Protein Displacement by Monoglyceride at the AirWater Interface

Jun 20, 2007 - Shear Rheology Combined with Brewster Angle Microscopy. Juan M. ...... Surface shear viscosities of pure protein and monoglyceride (pro...
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J. Phys. Chem. B 2007, 111, 8305-8313

8305

Protein Displacement by Monoglyceride at the Air-Water Interface Evaluated by Surface Shear Rheology Combined with Brewster Angle Microscopy Juan M. Rodrı´guez Patino,* Cecilio Carrera Sa´ nchez, Marta Cejudo Ferna´ ndez, and M. Rosario Rodrı´guez Nin˜ o Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, UniVersidad de SeVilla, c/o Prof. Garcı´a Gonza´ lez, 1, E-41012-SeVilla, Spain ReceiVed: March 12, 2007; In Final Form: April 30, 2007

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 milk protein (β-casein, caseinate, and β-lactoglobulin) and monoglyceride (monopalmitin and monoolein) mixed films spread and adsorbed 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 (π). In general, the greater the π values, the greater were 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 protein collapse, protein and monoglyceride coexist at the air-water interface. At surface pressures higher than that for the protein collapse, a squeezing of collapsed protein domains by monoglycerides was deduced. Near to the collapse point, the mixed film is dominated by the presence of the monoglyceride. Different proteins and monoglycerides show different interfacial structure, topography, and shear viscosity values, confirming the importance of protein and monoglyceride structure in determining the interfacial characteristics (interactions) of mixed films. The values of ηs are lower for disordered (β-casein or caseinate) than for globular (β-lactoglobulin) proteins and for unsaturated (monoolein) than for saturated (monopalmitin) monoglycerides in the mixed film. The displacement of the protein by the monoglycerides is facilitated under shear conditions.

Introduction 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.1-6 Interfacial rheology is also a very sensitive technique to monitor the interfacial structure and concentration of single emulsifiers at the interface or the relative concentration, the competitive adsorption, and the magnitude of interactions between different emulsifiers at the interface.7-9 Interfacial rheology can be defined 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.1-3,10-15 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.16,17 Interfacial shear rheology is most useful for polymer and mixed polymer surfactant adsorption layers and insoluble monolayers and gives access to interaction forces in two-dimensional layers.18,19 In addition, the shear may induce heterogeneity and segregation between protein and lipid domains at the interface during the flow of the monolayer. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers,20-25 but less is known about adsorbed films.25-27 * To whom all correspondence should be addressed. Tel: +34 95 4556446. Fax: +34 95 4557134. E-mail: [email protected].

Many real food dispersions (emulsions and foams) are formulated by a mixture of low-molecular weight emulsifiers (lipids, phospholipids, and surfactants) and biopolymers (proteins and some polysaccharides).28,29 These emulsifiers are amphiphilic in nature, which means that they have a tendency to adsorb at fluid interfaces. The distribution of emulsifiers (i.e., lipids and proteins) at fluid interfaces is determined by competitive or co-operative adsorption between the two types of emulsifiers, which in turn depend on interactions between emulsifier molecules.8,30 Interactions between molecules of emulsifiers could affect the most important properties of the adsorbed film (such as surface activity, kinetics of the film formation, structure, miscibility, superficial viscosity, etc.).7,8,16,17 Thus, the optimum use of these emulsifiers in relation to the stability, textural, and mechanical properties of dispersed food systems depends on the way in which the constituent emulsifiers adsorb and interact at fluid interfaces.28,29 In this paper we are concerned with the analysis of structural, topographical, and shear characteristics of mixed monolayers formed by a spread or adsorbed milk protein (β-casein or β-lactoglobulin) and a spread monoglyceride (monopalmitin or monoolein). β-Casein and β-lactoglobulin are food model proteins, which are distinguished by their good foaming and emulsifying properties, and for these reasons they are widely used in combination with monoglycerides in real food formulations.28,29,31 Monolayer technique has been used successfully for studying the properties of mixed emulsifiers spread8 and adsorbed and/or penetrated32 at the air-water interface.

10.1021/jp071994j CCC: $37.00 © 2007 American Chemical Society Published on Web 06/20/2007

8306 J. Phys. Chem. B, Vol. 111, No. 28, 2007 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% purity. β-Casein (>99%) was kindly supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Caseinate (a mixture of ≈38% β-casein, ≈39% Rs1-casein, ≈12% κ-casein, and ≈11% Rs2-casein) was kindly supplied and purified from bulk milk by Unilever Research (Colworth, U.K.). Whey protein isolate, a native protein with very high content of β-lactoglobulin (protein 92% ( 2%, β-lactoglobulin >95%, R-lactalbumin 99.8%) were used. The water used as 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 surface pressure (π)-area (A) isotherms of protein + monoglyceride mixed films at the air-water interface were performed on a fully automated modified Wilhelmy-type film balance, which integrates Brewster angle microscopy and a canal viscometer, as described previously.33 Before each measurement, the film balance was calibrated at 20 °C. Two different protocols were adopted, depending on whether the mixed films were formed by spreading or by adsorbing the protein at the fluid interface. For spread protein films, aliquots of aqueous solutions of protein at pH 7 were spread on the interface by means of a micrometric syringe. For adsorbed protein films from water, a protein solution was left in the trough and time was allowed for protein adsorption at the interface. The protein concentration was selected from previous data of the adsorption isotherm.34 At this protein concentration in solution, the surface pressure at equilibrium must be zero. Afterward, a monoglyceride solution in a hexane/ethanol mixture was spread at different points on the protein film. In preliminary experiments it was observed that the spreading solvent had no effect on the adsorbed protein monolayer. Mixtures of particular mass fractionssexpressed as the mass fraction of monopalmitin, XMP, or monoolein, XMO, in the mixtureswere studied. The compression rate was 49.5 cm2‚min-1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. The π-A isotherm was measured five times. The reproducibility of the results was better than (0.5 mN/m for surface pressure and (0.05 m2/mg for area. Brewster Angle Microscopy. 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.35,36 The surface pressure measurements, area, and imaging at specific surface pressures were carried out simultaneously by means of a device connected between the film balance and BAM. 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

Rodrı´guez Patino et al. 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.35,36 The imaging conditions were adjusted to optimize both image quality and quantitative measurement of reflectance (I) by increasing the shutter speed as the surface pressure or the protein content increased. Surface Shear Rheometry. To study the shear characteristics of adsorbed and spread films a homemade canal viscometer was used in this work. It is an analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past one another, and is described elsewhere.33 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 surface shear viscosity (ηs) was calculated from the rate of film flow (Q, square meters per second, recorded as a variation of the area associated with the movement of the barrier as a function of time) by a simplified equation (eq 1), which is analogous to the Poiseuille equation for the flow of liquids through capillary tubes.37 Strictly, eq 1 should be applicable only for Newtonian films. However, reliable surface shear data have presented data for lipids (monoglycerides) and disorder and globular proteins, for specific flow conditions.33

(π2 - π1)Wc3 ηS ) 12QL

(1)

The monolayer is allowed to flow through the canal (of width Wc and fixed length L ) 30 mm) 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. 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. Results and Discussion Structural, Topographical, and Shear Characteristics of Protein and Monoglyceride Films. Different structures can be deduced from π-A isotherm (Figure 1A) and BAM images for a monopalmitin monolayer as a function of surface pressure. A liquid expanded phase (LE, at π < 5 mN/m), liquid-condensed (LC, at 5 < π < 32 mN/m) (Figure 1c), and solid (S, at 32 < π < 53 mN/m) (Figure 1d) structures, and, finally, collapse and monolayer fracture (Figure 1e) at a surface pressure of about 53.1 mN/m (a surface pressure a little higher than the equilibrium surface pressure, πeMP ) 49.0 mN/m) were observed. The equilibrium surface pressure (πe) is the maximum surface pressure at which a spread monolayer can be compressed just before the monolayer collapse. The values of πe for pure components were taken from the literature.38 The evolution with the monolayer compression of the relative thickness (I) confirms the rich structural polymorphism of monopalmitin monolayers (Figure 1B). It can be seen that I increases as the monolayer is compressed, passes through a maximum at the end of the LC structure, and then decreases at the collapse point. The I peaks were observed as the circular monopalmitin domains with LC structure passed through the spot where this measurement was performed, denoting a heterogeneity in the monolayer structure at a microscopic level.

Interface Displacement of Protein by Monoglyceride

Figure 1. (A) Surface pressure-area isotherms (compression curves) for spread monolayers of monopalmitin, monoolein, β-casein, and β-lactoglobulin; and for adsorbed films of β-casein and β-lactoglobulin at the air-water interface. (B) Relative reflectivity (arbitrary units) as a function of surface pressure during the compression of monoglyceride and protein spread monolayers. Symbols (open symbols are for spread monolayers and solid symbols are for adsorbed films): (], - ‚ -) monopalmitin, (g, ‚‚‚) monoolein, (b, O, s) β-casein, and (2, 4, ---) β-lactoglobulin. The equilibrium surface pressures for monopalmitin (πeMP ) 49.0 mN/m), monoolein (πeMO ) 45.7 mN/m), β-casein (πeβ-CS ) 20.9 mN/m), and β-lactoglobulin (πeβ-LG ) 25.9 mN/m) are indicated by means of arrows.34 Visualization by Brewster angle microscopy: (a) protein aggregates in adsorbed films at the end of the first compression and at the beginning of a subsequent compression; (b) this image was observed for protein, monoolein, and monopalmitin (at π < 5 mN/m) spread films; (c) LC domains of monopalmitin at 5 < π < 32 mN/m; (d) solid domains of monopalmitin at π > 32 mN/m; and (e) fractures in monopalmitin monolayers at the collapse point indicated by an arrow. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm. Temperature 20 °C, pH 7.

Monoolein monolayer presents only the liquid expanded structure and collapses at the equilibrium surface pressure (πeMO ≈ 45.7 mN/m). BAM images corroborate that only the homogeneous LE phase is present during the compression of a monoolein monolayer up to the collapse point (Figure 1b). The values of I increase monotonically with π, which corroborates the fact that during the compression a denser monoolein film is formed but without any change in its structure (Figure 1B). The π-A isotherms (Figure 1A) and I-π plots (Figure 1B) confirm that β-casein monolayers at the air-water interface adopt two different structures and the collapse phase. At low surface pressures (π < 12-14 mN/m), β-casein molecules exist with a tail-train structure with amino acid segments located at the interface. At higher surface pressures, and up to the collapse point, amino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails. However, the residues of β-casein molecules at the air-water interface appeared to be of uniform thickness (Figure 1B) and isotropy

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Figure 2. Surface pressure dependence of surface shear viscosity for (A) spread and (B) adsorbed monoglyceride and protein films at the air-water interface. Symbols: (], - ‚ -) monopalmitin, (g, ‚‚‚) monoolein, (4, ---) β-casein, and (O, s) β-lactoglobulin. Temperature 20 °C, pH 7.

(Figure 1b). The relative thickness (Figure 1B) increased with the surface pressure and was a maximum at the collapse point (at πeβ-CS ) 20.9 mN/m). Contrary to β-casein, a flexible protein lacking secondary structure, β-lactoglobulin, a globular protein with a compact conformation, retains elements of the native structure, not fully unfolded at the interface. Thus, most amino acid residues in β-lactoglobulin adopt a 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 (at πeβ-LG ) 25.9 mN/m). The residues of β-lactoglobulin molecules at the air-water interface appeared to be of uniform reflectivity, suggesting homogeneity in thickness and film isotropy (Figure 1b). The relative thickness increases monotonically with the surface pressure and was a maximum at the collapse point (Figure 1B). The π-A isotherm deduced for adsorbed β-casein monolayer27 is more condensed than that obtained directly by spreading.33 However, β-lactoglobulin monolayers show ggood agreement between adsorbed and spread isotherms (Figure 1A).24 These results indicate that β-casein may be more unfolded during the spreading at the interface, whereas β-lactoglobulin can maintain part of its native structure (in the form of loops and tails) as it is spread or adsorbed at the interface. These differences are confirmed at microscopic level. In fact, even for a visible “homogeneous” interface of collapsed adsorbed β-casein domains, the presence of local heterogeneities was present at the interface during the monolayer expansion, at the lower surface pressures (Figure 1a). The domains of collapsed β-casein for adsorbed monolayers (Figure 1a) were not observed for spread monolayers (Figure 1b). Figure 2A shows the surface pressure dependence of ηs for monoglyceride and protein spread monolayers.33 It can be seen

8308 J. Phys. Chem. B, Vol. 111, No. 28, 2007 that over the overall range of surface pressures analyzed, the values of ηs for monoolein monolayers were very small and that this value practically did not depend on the surface pressure. These values of ηs were very similar to those for monopalmitin monolayers in the LE-LC phase transition at π < 20 mN/m. However, the value of ηs for monopalmitin monolayers was 17.5 times higher for an S than for an LC monopalmitin monolayer structure and was more than 7 times higher at the beginning than at the end of the LE to LC transition. These results confirm the extremely sensitive dependence of surface shear characteristics on the monolayer structure. That is, the ηs values of a monoglyceride (monopalmitin or monoolein) monolayer are directly linked with their structural and topographical characteristics (Figure 1). The lower values of ηs were observed for a liquid-expanded structure of the monolayer (Figure 2A). The values of ηs for β-lactoglobulin are also higher than those for β-casein under the same experimental conditions and increase with the surface pressure (Figure 2). The differences in solution structure between β-casein and β-lactoglobulin are reflected in their adsorption or spreading behavior at the airwater interface. Differences between surface shear viscosity for 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 (which can only form physical gels stabilized by intermolecular hydrogen bonds). The values of ηs are higher for spread (Figure 2A) than for adsorbed (Figure 2B) protein films.26,32,33 Thus, although the π-A isotherms for spread and adsorbed β-lactoglobulin monolayers are practically the same and adsorbed β-casein monolayer is more condensed than that obtained by spreading (Figure 1), the surface shear viscosity is different (Figure 2). These results confirm that the unfolding of the protein during the spreading at the interface is different for β-casein as compared to β-lactoglobulin. In summary, combined π-A isotherms, BAM (monolayer morphology and reflectivity), and surface shear measurements on the same monolayer provide complementary information on the characteristics of emulsifiers (monoglycerides and proteins) at fluid interfaces. The experiments have demonstrated the sensitive dependence of the interfacial shear viscosity (ηs) on the surface pressure of emulsifier (protein or monoglyceride) films at the air-water interface. In general, the greater the emulsifier surface pressure (i.e., at the higher surface density), the greater the values of ηs. At the same π, the value of ηs was higher for proteins than for monoglycerides. The higher ηs values were observed for β-lactoglobulin monolayers. Finally, the ηs value is also sensitive to the monolayer structure, as was observed for monoglycerides with a rich structural polymorphism (i.e., monopalmitin) and to the capacity of the protein to form physical (such as for β-casein) or covalent-disulfide (such as for β-lactoglobulin) interfacial gels, with the higher values of ηs for the latter. Structural Characteristics of Protein and Monoglyceride Spread and Adsorbed Mixed Films. The structural characteristics of protein and monoglyceride spread23,24 and adsorbed25,26 mixed films can be deduced from the π-A isotherms shown in Figure 3, for a particular mass fraction of monoglyceride in the mixture of 0.5 (as an example). Data for spread mixed films confirm that, at surface pressures lower than the equilibrium surface pressure of protein (at π < πeβ-CS or at π < πeβ-LG), both protein (β-casein or β-lactoglobulin) and monoglyceride (monopalmitin or monoolein) coexist at the

Rodrı´guez Patino et al.

Figure 3. Surface pressure-area isotherms for (A) spread (compression curves) and (B) adsorbed (compression-expansion curves) mixed films at the air-water interface. Symbols: (O) β-casein + monopalmitin, (s) β-casein + monoolein, (4) β-lactoglobulin + monopalmitin, and (- - -) β-lactoglobulin + monoolein. The equilibrium spreading pressure for β-casein (πeβ-CS) and β-lactoglobulin (πeβ-LG) is indicated by means of arrows. Protein displacement by monoglyceride or coexistence of protein and monoglyceride is deduced at surface pressures higher than or lower than the equilibrium spreading pressure of the protein, respectively. At every surface pressure the interactions between protein and monoglyceride are quite weak. For more information see the text. Temperature 20 °C, pH 7.

interface (Figure 3A). The structural polymorphism for protein + monopalmitin and the homogeneous LE structure for protein + monoolein mixed films can be seen. The coexistence of protein and monoglyceride is proved at a microscopic level by means of BAM images and film reflectivity.23,24 By means of the excess area and excess free energy, it can be also deduced that protein and monoglyceride form a mixed monolayer at the air-water interface with few attractive or repulsive interactions between film-forming components.23,24 At surface pressures higher than that for protein collapse (at π > πeβ-CS or at π > πeβ-LG), the π-A isotherm for mixed monolayers is parallel to that of monoglyceride and, finally, the collapse pressure of the mixed film coincides with that for pure monoglyceride. These results suggest that at the higher surface pressures: (i) the arrangement of the monoglyceride hydrocarbon chain in mixed monolayers is practically the same in the entire monoglyceride/protein fraction, (ii) the protein is displaced by the monoglyceride from the air-water interface, and finally, (iii) 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

Interface Displacement of Protein by Monoglyceride monolayers is similar to that of a pure monopalmitin monolayer (Figure 3A). Data for adsorbed mixed films confirm the same features as those deduced for spread mixed films. In fact, at π < πeβ-CS or at π < πeβ-LG, both protein (β-casein or β-lactoglobulin) and monoglyceride (monopalmitin or monoolein) coexist at the interface (Figure 3B). At surface pressures higher than that for protein collapse (at π > πeβ-CS or at π > πeβ-LG) the protein is displaced by the monoglyceride from the air-water interface. Upon expansion and further compression, the π-A isotherms were repeatable for both spread (data not shown) and adsorbed (Figure 3B) mixed films, which suggests that the protein displaced by monopalmitin during the compression (at π > πeβ-CS or at π > πeβ-LG) reenters the mixed monolayer upon expansion. This fact 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. However, some differences exist between spread and adsorbed mixed films: (i) Condensation of spread mixed film is similar no matter what the monolayer composition (β-lactoglobulin + monoolein mixed film is an exception because this monolayer is more expanded). (ii) β-Casein + monoglyceride adsorbed mixed films are more expanded than β-lactoglobulin + monoglyceride adsorbed mixed films. (iii) β-Casein + monopalmitin are more expanded than β-casein + monoolein mixed films, but the opposite was observed for β-lactoglobulin monoglyceride mixed films. (iv) For adsorbed protein + monoglyceride mixed films, a firstorder-like phase transition (with a degenerated plateau in the π-A isotherm) was observed upon the monolayer expansion at surface pressures close to the equilibrium surface pressure of the protein (at π ≈ πeβ-CS or at π ≈ πeβ-LG) (Figure 3B). These results suggest that for adsorbed mixed films the readsorption of previously displaced protein has a kinetic character, which was not evident for spread mixed films (data not shown). Topographical and Shear Characteristics of Protein and Monoglyceride Spread Mixed Films: (A) β-Casein + Monoglyceride Mixed Films. The effect of surface pressure on surface shear characteristics of β-casein-monopalmitin mixed films at XMP ) 0.5 is shown in Figure 4A.23 Similar results were observed for mixtures of monoglycerides and a real disordered protein (sodium caseinate).39 During the flow of the monolayer through the canal, the ηs value increases with time and tends to a “steady-state” plateau value. The “steadystate” 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 (anisotropy) of the mixed monolayer. The presence of circular LC monopalmitin domains on a homogeneous β-casein phase at 10 mN/m (Figure 4Aa) and the incipient squeezing out of β-casein by monopalmitin at 20 mN/ mswith many LC domains of monopalmitin (circular dark regions) floating over a sublayer of collapsed β-casein molecules (white image with a reflectivity similar to that for pure β-casein) (Figure 4Ab)scan be visualized by BAM. At π < πeβ-CS, the “steady-state” ηs value was of the same order of magnitude as those of pure film components (the “steady-state” ηs values for pure monopalmitin and β-casein monolayers are included in Figure 4A as reference), denoting the coexistence of both

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Figure 4. Surface pressure dependence of surface shear viscosity for (A) β-casein + monopalmitin and (B) β-casein + monoolein spread mixed films at the air-water interface. Surface pressure (in millinewtons per meter): (s) 10, (---) 20, and (‚‚‚) 40. Surface shear viscosities of pure protein and monoglyceride (protein/monoglyceride) adsorbed and spread films are included as reference. Visualization by Brewster angle microscopy: β-casein + monopalmitin spread mixed films at (Aa) 10, (Ab) 20, and (Ac) 40 mN/m, and β-casein + monoolein spread mixed films at (Ba) 10, (Bb) 20, and (Bc) 40 mN/m. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm. The presence of segregation (images Ac and Bc) in the monolayer is indicated by means of an arrow. Temperature 20 °C, pH 7.

components in the mixed film. At π > πeβ-CS, the ηs value is much lower than that for β-casein at the collapse point and tends to that for a pure monopalmitin monolayer, indicating that in this region the monolayer flow was dominated by the presence of LC domains of monopalmitin. However, the presence of segregated regions of LC monopalmitin domains and collapsed β-casein domains (Figure 4Ac) produces a reduction in the friction in the mixed film in relation to that for a pure monopalmitin monolayer. These phenomena are an indication that segregation in the mixed film was produced during the flow of monopalmitin and β-casein domains through the canal. The time evolution of ηs during the flow of a β-caseinmonoolein mixed monolayer at XMO ) 0.5 through the canal is shown in Figure 4B as a function of surface pressure.23 At 10 mN/m the values of ηs are very similar to those for a pure β-casein monolayer, which indicate the effect of β-casein on the shear characteristics of the mixed monolayer. However, at 20 mN/m some peaks in ηs are observed, which may be associated with the flow of large regions of collapsed β-casein through the canal. The peaks have 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. Thus, although the results of BAM corroborate the existence of isotropy in the mixed film (Figure 4Ba,Bb) the existence of segregated regions of collapsed β-casein was deduced from the

8310 J. Phys. Chem. B, Vol. 111, No. 28, 2007

Figure 5. Surface pressure dependence of surface shear viscosity for (A) β-lactoglobulin + monopalmitin and (B) β-lactoglobulin + monoolein spread mixed films at the air-water interface. Surface pressure (in millinewtons per meter): (s) 10, (---) 20, and (‚‚‚) 40. Surface shear viscosities of pure protein and monoglyceride (protein/ monoglyceride) adsorbed and spread films are included as reference. Visualization by Brewster angle microscopy: β-lactoglobulin + monopalmitin spread mixed films at (Aa) 10, (Ab) 20, and (Ac) 40 mN/m, and β-lactoglobulin + monoolein spread mixed films at (Ba) 10, (Bb) 20, and (Bc) 40 mN/m. Temperature 20 °C, pH 7.

film reflectivity (data not shown) and is the cause of the fluctuations observed in the ηs-π plot (Figure 4B). At 40 mN/m the values of ηs for the mixed monolayer are practically the same as those for a pure monoolein monolayer, which indicates both that β-casein is displaced from the interface by monoolein (Figure 4Bc) and that the shear characteristics of the mixed monolayer are dominated by the presence of monoolein. These phenomena are an indication that a shear-induced segregation was produced in the mixed monolayer, even for the flow of homogeneous monoolein and β-casein domains. (B) β-Lactoglobulin + Monoglyceride Mixed Films. The time evolution of ηs during the flow of a β-lactoglobulinmonoglyceride mixed monolayer through the canal is shown in Figure 5 as a function of surface pressure.24 The main features are (i) large fluctuations in ηs value are observed at π < πeβ-LG, which may be due to the flow of segregated β-lactoglobulin, LC monopalmitin (Figure 5Aa,Ab), and LE monoolein (Figure 5Ba,Bb) domains. In fact, in this region the values of ηs are between those of pure components but a little lower. These results indicate that segregated domains of β-lactoglobulin and monopalmitin (Figure 5A) or monoolein (Figure 5B) flow through the canal, but with few interactions between them, a phenomenon that produces a reduction in the friction in the mixed film in relation to that for pure monolayer components. (ii) At π > πeβ-LG, ηs values tend to those of pure monoglyceride monolayer, which corroborates the idea that β-lactoglobulin is displaced from the interface by monoglyceride (monopalmitin or monoolein) and the flow of the mixed film is dominated by the presence of monopalmitin (Figure 5Ac) or monoolein (Figure 5Bc).

Rodrı´guez Patino et al.

Figure 6. Surface pressure dependence of surface shear viscosity for (A) β-casein + monopalmitin and (B) β-casein + monoolein adsorbed mixed films at the air-water interface. Surface pressure (in millinewtons per meter): (s) 10, (---) 20, and (‚‚‚) 40. Surface shear viscosities of pure protein and monoglyceride (protein/monoglyceride) adsorbed and spread films are included as reference. Visualization by Brewster angle microscopy: β-casein + monopalmitin adsorbed mixed films at (Aa) 10, (Ab) 20, and (Ac) 40 mN/m, and β-casein + monoolein adsorbed mixed films at (Ba) 10, (Bb) 20, and (Bc) 40 mN/m. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm. The fracture (image Ac) and segregation (image Bc) in the monolayer are indicated by means of arrows. Temperature 20 °C, pH 7.

Topographical and Shear Characteristics of Protein and Monoglyceride Adsorbed Mixed Films. The time evolution of ηs during the flow of β-casein- and β-lactoglobulinmonoglyceride adsorbed mixed monolayers through the canal as a function of surface pressure is shown in Figures 6 and 7, respectively.25,26 Similar results were observed for mixtures of monoglycerides and sodium caseinate.27 It can be seen that (i) at π < πeβ-CS and at π < πeβ-LG (at 10 and 20 mN/m), the values of ηs are between those of pure monolayer components but a little lower. These results corroborate the coexistence of protein and monoglyceride in the mixed film but with few interactions between film-forming components. (ii) At π > πeβ-CS and at π > πeβ-LG (at 40 mN/m), the values of ηs tend to those of pure monopalmitin (Figures 6A and 7A) or monoolein (Figures 6B and 7B) but are a little lower. Thus, although the protein (β-casein or β-lactoglobulin) is displaced from the interface by monoglyceride (monopalmitin or monoolein) and the flow of the mixed film is dominated by the presence of monoglyceride, the presence of fractures in β-caseinmonopalmitin (Figure 6Ac) or segregation between collapsed β-casein-monoolein (Figure 6Bc), β-lactoglobulin-monopalmitin (Figure 7Ac), and β-lactoglobulin-monoolein (Figure 7Bc) domains produces a reduction in the friction during the flow of the mixed film through the canal. (iii) Over the range of surface pressure analyzed an absence of fluctuations in ηs-θ curves is observed, which denotes low segregation in adsorbed mixed films because the domains of monoglyceride and protein are of

Interface Displacement of Protein by Monoglyceride

Figure 7. Surface pressure dependence of surface shear viscosity for (A) β-lactoglobulin + monopalmitin and (B) β-lactoglobulin + monoolein adsorbed mixed films at the air-water interface. Surface pressure (in millinewtons per meter): (s) 10, (---) 20, and (‚‚‚) 40. Surface shear viscosities of pure protein and monoglyceride (protein/ monoglyceride) adsorbed and spread films are included as reference. Visualization by Brewster angle microscopy: β-lactoglobulin + monopalmitin adsorbed mixed films at (Aa) 10, (Ab) 20, and (Ac) 40 mN/m, and β-lactoglobulin + monoolein adsorbed mixed films at (Ba) 10, (Bb) 20, and (Bc) 40 mN/m. The presence of segregation (images Ac and Bc) in the monolayer is indicated by means of an arrow. Temperature 20 °C, pH 7.

J. Phys. Chem. B, Vol. 111, No. 28, 2007 8311

Final Considerations

Figure 8. Surface pressure dependence of surface shear viscosity for spread (open symbols) and adsorbed (solid symbols) mixed films of (A) protein + monopalmitin and (B) protein + monoolein at the airwater interface. Symbols: b, O) β-lactoglobulin + monopalmitin; (2, 4) β-casein + monoolein mixed films. The ηs-π dependence values for pure (---) monopalmitin, (‚‚‚) monoolein, (- ‚ -) β-casein, and (s) β-lactoglobulin films are included as reference. Temperature 20 °C, pH 7.

In preceding sections we have deduced that different proteins and monoglycerides show different interfacial structure and topography, confirming the importance of protein and emulsifier molecules in determining the mechanism of interfacial interactions in spread and adsorbed mixed films, with direct repercussions on the surface shear characteristics. The comparison between spread and adsorbed mixed films is important because the concentrations of the film-forming components are wellknown in the former. Thus, spread mixed films can be used for fundamental studies as reference for the analysis of adsorbed mixed films, because in the latter the concentrations of the filmforming components are unknown in experiments involving a film balance.32 The data presented in this paper confirm that the structural and topographical characteristics, shear-induced segregation, and squeezing-out phenomena of mixed proteins and monoglycerides at the air/water interface observed with protein (β-casein and β-lactoglobulin) and monoglyceride (monopalmitin and monoolein) mixed films appear to be generic. The reasons for these behaviors must be associated with the immiscibility between protein and monoglyceride at the air-water interface, with the protein displacement by the monoglyceride at surface pressures higher than that for the protein collapse, and with the shearinduced segregation in the mixed films, as analyzed in preceding

sections. These phenomena have significant repercussions on surface shear properties of spread and adsorbed mixed films. That is, the surface shear viscosity reflects the complex phenomena that occur in protein-monoglyceride mixed films under flow conditions. The surface pressure dependence of surface shear viscosity for mixed films analyzed in this paper is summarized in Figure 8. From these data we deduce the following conclusions: (i) The ηs values vary greatly with the surface pressure (or surface density) of the mixed film at the interface. For protein + monopalmitin mixed films (Figure 8A), the greater the surface pressure (i.e., at the higher surface density), the greater were the values of ηs, as might be expected on the basis of greater intermolecular interactions. That is, as the interactions in the mixed monolayer between monopalmitin molecules (with a LC or solid structure in the monolayer) or between collapsed protein are at a maximum, the values of ηs are higher than those with the minimum interactions between monopalmitin molecules (with an LE structure in the monolayer). For protein + monoolein mixed films (Figure 8B), the evolution of ηs with π is more complex. At π < πeβ-CS and at π < πeβ-LG (at 10 and 20 mN/m) the values of ηs increase with π, but the opposite was observed at π > πeβ-CS and at π > πeβ-LG (at 40 mN/m).

lower size (BAM images in Figures 6 and 7) as compared with those of spread films (BAM images in Figures 4 and 5).

8312 J. Phys. Chem. B, Vol. 111, No. 28, 2007 In fact, after the protein collapse the values of ηs are lower that those for a pure monoolein monolayer. (ii) For all protein + monoglyceride mixed films at π > πeβ-CS and at π > πeβ-LG, the values of ηs tend to those of pure monopalmitin (Figure 8A) or monoolein (Figure 8B), which confirms the protein displacement by the monoglyceride and the importance of the monoglyceride on the surface shear viscosity of the mixed film. (iii) At π < πeβ-CS and at π < πeβ-LG, significant fluctuations are observed in the values of ηs for both protein + monopalmitin (Figure 8A) and protein + monoolein (Figure 8B), which corroborates the coexistence of protein and monoglyceride domains at the interface and the flow of both film-forming components through the canal. The fluctuations in the values of ηs are higher for spread as compared with adsorbed mixed films, which indicates that the segregation between protein and monoglyceride during the flow of the mixed film is higher in the former. At π > πeβ-CS and at π > πeβ-LG, the fluctuations in the values of ηs disappear, coinciding with the flow of monoglyceride through the canal. (iv) At π < πeβ-CS and at π < πeβ-LG, the values of ηs are lower for adsorbed than for spread mixed films. The same phenomenon was observed for pure protein films (Figure 2), which confirms the importance of the protein in the surface shear viscosity of the mixed film. However, at π > πeβ-CS and at π > πeβ-LG, the values of ηs are the same for adsorbed and spread mixed films (β-lactoglobulin + monopalmitin spread mixed film at 40 mN/m is the only exception), which again confirms that the mixed films are practically dominated by the presence of monoglyceride. In summary, the fluctuations in surface shear viscosity (ηs) during the flow of the film through the canal, leading to phase separation, have consequences for anisotropic structure formation in protein + monoglyceride mixed films. These fluctuations in the values of ηs, implying unstable flow behavior, are observed at π < πe of the protein as both monoglyceride and protein coexist at the interface. Thus, at these surface pressures, fluctuations in ηs values imply the existence of segregated regions of monoglyceride and protein domains, resulting in phase separation, which become aligned in the shear direction. The fluctuations are less intense for monopalmitin-protein as compared with monoolein-protein films and for adsorbed than for spread mixed films. Consequently, shear-induced phase separation is favored for monoolein-protein spread mixed films. Thus, the larger domains observed by BAM for spread as compared with adsorbed mixed films are more influenced by the shear flow. At π > πe of the protein, these fluctuations disappear as a consequence of the demixing between protein and monoglyceride, which finally leads to the squeezing of the protein from the interface. Thus, the microscopic anisotropy of the mixed film at π < πeprotein becomes isotropic at π > πeprotein because an isotropic solid structure of monopalmitin domains or an isotropic liquid expanded structure of monoolein predominates at the interface. All these phenomena are a consequence of the weak interactions between protein and monoglyceride at the air-water interface, especially at the higher surface pressures after the protein collapse (π < πeβ-CS or π < πeβ-LG). These results also confirm the extremely sensitive dependence of surface shear characteristics on the immiscibility between film-forming components and protein displacement for monoglyceride, including the shear-induced phase segregation in protein + monoglyceride mixed films. Acknowledgment. This research was supported by CICYT through Grant AGL2004-01306/ALI.

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