Monoglycerides and β-Lactoglobulin Adsorbed Films at the Air−Water

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Langmuir 2007, 23, 7178-7188

Monoglycerides and β-Lactoglobulin Adsorbed Films at the Air-Water Interface. Structure, Microscopic Imaging, and Shear Characteristics Marta Cejudo Ferna´ndez, Cecilio Carrera Sa´nchez, Ma. Rosario Rodrı´guez Nin˜o, and Juan M. Rodrı´guez Patino* Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, UniVersidad de SeVilla, C/. Prof. Garcı´a Gonza´ lez 1, 41012-SeVille, Spain ReceiVed February 9, 2007. In Final Form: April 10, 2007 In this work we have analyzed the structural, topographical, and shear characteristics of mixed monolayers formed by adsorbed β-lactoglobulin (β-lg) and spread monoglyceride (monopalmitin or monoolein) on a previously adsorbed protein film. Measurements of the surface pressure (π)-area (A) isotherm, Brewster angle microscopy (BAM), and surface shear characteristics were obtained at 20 °C and at pH 7 in a modified Wilhelmy-type film balance. The π-A isotherm and BAM images deduced for adsorbed β-lactoglobulin-monoglyceride mixed films at π lower than the equilibrium surface pressure of β-lactoglobulin (π β-lg ) indicate that β-lactoglobulin and monoglyceride coexist at the e interface. However, the interactions between protein and monoglyceride are somewhat weak. At higher surface pressures (at π g π β-lg ) a protein displacement by the monoglyceride from the interface takes place. The surface shear viscosity e (ηs) of mixed films is very sensitive to protein-monoglyceride interactions and displacement as a function of monolayer composition (protein/monoglyceride fraction) and surface pressure. Shear can induce change in the morphology of monoglyceride and β-lactoglobulin domains, on the one hand, and segregation between domains of the film-forming components on the other hand. In addition, the displacement of β-lactoglobulin by the monoglycerides is facilitated under shear conditions.

Introduction Proteins and monoglycerides, being surface-active molecules, are successfully used as emulsifiers in many food dispersion formulations (emulsions and foams).1,2 Well-known examples of these food dispersions are ice-cream, whipped toppings, salad dressing, coffee creamers, confectionary and bakery products, etc.3-5 The chemical and physical properties of surface-active molecules are of great interest because they determine the colloidal stability of dispersed systems.1 The optimum use of emulsifiers depends on knowledge of their interfacial physical-chemical characteristics and the kinetics of film formation at fluid interfaces.1,2 In addition, the distribution of different emulsifiers (proteins and lipids) used in real food dispersions is determined by competitive and co-operative adsorption between these emulsifiers at the fluid-fluid interfaces and by the nature of protein-lipid interactions, both at the interface and in bulk phase. Interactions between molecules of emulsifiers could affect the structure, topography, and mechanical characteristics (surface rheological properties under shear or dilatation) in mixed films, with direct repercussions on the formation and stability of the dispersion.6-10 Thus, understanding the structural and mechanical * To whom all correspondence should be addressed. Tel.: +34 95 4556446. Fax: +34 95 4556447. E-mail: [email protected]. (1) Dickinson, E. An introduction to Food Colloids: Oxford University Press; Oxford, 1992. (2) McClements, D. J. Food Emulsions: Principles, Practice and Techniques, 2nd ed.; CRC Press: Boca Raton, FL, 2005. (3) Damodaran, S.; Paraf, A. Food Proteins and their Applications; Marcel Dekker: New York, 1997. (4) Hartel, R.; Hasenhuette, G. R. Food Emulsifiers and their Applications; Chapman and Hall: New York, 1997. (5) Friberg, S. E.; Larsson, K.; Sjo¨blom, J. Food Emulsions, 4th ed.; Marcel Dekker: New York, 2004. (6) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. Protein/emulsifier interactions. In Food Emulsions and their Applications; Hasenhuette, G. L., Hartel, R. W., Eds.; Chapman & Hall: New York, 1997; pp 95-146. (7) Bos, M. A.; van Vliet, T. AdV. Colloid Interface Sci. 2001, 91, 437.

characteristics of emulsifiers at fluid interfaces is potentially important in order to improve structure, stability, texture, taste, and other organoleptic properties of food colloid formulations.11,12 Recently we have analyzed how the structural characteristics of mixed monolayers formed by milk proteins and monoglycerides differ depending on the method used for the formation of the mixed film at the air-water interface (spreading, penetration, or by spreading of a lipid into a previously adsorbed protein film).13 Thus, further information about these phenomena would be very helpful in the prediction of optimized formulations for food foams and emulsions. In this work we are concerned with the analysis of structural, topographical, and shear characteristics of mixed films formed by an adsorbed globular milk protein (β-lactoglobulin) and a spread monoglyceride (monopalmitin or monoolein). These emulsifiers have been selected because of their good foaming and emulsifying properties and because the interfacial characteristics of spread mixed films are well-known.14,15 However, little is known about the interfacial characteristics of adsorbed mixed films,13 although from a technological point of view adsorbed films are more interesting than spread films. The (8) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R.; Carrera, C. Curr. Opin. Colloid Interface Sci. 2003, 8, 387-395. (9) Wilde, P. J.; Mackie, A. R.; Husband, F.; Gunning, P.; Morris, V. AdV. Colloid Interface Sci. 2004, 108-109, 63. (10) Pugnaloni, L. A.; Dickinson, E.; Ettelaie, R.; Mackie, A. R.; Wilde, P. J. AdV. Colloid Interface Sci. 2004, 107, 27. (11) Leser, M. E.; Michael, M.; Watzke, H. J. Food goes nano-New horizons for food structure research. In Food Colloids: Biopolymers and Materials; Dickinson, E., van Vliet, T., Eds.; Royal Society Chemistry: Cambridge, 2003; pp 3-13. (12) Rodrı´guez Patino, J. M.; Lucero, A.; Rodrı´guez Nin˜o, Ma. R.; Mackie, A. R.; Gunning, A. P.; Morris, V. J. Food Chem. 2007, 102, 532. (13) Cejudo, M.; Carrera, C.; Rodrı´guez Nin˜o; M. R., Rodrı´guez Patino, J. M. Food Hydrocollooids 2007, 21, 906. (14) Rodrı´guez, Patino, J. M.; Rodrı´guez, Nin˜o, Ma. R.; Carrera, C.; Cejudo, M. Langmuir 2001, 17, 7545. (15) Carrera, C.; Rodrı´guez Patino, J. M. Colloids Surf., B 2004, 36, 57.

10.1021/la7003497 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/19/2007

Monoglycerides and β-Lactoglobulin Adsorbed Films

structural and topographical characteristics of emulsifiers at interfaces are important from a fundamental point of view because these properties determine their application in food dispersion formulations.8 On the other hand, interfacial rheology is important for food colloids because the structural and mechanical properties of food emulsifiers at fluid interfaces have an influence on the stability and texture of the product.7,16,17 In addition, shear viscosity may contribute appreciably to the long-term stability of dispersions7,17 and is sensitive to the precise form of the displacement of protein and lipids at the interface.18,19 Experimental Section Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90) and 1-mono-(cis-9-octadecanoyl) glycerol (monoolein, RYLO MG 19) were kindly supplied by Danisco Ingredients (Brabran, Denmark) with over 95-98% of purity. Whey protein isolate, a native protein with a very high content of β-lactoglobulin (protein 92 ( 2%, β-lactoglobulin >95%, R-lactalbumin 99.8%) were used. The water used as the subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl, Sigma, >99.5%) was used to achieve pH 7. Ionic strength was 0.05 M in all the experiments. Surface Film Balance. Measurements of the surface pressure (π) versus average trough area (A) were performed on a modified Wilhelmy-type film balance, which integrates BAM and surface shear rheometry, as described elsewhere.20 Before each measurement, the film balance was calibrated at 20 °C. For β-lactoglobulin adsorbed films from water, a β-lactoglobulin solution at 1 × 10-6 - 5 × 10-6 % (wt/wt) was left in the trough and time was allowed for protein adsorption at the interface. These protein concentrations were selected from previous data of the adsorption isotherm.21 At this protein concentration in solution the surface pressure at equilibrium is zero. In fact, after 24 h the surface pressure (π) at the maximum area of the trough was practically zero, and the monoglyceride (1.4 × 10-4 - 1.5 × 10-4 mg/µL) was spread at different points on the previously adsorbed β-lactoglobulin film. For pure adsorbed protein films, the maximum protein concentration in the bulk phase should be selected in order to obtain a reasonable rate of adsorption at the interface, while maintaining the equilibrium surface pressure as zero.21 On the other hand, for mixed films, at low protein concentrations in the aqueous phase we cannot observe the collapse point, especially for low monoglyceride concentrations. Thus, in these experiments we have selected optimum conditions in order to obtain the complete π-A isotherm of the mixed film, from the more expanded monolayer (at the higher areas) to the more condensed monolayer, at the collapse point (at the lower areas). Mixtures of particular mass fractionss expressed as the mass fraction of monoglyceride in the mixture, Xswere studied. The compression rate was 3.3 cm min-1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. To allow for β-lactoglobulinmonoglyceride interactions, 30 min was allowed to elapse before (16) Dickinson, E. Colloids Surf., B 1999, 15, 161. Dickinson, E. Colloids Surf., B 2001, 20, 197. (17) Murray, B. S. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 179-220. (18) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (19) Mackie, A. R.; Gunning, P. A.; Pugnaloni, L. A.; Dickinson, E.; Wilde, P. J.; Morris, V. J. Langmuir 2003, 19, 6032. (20) Rodrı´guez Patino, J. M.; Carrera, C. Langmuir 2004, 20, 4530. (21) Rodrı´guez Nin˜o, Ma. R.; Carrera, C.; Cejudo, M.; Rodrı´guez Patino, J. M. J. Amer. Oil Chem. Soc. 2001, 78, 873.

Langmuir, Vol. 23, No. 13, 2007 7179 compression was performed. After 30 min at the maximum area, measurements of compression-expansion cycles were performed with 30 min of waiting time between each expansion-compression cycle. The measurement of the π-A isotherm was performed before the surface shear rheology experiments. This isotherm was recorded with the maximum width of the canal at which the surface pressure at both sides of the canal was the same (i.e., ∆π was zero during the monolayer compression). The initial surface pressure of the mixed film at the maximum area before compression was zero. The reproducibility of the results was better than (0.5 mN/m for surface pressure and (0.05 m2/mg. Brewster Angle Microscopy (BAM). A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography of the monolayer. The BAM was positioned over the film balance. Further characteristics of the device and operational conditions have been described elsewhere.22,23 The surface pressure measurements, area, and BAM images as a function of time were carried out simultaneously by means of a device connected between the film balance and BAM. These measurements were performed during the flow of the monolayer through the canal. The imaging conditions were adjusted to optimize image quality. Thus, generally as the surface pressure or the protein content increased, the shutter speed was also increased. Some images were recorded in the absence of the automatic gain control in order to distinguish between protein domains (with high reflectivity), which saturate the camera with a completely white image, and monoglyceride domains (with low reflectivity). Surface Shear Rheometry. To study the shear characteristics of spread films, a homemade canal viscometersan analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past one anothersas described elsewhere20 was used in this work. Briefly, surface shear viscosity measurements were taken on a modified Wilhelmy-type film balance, with two surface pressure sensors located at both sides of the canal in the center of the trough. The canal was constructed of two Teflon bars to render them hydrophobic and was mounted on the trough. The separation between the two bars can be varied and adjusted by means of a precision screw. The monolayer is allowed to flow through a canal of a width Wc and fixed length, L ) 30 mm, and at a constant flow (Q, m2 s-1, recorded as a variation of the area associated with the movement of the barrier as a function of time). The surface shear viscosity (ηs) was calculated from the rate of film flow Q by eq 1, which is analogous to the Poiseuille equation for the flow of liquids through capillary tubes.24 ηs )

(π2 - π1)Wc3 12QL

(1)

The monolayer was compressed to the desired surface pressure and then the flow of the monolayer through the canal was facilitated by the movement of the two barriers in the same direction, from a region of higher surface pressure (π2) to a region of lower surface pressure (π1). The value of ∆π ) π2 - π1 was deduced during the flow of the monolayer. As for pure components,20 an optimization of Wc and Q is necessary in order to obtain the best flow conditions of the mixed monolayer through the canal. During these experiments practically all the monolayer (ca. 90%) flows through the canal, thus the data presented in this work represent the overall behavior of the monolayer under shear conditions. The subphase temperature was controlled at 20 °C by water circulation from a thermostat, within an error range of (0.3 °C. The temperature was measured by a thermocouple located just below the air-water interface. Data were obtained after a minimum of three measurements, and the repeated results prove the reproducibility of the method. (22) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Langmuir 1999, 15, 2484. (23) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Food Hydrocolloids 1999, 13, 401. (24) Harkins, W. D.; Kirkwood, J. G. Nature (London) 1938, 141, 38.

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Figure 1. Surface pressure-area isotherms (compression-expansion curves) for adsorbed β-lactoglobulin-monopalmitin mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (O) 0, (s) 0.2, (- - -) 0.4, (‚‚‚‚) 0.5, and (- ‚ -) 0.6. The equilibrium surface pressures of β-lactoglobulin (π β-lg ) and monopalmitin (π MP e e ) are indicated by means of arrows.

Results and Discussion Structural, Topographical, and Shear Characteristics of β-Lactoglobulin-Monopalmitin Mixed Monolayers Adsorbed at the Air-Water Interface. Structural Characteristics. The π-A isotherm for adsorbed β-lactoglobulin monolayer was obtained as a control at the beginning of each experiment (Figure 1). This isotherm is practically the same as those obtained previously for β-lactoglobulin adsorbed films13,25,26 and that obtained directly by spreading.27 Thus, the structures of the monolayers formed in the two different ways must be identical, at least for adsorption from low bulk protein concentrations. The results of the π-A isotherms (Figure 1) confirm that a β-lactoglobulin monolayers at the air-water interface adopt a liquid expanded-like structure and the collapse phase. However, according to Graham and Phillips,28 β-lactoglobulin retains elements of the native structure, not fully unfolded at the interface. Thus, most amino acid residues in β-lactoglobulin adopt loop conformation at the air-water interface. But the loop conformation is more condensed at higher surface pressures and is displaced toward the bulk phase at the collapse point. The monolayer collapses at a surface pressure a little higher than the equilibrium surface pressure,21 which is indicated in Figure 1 by means of ≈ 25.9 mN/m). an arrow (π β-lg e Mixtures of particular mass fractionssranging between 0 and 0.6, expressed as the mass fraction of monoglyceride in the mixture, Xswere studied. The amount of spread monoglyceride was calculated on the basis of the mass of previously adsorbed β-lactoglobulin, which was calculated form the adsorbed π-A isotherm. It must be emphasized that, due to this assumption, in Figure 1 the area in the X-axis is not the true area per unit mass of mixed film but the trough (apparent) area (AAPPARENT). On the other hand, as opposed to spread monolayers,14 for adsorbed monolayers the mixtures with mass fractions higher than X ) 0.6 saturate the interface, under the experimental conditions used in this work. From the π-A isotherms for β-lactoglobulin + monopalmitin mixed films (Figure 1) it can be seen that there was a monolayer expansion as the monopalmitin concentration in the mixture was increased, especially at higher surface pressures (at π > π β-lg ). e That is, the π-A isotherm is displaced toward higher A as the concentration of monopalmitin in the mixture increases. These (25) Rodrı´guez Patino, J. M.; Cejudo, M. Langmuir 2004, 20, 4515. (26) Rodrı´guez Patino, J. M.; Cejudo, Rodrı´guez Nin˜o, M. R.; Carrera, C. Biomacromolecules 2006, 45, 7510. (27) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (28) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427.

Cejudo Ferna´ ndez et al.

data are in agreement with those deduced for spread β-lactoglobulin-monopalmitin.14,29 At surface pressures lower than the equilibrium surface pressure of β-lactoglobulin (at π < π β-lg ), e both β-lactoglobulin and monopalmitin coexist at the interface. In fact, we can see the main transition between liquid expanded (LE) and liquid condensed (LC) phases, which is typical of monopalmitin monolayers at low surface pressures (at π ≈ 5 mN/m).22 At surface pressures higher than that for β-lactoglobulin collapse (at π > π β-lg ), the π-A isotherm for mixed monolayers e was parallel to that of monopalmitin. These results suggest that at the higher surface pressures the arrangement of the monopalmitin hydrocarbon chain in mixed monolayers is practically the same in the entire β-lactoglobulin/monopalmitin fraction. In a previous paper we deduced by means of the excess area that adsorbed β-lactoglobulin and monopalmitin form a mixed monolayer at the air-water interface with few repulsive interactions between film-forming components at low monopalmitin concentrations in the mixed film and at surface pressures lower than that for the β-lactoglobulin collapse (at π < π β-lg ), e as the film-forming components adopt a similar liquid-like structure.25 However, these interactions become attractive at higher monopalmitin concentrations in the mixed film (X > 0.5). At the highest surface pressures (at π > π β-lg ) at the collapse e point of the mixed film, immiscibility between monolayer-forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monopalmitin monolayer (Figure 1). This collapse pressure is produced at a surface pressure a little higher than the equilibrium surface pressure for monopalmitin.22 The fact that, upon expansion and further compression, the π-A isotherms were repetitive (data not shown) suggests that the protein displaced by monopalmitin during the compression (at π > π β-lg ) re-enters the mixed monolayer upon expansion e and supports the idea that the protein remains underneath the monoglyceride film either through hydrophobic interactions between protein and lipid or by local anchoring through the monoglyceride layer.14,25,30 However, for adsorbed β-lactoglobulin-monopalmitin mixed monolayers a first-order-like phase transition was observed upon the monolayer expansion (Figure 1) at surface pressures close to the equilibrium surface pressure of β-lactoglobulin (at π ≈ π β-lg )swith a degenerated e plateau in the π-A isotherm. These results suggest that the readsorption of previously displaced β-lactoglobulin has a kinetic character, which was not evident for spread mixed films.14,25 Topographical Characteristics. The evolution with the surface pressure of BAM images (Figure 2) corroborates, at a microscopic level, the structural characteristics and interactions of adsorbed β-lactoglobulin-monopalmitin mixed monolayers, as deduced from π-A isotherms (Figure 1). The morphology of β-lactoglobulin monolayers at the air-water interface appeared to be of uniform reflectivity (data not shown), suggesting homogeneity in thickness and film isotropy. This morphology is characteristic for this protein20,25,26 and is independent of the experimental conditions adopted, either for spread or adsorbed monolayers.25 However, at the end of the expansion, at the lowest surface pressure (at π ≈ 0 mN/m), and at the beginning of the recompression of the monolayer (at π ≈ 0 mN/m), interfacial regions with aggregations of collapsed β-lactoglobulin, which were formed during the first compression at π g π β-lg , were e (29) Cornec, M.; Narsimhan, G. Langmuir 2000, 16, 1216. (30) Li, J. B.; Kra¨gel, J.; Makievski, A. V.; Fainermann, V. B.; Miller, R.; Mo¨hwald, H. Colloids Surf., A 1998, 142, 355.

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Figure 2. Visualization of adsorbed β-lactoglobulin-monopalmitin mixed monolayers by Brewster angle microscopy at 20 °C: (a and b) β-lactoglobulin-monopalmitin mixed films at π ≈ 0 (at the beginning of the monolayer recompression or at the end of the expansion), shutter 1/50 s; (c) β-lactoglobulin-monopalmitin mixed films at 15 mN/m and shutter 1/50 s; (d) β-lactoglobulin-monopalmitin mixed film at 26.1 mN/m and shutter 1/125 s; (e) β-lactoglobulin-monopalmitin mixed films at 27.2 mN/m and shutter 1/125 s; (f) β-lactoglobulin-monopalmitin mixed films at 21 mN/m (during the monolayer expansion) and shutter 1/50 s; (g) β-lactoglobulin-monopalmitin mixed films at 32.7 mN/m and shutter 1/250 s; (h) β-lactoglobulin-monopalmitin mixed films at 11.5 mN/m (during the monolayer expansion) and shutter 1/50 s. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

observed at the interface (Figure 2a). In this region we also observed isolated interfacial domains of collapsed β-lactoglobulin (Figure 2b). For β-lactoglobulin-monopalmitin mixed films, islands of β-lactoglobulin and monopalmitin do exist at the air-water interface, depending on the surface pressure and the composition of the mixed film. At surface pressures lower than that for β-lactoglobulin collapse, a mixed monolayer of monopalmitin and β-lactoglobulin may exist with small domains of monopalmitin (with a liquid condensed structure at π > 5 mN/m) uniformly distributed on the homogeneous β-lactoglobulin layer (Figure 2c). These domains were more numerous as the surface pressure increased. At surface pressures higher than that for β-lactoglobulin collapse (at π g π β-lg ), the mixed monolayers e

were practically dominated by collapsed β-lactoglobulin residues (bright region), which are displaced from the interface by darkcircular monopalmitin domains floating over a sublayer of collapsed residues of β-lactoglobulin (Figure 2d). However, the topography of the mixed film also proves the existence of some folds of collapsed β-lactoglobulin domains (Figure 2e). These structures define a shear plane observed during the monolayer expansion (Figure 2f). The squeezing of β-lactoglobulin by monopalmitin was more intense as the surface pressure increased ) and/or at high content of monopalmitin in the (at π g π β-lg e mixture (Figure 2g). Another topographical characteristic of the adsorbed film was the presence of some fractures in the monolayer at the collapse point of the mixed film,25 which are more evident during the film expansion (Figure 2h).

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Figure 3. Difference in surface pressure between the limits of the canal during the flow of β-lactoglobulin adsorbed monolayers at the air-water interface as a function of surface pressure (mN/m): (A) 5, (B) 10, (C) 15, and (D) 20. Temperature was 20 °C, and pH ) 7. Effect of width of canal (Wc, mm) and β-lactoglobulin flow through the canal (Q, m2 s-1). A and B: (s) Q ) 3.75 × 10-6 and Wc ) 1.4, (- - -) Q ) 6.25 × 10-6 and Wc ) 1.4, (‚‚‚‚) Q ) 1.25 × 10-5 and Wc ) 1.4, and (- ‚ -) Q ) 6.25 × 10-6 and Wc ) 0.9. C and D: (s) Q ) 6.25 × 10-6 and Wc ) 1.4, (- - -) Q ) 6.25 × 10-6 and Wc ) 0.9, (‚‚‚‚) Q ) 3.75 × 10-5 and Wc ) 0.9, (- ‚ -) Q ) 1.25 × 10-6 and Wc ) 0.9, and (- ‚‚ -) Q ) 3.75 × 10-6 and Wc ) 1.4.

Shear Characteristics. The time evolution of ∆π during the flow of an adsorbed β-lactoglobulin monolayer depends on the β-lactoglobulin flow through the canal and the surface pressure (Figure 3). An optimization of Wc and Q was necessary in order to obtain the best flow conditions of a protein monolayer through the canal, with few fluctuations in the ∆π-time flow curve, and more reproducible data. The steady state in ∆π was reached relatively quickly during the flow of the β-lactoglobulin monolayer at low surface pressures, but more time is necessary at high surface pressure. From the ∆π the surface shear viscosity (ηs) was determined by means of eq 1. The steady state ∆π value can be associated with the equilibrium shear viscosity in stationary flow. At this point there exists a balance between the formation and breaking of the monolayer structure or a constant friction within the β-lactoglobulin monolayer domains along the canal. The fluctuations in the ∆π value may be due to friction between β-lactoglobulin domains at the higher surface pressures, a phenomenon that denotes the heterogeneity of the monolayer. From the ∆π at steady state, the surface shear viscosity was determined as a function of surface pressure (Figure 4). Over the overall range of surface pressures the values of ηs did not depend on the flow Q (Figure 4). The values of ηs increased with surface pressure, especially as the residues of β-lactoglobulin adopt the more condensed conformation of loops at the air-water interface. At high surface pressures, as amino acid segments are extended into the underlying aqueous solution adopting the form of loops, an increase in the viscous drag caused by the subphase may explain the higher values of ηs. The values of ηs are higher for spread20 than for adsorbed β-lactoglobulin films (insert in Figure 4A). Thus, although the π-A isotherms for adsorbed β-lactoglobulin monolayer are practically the same as those obtained directly by spreading,25 the surface shear viscosity is different. One speculation is that β-lactoglobulin may be spread at the interface maintaining a more unfolded structure as compared

Figure 4. Effect of surface pressure on surface shear viscosity for adsorbed β-lactoglobulin at the air-water interface. Symbols: (O, b) Q ) 3.75 × 10-6, (4, 2) Q ) 1.25 × 10-5, and (3, 1) Q ) 6.25 × 10-6; open symbols correspond to Wc ) 0.9 mm and closed symbols Wc ) 1.4 mm. The surface shear viscosity for pure (], - - -) monopalmitin and (3, ‚‚‚‚) monoolein spread monolayers20 are included as a reference in plots A and B. The surface shear viscosity for pure β-lactoglobulin (O) spread20 and (b) adsorbed films are included in the insert of plot A and in plot B. Temperature was 20 °C, and pH ) 7.

Monoglycerides and β-Lactoglobulin Adsorbed Films

Figure 5. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monopalmitin at XMP ) 0.2 and at (A) 10 mN/ m, (B) 20 mN/m, and (C) 25 mN/m. The surface shear viscosity for a pure monopalmitin monolayer at 25 mN/m is included as a reference (the ηs values are represented by the striped pattern). Symbols: (s) Q ) 6.25 × 10-6 and Wc ) 1.4, (- - -) Q ) 1.25 × 10-6 and Wc ) 1.4, (‚‚‚‚) Q ) 1.25 × 10-5 and Wc ) 0.9, (- ‚ -) Q ) 6.25 × 10-6 and Wc ) 0.9, and (- ‚‚ -) Q ) 3.75 × 10-6 and Wc ) 1.4. Temperature was 20 °C, and pH ) 7.

with adsorbed films. Thus, the interactions between unfolded β-lactoglobulin residues must be higher for spread than for adsorbed β-lactoglobulin films, a phenomenom that explains the higher value of ηs for β-lactoglobulin spread films as compared with adsorbed one. The surface shear viscosity for pure monopalmitin and monoolein spread monolayers20 is included in Figure 4 as a reference. At surface pressures lower than 15 mN/m, the values of ηs were similar for adsorbed β-lactoglobulin and for monoglycerides (Figure 4A). The differences observed in ηs values for β-lactoglobulin and monopalmitin at π > 15 mN/m can be of utility in the analysis of the shear characteristics of adsorbed mixed films. The effect of surface pressure on surface shear characteristics of adsorbed β-lactoglobulin-monopalmitin mixed monolayers under different operational conditions and at XMP ) 0.2, 0.4, 0.5, and 0.6 are shown in Figures 5, 6, 7, and 8, respectively. As can be observed, the ηs-time curves are dependent on the monolayer flow through the canal (Q) and the width of the canal (Wc). For some flow conditions, the ηs-time curve develops a maximum (see Figures 6A, 6B, and 8D) and then ηs decreases with time, before the ηs “steady state” value is attained. These phenomena

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Figure 6. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monopalmitin at XMP ) 0.4 and at (A) 10 mN/ m, (B) 20 mN/m, and (C) 30 mN/m. The surface shear viscosity for a pure monopalmitin monolayer at 30 mN/m is included as a reference (the ηs values are represented by the striped pattern). Symbols: (s) Q ) 1.25 × 10-5 and Wc ) 1.4, (- - -) Q ) 1.25 × 10-5 and Wc ) 0.9, (‚‚‚‚) Q ) 1.25 × 10-5 and Wc ) 1.4, (- ‚ -) Q ) 6.25 × 10-6 and Wc ) 2.05, and (- ‚‚ -) Q ) 3.75 × 10-6 and Wc ) 1.4. Temperature was 20 °C, and pH ) 7.

were also described by another author in relation to stressstrain curves for proteins using a Couette-type interfacial viscometer.31 According to this author, the maximum can be associated with the strength of the monolayer, whereas the decrease in ηs after the maximum may represent a structure breakdown of the mixed film under shear. The breaking of LC domains was evident at a microscopic level by means of BAM. In fact, the size of the LC domains was lower under shear (Figure 2c,d) than with the open canal (data not shown). However, we have also observed that there exists a deformation of the domains and/or a segregation of β-lactoglobulin and monopalmitin domains at the interface during the flow of the monolayer. That is, the shear may induce heterogeneity in the structure of the mixed monolayer at a microscopic level along the BAM image. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers15,32-36 (31) Izmailova, V. N. Prog. Surf. Membr. Sci. 1979, 13, 141. (32) Murayama, T.; Fuller, G.; Frank, C.; Robertson, C. Science 1996, 274, 233. (33) Murayama, T.; Lauger, J.; Fuller, G.; Frank, C.; Robertson, C. Langmuir 1998, 14, 1836. (34) Igne´s-Mullol, J.; Schwartz, D. K. Nature 2001, 410, 348-351. (35) Carrera, C; Rodrı´guez Patino, J. M. Langmuir 2004, 20, 6327.

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Figure 7. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monopalmitin at XMP ) 0.5 and at (A) 10 mN/m, (B) 20 mN/m, (C) 30 mN/m, and (D) 40 mN/m. The surface shear viscosity for a pure monopalmitin monolayer at 30 and 40 mN/m is included as a reference (the ηs values are represented by the striped pattern). Symbols: (s) Q ) 1.25 × 10-5 and Wc ) 0.9, (- - -) Q ) 1.25 × 10-5 and Wc ) 1.4, (‚‚‚‚) Q ) 6.25 × 10-6 and Wc ) 2.05, and (- ‚ -) Q ) 3.75 × 10-6 and Wc ) 1.4. Temperature was 20 °C, and pH ) 7.

Figure 8. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monopalmitin at XMP ) 0.6 and at (A) 10 mN/m, (B) 20 mN/m, (C) 30 mN/m, and (D) (1) 40 mN/m and (2) at the collapse point. The surface shear viscosity for a pure monopalmitin monolayer at the collapse and at 40 mN/m is included as a reference (the ηs values are represented by the striped pattern). Symbols: (s) Q ) 1.25 × 10-5 and Wc ) 1.4, (- - -) Q ) 1.25 × 10-5 and Wc ) 0.9, (‚‚‚‚) Q ) 6.25 × 10-6 and Wc ) 2.05, and (- ‚ -) Q ) 3.75 × 10-6 and Wc ) 2.05. Temperature was 20 °C, and pH ) 7.

and may be the cause of the ηs peaks observed during the flow of the monolayer (Figures 5B, 6B, 7B, and 8B). The “steady state” ηs value can be associated with the existence of a balance between the formation and breaking of monolayer structures or with a constant friction within the mixed monolayer domains along the canal. The fluctuations in the “steady state” ηs value may be due to friction between LC monopalmitin domains or (36) Rodrı´guez Patino, J. M.; Cejudo, M.; Rodrı´guez Nin˜o, M. R.; Carrera, C. Ind. Eng. Chem. Res. 2006, 45, 1886.

between segregated regions of monopalmitin and β-lactoglobulin, both of these phenomena denoting the heterogeneity of the mixed monolayer. There was a general tendency for ηs to increase as the surface pressure increased, as might be expected on the basis of greater intermolecular interactions. The “steady state” ηs values for pure monopalmitin and β-lactoglobulin monolayers are included in Figures 5-8 as reference. However, there were significant differences in ηs during the flow of the mixed film through the , the ηs canal, depending on the film composition. At π < π β-lg e

Monoglycerides and β-Lactoglobulin Adsorbed Films

Figure 9. Surface pressure-area isotherms (compression-expansion curves) for adsorbed β-lactoglobulin-monoolein mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (O) 0, (s) 0.2, (- - -) 0.4, (‚‚‚‚) 0.5, and (- ‚ -) 0.6. The πe of β-lactoglobulin (π β-lg ) is indicated e by means of an arrow.

values of the mixed films are lower than those for a pure component (monopalmitin and β-lactoglobulin) monolayer, regardless of the composition of the mixed films, at XMP ) 0.2 (Figure 5A,B), XMP ) 0.4 (Figure 6A,B), XMP ) 0.5 (Figure 7A,B), and XMP ) 0.6 (Figure 8A,B). These results indicate that, in this region, β-lactoglobulin and liquid-condensed (LC) domains of monopalmitin flow through the canal but with few interactions between them, producing a reduction in the friction in the mixed monolayer in relation to that for pure component monolayers and fluctuations in ηs values. After the β-lactoglobulin collapse, at surface pressures of 25, 30, and 40 mN/m and at the collapse of the mixed film (see Figures 5-8), the ηs values of the mixed film were similar to those for a pure monopalmitin monolayer, with a few exceptions (see Figures 6C and 8C). These exceptions may have coincided with less than optimum conditions for the flow of the monolayer because the values of Q and Wc were not the most appropriate. These results suggest that, in this region, the mixed monolayer

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was dominated by the presence of monopalmitin at the interface. In fact, at the higher surface pressures a squeezing of β-lactoglobulin protein by monopalmitin takes place, as was observed for the topography of the mixed film (see Figure 2g). The segregation of β-lactoglobulin and monopalmitin domains at the interface during the flow of the monolayerswhich is evident (Figure 2e) and during the monolayer compression at π > π β-lg e during the expansion after the collapse, with the presence of some fractures of collapsed β-lactoglobulin aligned in the direction of the flow (Figure 2f)scan explain the fact that the ηs values of the mixed film were co-incident with those in the lower limit for a pure monopalmitin monolayer or even a little lower (Figures 7C,D and Figure 8D). Structural, Topographical, and Shear Characteristics of β-Lactoglobulin-Monoolein Mixed Monolayers Adsorbed at the Air-Water Interface. Structural Characteristics. The structural characteristics of adsorbed β-lactoglobulin-monoolein mixed monolayers were essentially different to those of monopalmitin in the mixture, as deduced from π-A isotherms (Figure 9). Briefly, as expected,14,36 β-lactoglobulin-monoolein mixed films at surface pressures lower than that for β-lactoglobulin collapse (at π < 25.9 mN/m) adopt a liquid-like-expanded structure, as for pure components. There was a monolayer expansion due to the presence of monoolein in the mixture. At surface pressures higher than that for β-lactoglobulin collapse, the π-A isotherm for mixed monolayers was practically parallel to that of monoolein. At the highest surface pressures, at the collapse point of the mixed film, immiscibility between monolayer-forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of a pure monoolein monolayer (Figure 9). Topographical Characteristics. BAM images for adsorbed β-lactoglobulin-monoolein mixed monolayers (Figure 10) were different than those described above for adsorbed β-lactoglobulin-monopalmitin mixed monolayers (Figure 2). In fact, at surface pressures lower than that for β-lactoglobulin collapse (at π < 25.9 mN/m), the topography of pure components and

Figure 10. Visualization of adsorbed β-lactoglobulin-monoolein mixed monolayers by Brewster angle microscopy at 20 °C: (a) pure β-lactoglobulin monolayer, pure monoolein monolayer, or β-lactoglobulin-monoolein mixed films at π < π β-lg and shutter speed 1/50 s; e (b) pure β-lactoglobulin monolayer at 19 mN/m and shutter speed 1/50 s; (c) β-lactoglobulin-monoolein mixed films at 34.4 mN/m and shutter speed 1/250 s; (d) β-lactoglobulin-monoolein mixed films at 44.6 mN/m and shutter speed 1/50 s. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

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Figure 11. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monoolein at XMO ) 0.2 and at (A) 10 mN/m and (B) at (‚‚‚, ---, -‚-) 20 and (-‚‚-) 25 mN/m. The surface shear viscosity values for a pure monoolein monolayer and β-lactoglobulin are included as a reference (the ηs values for monoolein and β-lactoglobulin are represented by the striped and checkered patterns, respectively). Symbols: (s) Q ) 6.25 × 10-6 and Wc ) 0.9, (- - -) Q ) 1.25 × 10-5 and Wc ) 1.4, (‚‚‚‚) Q ) 1.25 × 10-5 and Wc ) 0.9, (- ‚ -) Q ) 6.25 × 10-6 and Wc ) 1.4, and (- ‚‚ -) Q ) 6.25 × 10-6 and Wc ) 1.4. Temperature was 20 °C, and pH ) 7.

that of the mixed monolayer are practically identical because in this region both components and the mixed monolayer form an isotropic (homogeneous) monolayer without any difference in the domain topography (Figure 10a). The presence of isolated bright β-lactoglobulin domains can also be distinguished in this region (Figure 10b), denoting the segregation of monoolein and β-lactoglobulin at the interface. At surface pressures near and after β-lactoglobulin collapse, BAM images demonstrated the squeezing out of collapsed β-lactoglobulin by monoolein, with some white regions which correspond to the presence of a thicker β-lactoglobulin collapsed-monolayer (Figure 10c). At the higher surface pressures, and especially at the collapse point, the topography of the mixed monolayer was dominated by the presence of small domains of collapsed β-lactoglobulin (bright regions) and monoolein (dark regions) at the interface (Figure 10d). Shear Characteristics. The time evolution of ηs during the flow through the canal of β-lactoglobulin-monoolein mixed monolayers at XMO ) 0.2, 0.4, 0.5, and 0.6 is shown in Figures 11, 12, 13, and 14, respectively, as a function of surface pressure. The presence of a maximum in the ηs-time curve or the existence of large fluctuations in the ηs value for adsorbed β-lactoglobulinmonoolein mixed monolayers was observed for some systemss at XMO ) 0.2 (Figure 11) and at XMO ) 0.4 (Figure 12B), at π . This phenomenon can be associated with the segre< π β-lg e gation between β-lactoglobulin and monoolein domains at the interface during the flow of the monolayer15,34sbecause at these surface pressures β-lactoglobulin and monoolein coexist at the air-water interface14,26sand/or for the use of inappropriate flow conditions.15,20,35 It can be seen that, over the overall range of surface pressures analyzed, the values of ηs for the mixed monolayer were very small and that these values were practically independent of the

Figure 12. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monoolein at XMO ) 0.4 and at (A) 10 mN/m, (B) 25 mN/m, and (C) 30 mN/m. The surface shear viscosity values for a pure monoolein monolayer and β-lactoglobulin are included as a reference (the ηs values for monoolein and β-lactoglobulin are represented by the striped and checkered patterns, respectively). Symbols: (s) Q ) 1.25 × 10-5 and Wc ) 1.4, (- - -) Q ) 1.25 × 10-5 and Wc ) 0.9, and (‚‚‚‚) Q ) 6.25 × 10-6 and Wc ) 1.4. Temperature was 20 °C, and pH ) 7.

surface pressure (see Figures 11-14). From these results we , the values of ηs were concluded the following: (i) At π < π β-lg e very similar to those for a pure adsorbed β-lactoglobulin or monoolein monolayer, especially at low concentrations of monoolein in the mixture (at XMO ≈ 0.2-0.4) (see Figures 11 and 12). These results indicate that the shear characteristics of the mixed monolayer are dependent on the presence of adsorbed β-lactoglobulin or monoolein at the interface at π < π β-lg . e However, in this region (at π < π β-lg ) the interface is dominated e by monoolein at high monoolein concentrations (Figures 13B and 14A). (ii) At π > π β-lg , and, especially, at the higher surface e pressures or at the collapse point of the mixed films, the ηs values were lower than that for a pure β-lactoglobulin adsorbed film, especially at XMO > 0.2 (see Figures 12C, 13C,D, and 14B), which may be associated with the flow of LE monoolein domains through the canal. These data corroborate the hypothesis that β-lactoglobulin is displaced from the interface by monoolein at π > π β-lg . (iii) For some systems, at the collapse of the mixed e film, the ηs values of β-lactoglobulin-monoolein mixed monolayers were at the lower limit of that for a pure monoolein monolayer (see Figures 12C, 13D, and 14B). This phenomenon

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Figure 13. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monoolein at XMO ) 0.5 and at (A) 10 mN/m, (B) 20 mN/m, (C) 30 mN/m, and (D) 40 mN/m. The surface shear viscosity value for a pure monoolein monolayer is included as a reference (the ηs value for monoolein is represented by the striped pattern). Symbols: (s) Q ) 1.25 × 10-5 and Wc ) 1.4 and (- - -) Q ) 1.25 × 10-5 and Wc ) 0.9. Temperature was 20 °C and pH ) 7.

Figure 14. Surface shear viscosity for an adsorbed mixed film of β-lactoglobulin and monoolein at XMO ) 0.6 and (A) at (s) 10 mN/m, (- - -) at 20 mN/m, and (‚‚‚‚) at 30 m/m; and (B) at (- ‚ -) 40 and (- ‚‚ -) at the collapse point. The surface shear viscosity value for a pure monoolein monolayer is included as a reference (the ηs value for monoolein is represented by the striped pattern). Symbols: (s) Q ) 1.25 × 10-5 and Wc ) 0.9, (- - -) Q ) 1.25 × 10-5 and Wc ) 0.9, (- - - and ‚‚‚‚) Q ) 1.25 × 10-5 and Wc ) 0.9, (- ‚ -) Q ) 1.25 × 10-6 and Wc ) 0.9, and (- ‚‚ -) Q ) 1.25 × 10-6 and Wc ) 0.9. Temperature was 20 °C and pH ) 7.

can be associated with the presence of small fractures in the collapsed film. Final Considerations. In the preceding sections we discussed structural, topographical, and shear characteristics of β-lactoglobulin and monoglyceride mixed adsorbed films at the air-

water interface. The same thermodynamic and dynamic characteristics have been analyzed recently for a model disordered milk protein (β-casein) using the same experimental techniques26 and for protein monoglyceride spread films.15,20 Thus, a comparison between these proteins in adsorbed and spread mixed films with monoglycerides would be of interest to see how the surface shear characteristics (derived from the interactions and displacement of proteins by monoglycerides) depend on the chemistry of the protein or monoglyceride molecule and the formation of a mixed film at the air-water interface. Adsorbed and Spread Pure Protein Films. The flow of a β-lactoglobulin adsorbed film through the canal is more difficult than for β-casein,26 as reflected in the time taken to attain the maximum value of ∆π and the level of the fluctuations in ∆π during the flow, which are higher for β-lactoglobulin (Figure 3) than for β-casein.26 The values of ηs for β-lactoglobulin are also higher (Figure 4) than those for β-casein26 under the same experimental conditions. The same behavior was reported by other authors.20,37,38 The differences in solution structure between β-casein, a flexible protein lacking secondary structure, and β-lactoglobulin, a globular protein with a compact conformation, are reflected in their adsorption or spreading behavior at the air-water interface.39 The high values of ηs for β-lactoglobulin correlate well with the capacity of β-lactoglobulin for interfacial gelation.20 The interfacial gelation by cross-linking of disulfide residues in β-lactoglobulin films and the formation of interfacial aggregates of significant sizes may be the causes of the high values of ηs (Figure 4) and the fluctuations in ∆π during the flow (37) Dickinson, E.; Matsumura, Y. Int. J. Biol. Macromol. 1991, 13, 26. (38) Benjamins, J. Static and Dynamic Properties of Proteins Adsorbed at Liquid Interfaces; Ph.D. Dissertation, Wageningen University: Wageningen, The Netherlands, 2000. Benjamins, J.; Lucassen-Reynders, E. H. In Proteins at Liquid Interface; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 341384. (39) Horne, D. S.; Rodrı´guez Patino, J. M. Adsorbed biopolymers: behavior in food applications. In Biopolymers at Interfaces, 2nd ed.; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; pp 857-900.

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of the monolayer though the canal (Figure 3),40 as compared with β-casein, which can only form physical gels stabilized by intermolecular hydrogen bonds.20 Therefore, differences between surface shear viscosity of adsorbed and spread protein films reflect differences, among others, in the protein structure and the potential for the formation of disulfide cross-linking and the formation of interfacial aggregates of significant sizes (such as for β-lactoglobulin) as compared with β-casein. Adsorbed Protein-Monoglyceride Mixed Films. The values of ηs for β-casein-36 and β-lactoglobulin-monoolein (Figures 11-14) mixed adsorbed films are similar, and (for some systems) the values of ηs for β-lactoglobulin-monoolein are even 1 order of magnitude lower than those for β-casein-monoolein mixed films. These results corroborate the idea that the presence of monoolein in the mixed systems has a significant effect on the values of ηs, because the values of ηs are higher for pure β-lactoglobulin than for β-casein adsorbed films.26 The fact that the values of ηs for β-casein-36 and β-lactoglobulin-monopalmitin mixed films are similarsat XMP ) 0.2 (Figure 5) and at π < πesor that the values of ηs are higher for β-lactoglobulin- than for β-casein-monopalmitin mixed filmss at XMP ) 0.2 (Figure 5) and at π > πe, XMP ) 0.4 and at π > 20 mN/m (Figure 6), and XMP ) 0.5 at every π (Figure 7)s corroborates the idea that (i) the presence of protein (βlactoglobulin or β-casein) in the mixed systems has a significant effect on the flow of the film and that (ii) the protein is not completely displaced by monopalmitin and has an effect on the flow of mixed films at high surface pressures. For both β-casein-36 and β-lactoglobulin-monoglyceride (Figures 5-8 and 11-14) mixed films the values of ηs are higher for protein-monopalmitin than for protein-monoolein, especially at high π, which confirms the importance for the ηs values of the presence of the monoglyceride (monopalmitin or monoolein) in the mixed film, because ηsMONOPALMITIN is higher than ηsMONOOLEIN. Adsorbed and Spread Protein-Monoglyceride Mixed Films. In this work we have analyzed the structural (Figures 1 and 9), topographical (Figures 2 and 10), and shear (Figures 5-8 and 11-14) characteristics of β-lactoglobulin and monoglyceride (monopalmitin or monoolein) mixed films adsorbed at the airwater interface as a function of β-lactoglobulin/monoglyceride mass fraction and surface pressure. The same interfacial characteristics have been determined recently for spread mixed monolayers using the same experimental techniques.14,15 Some differences between adsorbed and spread mixed films are as follows: (i) Although the miscibility between β-lactoglobulin and monoglyceride is weak, the interactions between film-forming components are higher for adsorbed than for spread mixed films. (ii) During the flow of the mixed film, the segregation of filmforming components is higher and the flow is facilitated (the values of ηs are lower) for adsorbed films compared with spread (40) Wijmans, C. M.; Dickinson, E. Langmuir 1999, 15, 8344.

Cejudo Ferna´ ndez et al.

films. (iii) The topography of the mixed film confirms that the segregation between film-forming components is lower for spread than for adsorbed films because the β-lactoglobulin and monoglyceride domains are smaller in the latter. (iv) The readsorption of previously displaced β-lactoglobulin is slower for adsorbed than for spread films. All these phenomena are a consequence of the increased interactions between film-forming components at the interface in mixed adsorbed films compared with spread films. Finally, in the absence of shear we have observed that monoolein has a lower capacity than monopalmitin for β-lactoglobulin displacement from the air-water interface.14 However, under shear conditions these differences are attenuated both for spread15 and adsorbed (this work) mixed films, a phenomenon that can be attributed to the film segregation under shear conditions.

Conclusions From the results derived from these experiments it can be concluded that (i) surface pressure-area isotherms coupled with BAM and surface shear rheology are useful tools to analyze structure, topography, and shear characteristics of adsorbed protein-monoglyceride mixed films at the air-water interface. (ii) Interactions, topography, and displacement of β-lactoglobulin by monoglycerides in adsorbed mixed monolayers at the airwater interface depend on the particular β-lactoglobulinmonoglyceride system, β-lactoglobulin/monoglyceride fraction, and surface pressure. (iii) β-lactoglobulin and monoglyceride coexist at the air-water interface at low concentrations of monoglyceride in the mixture and at surface pressures lower than that for the β-lactoglobulin collapse (at π < 25.9 mN/m). At higher surface pressures, the collapsed β-lactoglobulin is displaced from the interface by monoglyceride (either monopalmitin or monoolein). The β-lactoglobulin displacement by monoglycerides is not total at the highest surface pressure, at the collapse point of the mixed monolayer, at the monoglyceride mass fractions studied in this work (lower than 0.6). (iv) Immiscibility between monolayer-forming components is also deduced at the highest surface pressures, due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monopalmitin or monoolein monolayers. Surface shear characteristics of the film are sensitive to interactions, topography, and displacement of protein by monoglyceride, including shearinduced segregation in the mixed films. All these phenomena depend on the chemistry of the emulsifier molecule (protein or monoglyceride), the protein/monoglyceride fraction, the surface pressure, and the film formation (either by spreading or by adsorption). Acknowledgment. This research was supported by CICYT through Grant AGL2004-01306/ALI. LA7003497