Whey Protein IsolateâMonoglyceride Mixed Monolayers at the Air

Langmuir 2001, 17, 7545-7553

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Whey Protein Isolate-Monoglyceride Mixed Monolayers at the Air-Water Interface. Structure, Morphology, and Interactions Juan M. Rodrı´guez Patino,* Ma. Rosario Rodrı´guez Ninõ, Cecilio Carrera Sańchez, and Marta Cejudo Fernańdez Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/ Profesor Garcıá Gonza´ lez, s/nu´ m, 41012-Seville, Spain Received February 22, 2001. In Final Form: May 2, 2001 The surface pressure (π)-area (A) isotherms and Brewster angle microscopy (BAM) of monoglyceride and whey protein isolate (WPI) mixed films spread on buffered water at pH 5 and 7 and at 20 °C were determined as a function of the mass fraction (X) of monoglyceride (monopalmitin or monoolein) in the mixture. From the X and π-dependence on excess area, free energy, collapse pressure, BAM images, and the evolution with π of the relative reflectivity (I) of BAM images, it was deduced that the structural characteristics, miscibility, and morphology of monoglyceride-WPI mixed films are very dependent on surface pressure and monolayer composition. The monolayer was more expanded as the monoglyceride concentration in the mixture was increased. Over the overall range of existence of the mixed film, the monolayer presents some heterogeneities. At higher π, after the WPI collapse, characteristic squeezing-out phenomena were observed. At the monoglyceride monolayer collapse, the mixed film was practically dominated by the presence of monoglyceride. However, some degree of interactions exists between monoglyceride and WPI in the mixed film, and these interactions are more pronounced as the monolayer is compressed at the highest surface pressures.

Introduction Food dispersions (emulsions and foams) are complicated multicomponent systems containing many emulsifiers which may show surface activity by themselves (proteins and low-molecular-weight emulsifiers) or by associating with each other.1-5 Whey proteins and monoglycerides are widely used in the food industry as emulsifiers and stabilizers in foams and emulsions. To control the production, stability, and other organoleptic and textural properties of food dispersions, detailed knowledge of the role of emulsifiers at fluid interfaces is required. Food proteins are distinguished by their good foaming and emulsifying properties and, for these reasons, are widely used in food formulations.6,7 Proteins show a strong tendency to adsorb to fluid interfaces (air-water and oilwater), and thus they find an important use in the manufacture of stable emulsions (i.e., ice cream, cream liqueurs, whipped toppings, coffee whiteners, and products for infant nutrition, etc.), where long-term emulsion stability is essential.8 On the other hand, in many food formulations, proteins are not the only emulsifiers present, because small molecule emulsifiers (monoglycerides, phospholipids, surfactants, etc.) are also incorporated in the formulation.3-5 The small molecule emulsifiers can * To whom correspondence should be addressed. Tel: +34 5 4557183. Fax: +34 5 4557134. E-mail: [email protected]. (1) Sjo¨blom, J. Emulsions and Emulsion Stability; Marcel Dekker: New York, 1996. (2) Damodaran, S.; Paraf, A. Food Proteins and their Applications; Marcel Dekker: New York, 1997. (3) Friberg, S. E.; Larsson, K. Food Emulsions, 3rd ed.; Marcel Dekker: New York, 1997. (4) Hartel, R.; Hasenhuette, G. R. Food Emulsifiers and their Applications; Chapman and Hall: New York, 1997. (5) Gunstone, F. D.; Padley, F. B. Lipid Technologies and Applications; Marcel Dekker: New York, 1997. (6) Halling, P. J. Crit. Rev. Food Sci. Nutr. 1981, 13, 155. (7) Dickinson, E. J. Chem. Soc., Faraday Trans. 1998, 80, 1657. (8) Dickinson, E. Colloids Surf., B 1999, 15, 161.

cover the interface that proteins do not, resulting in an emulsion with smaller particles, leading to greater stability. However, the effect of the small molecules in destabilizing the emulsion is more important in some products.9 Thus, an important effect of the small molecule emulsifiers is to promote the displacement of proteins from the interface.10 In particular, knowledge is required about the interactions between different emulsifiers at fluid interfaces, because when a mixture of proteins and lowmolecular-weight emulsifiers exists at the interface, a reduction in stability is often observed.11 This is believed to be because low-molecular-weight emulsifiers and proteins stabilize interfaces by different mechanisms.11,12 This work is an extension of previous studies of proteinlipid mixed monolayers spread at the air-water interface,13-16 which incorporate the application of a new noninvasive microscopic technique, namely Brewster angle microscopy (BAM). These studies led to the conclusions that monoglyceride (monopalmitin and monoolein) and proteins (β-casein and caseinate) form practically immiscible and heterogeneous mixed films at the airwater interface and that monoglyceride was unable to completely displace β-casein13,14 and caseinate15 from the interface. Recent work has allowed the indirect study by atomic force microscopy (AFM) of competitive adsorption (9) Goff, H. D.; Jordan, W. K. J. Dairy Sci. 1989, 72, 18. (10) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (11) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsions and their Applications; Hasenheutte, G. L.; Hartel, R. W., Eds.; Chapman and Hall: New York, 1997; p 95. (12) Coke, M.; Wilde, P. J.; Russell, E. J.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489. (13) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma. J. Agric. Food Chem. 1999, 47, 4998. (14) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma. R. Langmuir 1999, 15, 4777. (15) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, Ma. R.; Carrera, C. S.; Cejudo, F. M. J. Colloid Interface Sci., in press. (16) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Rodrı´guez Patino, J. M. Biomacromolecules 2001, 2, 1001.

10.1021/la0102814 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/03/2001

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of proteins and surfactants at a molecular level.17-19 This has led to the proposal that proteins are displaced by an “orogenic” mechanism.17 BAM images make it possible to visualize this orogenic displacement of proteins by surfactants directly at the interface.16 Experimental Section Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin) and 1-mono(cis-9-octacenoyl)glycerol (monoolein) were supplied by Danisco Ingredients with over 95-98% purity according to the supplier. 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. Whey protein isolate (WPI), a native protein with a high content of β-lactoglobulin (protein 92 ( 2%, β-lactoglobulin > 95%, R-lactalbumin < 5%) obtained by fractionation, was supplied by Danisco Ingredients (Brabran, Denmark). The sample was stored below 0 °C, and all work was done without further purification. Samples for interfacial characteristics of WPI films were prepared using Milli-Q ultrapure water and were buffered at pH 5 and 7. Analytical-grade acetic acid, sodium acetate, and Trizma [(CH2OH)3CNH2/(CH2OH)3CNH3Cl] for the buffered solution were used as supplied by Sigma (>95%) without further purification. Ionic strength was 0.05 M in all the experiments. Methods. Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on a fully automated Langmuir-type film balance, as described elsewhere.20,21 Mixtures of particular mass ratios (ranging between 0 and 1, expressed as the mass fraction of monoglyceride in the mixture, X) were studied. Aliquots of WPI (1.543 × 10-4 mg/µL) at pH 7 were spread on the interface by the Trurnit method.22 The subphase temperature was controlled at 20 °C by water circulation from a thermostat, within an error range of (0.3 °C. Each isotherm was measured at least 5 times. The reproducibility of the surface pressure results was better than (0.5 mN/m. Brewster Angle Microscope (BAM). A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the morphology of the monolayer, as described elsewhere.23,24 The relative thickness of the film was determined from the gray level (GL), according to a procedure described previously.23,24 These parameters can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light.25 At Brewster angle I ) |Rp|2 ) Cδ2, I is the relative reflectivity, C is a constant, δ is the film thickness, and Rp is the p component of the light.

Results Surface Pressure-Area Isotherms. Monopalmitin-WPI Mixed Films. Figure 1A shows the π-A isotherm for compression of monopalmitin-WPI mixed monolayers at 20 °C and pH 7. The monopalmitin monolayer adopts different structures as a function of surface pressure. A liquid-expanded (LE) structure was observed at π < 5 mN/m. A degenerate first-order phase (17) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (18) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. (19) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 8176. (20) Rodrı´guez Patino, J. M.; Ruı´z, M.; de la Fuente, J. J. Colloid Interface Sci. 1992, 154, 146. (21) Carrera, S. C.; Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 175. (22) Trurnit, H. J. J. Colloid Sci. 1960, 15, 1. (23) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma. R. Langmuir 1999, 15, 2484. (24) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma. R. Food Hydrocolloids 1999, 13, 401. (25) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1992.

Figure 1. (A) Surface pressure-area isotherms (compression curve) for monopalmitin-WPI mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (4) 0, (-) 0.2, (- - -) 0.4, (-‚-) 0.6, (-‚‚-) 0.8, and (O) 1. (B) Surface pressure-area isotherms (compression curve) for monopalmitin-WPI mixed monolayers. The molecular area was calculated on the basis that only the monopalmitin was adsorbed on the interface.

transition between liquid-expanded and liquid-condensed (LC) structures was observed at 5 < π < 30 mN/m, due to the fact that this transition does not occur at constant surface pressure. This transition agrees well with previous results23 and is confirmed28 by a change in the slope of the π-A isotherm (data not shown), which is a measure of the monolayer elasticity [E ) -A(dπ/dA)T]. The liquidcondensed structure was observed at π > 30 mN/m. Finally, the collapse phase was observed at a surface pressure of about 53.1 mN/m. However, WPI monolayers have a liquid-expanded-like structure under these experimental conditions. At a surface pressure of ca. 31 mN/ m, the WPI monolayer collapses. These results were reproducible within the experimental error as were those obtained previously with monopalmitin,23,26 WPI,27 and β-lactoglobulin.26 (26) Cornec, M.; Narsimhan, G. Langmuir 2000, 16, 1216. (27) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma.; Cejudo F. M. In Food Colloids: Fundamentals and Applications; Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: Cambridge (UK), 2001; p 22. (28) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interface; Wiley: New York, 1966.

Whey Protein Isolate-Monoglyceride Monolayers

The monolayer structure in monopalmitin-WPI mixed films (Figure 1A) at surfaces pressures lower than that for WPI collapse (π = 31 mN/m) adopts a structural polymorphism, as for monopalmitin. Moreover, it can be seen that there was a monolayer expansion as the monopalmitin content in the mixture was increased. That is, the π-A isotherm is displaced toward higher A as the content of monopalmitin in the mixture increases. At surface pressures higher than that for WPI collapse, the π-A isotherms for mixed monolayers were parallel to that of monopalmitin. These data are in agreement with those deduced for β-lactoglobulin-monopalmitin mixed films.26 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 monopalmitin-WPI ratio. In fact, we present in Figure 1B hypothetical π-A isotherms for mixed monolayers calculated on the basis that only monopalmitin is present at the air-water interface. We can see that, supposing that the mixed monolayers are dominated by monopalmitin (Figure 1B), the π-A isotherms for monopalmitin and monopalmitin-WPI mixed monolayers at X > 0.2 and at surface pressures higher than that for the WPI collapse are practically coincident. In contrast to the above data, π-A isotherms calculated again on the basis that the mixed monolayers are dominated by WPI (data not shown) are totally different from those for pure components under all experimental conditions. On the other hand, it can be seen that the collapse pressure for monopalmitin-WPI mixed monolayers is similar to that for monopalmitin and does not depend on the monolayer composition (Figure 1A). The interactions between components in the mixed monolayer can be studied from the point of view of miscibility between components on the mixed monolayer by means of the excess area, Aexc,28 and the excess free energy, ∆Gexc.29,30 For monopalmitin-WPI mixed monolayers (data not shown), the excess area was negative at surface pressures higher than 5 mN/m and for X < 0.8. Moreover, there was a minimum in the ∆Gexc-X relationship, which corresponds to a monopalmitin ratio in the mixture of 0.2. At higher surface pressures, the absolute value of the minimum in the ∆Gexc-X relationship is highest. The effect of pH on monopalmitin, WPI, and monopalmitin-WPI mixed monolayers can be deduced from the π-A isotherms at pH 7 (Figure 1A) and pH 5 (Figure 2A). It can be seen that the monopalmitin and WPI monolayer structure is practically independent of the aqueous-phase pH (Figures 1A and 2A). For monopalmitin-WPI mixed monolayers, the monolayer structure is also practically independent of the aqueous-phase pH (Figures 1A and 2A). That is, there are no structural changes after mixing as a function of aqueous-phase pH, but the attractive interactions between film-forming components are higher on acidic subphase as was deduced from the Aexc-X and ∆Gexc-X relationships (data not shown). Monoolein-WPI Mixed Films. The structural characteristics of the monoolein monolayer at pH 7 (Figure 3A) were different from that for monopalmitin as deduced from π-A isotherms. As expected,13 monoolein monolayers had a liquid-expanded structure under all experimental conditions, as did WPI monolayers. However, the collapse (29) Goodrich. F. C. In Proc. 2nd International Congress on Interface Activity; Butterworth: London, Academic Press: New York, ?; Vol. 1, p 85. (30) Pagano, R. E.; Gershfeld, N. L. J. Phys. Chem. 1972, 76, 1238.

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Figure 2. Surface pressure-area isotherms (compression curve) for (A) monopalmitin-WPI and (B) monoolein-WPI mixed monolayers on buffered water at pH 5 and at 20 °C. Mass fraction of lipid (monopalmitin or monoolein) in the mixture (X): (4) 0, (-) 0.2, (- - -) 0.4, (-•-) 0.6, (-‚‚-) 0.8, and (O) 1.

pressure for monoolein monolayers at 20 °C is lower (44.3 mN/m) than that for monopalmitin, which indicates that hydrophobic interactions between hydrocarbon chains for monoolein are lower than those for monopalmitin monolayers. In fact, monopalmitin and, to a large extent, monoolein could be partially submerged in water at the interface.21 The behavior of monoolein-WPI mixed monolayers at pH 5 and 7 was essentially different than that of monopalmitin in the mixture, as deduced from the π-A isotherm (Figures 2B and 3A), excess area, and excess free energy (data not shown). Briefly, monoolein-WPI mixed films (Figures 2B and 3A) at surface pressures lower than that for WPI collapse (π = 31 mN/m) adopt a liquidlike-expanded structure, as for pure components. There was a monolayer expansion as the monoolein content in the mixture was increased (Figures 2B and 3A). At surface pressures higher than that for WPI collapse, the π-A isotherms for mixed monolayers were parallel to that of monoolein. At these experimental conditions, the π-A isotherms for monoolein-WPI mixed monolayers (Figure 3A) and the hypothetical π-A isotherms for mixed monolayers calculated on the basis that only monoolein is present at the air-water interface (Figure 3B), at X > 0.2, are practically coincident. The collapse pressure for mixed monolayers is similar to that

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Figure 3. (A) Surface pressure-area isotherms (compression curve) for monoolein-WPI mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (4) 0, (-) 0.2, (- - -) 0.4, (-‚-) 0.6, (-‚‚-) 0.8, and (O) 1. (B) Surface pressure-area isotherms (compression curve) for monopalmitin-WPI mixed monolayers. The molecular area was calculated on the basis that only the monopalmitin was adsorbed on the interface.

for monoolein and did not depend on the monolayer composition or pH (Figures 2B and 3A). Brewster Angle Microscopy. BAM images were taken in different regions of monopalmitin-WPI (Figure 4) and monoolein-WPI (Figure 5) mixed monolayers on aqueous solutions at pH 5 and 7. The images for monopalmitin-WPI mixed monolayers (Figure 4) clearly indicate that at low surface pressures there exists a coexistence between small circular liquidcondensed (LC) monopalmitin domains and liquidexpanded (LE) domains of monopalmitin and probably of WPI (Figure 4A and B). The existence of LC monopalmitin domains was evident in the mixed monolayer even at the lower lipid concentrations (Figure 4C-E). The LC monopalmitin domains grow in size at the expense of the LE ones (Figure 4B, C, and E). At the beginning of the LE to LC transition, the LC domains adopted a dentritic shape (Figure 4B). The dentritic LC domains grew in size and adopted a circular shape as the surface pressure was increased, no matter what the interfacial composition was (Figure 4B, C, and E). The LC domains of monopalmitin in the mixed film did not have a uniform intensity (Figure 4B, C, and E), and this intensity changed with the analyzer angle (data not shown), a typical behavior of crystallinelike domains at the air-water interface. However, we are unable to distinguish between liquid-expanded monopalmitin domains and WPI in the continuous homogeneous phase. By the movement of BAM along the film balance, some regions with small LC monopalmitin domains (Figure 4D) and the frontier between regions with higher


and smaller LC monopalmitin domains (Figure 4C) can be observed. At higher surface pressures, near and after the WPI collapse (25 < π < 37 mN/m), the squeezing-out of WPI by monopalmitin can be distinguished (Figure 4F-K) in a region with LC domains of monopalmitin (dark area) over a sublayer of collapsed protein (bright area), with different morphologies but independent of the monolayer composition and pH. At the lipid collapse, the monopalmitin domains were so closely packed that the monolayer morphology acquired a high homogeneity (Figure 4L). However, in this region of highest surface pressure, different regions of collapsed WPI were observed on the interface (Figure 4M and N). During the expansion, the monolayer morphology of the region rich in collapsed monopalmitin, which occupies the majority of the interface, was similar (images not shown) to those described above for the monolayer compression. However, in the zone where collapsed WPI was observed, the regions of collapsed WPI (Figure 4O) and monolayer fractures (Figure 4P) were open and then desegregated into small domains of collapsed WPI. As the expansion progressed, the characteristic morphology of the mixed films was the formation of a 2-D foam (Figure 4Q and R) due to the break-up of the WPI collapsed structure. After 30 min of waiting time at the maximum area, the monolayer recovered its original morphology, according to the conclusion of reversibility of the monolayer structure deduced from the π-A isotherm during continuous compression-expansion cycles. BAM images for monoolein-WPI mixed monolayers (Figure 5) were different to those described above for monopalmitin-WPI mixed monolayers (Figure 4), especially during the monolayer compression. In fact, at surface pressures lower than that for WPI collapse (π ≈ 31 mN/ m), we were unable to deduce any difference from BAM images between pure components and the mixed monolayer because in this region both components and the mixed monolayer as well form an isotropic (homogeneous) monolayer without any difference in the domain morphology (images not shown). At surface pressures near to and after the WPI collapse, BAM images (Figure 5A-F) demonstrated that monoolein and WPI molecules adopted an isotropic structure in the mixed monolayer with some holes (Figure 5D-F) associated with the presence of monoolein at the interface and some white regions (Figure 5A-E) which correspond to the presence of a thicker WPI collapsed monolayer. At the higher surface pressures, and especially at the collapse point of the pure monoolein monolayer, the mixed monolayer was dominated by the presence of monoolein at the interface (Figure 5I). However, BAM images (Figure 5G and H) recorded by the movement of BAM along the film balance also demonstrated that, even at the highest surface pressures, a monoolein monolayer was unable to completely displace WPI molecules from the air-water interface. During the expansion, the morphology of the monoolein-WPI mixed monolayer was practically homogeneous (images not shown), except in the regions where islands of collapsed WPI were observed. In fact, in these zones, some regions of collapsed WPI residues (observed as a white path in the image) with holes of stacked monoolein domains were recorded (Figure 5J-L). These structures were open and then desegregated into small domains of collapsed WPI, as the expansion progressed. Finally, the characteristic morphology of the mixed films at the maximum area was the formation of a 2-D foam


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Figure 4. Visualization of monopalmitin-WPI mixed monolayers by Brewster angle microscopy at 20 °C. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Key: (A) π ) 9.0 mN/m (compression), XMONOPALMITIN ) 0.5, pH 7; (B) π ) 10.0 mN/m (compression), XMONOPALMITIN ) 0.8, pH 7; (C) π ) 15.0 mN/m (compression), XMONOPALMITIN ) 0.2, pH 7; (D) π ) 15.0 mN/m (compression), XMONOPALMITIN ) 0.2, pH 7; (E) π ) 19.0 mN/m (compression), XMONOPALMITIN ) 0.2, pH 7; (F) π ) 25.0 mN/m (compression), XMONOPALMITIN ) 0.5, pH 7; (G) π ) 26.4 mN/m (compression), XMONOPALMITIN ) 0.25, pH 5; (H) π ) 26.2 mN/m (compression), XMONOPALMITIN ) 0.5, pH 5; (I) π ) 30.3 mN/m (compression), XMONOPALMITIN ) 0.5, pH 5; (J) π ) 32.0 mN/m (compression), XMONOPALMITIN ) 0.2, pH 7; (K) π ) 37.1 mN/m (compression), XMONOPALMITIN ) 0.5, pH 5; (L) π ) 50.0 mN/m (compression), XMONOPALMITIN ) 0.5, pH 7; (M) π ) 50.0 mN/m (compression), XMONOPALMITIN ) 0.8, pH 7; (N) π ) 48.0 mN/m (compression), XMONOPALMITIN ) 0.2, pH 7; (O) π ) 26.0 mN/m (expansion), XMONOPALMITIN ) 0.25, pH 5; (P) π ) 21.7 mN/m (expansion), XMONOPALMITIN ) 0.5, pH 5; (Q) π ) 6.0 mN/m (expansion), XMONOPALMITIN ) 0.5, pH 7; (R) π ) 0.7 mN/m (expansion), XMONOPALMITIN ) 0.5, pH 5.

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Figure 5. Visualization of monoolein-WPI mixed monolayers by Brewster angle microscopy at 20 °C. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Key: (A) π ) 24.0 mN/m (compression), XMONOOLEIN ) 0.2, pH 7; (B) π ) 29.0 mN/m (compression), XMONOOLEIN ) 0.25, pH 5; (C) π ) 33.2 mN/m (compression), XMONOOLEIN ) 0.25, pH 5; (D) π ) 33.0 mN/m (compression), XMONOOLEIN ) 0.25, pH 5; (E) π ) 32.7 mN/m (compression), XMONOOLEIN ) 0.75, pH 5; (F) π ) 35.0 mN/m (compression), XMONOOLEIN ) 0.2, pH 7; (G) π ) 41.8 mN/m (compression), XMONOOLEIN ) 0.75, pH 5; (H) π ) 30.0 mN/m (compression), XMONOOLEIN ) 0.5, pH 5; (I) π ) 42.1 mN/m (compression), XMONOOLEIN ) 0.5, pH 5; (J) π ) 34.4 mN/m (expansion), XMONOOLEIN ) 0.5, pH 5; (K) π ) 31.9 mN/m (expansion), XMONOOLEIN ) 0.25, pH 5; (L) π ) 30.0 mN/m (expansion), XMONOOLEIN ) 0.25, pH 5; (M) π ) 0.6 mN/m (expansion), XMONOOLEIN ) 0.5, pH 5; (N) π ) 0.4 mN/m (expansion), XMONOOLEIN ) 0.75, pH 5; (O) π ) 0.6 mN/m (expansion), XMONOOLEIN ) 0.5, pH 5.

(Figure 5M-O), as discussed above for monopalmitinWPI mixed monolayers. Relative Monolayer Thickness. The I-π plots for compression of the monopalmitin-WPI mixed monolayer at pH 5 and 7, and at 0.5 in mass fraction of monopalmitin in the mixture (as an example), are shown in Figure 6. For comparison purposes, a continuous line was deduced for pure components (monopalmitin and WPI) by fitting the I-π data derived from repeated measurements. These

results reflect the higher relative thickness of WPI with respect to monopalmitin at higher surface pressures, a phenomenon that can be used to analyze the application of BAM to monoglyceride-WPI mixed monolayers. During the monolayer compression at low surface pressures, the I-π plots for the mixed film were essentially the same as for pure monopalmitin and WPI monolayers. At π > 5 mN/m, the existence of some I peaks was observed when liquid-condensed domains of monopalmitin in a homo-


Figure 6. Relative reflectivity (arbitrary units) as a function of surface pressure during the compression for monopalmitinWPI mixed monolayers on buffered water at (A) pH 7 and (B) pH 5. Monopalmitin mass fraction in the mixture: 0.5. The line-fit for the monopalmitin (continuous line) and WPI (discontinuous line) pure monolayers is included in plots (A) and (B). Temperature: 20 °C. Shutter speed (s): (O) 1:50, (4 and 3) 1:250.

geneous liquid-expanded environment of monopalmitin, and probably of a homogeneous WPI film as well, passed through the spot where this measurement was performed. At higher surface pressures (at π ) 20-31 mN/m), near the WPI collapse, the relative intensity of some spots was the same as for pure WPI. This is due to the fact that in this region, domains of pure WPI (with high I) and monopalmitin (with low I) passed alternatively through the spot where these measurements were performed. In the region after the WPI collapse (π > 31 mN/m), the camera was saturated with some spots of high I values, characteristic of WPI collapsed monolayers. However, the intensity of the I peaks decreased at the higher content of monopalmitin in the mixture. Thus, at X ) 0.8, the I-π plot at the highest surface pressures follows the same trend as that of pure monopalmitin (data not shown). The I-π plots for the monoolein-WPI mixed monolayers at pH 5 and 7, and at 0.5 in mass fraction of monoolein in the mixture (as an example), are shown in Figure 7. The continuous lines were deduced by fitting the I-π data derived from repeated measurements with pure components (monoolein and WPI). From these results, some differences can be deduced as compared to those recorded for monopalmitin-WPI mixed monolayers (Figure 6). Namely, at the lower content of monoolein in the mixture (data not shown) and for mixed monolayers at 0.5 and at pH 5 (Figure 7B), at higher surface pressures, near to and after the WPI collapse, the relative intensity of some spots

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Figure 7. Relative reflectivity (arbitrary units) as a function of surface pressure during the compression for monooleinWPI mixed monolayers on buffered water at (A) pH 7 and (B) pH 5. Monoolein mass fraction in the mixture: 0.5. The line-fit for the monoolein (continuous line) and WPI (discontinuous line) pure monolayers is included in plots (A) and (B). Temperature: 20 °C. Shutter speed (s): (O) 1:50, (4) 1:250.

was the same as for pure WPI. For mixed monolayers at pH 7 (Figure 7A) and at the higher content of monoolein in the mixture (data not shown), the I-π plots were practically the same as those for pure monoolein, over the overall range of surface pressures, which strengthens the conclusion that monoolein, with the lower relative thickness, predominates at the interface. However, the pH had no significant influence on the relative film thickness of monoolein-WPI mixed monolayers, as was observed for pure monoolein and WPI monolayers. Discussion In this paper, we report on structural and morphological characteristics of monopalmitin-WPI and monooleinWPI mixed monolayers spread on water at pH 5 and 7 and at 20 °C. The structural characteristics and morphology of monoglyceride-WPI mixed films are dependent on surface pressure, monolayer composition, and, to a minor extent, on the aqueous phase pH. From the π-A isotherm of mixed monolayers (Figures 1-3), including the application of the additivity rule on miscibility and the quantification of interactions between monolayer components by the excess free energy (data not shown) and according to results for other systems,31 it has been shown (31) de la Fuente, J.; Rodrı´guez Patino, J. M. AIChE J. 1995, 41, 1955. de la Fuente, J.; Rodrı´guez Patino, J. M. AIChE J. 1996, 42, 1416. de la Fuente, J.; Rodrı´guez Patino, J. M. Langmuir 1995, 11, 2163. de la Fuente, J.; Rodrı´guez Patino, J. M. Colloids Surf., A 1995, 104, 29.

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that, at a macroscopic level, monoglyceride-WPI mixed monolayers form a mixed monolayer at the air-water interface with few interactions between film-forming components, at surface pressures lower than that for the WPI collapse (π ≈ 31 mN/m). At the highest surface pressures, at the collapse point of the mixed film, immiscibility between monolayer-forming components is deduced32,33 due to the fact that the collapse pressure of mixed monolayers is similar to that of the pure monoglyceride monolayer (Figures 1-3). At higher surface pressures, the collapsed WPI is displaced from the interface by monoglyceride (either monopalmitin or monoolein). The WPI displacement by the monoglyceride is practically total at the highest surface pressure, at the collapse point of the mixed monolayer, especially for monoglyceride-WPI mass fractions higher than 0.2 (Figures 1B and 3B). These results also show weak interactions between monoglycerides and WPI at the air-water interface, in particular, at the collapse point. The same behavior was observed for monopalmitin-βcasein,13 monoolein-β-casein,14 and monoglyceridecaseinate15 mixed films. However, the fact that upon expansion and further compression, the π-A isotherms were repetitive suggests that the protein reenters the mixed monolayer and supports the idea that the protein remains underneath the monoglyceride film26 either through hydrophobic interactions between the protein and the lipid or by local anchoring through the monoglyceride layer.26,34 The evolution with the surface pressure of BAM images (Figures 4 and 5) and relative reflectivity (Figures 6 and 7) gives complementary information, at a microscopic level, on the structural characteristics and interactions of spread monoglyceride-WPI mixed monolayers, as deduced from π-A isotherms (Figures 1-3). In summary, the results reported here and in previous works13-15 suggest that in monoglyceride-protein mixed films, islands of protein and monoglyceride do exist at the air-water interface, on a microscopic level, but with few interactions between them, depending on the surface pressure. At surface pressures lower than that for protein collapse, a mixed monolayer of monoglyceride and protein may exist. However, at surface pressures higher than that for protein collapse, the mixed monolayers were practically dominated by monoglyceride molecules. That is, at higher surface pressures, collapsed protein residues may be displaced from the interface by monoglyceride molecules. Over the overall range of existence of the mixed film, the monolayer presents some heterogeneity due to the fact that domains of monoglyceride and protein residues are present during the monolayer compression, giving I peaks of collapsed WPI with high relative film thickness. Interactions, miscibility, and displacement of proteins by monoglycerides from the air-water interface depend on the particular protein-monoglyceride system. Different proteins show different interfacial morphology, confirming the importance of a protein secondary structure in determining the mechanism of interfacial interactions. For the more disordered milk proteins (β-casein and caseinate), three different well-defined regions can be defined (Figure 8A) over the heterogeneous morphology of the interface:13-15 an isotropic protein-rich region forming a thicker film with high I, an anisotropic monopalmitin-rich or isotropic monoolein-rich region forming (32) Carrera, C.; de la Fuente, F.; Rodrı´guez Patino, J. M. Colloids Surf., A 1998, 143, 477. (33) Joos, P.; Demel, R. A. Biochim. Biophys. Acta 1969, 183, 447. (34) Li, J. B.; Kra¨gel, J.; Makievski, A. V.; Fainermann, V. B.; Miller, R.; Mo¨hwald, H. Colloids Surf., A 1998, 142, 355.


Figure 8. Molecular models for (A) monoglyceride-disordered proteins and (B) monoglyceride-globular proteins mixed films at the air-water interface, showing the protein-rich region (bright thicker region), the monoglyceride-rich region (dark thinner region), and the frontier between protein-rich and monoglyceride-rich regions.

a thinner film with low I, and the frontier between these regions where monoglyceride-protein interactions take place. For monopalmitin-β-casein and monopalmitincaseinate mixed films, the size of the LC monopalmitin domains in the frontier is lower than that in the monopalmitin-rich region, a phenomenon that we associate with the existence of monopalmitin-protein interactions. However, for the more ordered WPI, the morphology of the interface is characterized by numerous smaller segregated regions of proteins and monoglyceride domains distributed homogeneously over the interface (Figure 8B). The model shown in Figure 8 suggests the idea that interactions between proteins and monoglycerides are higher for globular (WPI) than for disordered (β-casein and caseinate) proteins, as was observed experimentally from data deduced from the π-A isotherms13-15 (Figures 1-3). From these studies, the idea also emerges that the displacement of disordered (β-casein and caseinate) and globular (WPI) proteins by monoglyceride from the airwater interface depends on the protein-monoglyceride system. From the hypothetical isotherms shown in Figures 1B and 3B, we define a displacement surface pressure (πd) as the minimum surface pressure above which the π-A isotherms for monoglyceride and monoglycerideprotein mixed monolayers are coincident. Thus, at low πd, the protein displacement by the monoglyceride is facilitated. In Figure 9, we show the πd as a function of the mixture composition for different monoglyceride-protein mixed films. The protein displacement by monopalmitin


Figure 9. The displacement surface pressure of (0) β-casein, (O) caseinate, and (4) WPI by (A) monopalmitin and (B) monoolein from spread mixed monolayers at the air-water interface as a function of the mass fraction of monoglyceride in the mixture. The horizontal lines represent the equilibrium surface pressures of (-, πe MP) monopalmitin, (‚‚‚‚, πe MO) monoolein, (- - - -, πe BC) β-casein, (-‚-, πe CS) caseinate, and (-‚‚-, πe WPI) WPI pure monolayers at 20 °C and at pH 7.

is easier for β-casein than it is for caseinate and WPI, in this order (Figure 9A). In fact, at X > 0.2, the πd value for monopalmitin-β-casein mixed films is lower than those for monopalmitin-caseinate and monopalmitin-WPI mixed films. β-Casein displacement by monopalmitin at X ) 0.8 was deduced even for surface pressures close to that of β-casein collapse (Figure 9A). However, for caseinate and WPI, higher surface pressures are necessary for a quantitative displacement of the protein from the interface, in particular at lower monopalmitin concentrations in the mixture. That is, the existence of low protein interactions in disordered proteins, as deduced from dilatational properties for β-casein and caseinate,35 fa-

Langmuir, Vol. 17, No. 24, 2001 7553

cilitates the protein displacement by monopalmitin from the air-water interface. These results are in agreement with the opinion that the displacement of proteins by surfactants depends on the extent of protein-protein interactions.36,37 On the other hand, the lower surface activity of monoolein justifies the idea that this lipid has a lower capacity than monopalmitin for protein displacement. In fact, monoolein (Figure 9B) requires higher surface pressures than does monopalmitin (Figure 9A) for protein displacement from the air-water interface. Until recently,17 the exact mechanism for protein displacement was unclear. Recent studies16-19 have shown that the displacement of milk proteins from the air-water interface by water-soluble surfactants (Tween 20 and sodium dodecyl sulfate) involves a novel orogenic mechanism. Such a model allows an explanation of the displacement of protein by surfactants, but there exist differences between adsorbable water-soluble surfactants and spread water-insoluble monoglycerides. The results suggest that for spread monoglycerides, the first stage of the orogenic mechanism, which occurs at surface pressures lower than the equilibrium surface pressure of the protein, involves a displacement front of monoglyceride domains instead of the adsorption of water-soluble surfactant molecules at defects in the protein network. The second stage, which occurs at surface pressures near to and above the equilibrium surface pressure of the protein, involves a buckling of the monolayer and reordering of the molecules as the protein film gets thicker in response to the decreasing surface coverage. Finally, at sufficiently high surface pressures, the protein network begins to fail, freeing proteins which then desorb from the interface.16-19 But, for spread monoglyceride monolayers, the protein displacement is not total even at the highest surface pressure, at the collapse point of the mixed film. The orogenic displacement mechanism is a consequence of the existence of a few interactions between proteins and monoglycerides at the air-water interface. Acknowledgment. This research was supported by the European Community through grant FAIR-CT961216, by CICYT through grant ALI97-1274-CE, and by DGICYT through grant PB97-0734. Note Added after ASAP Posting. This article was released ASAP on 11/3/2001 with an error in the caption of Figure 5. The correct version was posted on 11/6/2001. LA0102814 (35) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma.; Cejudo, F. M. J. Colloid Interface Sci. 2001, 242, 141. (36) Chen, J.; Dickinson, E. J. Sci. Food Agric. 1993, 62, 283. (37) Dickinson, E.; Hong, S. K. J. Agric. Food Chem. 1994, 42, 1602.

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