Flow-Induced Molecular Segregation in β-Casein−Monoglyceride

Langmuir 2004, 20, 6327-6334

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Flow-Induced Molecular Segregation in β-Casein-Monoglyceride Mixed Films Spread at the Air-Water Interface Cecilio Carrera Sa´nchez 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 12, 2004. In Final Form: April 26, 2004 In this work, we have used different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial shear rheology) to analyze the static (structure, topography, reflectivity, miscibility, and interactions) and flow characteristics (surface shear characteristics) of β-casein and monoglyceride (monopalmitin and monoolein) mixed films spread on the air-water interface. The structural, topographical, and shear characteristics of the mixed films depend on the surface pressure and on the composition of the mixed film. The surface shear viscosity (ηs) varies greatly with the surface pressure. In general, the greater the surface pressure, the greater the values of ηs. At higher surface pressures, collapsed β-casein residues may be displaced from the interface by monoglyceride molecules with important repercussions on the shear characteristics of the mixed films. A shear-induced change in the topography of monoglyceride and β-casein domains, on one hand, and a segregation between domains of the film-forming components, on the other hand, were also observed. The displacement of the β-casein by the monoglycerides is facilitated under shear conditions, especially for β-casein-monoolein mixed films.

Introduction Emulsifiers used in commercial food formulations typically consist of a mixture of surface-active derivatives because they can be produced at a relatively lower cost than pure emulsifiers. In addition, in many emulsifier applications, mixtures of different emulsifiers (mainly polar lipids and proteins) often exhibit properties superior to those of the individual emulsifier alone due to synergistic interactions between emulsifier molecules.1-4 In previous works with mixtures of monoglycerides and milk proteins, we have observed that when molecules of both emulsifiers are spread at the air-water interface they are more expanded or packed more closely together than when either emulsifier is present alone, indicating some form of association.5 Interactions between molecules of emulsifiers could affect not only the film structure and topography but also the dynamic behavior of mixed films, which is recognized as being of importance in the formation and stability of food. The study of such dynamic behavior can be described by interfacial rheology. Interfacial rheology is important for food colloids (emulsions and foams) because the structural and mechanical properties of food emulsifiers at fluid interfaces have an influence on the stability and texture of the product.4,6-8 In addition, interfacial rheology is a very sensitive technique to monitor the interfacial structure and concentration of single emulsifiers at the interface or * To whom correspondence should be addressed. Tel: +34 95 4556446. Fax: +34 95 4557134. E-mail: [email protected]. (1) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsions and their Applications; Hasenhuette, G. L., Hartel, R. W., Eds.; Chapman and Hall: New York, 1997; pp 95-146. (2) Nylander, T. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 365-431. (3) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (4) Dickinson, E. Colloids Surf., B 2001, 20, 197. (5) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R.; Carrera, C. Curr. Opin. Colloid Interface Sci. 2003, 8, 387. (6) Dickinson, E. Colloids Surf., B 1999, 15, 161. (7) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437. (8) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2002, 7, 426.

the relative concentration, the competitive adsorption, and the magnitude of interactions between different emulsifiers at the interface.5 Interfacial rheology can be defined9,10 for both compressional deformation (dilatational rheology) and shearing motion of the interface (shear rheology). While shear viscosity may contribute appreciably to the long-term stability of dispersions, dilatational rheology plays an important role in short-term stability.7,11,12 Moreover, the ability of the protein to resist displacement by emulsifiers is closely linked to the surface dilatational rheology, whereas the precise form of the displacement is considered to be more closely related to the surface shear behavior.3,8,13,14 The aim of this contribution was to analyze the static (structure, topography, and relative reflectivity) and surface shear properties of mixed food emulsifiers (β-casein and monoglycerides) at the air-water interface. In this work, we have used for the first time a unique device that incorporates different interfacial techniques, such as surface film balance, Brewster angle microscopy, and interfacial shear rheology, to analyze the behavior of mixed films spread at the air-water interface. There have been a few studies of interfacial rheological properties of oilsoluble emulsifiers and proteins spread at the air-water interface. However, as far as we know, the study of shear characteristics of milk proteins and monoglycerides has not been performed so far, although in practice mixtures (9) Murray, B. S.; Dickinson, E. Food Sci. Technol. Inst. 1996, 2, 131. (10) Murray, B. S. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 179-220. (11) Benjamins, J.; Lucassen-Reynders, E. H. In Proteins at Liquid Interface; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 341-384. (12) Lucassen-Reynders, E. H.; Benjamins, J. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, 1999; pp 195-206. (13) Roth, S.; Murray, B. S.; Dickinson, E. J. Agric. Food Chem. 2000, 48, 1491. (14) Mackie, A. R.; Gunning, P. A.; Pugnaloni, L. A.; Dickinson, E.; Wilde, P. J.; Morris, V. J. The growth of surfactant domains in protein films. Langmuir 2003, 19, 6032.

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of these emulsifiers are usually used in order to achieve an optimal effect in food formulations.15 Materials and Methods Materials. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90) and 1-mono(cis-9-octadecanoyl)glycerol (monoolein, RYLO MG 19) were supplied by Danisco Ingredients with over 95-98% purity. To form the surface film, monoglyceride was spread in the form of a solution, using hexane/ ethanol (9:1, v:v) as a spreading solvent. Analytical grade hexane (Merck, 99%) and ethanol (Merck, >99.8%) were used without further purification. β-Casein (99% pure) was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Samples for interfacial characteristics of β-casein films were prepared using Milli-Q ultrapure water at pH 7. The water used as the subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution (Sigma, >99.5%) called Trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) was used to adjust the subphase at pH 7. The ionic strength was 0.05 M in all the experiments. Surface Shear Rheometry. To study the shear characteristics of spread films, a homemade canal viscometer (an analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past one another) as described elsewhere16 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 it 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 a simplified equation (eq 1) which is analogous to the Poiseuille equation for the flow of liquids through capillary tubes.17

ηs )

(π2 - π1)Wc3 12QL

Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on the same modified Wilhelmy type film balance, as described elsewhere.18,19 The measurement of the π-A isotherm was performed before the experiments of surface shear rheology. 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 mean deviation was within (0.1 mN/m for surface pressure and (0.125 × 10-3 m2 per mg for area. 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. Brewster Angle Microscopy (BAM). For microscopic observation of the monolayer structure, a Brewster angle microscope, BAM 2 plus (NFT, Germany), was used as described elsewhere.20,21 The Brewster angle microscope was located at the exit of the canal. To measure the relative thickness of the film (I ) Cδ2, where C is a constant and δ is the film thickness) in the absence of shear, a previous camera calibration is necessary in order to determine the relationship between the gray level (GL) and the relative reflectivity (I), according to a procedure described previously.20,21 In all BAM images under the flow of the monolayer, the imaging conditions were adjusted to optimize image quality and not to enable quantitative measurement of reflectance (I). Thus, the reflectance of each image was quantified by the gray level in a direction diagonal to the interface. The gray level as a function of distance gives complementary information about the heterogeneity of each image (i.e., the relative film thickness is proportional to the square root of the gray level for the proper monolayer) along the diagonal but cannot be used to compare the BAM images with each other at different experimental conditions. All BAM images under shear conditions are accompanied by the evolution of the gray level with the distance in the direction of the diagonal in the image, which is indicated by means of a bar. The application of different devices (Wilhelmy type film balance, Brewster angle microscopy, and surface dilatational rheology) to the same monolayer makes possible the characterization of the monolayer under shear conditions and at equilibrium, at a macroscopic and at a microscopic scale.

(1)

The monolayer is allowed to flow through a canal without compression by the movement in the same direction of two barriers 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. Mixtures of a particular mass fraction of monoglyceride in the mixture of X ) 0.5 were studied, as a function of the surface pressure and monolayer composition. Aliquots of aqueous solutions of β-casein (1.543 × 10-4 mg/µL) at pH 7 were spread on the interface by means of a micrometric syringe. Afterward, a monoglyceride solution in a hexane/ethanol mixture was spread at different points on the β-casein film. To allow for spreading and β-casein-monoglyceride interactions, 30 min was allowed to elapse before compression was performed. To ensure interactions and homogeneity, the mixed film was compressed near the collapse point of the mixture and then expanded immediately to avoid the collapse. After 30 min at the maximum area, 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. As for pure components,16 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. These data were obtained after a minimum of three measurements, and the repeated results prove the reproducibility of the method. (15) Dickinson. E. An Introduction to Food Colloids; Oxford University Press: Oxford, U.K., 1992. (16) Rodrı´guez Patino, J. M.; Carrera, C. Langmuir 2004, 20, 4530. (17) Harkins, W. D.; Kirkwood, J. G. Nature (London) 1938, 141, 38.

Results and Discussion Structural, Topographical, and Surface Shear Characteristics of β-Casein-Monopalmitin Mixed Monolayers at the Air-Water Interface. Structural and Topographical Characteristics. Results derived from π-A isotherms (Figure 1A) in the modified Wilhelmy type trough are in good agreement with those obtained in the same unmodified trough with pure β-casein22 and monopalmitin23 monolayers and in the Langmuir type trough with the same β-casein, monopalmitin, and β-caseinmonopalmitin mixed films.24,25 Briefly, the results of π-A isotherms confirm that β-casein monolayers at the airwater interface adopt two different structures and the collapse phase. At low surface pressures, β-casein molecules may exist as trains with all amino acid segments located at the interface.26 This structure was observed at surface pressures lower than ca. 10 mN/m (Figure 1A). At (18) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; Cejudo, M. Langmuir 2001, 17, 4003. (19) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (20) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Nin˜o, Ma. R. Langmuir 1999, 15, 2484. (21) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Food Hydrocolloids 1999, 13, 401. (22) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Nin˜o, Ma. R.; Cejudo, M. Langmuir 2001, 17, 4003. (23) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Nin˜o, Ma. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (24) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. J. Agric. Food Chem. 1999, 47, 4998. (25) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R.; Carrera, C. J. Agric. Food Chem. 2003, 51, 112.

Flow-Induced Molecular Segregation

Figure 1. (A) Surface pressure (π)-area (A) isotherm of (solid line) β-casein, (dashed line) monopalmitin, and (open circles) β-casein-monopalmitin mixed film at XMP ) 0.5. The arrows indicate the equilibrium surface pressure of β-casein (πe (BC)) and the collapse pressure of monopalmitin (πc (MP)). (B) Relative reflectivity as a function of surface pressure for (solid line) β-casein, (dashed line) monopalmitin, and (open circles) β-casein-monopalmitin mixed film at XMP ) 0.5. Temperature ) 20 °C. pH ) 7.

higher surface pressures and up to the equilibrium surface pressure (πe ≈ 21 mN/m), which is indicated in Figure 1A by means of an arrow, amino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails. The equilibrium surface pressure (πe) is the maximum surface pressure to which a spread monolayer may be compressed without the possibility of monolayer collapse. Different structures can be deduced for the monopalmitin monolayer as a function of surface pressure (Figure 1A). The liquid-expanded phase (LE) (at π < 5 mN/m), a first-order phase transition between liquidcondensed (LC) and liquid-expanded structures (at 5 < π < 30 mN/ m), the liquid-condensed structure (at π > 30 mN/m), and finally the solid (S) structure near to the monolayer collapse at a surface pressure of about 53.1 mN/m were observed. β-Casein-monopalmitin (Figure 1A), at a monopalmitin mass fraction of XMP ) 0.5 (as an example) and at surface pressures lower than that for β-casein collapse (πC = 21 mN/m), adopts a structural polymorphism, as for pure components. In this region, a mixed monolayer of monoglyceride and β-casein may exist. However, at surface pressures higher than that for β-casein collapse, the π-A isotherm for the mixed monolayer at XMP ) 0.5 was parallel to that of monopalmitin, which demonstrates that the arrangement of the monopalmitin hydrocarbon chain in mixed monolayers is practically the same.24,25 That is, at higher surface pressures, collapsed β-casein residues may be displaced from the interface by monopalmitin molecules. Moreover, the mixed film collapses at a surface pressure similar to that for monopalmitin, which is an indication of the immiscibility between film-forming components at the highest surface pressures. (26) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427.

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Results of BAM, in particular the relative reflectivity as a function of surface pressure (Figure 1B) and topography (data not shown), obtained with β-casein, monopalmitin, and a β-casein-monopalmitin mixed film at XMP ) 0.5 in the absence of shear clearly show at a microscopic level the same structural characteristics as those deduced from the π-A isotherms. Briefly, at π < 20 mN/m, the I-π plots were the same as for pure components. In this region, LC domains of monopalmitin existed in an environment of homogeneous LE monopalmitin and LE-like β-casein domains, during the compression. The main difference was observed in the region after the β-casein collapse (20 mN/m < π < 45 mN/m), because the relative reflectivity of the mixed film was lower than for pure β-casein. This suggests that monopalmitin is able to displace β-casein residues from the interface toward a sublayer beneath the monopalmitin monolayer.24,25 The peaks in the relative reflectivity are due to the fact that in this region domains of pure β-casein (with high I) and monopalmitin (with low I) passed alternatively through the spot where this measurement was performed. Near to the collapse of the mixed films (at 45 mN/m < π < 52 mN/m), the peaks in relative reflectivity practically disappeared and the I values decreased to those typical for a pure monopalmitin monolayer. This suggests that in this region monopalmitin predominates at the interface. The Effect of Surface Pressure on Surface Shear Characteristics. The surface shear characteristics of a β-casein-monopalmitin mixed monolayer at XMP ) 0.5, at 10 mN/m, and at different operational conditions, at a constant width of the canal (Wc ) 1.5 mm) and two different flows of the monolayer (3.75 × 10-6 and 1.25 × 10-5 m2 s-1), have been determined (Figure 2B). Different β-caseinmonoglyceride mixtures (X ) 0.25 and 0.75) behaved in a similar way under shear conditions (data not shown). As can be seen, the ηs-θ curves depend on the monolayer flow through the canal (Q). At the higher value of Q, the ηs-θ curve develops a maximum, and then ηs decreases with time before the ηs “steady state” value is attained. The maximum disappears as the flow decreases. These phenomena were also described by other authors in relation to stress-strain curves for proteins using a Couette type interfacial viscometer.27 According to these authors, 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, similar to the overshoot observed with thixotropic fluids in bulk rheology. The breaking of LC domains was evident at a microscopic level by means of BAM (Figure 2, image A1). In fact, the size of the LC domains was lower under shear 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-casein and monopalmitin domains at the interface during the flow of the monolayer (Figure 2, image A2). That is, the shear may induce a heterogeneity in the structure of the mixed monolayer at a microscopic level as observed by the evolution of the gray level (i.e., the film thickness) along the BAM image. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers28-30 and may be the cause of the ηs peaks observed during the flow of the monolayer (Figure 2B). (27) Izmailova, V. N. Prog. Surf. Membr. Sci. 1979, 13, 141. (28) Murayama, T.; Fuller, G.; Frank, C.; Robertson, C. Science 1996, 274, 233. (29) Murayama, T.; Lauger, J.; Fuller, G.; Frank, C.; Robertson, C. Langmuir 1998, 14, 1836. (30) Igne´s-Mullol, J.; Schwartz, D. K. Nature 2001, 410, 348.

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Figure 2. (A1,A2) Visualization of β-casein and monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The evolution of the gray level along the interface is included for images A1 and A2. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monopalmitin at XMP ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal, Q (m2 s-1): (Timea, solid line) 3.75 × 10-6 and (Timeb, crosses) 1.25 × 10-5. The surface shear viscosity for pure β-casein (striped pattern) and monopalmitin (checked pattern) monolayers is included as a reference. The arrows (marked A1 and A2) correspond to the times at which A1 and A2 BAM images were taken. Surface pressure ) 10 mN/m. Temperature ) 20 °C. pH ) 7.

The steady state ηs value was reached at a time halfway between those for pure monopalmitin and β-casein monolayers. However, the steady state ηs value was similar to that for a pure monopalmitin monolayer (Figure 2B; the steady state ηs values for pure monopalmitin and β-casein monolayers are included in Figure 2B as a reference), denoting the importance of the monoglyceride on the shear characteristics of the mixed film. The steady state ηs value can be associated with the existence of a balance between the formation and breaking of monolayer structure or 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 domains or between segregated regions of monopalmitin and β-casein, both of these phenomena denoting the heterogeneity of the mixed monolayer. Finally, the values of ηs at the steady state did not depend on the flow Q within the experimental error, which is an indication that the mixed monolayer displays Newtonian behavior under these experimental conditions. There was a general tendency for ηs to increase as the surface pressure increased at 20 mN/m (Figure 3B), as might be expected on the basis of greater intermolecular interactions. However, there were significant differences in ηs during the flow of the mixed film through the canal. At the beginning of the flow (after the ηs values reached

Sa´ nchez and Rodrı´guez Patino

Figure 3. (A1,A2) Visualization of β-casein and monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The evolution of the gray level along the interface is included for images A1 and A2. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monopalmitin at XMP ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal, Q (m2 s-1): (Timea, solid line) 3.75 × 10-6 and (Timeb, crosses) 1.25 × 10-5. The surface shear viscosity for pure β-casein (striped pattern) and monopalmitin (checked pattern) monolayers is included as a reference. The arrows (marked A1 and A2) correspond to the time at which A1 and A2 BAM images were taken. Surface pressure ) 20 mN/m. Temperature ) 20 °C. pH ) 7.

a steady state), the ηs value for the mixed film was similar to that for a pure monopalmitin monolayer. However, at long-term flow great fluctuations in ηs values were also observed. These fluctuations in ηs values were more significant and extended in time (Figure 3B) than during the first period and may be associated with the flow of large regions of collapsed β-casein through the canal instead of film heterogeneities associated with the presence of LC monopalmitin domains. Interestingly, the peaks had ηs values lower than that for a pure β-casein monolayer, indicating that in this end region the monolayer flow was dominated by the presence of β-casein but the LC domains of monopalmitin produced a reduction in the friction in the mixed monolayer in relation to that for a pure β-casein monolayer. The results of BAM corroborate these hypotheses. In fact, at 20 mN/m, close to the β-casein collapse (see Figure 1A), images A1 and A2 (Figure 3) show a characteristic squeezing-out phenomenon with many LC domains of monopalmitin (the circular dark regions) floating over a sublayer of collapsed β-casein molecules (characterized by a completely white image with a reflectivity similar to that for pure β-casein). As a consequence of this topog-

Flow-Induced Molecular Segregation

Figure 4. (A1,A2) Visualization of β-casein and monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The evolution of the gray level along the interface is included for images A1 and A2. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monopalmitin at XMP ) 0.5: (dashed line) at 30 mN/m and Wc ) 1.5 mm; (solid line, crosses) at 40 mN/m and Wc ) 2.0 mm, with (solid line) Q ) 3.75 × 10-6 and (crosses) Q ) 6.25 × 10-6 m2 s-1. The surface shear viscosity for a pure monopalmitin monolayer at 30 mN/m (checked pattern) and at 40 mN/m (striped pattern) is included as a reference. The arrows (marked A1 and A2) correspond to the time at which A1 and A2 BAM images were taken. Temperature ) 20 °C. pH ) 7.

raphy, the gray level (i.e., the monolayer film thickness) presented significant fluctuations along the interface. These phenomena were an indication that a segregation in the mixed monolayer was produced during the flow of LC monopalmitin domains with low ηs values, at the beginning of the flow, followed by the flow of a β-caseinmonopalmitin mixed monolayer depleted in monopalmitin content. There are significant differences in the shear characteristics of the mixed film after the β-casein collapse, at surface pressures of 30 and 40 mN/m (Figure 4B). In fact, at these surface pressures the ηs values of the mixed film were similar to those for a pure monopalmitin monolayer, with the presence of fluctuations in the ηs values which were due to the heterogeneity of the topography of the film at a microscopic level (Figure 4, images A1 and A2). At these experimental conditions, the ηs value of a pure β-casein collapsed monolayer was similar to that observed at 20 mN/m (Figure 3B). As at lower surface pressures, the size of the LC domains was lower under shear (Figure 4, image A1) than with the open canal (in the absence of shear). But we have also observed a segregation of β-casein and monopalmitin domains at the interface during the

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Figure 5. (A1,A2) Visualization of β-casein and monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The evolution of the gray level along the interface is included for images A1 and A2. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monopalmitin at XMP ) 0.5 and (solid line) at 45 mN/m and (crosses) at the monolayer collapse. The surface shear viscosity for a pure monopalmitin monolayer at 45 mN/m (striped pattern) and at the collapse point (checked pattern) is included as a reference. The arrows (marked A1 and A2) correspond to the time at which A1 and A2 BAM images were taken. Width of canal Wc ) 2.0 mm. Monolayer flow through the canal Q ) 3.75 × 10-6 m2 s-1. Temperature ) 20 °C. pH ) 7.

flow of the monolayer. In Figure 4, image A2 shows the frontier between regions of collapsed β-casein domains and LC domains of monopalmitin separated by a shear plane, which is indicated in the figure by means of two arrows. The existence of these isolated spots with collapsed β-casein residues was the cause of the peaks observed in the I-π plot (Figure 1B). Finally, near to the monolayer collapse (at 45 mN/m) or at the collapse point, the ηs values of a β-caseinmonopalmitin mixed monolayer at XMP ) 0.5 were similar to those for a pure monopalmitin monolayer (Figure 5B). These results suggest that in this region the mixed monolayer was dominated by the presence of monopalmitin at the interface. In fact, at the higher surface pressures, the topography was dominated for monopalmitin with a solid structure (Figure 5, image A1) or collapsed monopalmitin film with the presence of some fractures of collapsed β-casein (which were not representative of the overall topography of the mixed film) aligned in the direction of the flow as typical features of the mixed film (Figure 5, image A2). This topography under shear conditions is in good agreement with the absence of reflectivity peaks in the I-π plot and with the decrease

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Figure 6. (A) Surface pressure (π)-area (A) isotherm of (solid line) β-casein, (dashed line) monoolein, and (open circles) β-casein-monoolein mixed film at XMO ) 0.5. The arrows indicate the equilibrium surface pressure of β-casein (πe (BC)) and the collapse pressure of monoolein (πc (MO)). (B) Relative reflectivity as a function of surface pressure for (solid line) β-casein, (dashed line) monoolein, and (open circles) β-caseinmonoolein mixed film at XMO ) 0.5. Temperature ) 20 °C. pH ) 7.

in relative reflectivity to a value close to that for a pure monopalmitin monolayer (Figure 1B). Structural, Topographical, and Surface Shear Characteristics of β-Casein-Monoolein Mixed Monolayers at the Air-Water Interface. Structural and Topographical Characteristics. The π-A isotherms for monoolein, β-casein, and a β-casein-monoolein mixed monolayer at a mass fraction of monoolein in the mixture of XMO ) 0.5 (as an example) are shown in Figure 6A. As expected,31 monoolein monolayers had a liquid-expanded structure under all experimental conditions. The collapse of monoolein monolayers occurred at a surface pressure close to the equilibrium surface pressure (πe = 45.7 mN/ m). The monolayer structure in a β-casein-monoolein mixed monolayer at XMO ) 0.5 was liquid-expanded-like, as for pure monolayer components. At surface pressures higher than that for β-casein collapse, the π-A isotherm for the mixed monolayer was parallel to that of pure monoolein. These results suggest that the composition of the mixed monolayer was very dependent on the surface pressure. At surface pressures lower than that for β-casein collapse, a mixed monolayer of monoolein and β-casein could exist, β-casein molecules adopting different structures in the isotropic liquid-expanded monoolein monolayer, depending on the surface pressure, as described above. However, at surface pressures higher than that for β-casein collapse, the mixed monolayer was practically dominated by monoolein molecules. That is, at higher surface pressures, collapsed β-casein residues may be displaced from the interface by monoolein molecules.5 The I values (Figure 6B) for a β-casein-monoolein mixed film at XMO ) 0.5 were similar to that for β-casein or monoolein as the surface pressures are lower than 10 (31) Rodrı´guez Nin˜o, Ma. R.; Rodrı´guez Patino, J. M.; Carrera, C.; Cejudo, M.; Navarro, J. M. Chem. Eng. Commun. 2003, 190, 15.

Sa´ nchez and Rodrı´guez Patino

Figure 7. (A) Visualization of β-casein and monoolein mixed monolayers at XMO ) 0.5 by BAM. The evolution of the gray level along the interface is included for image A. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monoolein at XMO ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal, Q (m2 s-1): (solid line) 3.75 × 10-6 and (crosses) 1.25 × 10-5. The surface shear viscosity for pure β-casein (striped pattern) and monoolein (checked pattern) monolayers is included as a reference. Surface pressure ) 10 mN/m. Temperature ) 20 °C. pH ) 7.

(as β-casein molecules exist as trains with all amino acid segments located at the interface) or in the range of 10 < π < 20 mN/m (as β-casein amino acid segments adopt the form of loops and tails). In the range of 20 < π < 30 mN/m, the I values for some intensity peaks were similar to those for a collapsed β-casein monolayer, indicating that at a microscopic level domains of collapsed β-casein were present at the interface. At surface pressures higher than 30 mN/m, the I-π plot for the mixed film approached that for the pure monoolein monolayer, denoting the displacement of β-casein by monoolein from the air-water interface. Finally, the mixed film collapsed at a surface pressure similar to that for monoolein. These results suggests that β-casein and monoolein are practically immiscible with regions of monoolein or β-casein alternating at the air-water interface, depending on the surface pressure. The Effect of Surface Pressure on Surface Shear Characteristics. The time evolution of ηs during the flow of a β-casein-monoolein mixed monolayer at XMO ) 0.5 through the canal is shown in Figures 7-9 as a function of surface pressure. In contrast with β-casein-monopalmitin monolayers, the steady state in ηs values was reached more quickly for a monoolein- than for a monopalmitin-β-casein mixed monolayer, as was observed for pure monoglyceride monolayers.16 On the other hand, at the steady state the fluctuations in ηs value for a β-casein-monoolein mixed monolayer were only observed for a specific range of surface pressures near to the collapse of β-casein (Figure 8B). Over the overall range of surface pressures analyzed, the values of ηs did not depend on the flow Q within the experimental error, which is an indication that β-casein-monoolein mixed mono-

Flow-Induced Molecular Segregation

Figure 8. (A1-A3) Visualization of β-casein and monoolein mixed monolayers at XMO ) 0.5 by BAM. Image A3 corresponds to image A2 but with the automatic gain control switched off. The evolution of the gray level along the interface is included for images A1 and A2. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monoolein at XMO ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal, Q (m2 s-1): (Timea, solid line) 3.75 × 10-6 and (Timeb, crosses) 1.25 × 10-5. The surface shear viscosity for pure β-casein (striped pattern) and monoolein (checked pattern) monolayers is included as a reference. The arrows (marked A1 and A2) correspond to the time at which A1 and A2 BAM images were taken. Surface pressure ) 20 mN/m. Temperature ) 20 °C. pH ) 7.

layers display Newtonian behavior. The behavior of the mixed monolayer at 20 mN/m is an exception (Figure 8B). 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 practically did not depend on the surface pressure (Figures 7-9). Interestingly, the values of ηs were very similar to those for a pure monoolein monolayer. These results indicate that the shear characteristics of the mixed monolayer are dominated by the presence of monoolein at the interface. However, at 20 mN/m some peaks in ηs were observed and extended in time (Figure 8B), which may be associated with the flow of large regions of collapsed β-casein through the canal. The peaks had ηs values lower than that for a pure β-casein monolayer, indicating that the monolayer flow was dominated by the presence of β-casein, but the LE domains of monoolein produced a reduction in the friction in the mixed monolayer in relation to that for a pure β-casein monolayer. The results of BAM corroborate these hypotheses. In fact, close to the β-casein collapse (at 20 mN/m), images

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Figure 9. (A1,A2) Visualization of β-casein and monoolein mixed monolayers at XMO ) 0.5 by BAM. The evolution of the gray level along the interface is included for images A1 and A2. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of β-casein and monoolein at XMO ) 0.5 and at (solid line) 40 mN/m and at (crosses) the monolayer collapse, at Wc ) 0.9 mm, and at Q ) 1.25 × 10-5 m2 s-1. The surface shear viscosity for a pure monoolein monolayer at 40 mN/m and at the collapse point (checked pattern) is included as a reference. Temperature ) 20 °C. pH ) 7.

A1 and A2 (Figure 8) show regions of monoolein (A1) and β-casein (A2) domains, respectively, during the flow of the mixed film. The presence of β-casein domains was corroborated by the saturation of the camera shown in image A3, which corresponds to image A2 but with the automatic gain control switched off. As a consequence of this topography, the gray level (monolayer film thickness) presented minor fluctuations along the interface. The existence of these segregated spots was the cause of the peaks observed in the I-π plot (Figure 6B). These phenomena were an indication that a shear-induced segregation was produced in the mixed monolayer, with the flow of homogeneous LE monoolein domains (Figure 8, image A1) and collapsed β-casein domains (Figure 8, image A2). Surprisingly, after the β-casein collapse, at 30 mN/m (data not shown), at 40 mN/m (Figure 9), and at the collapse point of the mixed film (Figure 9), the ηs values of β-casein-monoolein mixed monolayers were similar to those for a pure monoolein monolayer. In fact, in the absence of shear we have observed that the lower surface activity of monoolein justified the idea that this monoglyceride has a lower capacity than monopalmitin for protein displacement,5 a phenomenon that was not observed in this work under shear conditions. The homogeneous topography of the mixed monolayer (see images A1 and A2

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in Figure 9) was also characteristic of a monoolein monolayer,20 with the presence of some fractures of collapsed β-casein (which were not representative of the overall topography of the mixed film) aligned in the direction of the flow as typical features of the mixed film (Figure 9, image A2). However, the presence of small spots of collapsed β-casein have no significant repercussion on the ηs values of the mixed film under these experimental conditions. Clearly, β-casein displacement by monoolein was facilitated by the monolayer flow. The ηs values of β-casein-monoolein mixed monolayers are directly linked with their structural and topographical characteristics (Figure 6). The lower values of ηs were observed for a liquid-expanded structure of the monoolein monolayer, coinciding with a more homogeneous flow of the monolayer through the canal, even at the higher values of Q. These results confirm again the extremely sensitive dependence of surface shear characteristics on the monolayer structure, including the shear-induced segregation in the mixed film. All these phenomena are a consequence of the practical immiscibility between film-forming components.5 Conclusions In this work, we have used different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial shear rheology) to analyze the static (structure, topography, reflectivity, miscibility, and interactions) and flow characteristics (surface shear characteristics) of β-casein and monoglyceride (monopalmitin and monoolein) mixed films spread on the air-water interface. The structural and topographical characteristics obtained in a modified Wilhelmy type trough in the absence of shear are in good agreement with those obtained in the same unmodified trough and in the Langmuir type trough with the same β-casein, monoglyceride (monopalmitin or monoolein), and β-caseinmonoglyceride mixed films. Monopalmitin monolayers present a structural polymorphism as a function of surface pressure, while monoolein and β-casein only present a liquid-expanded-like structure over the overall range of surface pressures. At surface pressures lower than that for β-casein collapse, β-casein and monoglyceride domains are present at the interface in the mixed film, but with few interactions between them. However, at surface

Sa´ nchez and Rodrı´guez Patino

pressures higher than that for β-casein collapse the structural and topographical characteristics of the mixed film were dominated by the presence of the monoglyceride at the interface. The shear characteristics of pure and β-casein-monoglyceride mixed films are sensitive to the structural characteristics of the monolayer. The ηs values were higher for β-casein than for monoglyceride, with the lower ηs values for monoolein. The differences observed between monoglyceride and β-casein monolayers in shear rheology justify the use of this technique to analyze the interfacial characteristics (structure, interactions, miscibility, squeezing-out phenomena, etc.) of β-caseinmonoglyceride mixed films at the air-water interface and thus enable a global consistency check on the same monolayer. The ηs value varies greatly with the surface pressure (or surface density) of the mixed monolayer at the interface. In general, the greater the surface pressure (i.e., at the higher surface density), the greater the values of ηs. Moreover, the ηs value is also sensitive to the miscibility and/or displacement of film-forming components at the interface. At surface pressures lower than that for β-casein collapse, a shear-induced change in the topography of monopalmitin and β-casein domains was observed. At surface pressures higher than that for the β-casein, the above-mentioned changes in the topography of the mixed monolayer are accompanied by the squeezing of collapsed β-casein domains by monoglycerides. Near to the collapse point, the mixed film is dominated by the presence of the monoglyceride. Different proteins and monoglycerides show different interfacial topography, confirming the importance of protein and emulsifier structure in determining the interfacial interactions.32 But this work has demonstrated that there exist differences in β-casein displacement by monoglycerides (monopalmitin or monoolein) in the absence or in the presence of shear. In fact, under shear conditions the additional segregation of the monolayer facilitates the displacement of the β-casein by the monoglyceride, especially for β-casein-monoolein mixed films. Acknowledgment. The authors acknowledge the support of CICYT through Grant AGL2001-3843-C02-01. LA049625R (32) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R.; Carrera, S. C.; Cejudo, M. Langmuir 2001, 17, 7545.