(19) Mitchell, J. R. Dev. Food Proteins 1986, 4, 291.
(20) Williams, A.; Prins, A. Colloids Surf., A 1996, 4, 267.
(21) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M.
J. Chem. Soc., Faraday Trans 1995, 91, 2847.
Mixed Protein Displacement
Figure 2. Shear elastic modulus plotted as a function of time
for adsorbing 1 µM β-casein (line 1), 1 µM β-lactoglobulin (line
2), and a 1:1 mixture of the two proteins at a total protein
concentration of 1 µM (line 3).
surface pressure of the film was still steadily increasing
after 105 min (data not shown). The mixed system showed
features of both proteins. The peak from the partial
collapse of the β-casein is present although shifted to
slightly longer times. The much broader peak from
β-lactoglobulin was also visible but reduced by nearly a
factor of 4 to 22 mN/m. Subsequently the dilatational
elasticity climbed very slowly to 25.4 mN/m after nearly
4 h. The surface pressure of the mixed film took about 30
min to reach equilibrium (data not shown). The data
suggest that both proteins were observed to adsorb,
resulting in a mixed β-lactoglobulin/β-casein interface.
However, the surface elasticity of the mixed system
suggests that β-casein was the dominant protein at this
stage in the adsorption. This agrees with previous work
showing the more rapid adsorption of β-casein. If, rather
simplistically, one assumes the surface dilatational elastic
modulus of the separate components to be merely additive,
then the curve for an interface containing 85% casein fits
the data reasonably well, especially in the latter stages.
Further investigation will be required in order to fully
ascertain the true nature of the rheological behavior of
the mixed interfacial film. Especially in terms of the degree
to which such immobile systems may become phase
separated.
The data from the measurement of surface shear
elasticity are shown in Figure 2. Again, the figure shows
data for the individual proteins and the mixed system.
The film formed by β-casein was so weak as to be almost
immeasurable while the film formed by β-lactoglobulin
gave quite high values. The values for β-lactoglobulin rose
to a peak of 4.9 mN/m after 50 min and then decreased
in elasticity to about 2 mN/m after 3 h. The mixed film
shows a gradual slow rise to 1 mN/m after 3 h. The results
show a similar trend to the surface dilatational elasticity,
in that β-casein was clearly the dominant protein at these
concentrations. However, the surface shear elasticity
would suggest that there was almost no adsorbed β-lac-
toglobulin. The difference between the results from the
surface shear and the surface dilatational rheology can
be put down to the different methodologies. Surface
dilatational rheology measures the changes in surface
pressure during compression and expansion of the inter-
face whereas surface shear rheology measures directly
the mechanical properties of the interface under shear.
Both methods are sensitive to protein structure and the
number and strength of intermolecular interactions.
However, surface shear tends to be more sensitive to
protein interactions and surface dilatation more sensitive
to adsorbed structure and composition. Hence, the surface
dilatation reveals that under these conditions β-casein
Langmuir, Vol. 17, No. 21, 2001 6595
Figure 3. A series of fluorescence images following the
displacement of a mixed, dual stained protein film. β-Casein
was labeled green and lactoglobulin was labeled red. All images
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
the mixed film at various stages of displacement transferred
at 27 (b), 28.5 (c), and 29.5 mN/m (d).
was the predominant protein at the surface but a small
amount of β-lactoglobulin was present. Despite this
presence, the mechanical strength of the adsorbed film
(as measured by the surface shear elasticity) was almost
totally dominated by the β-casein component.
Therefore both surface rheological methods suggest that
at these equal concentrations β-casein adsorbed more
rapidly than β-lactoglobulin and dominated the surface
rheological properties. To investigate further, fluorescence
microscopy was used to see if there was a surface
concentration excess of β-casein and to observe any phase
separation which may occur.
Phase Separation. The aim here was to determine
the spatial distribution of the two proteins at the interface
and thus to ascertain if the proteins were evenly mixed
or if phase separation occurred and how the protein
distribution could help us understand the surface rheo-
logical behavior. Previous work on protein/surfactant
mixed interfaces has shown, using AFM, how the sur-
factant phase separates into domains to displace the
protein. AFM was ideally suited to these investigations
because it could easily distinguish between the separate
protein and surfactant domains. AFM cannot however
distinguish between two different proteins. To determine
the spatial distribution of the two proteins in the mixed
film, fluorescence microscopy was employed.
In these experiments, the two proteins of interest were
labeled with fluorescent probes, β-casein with FITC and
β-lactoglobulin with rhodamine 6G. In both cases the
extent of labeling was approximately 1:1 protein to dye.
The total protein concentration in the Langmuir trough
was 0.5 µM, and adsorption was allowed to continue for
3 days. Periodically, sections of the interface were
transferred onto glass coverslips by the Langmuir-
Blodgett method. Figure 3a is from an interface after
adsorption for 2 days (surface pressure ) 21.5 mN/m) and
shows a rather homogeneous deposition of protein. The
dominant protein was the FITC-labeled β-casein. There
was a low level of very localized heterogeneity observed
in all the images throughout the duration of the experi-
ments. To check whether the heterogeneity seen in this
image was representative of a small amount of phase
separation of the proteins on the microscopic scale, a
similar experiment was performed using only the fluo-
6596 Langmuir, Vol. 17, No. 21, 2001 Mackie et al.

rescent β-lactoglobulin. When the autocorrelation func-

tions of the two image sets were compared (data not
shown), they were found to be very similar. This suggests
that the small-scale heterogeneities were due simply to
random adsorption of the protein. There were no changes
in the spatial distribution of the two proteins and no phase
separation processes were observed over the experimental
period of 3 days. This behavior is very different from the
phase separation observed with the surfactant/protein
systems studied10 that could show phase separation in
less than 1 h. The main differences between adsorbed
proteins and surfactants are that (a) adsorbed surfactant
is able to exchange with nonadsorbed surfactant and (b)
adsorbed surfactant can freely diffuse at the interface. In
contrast, proteins, once adsorbed and interacting with
neighboring proteins, can neither exchange with the bulk
nor diffuse at the interface. The mobility of a surfactant
allows molecular segregation to occur and the resultant
domains to expand and deform. The distinct lack of
mobility of the adsorbed protein molecule arrests any
segregation process, and consequently there is no driving
force for phase separation. Hence the results of the
Figure 4. AFM images showing mixed β-casein and β-lacto-
fluorescence experiments shown in Figure 3a demonstrate globulin films being displaced by Tween 20. The films were
that no phase separation was observed, even after 3 days. transferred at surface pressures of 21.6 (a) and 25.0 mN/m (b).
Any local heterogeneity in the surface distribution of The image sizes are 4.0 and 5.0 µm, respectively. Also shown
protein is due to the initial adsorption process, after which are AFM images of a β-casein film being displaced by Tween
no measurable redistribution was observed. The lack of 20 at a surface pressure of 20.7 mN/m (c) and a β-lactoglobulin
rearrangement and phase separation observed here is in film being displaced by Tween 20 at a surface pressure of 26.1
contrast to the work published recently by Sengupta and mN/m (d). The image sizes are 9.0 and 4.0 µm, respectively.
Damodaran.14 We had observed previously that excessive
fluorescent labeling could affect the surface properties of
the protein. Measurements made on the surface rheology
of the fluorescent-labeled mixed protein film in the present
study showed it to be consistent with the unlabeled system.
This indicates that the labeling had not significantly
affected the strength of the film.
In summary, fluorescent microscopy demonstrated that
β-casein was indeed the dominant adsorbed protein under
these conditions and that even after 3 days, no segregation
or phase separation of the adsorbed proteins was observed.
Displacement. The displacement of the mixed β-lac-
toglobulin/β-casein interface by the nonionic surfactant
Tween 20 was studied. The aim was to see if the structure
of the mixed protein interface influenced how the indi-
vidual proteins were displaced. This is important for
understanding how real protein mixtures interact with Figure 5. The surface area (%) occupied by protein is plotted
lipids and surfactants in food foams and emulsions. as a function of the surface pressure at which the LB film was
The displacement was measured first on unlabeled transferred. Data are shown for films of β-casein alone ([),
protein using AFM. Figure 4 shows AFM images of a mixed β-lactoglobulin alone (9), and the 1:1 mixed film (2). Also shown
are data from fluorescent-labeled films displaced after aging
β-lactoglobulin/β-casein film at various stages of displace- for 3 days (b).
ment by Tween 20. It can be observed that the appearance
of the protein films changed as displacement progressed. with the isotropic compression of the β-casein nor as
The light areas are where the protein is located, and the irregular as those seen in the β-lactoglobulin film. The
dark areas are where surfactant occupied the surface. shape of the displacement patterns gives a clear indication
Figure 4a shows a mixed film transferred at a surface that there was indeed a mixed film at the interface.
pressure of 21.6 mN/m where protein covered 69% of the However, these images alone do not provide enough
surface area. The second image (Figure 4b) shows the information to draw firm conclusions about the spatial
mixed film after further displacement. This image is of a distribution of the individual proteins within the film.
film transferred at a surface pressure of 25.0 mN/m and All AFM images were analyzed to provide information
shows protein film occupying an area of 30.5%. For about both the surface area occupied by the protein and
comparison panels c and d of Figure 4 show pure β-casein the thickness of the protein layer. Figures 5 and 6 show
and pure β-lactoglobulin films, respectively. The adsorbed the protein area and thickness, respectively, as a function
β-casein film was transferred at a surface pressure of 20.7 of the surface pressure at which the film was transferred.
mN/m, and the film had a mean protein area of 51%. The For comparison both figures show data on films of pure
adsorbed β-lactoglobulin film was transferred at a surface β-casein and of pure β-lactoglobulin. The displacement of
pressure of 26.1 mN/m, and the protein occupied an area the mixed film occurred over a range of some 7 mN/m and
of 49%. It is clear that the holes visible in the mixed protein falls between the displacement curves for the individual
film show characteristics of both single-component protein proteins (Figure 5). The fact that the displacement falls
films. The shapes of the holes were neither circular as linearly between the two curves for the individual proteins
Mixed Protein Displacement
Figure 6. The film thickness of the protein layer is plotted as
a function of the surface pressure at which the LB film was
transferred. Data are shown for films of β-casein alone ([),
β-lactoglobulin alone (9), and the 1:1 mixed film (2).
suggests that there has been no phase separation between
the proteins in the mixed film. This mixed film required
a higher surface pressure to displace than a β-casein film
but a lower surface pressure than a β-lactoglobulin film.
If there had been a high degree of phase separation, one
might have expected to see the displacement for the mixed
system follow the curves for the single proteins at least
over part of the range. In the early stages the β-casein
domains would have been displaced; thus the mixed film
would have followed the displacement curve for the
β-casein alone. In the latter stages only the β-lactoglobulin
would have remained at the interface, and thus the mixed
film displacement curve would have resembled the β-lac-
toglobulin displacement curve. The other possibility is
that there was very little β-casein on the surface because
it had already been displaced by the β-lactoglobulin. This
would mean that at the lowest surface pressure at which
the mixed surfactant protein film was measured (21.2
mN/m), any domains of purely β-casein would have already
collapsed and been replaced by either Tween 20 or
β-lactoglobulin.
The film thickness data showed a similar trend to the
area data. However, Figure 6 is not quite as simple to
interpret because the thickening of the mixed film occurs
at surface pressures where a pure β-casein film would not
exist. Despite this the mixed film data seem to show a
transition from the β-casein data to the β-lactoglobulin
data with an apparent decrease in thickness during this
transition. Above about 24 mN/m the mixed film shows
essentially the same thickness as the equivalent β-lac-
toglobulin film.
To gain more information about the displacement
process of the mixed proteins, the interface formed using
the fluorescent-labeled protein was displaced by the
addition of Tween 20. The interface had been aged for 3
days, and the displacement data are shown in Figure 5.
The aging clearly makes the protein more difficult to
displace due to the development of interprotein interac-
tions over long adsorption times. This aging phenomenon
is a feature of protein films and has been noted by others.18
Initially the subphase concentration was 1 µM Tween 20,
but this was increased to 2 µM after 1 h. Panels b-d of
Figure 3 show the progressive displacement of the protein
film by the adsorbing Tween 20. Figure 3b is an image of
a film transferred at a surface pressure of 27 mN/m and
shows the protein film still occupying 66% of the total
area. The image showed slightly less green and slightly
more red than the previous image making it appear more
Langmuir, Vol. 17, No. 21, 2001 6597
Figure 7. Histograms of the proportion of the total measure
fluorescence in the images in Figure 3 that comes from the
FITC-labeled β-casein. Line 1 is for Figure 3a, line 2 is for
Figure 3b, line 3 is for Figure 3c and line 4 is for Figure 3d.
yellow. This would seem to suggest that the film was
compressed at the expense of the FITC-labeled β-casein.
Figure 3c shows an image of a film transferred at a surface
pressure of 28.5 mN/m. The protein film in this image
occupies only 48% of the total area, and again the image
appeared more yellow than the previous indicating that
more FITC-labeled β-casein has been lost from the
interface. The final image in the series, Figure 3d, was
transferred at a surface pressure of 29.5 mN/m and shows
the protein film occupying only 42% of the total surface
area. The protein film in this image appeared to be very
similar in color to the previous image. To quantify these
color changes, each image was separated into hue (color),
saturation (color density), and intensity (brightness). The
hue, or color scale, was then adjusted to cover only red to
green, and histograms of the images were calculated.
These histograms are plotted in Figure 7. This shows that
there was a significant change in the color of the
fluorescent protein film as the displacement progressed.
The color change indicated that the ratio of FITC β-casein
to rhodamine 6G β-lactoglobulin decreased. This in turn
suggests that the Tween 20 preferentially displaced the
β-casein from the interface. Thus, either the displacement
was not entirely “orogenic” as protein appears to have
been lost from the interface at all stages of displacement
or there were local heterogeneities in the surface protein
distribution resulting in very small domains of almost
pure β-casein in a continuum of mixed β-casein/β-lacto-
globulin.
Conclusions
In this work we have used various methods to probe the
interactions between β-casein and β-lactoglobulin. Surface
rheological measurements demonstrated that β-casein
adsorbs to the interface more rapdly than β-lactoglobulin.
Thus, the interface is dominated by the β-casein in the
early stage of protein film development. It is evident from
the microscopy that the β-lactoglobulin that subsequently
adsorbs does not cause any form of large-scale phase
separation. This is almost certainly due, at least in part,
to the lack of diffusion of the protein molecules within the
mixed film. The rheological data suggest that at the
concentrations used in this study, a 1:1 molar solution
leads to a film containing about 80% β-casein. The evidence
from both the AFM and the fluorescence microscopy
suggests that the two proteins form a homogeneously
mixed film which because of the high surface concentration
ratio of β-casein to β-lactglobulin, contained small (<200
nm) domains of almost pure β-casein.
6598 Langmuir, Vol. 17, No. 21, 2001
From the displacement studies we have seen that the
β-casein is displaced from the interface in preference to
the β-lactoglobulin. In the final stages of displacement,
the shapes of the holes in the film resemble those in a
β-lactoglobulin film and the surface pressure required to
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
film. The early stages of formation were dominated by the
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.
Mackie et al.
The use of different surface rheological techniques
together with imaging methods allows a more complete
understanding of the influence of protein composition and
interactions on the mechanical properties of interfacial
films formed by complex mixtures of food proteins.
Acknowledgment. This work was funded by the
BBSRC as part of the core grant to the institute.
LA010687G