Dynamic Properties of Î²-CaseinâMonoglyceride Mixed Films at the Air

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Dynamic Properties of β-Casein-Monoglyceride Mixed Films at the Air-Water Interface. Long-Term Relaxation Phenomena Juan M. Rodrı´guez Patino,* M. Rosario Rodrı´guez Ninõ, and Cecilio Carrera Sańchez Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/Professor Garcıá Gonza´ lez, s/nu´ m. 41012-Seville, Spain Received February 26, 2002. In Final Form: July 30, 2002 Long-term relaxation phenomena in β-casein-monoglyceride mixed films at the air-water interface were studied using a surface film balance and Brewster angle microscopy. Relaxation in surface area at a constant surface pressure (at 20 mN/m), just below the β-casein collapse, or at a constant area at the collapse point of the mixed film has been analyzed according to models for desorption, collapse, and/or organization/reorganization changes. At a constant surface pressure, the organization/reorganization change of β-casein molecules in monopalmitin- and monoolein-β-casein mixed films is the mechanism that controls the relaxation process. At the collapse point of the mixed film, the relaxation phenomena may be due either to nucleation and growth of critical nuclei of monoglyceride or to a complex mechanism including competition between desorption and monolayer collapse. For β-casein-monolaurin mixed films, the relaxation phenomena are mainly due to the irreversible loss of monolaurin molecules by desorption into the bulk aqueous phase.

Introduction Many real food formulations are complex dispersions such as foams and emulsions. These dispersions are inherently unstable systems because of their large interfacial area. That is, in addition to formation, stability is one of the most important properties of such dispersions. From a practical point of view, food dispersions need to be stable because many of them are designed to have a long shelf life. The stability of food emulsions is complex because it covers both a number of phenomena and a variety of systems with different contents.1-3 For this reason, food dispersions contain many emulsifiers. Oilin-water emulsions (i.e., ice cream, cream liqueurs, mayonnaise, etc.) are stabilized by the adsorption of smallmolecular-weight emulsifiers (i.e., polar lipids, such as mono- and diglycerides), protein molecules (milk and egg proteins), or aggregates of protein molecules (i.e., casein micelles), or by a mixture of these.4 For recent reviews, readers are directed to recent references.2,3,5-9 Proteins and polar lipids typically coexist in food dispersions (emulsions and foams), sometimes unasso* To whom correspondence should be addressed. Tel: +34 95 4557183. Fax: +34 95 4557134. E-mail: [email protected]. (1) Dickinson E. An Introduction to Food Colloids; Oxford University Press: Oxford, 1992. (2) Friberg, S. E.; Larsson, K. Food Emulsions, 3rd ed.; Marcel Dekker: New York, 1997. (3) Sjo¨blom, J. Emulsions and Emulsion Stability; Marcel Dekker: New York, 1996. (4) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsions and their Applications; Hasenhuette, G. L., Hartel, R. W., Eds.; Chapman & Hall: New York, 1997; p 95. (5) Damodaran, S.; Paraf, S. Food Proteins and their Applications; Marcel Dekker: New York, 1997. (6) Hartel, R.; Hasenhuette, G. R. Food Emulsifiers and their Applications; Chapman and Hall: New York, 1997. (7) Gunstone, F. D.; Padley, F. B. Lipid Technologies and Applications; Marcel Dekker: New York, 1997. (8) McDonald, R. E.; Min, D. B. Food Lipids and Health; Marcel Dekker: New York, 1996. (9) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R. Colloids Surf., B 1999, 15, 235.

ciated with each other but also in association, with specific functions in the processing and properties of the final product.4 Proteins and lipids have an important physical property in common, their amphiphilic nature. This property provides the possibility for association, adsorption, and reorientation at fluid-fluid interfaces, depending on the properties of the components and the protein-lipid ratio.10-13 However, more important in some products is the effect of the small-molecule emulsifiers in destabilizing the emulsion.14 In the formulation of ice cream, the small-molecule emulsifier (typically, mono- and diglycerides) is added to break the adsorbed layer of protein and allow the adsorption of fat to the surface of the air bubble. Thus, an important action of the small-molecule emulsifiers is to promote the displacement of proteins (mainly caseins) from the interface. The competitive adsorption and displacement between lipids and proteins at fluid-fluid interfaces have been studied in detail in several investigations. For further information concerning the interfacial characteristics of food emulsifiers (proteins and lipids), the reader is referred to recent reviews.2-5,15-19 However, so far, little is known about the structure that proteins and lipids adopt at fluid-fluid interfaces. (10) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M. J. Am. Oil Chem. Soc. 1998, 75, 1233. (11) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M., J. Am. Oil Chem. Soc. 1998, 75, 1241. (12) Rodrı´guez Ninõ, M. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 1998, 46, 2177. (13) Rodrı´guez Ninõ, M. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Langmuir 1998, 14, 2160. (14) Goff, H. D.; Jordan, W. K. J. Dairy Sci. 1989, 72, 18. (15) Cayot, P.; Lorient, D. In Food Proteins and their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997; p 225. (16) Dalgleish, D. In Emulsions and Emulsion Stability; Sjo¨blom, J., Ed.; Marcel Dekker: New York, 1996; p 287. (17) Dalgleish, D. G. In Food Proteins and their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997; p 199. (18) Nylander, T.; Ericsson, B. In Food Emulsions; Friberg, S. E., Larsson, K., Eds.; Marcel Dekker: New York, 1997; p 189. (19) Rodrı´guez Ninõ, M. R.; Carrera, C.; Rodrı´guez Patino, J. M.; Cejudo, M.; Garcıá, J. M. Chem. Eng. Commun., in press.

10.1021/la0202071 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/19/2002

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The highly complex mechanisms involved in the formation and stabilization/destabilization of food dispersions make fundamental studies in applied systems difficult. Thus, the study of characteristics of food emulsifier monolayers at the air-water interface as a food model presents several advantages. In fact, relationships between structural characteristics of emulsifier monolayers on fluid-fluid interfaces and dispersion stability have been established, as have those between condensed film formation and emulsifier association in the bulk phase.20-22 The aim of this work is the study of the long-term relaxation phenomena in β-casein and monoglyceride (monopalmitin, monoolein, and monolaurin) mixed monolayers spread at the air-water interface as a function of processing variables (surface composition and surface pressure). This work is an extension of previous studies on relaxation phenomena in monoglyceride23 and protein24 monolayers at the air-water interface. As far as we know, long-term relaxation phenomena in protein-monoglyceride mixed monolayers have not been studied so far. Nonequilibrium processes occurring in systems containing fluid-fluid interfaces with a surfactant present are of great practical significance.25-29 Experimental Section Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90), 1-mono(cis-9-octadecanoyl) glycerol (monoolein, RYLO MG 19), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMODAN ML 90) were supplied by Danisco Ingredients with over 95-98% purity. β-Casein (99% pure) was supplied and purified from bulk milk from the Hannah Research Institute, Ayr, Scotland.30 The sample was stored below 0 °C, and all work was done without further purification. Samples for interfacial characteristics of β-casein films were prepared using Milli-Q ultrapure water and were buffered at pH 7. 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. The water used as a subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution called trizma ((CH2OH)3CNH2/ (CH2OH)3CNH3Cl) was used for pH 7. All these products were supplied by Sigma (>99.5%). Ionic strength was 0.05 M in all the experiments. Surface Film Balance. Measurements of surface pressure (π)-area (A) isotherms and surface relaxation in β-caseinmonoglyceride mixed films at the air-water interface were performed on a fully automated Langmuir type film balance. The method has been described previously for pure23,24 and mixed components.31,32 Two kinds of experiment were used for the analysis of relaxation in β-casein-monoglyceride mixed monolayers. First, the surface pressure (π) is kept constant and the area A is measured as a function of time. This relaxation (20) Dickinson, E. Colloids Surf., B 1999, 15, 161. (21) Dickinson, E. Colloid Surf., B 2001, 20, 197. (22) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (23) Carrera, C.; Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 175. (24) Rodrı´guez Ninõ, M. R.; Carrera, C.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 161. (25) Baszkin, A.; Norde, W. Physical Chemistry of Biological Interfaces; Marcel Dekker: New York, 2000. (26) Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1985. (27) Dukhin, S. S.; Kretzschmar, G.; Miller, R. Dynamic of Adsorption at Liquid Interfaces; Elsevier: Amsterdam, 1995. (28) Malhotra, A. K.; Wasan, D. T. Thin Liquid Films; Surfactant Science Series Vol. 29; Marcel Dekker: New York, 1988; p 829. (29) Mittal, K. L.; Kumar, P. Emulsions, Foams, and Thin Films; Marcel Dekker: New York, 2000. (30) Atkinson, P. J.; Dickinson, E.; Horne, D.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 1, 2847. (31) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. J. Agric. Food Chem. 1999, 47, 4998. (32) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 4777.

Rodrı´guez Patino et al.

Figure 1. Relaxation mechanisms in spread monolayers at the air-water interface, where π and πe are the surface pressure and the equilibrium surface pressure, respectively. experiment is the usual, preferred method and is capable of being interpreted kinetically.33 In the second type of experiment, the area is kept constant (at the collapse) and the surface pressure decreases. This decrease is measured as a function of time. To analyze the reversibility of the relaxation phenomena, a π-A isotherm was performed 30 min after the relaxation study with the film at the maximum area. The experiments (π-A isotherm and relaxation phenomena in mixed films) were measured at least three times. The reproducibility of the results was better than (0.5 mN/m for surface pressure and (0.005 nm2/molecule for area. All isotherms were recorded continuously by a device connected to the film balance and then analyzed off-line. Data Analysis. Various relaxation mechanisms can be fitted to the results derived from the above-mentioned experiments, as is shown in Figure 1. Relaxation mechanisms other than desorption and collapse are difficult to quantify in typical relaxation experiments such as those used in this work. Desorption of a spread monolayer at any constant surface pressure involves two stages.34 The first is dissolution into the bulk aqueous phase to form a saturated aqueous layer. During the initial nonsteady-state period of desorption, the rate of monolayer molecular loss can be expressed by eq 1. The second stages occur when, after a time, the concentration gradient within the diffusion layer becomes constant and desorption reaches a steady state. The rate of monolayer molecular loss is then given by eq 2. In eqs 1 and 2, A and A0 are the molecular area at time θ and at the initial moment, and coefficients A1 and A2 account for the rate of dissolution and diffusion, respectively. At a surface pressure higher than the equilibrium surface pressure (πe), with insoluble monolayers, the relaxation phenomena are due to the transformation of a homogeneous monolayer phase into a heterogeneous monolayer-collapse phase system. Monolayer collapse may occur either due to a macroscopic film fracture or by a process of nucleation and the growth of bulk surfactant fragments, whenever a characteristic surface pressure is exceeded.35 The collapse rate should follow eq 3, where B1 and B2 account for the formation of nuclei and the growth rate of nuclei, respectively.

-Log(A/A0) ) A1θ1/2

(1)

-Log(A/A0) ) A2θ

(2)

-Log(A/A0) ) B1θ + B2θ2

(3)

The modeling of monolayer collapse by homogeneous nucleation and the growth of bulk surfactant nuclei can also be analyzed (33) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interface; Wiley: New York, 1996. (34) Ter Minassian-Saraga, L. J. Colloid Sci. 1956, 11, 398. (35) Smith, R. D.; Berg, J. C. J. Colloid Interface Sci. 1980, 74, 273.

Properties of β-Casein-Monoglyceride Mixed Films

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by applying the Prout-Tompkins36 equation (eq 4) to the data derived from relaxation experiments at constant surface collapse area.

Log[(π0 - π)/π] ) C1 Log θ + C2

(4)

where π and π0 are the surface pressures at time θ and at the initial moment, respectively, and C1 and C2 are coefficients, which depend on the experimental conditions. For proteins, various relaxation mechanisms ascribed to desorption or collapse of a spread monolayer, at a surface pressure higher than πe, were applied to the results derived from relaxation data.24 However, the best fit of the results was obtained by means of two exponential equations, eqs 5 and 6, for relaxation kinetics of β-casein monolayers at constant surface pressure and at constant surface area, respectively. These relaxation phenomena were associated with conformation-organization change and hydrophilic group hydration,24 with repercussions in surface dilatational properties.37

(A/A0) ) (A/A0)0 + P1e(-θ/τ1) + P2e(-θ/τ2)

(5)

π/π0 ) (π/π0)0 + P3e(-θ/τ1) + P4e(-θ/τ2)

(6)

where (A/A0)0 and (π/π0)0 are the amplitude of the relative area and relative surface pressure at the initial moment, respectively, τ1 and τ2 are the relaxation times, and P1, P2, P3, and P4 are constants. Rheological interfacial properties have a role in the long-term stability of dispersed systems (emulsions and foams) and the stability of the emulsion or foam in the production stage.20-22,38 The surface rheology is of interest because of its extreme sensitivity to the nature of intermolecular interactions at the interface11,39-41 and/or because of its relationships with relaxation phenomena at the interface.37,42 In fact, different surface dilatational characteristics, including nonlineal viscoelastic behavior, were observed for monoglyceride42 and protein37 spread films depending on the amplitude and the frequency of the oscillation. At the temperature used in these experiments, it is unlikely that evaporation contributes significantly to the relaxation phenomena observed with monoglyceride monolayers.23 Moreover, as the same relationship (eq 2) fits the relaxation of relative molecular area by evaporation and diffusion in the processing of the experimental data it is convenient to consider evaporation to be included in diffusion. Brewster Angle Microscopy (BAM). The development of new techniques for microscopic observation and characterization of monolayers at the air-water interface has been used advantageously to clarify the structural characteristics of amphiphilic substances at fluid-fluid interfaces. BAM, coupled with relaxation in surface pressure (π) at constant area or relaxation in area (A) at constant surface pressure, was used in this work for the first time to visualize and determine structural changes during relaxation phenomena of mixed emulsifier monolayers at the air-water interface. Further characteristics of the device and operational conditions have been described elsewhere.43,44 The relative film reflectivity can be measured by determining (36) Prout, E. G.; Tompkins, F. C. Trans. Faraday Soc. 1944, 40, 488. (37) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (38) Walstra, P. Chem. Eng. Sci. 1993, 48, 333. (39) 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; p 195. (40) Rodrı´guez Ninõ, M. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Ind. Eng. Chem. Res. 1996, 35, 4449. (41) Rodrı´guez Ninõ, M. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 1998, 46, 2177. (42) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. Langmuir 2001, 17, 4003. (43) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 2484. (44) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Food Hydrocolloids 1999, 13, 401.

Figure 2. Relaxation at constant surface pressure (π ) 20 mN/m) of β-casein-monopalmitin mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): (O) 0, (solid line) 0.2, (dashed line) 0.4, (dotted line) 0.6, (dashed-dotted line) 0.8, and (4) 1. Table 1. Characteristic Parameters [Equation 5] for Relaxation of β-Casein-Monopalmitin and β-Casein-Monoolein Mixed Films at Constant Surface Pressure (π ) 20 mN/m) and at 20 °C β-casein-monopalmitin

β-casein-monoolein

X

τ1

τ2

(A/A0)60

τ1

τ2

(A/A0)60

0 0.2 0.4 0.6 0.8 1

2.3 4.9 8.4 7.0 7.6 2.1

5.7 6.7 5.5 11.7 9.2 6.2

0.86 0.93 0.93 0.95 0.96 0.88

2.3 8.9 3.0 12.4 2.2

5.7 10.8 15.4 4.7 5.3

0.86 0.95 0.97 0.95 0.95 0.90

the light intensity at the camera and analyzing the polarization state of the reflected light through the method described elsewhere.43,44 In all BAM images, the light regions are protein rich and the dark regions are monoglyceride rich. The imaging conditions were adjusted to optimize both image quality and quantitative measurement of reflectivity. Thus, generally as the surface pressure or the protein content increased the shutter speed was also increased.

Results and Discussion Relaxation Phenomena in β-Casein-Monopalmitin Mixed Films. Relaxation Phenomena at Constant Surface Pressure. Figure 2 shows the relaxation in relative molecular area at 20 °C and at 20 mN/m for β-caseinmonopalmitin mixed films. For β-casein-monopalmitin mixed films, the (A/A0) relaxation has an exponential time dependence as expected for conformation/reorganization changes within the spread molecules. The best fits of the experimental data were obtained by means of eq 5 with two relaxation times, which means that the relaxation of β-casein-monopalmitin mixed films is not a simple process. In Table 1, the relaxation times and the value of relative area at 60 min of relaxation time, (A/A0)60, are shown. It can be seen that the amplitude of the (A/A0)60 relaxation is lower for pure β-casein than for β-caseinmonopalmitin mixed films. However, what is more difficult to establish is a relationship between the relaxation times and the mixture composition, although the fit of the experimental data by means of eq 5 is excellent. These phenomena may be due, among other things, to a looping of the amino acid segments of β-casein into the underlying aqueous phase after the compression up to 20 mN/m. Under these experimental conditions (at a surface pressure of 20 mN/m), whole protein molecules or segments of the protein molecules are probably squeezed down into the subphase solution, and what is most likely is that

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Figure 3. Compression-expansion cycles for β-casein-monopalmitin mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): (A) 0.2, (B) 0.4, (C) 0.6, and (D) 0.8. The first cycle was at 30 min after spreading, at the maximum area. The second cycle was at 30 min after the first one, at the maximum area. The third cycle was at 30 min after the relaxation experiment at constant surface pressure (π ) 20 mN/m), at the maximum area.

this structure adopts the form of loops and tails.24 This structure exists at surface pressures between 10 mN/m and the equilibrium surface pressure (πe), as the monolayer collapses. From the π-A isotherm of mixed monolayers,31 including the application of the additivity rule on miscibility and the quantification of interactions between monolayer components by the excess free energy, and according to results from Brewster angle microscopy, including the monolayer morphology and the relative reflectivity, it has been shown that β-casein-monoglyceride mixed monolayers form a mixed monolayer at the air-water interface with few interactions between filmforming components, at surface pressures lower than that for the β-casein collapse (π ≈ 20.9-21.8 mN/m). However, the data in Table 1 also show that the presence of monopalmitin in the mixture has an effect on the magnitude of relaxation in the mixed film, but not on their mechanisms. It can be seen that the amplitudes of the relative area are the same for the mixed films no matter what the composition is. Moreover, these amplitudes are lower for pure β-casein than for the mixed films. The relaxation phenomena at 20 mN/m for β-caseinmonopalmitin mixed films are reversible processes, as can be deduced from data presented in Figure 3. It can be seen that the π-A isotherms are repetitive after different compression-expansion cycles, the last one being registered after the relaxation experiment at 20 mN/m. These data confirm that the relaxation phenomena observed with β-casein-monopalmitin mixed monolayers at constant surface pressures lower than πe are not due to a monolayer molecular loss, but it is more likely that these phenomena could be related to the kinetics of the molecular reorganization during the looping of the amino acid residues. Interestingly, the hysteresis in π-A isotherms during the compression-expansion cycle decreased with the β-casein proportion in the mixture. This is an indication of the importance of the β-casein content

in the mixture in the dynamic relaxation phenomena. In fact, the higher the β-casein/monopalmitin ratio in the mixture, the higher the hysteresis, because pure β-casein monolayers have higher viscoelastic characteristics37,42 and lower superficial diffusion and mobility4 than monopalmitin. At the higher content of monopalmitin in the mixture (Figure 3D), the hysteresis in the π-A isotherm was the same as for the pure monopalmitin monolayer (data not shown). Further information about the mechanism or mechanisms that control the relaxation phenomena in β-caseinmonopalmitin mixed films at constant surface pressure (at 20 mN/m) can be deduced from the time evolution of surface pressure and relative reflectivity during a compression-expansion cycle (Figure 4A) for a mass fraction of monopalmitin in the mixture of 0.5 (as an example). It can be seen that at 20 mN/m, monopalmitin in the mixture has a liquid-condensed structure, as can be deduced from the existence of the characteristic main transition between the liquid-expanded and liquid-condensed phases at about 5 mN/m and from the reflectivity peaks, but the presence of β-casein in the mixture can be deduced because some peaks have a reflectivity similar to that for pure β-casein. These data also support the hypothesis that β-casein and monopalmitin form a practically immiscible monolayer at the air-water interface. In Figure 5, we observe some interesting features of the evolution of the reflectivity for a mixed monolayer of β-casein + monopalmitin at a mass fraction of monopalmitin in the mixture of 0.5 (as an example) during the relaxation at 20 mN/m. During the relaxation at constant surface pressure, the mixed film presents the existence of some heterogeneity, which is typical of monoglycerideprotein mixed films at the air-water interface.31,32,46 This (45) Wilde, P. J.; Rodrı´guez Ninõ, M. R.; Clark, D. C.; Rodrı´guez Patino, J. M. Langmuir 1997, 13, 7151.


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Figure 5. The time evolution of (O) surface pressure and (4) relative reflectivity (at a shutter speed of 1/250 s) during the relaxation at constant surface pressure (π ) 20 mN/m) for a β-casein-monopalmitin mixed monolayer on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture) ) 0.5. The lined area represents the relative reflectivity of the pure β-casein or monopalmitin monolayer at 20 mN/m. Key: (A) domains of monopalmitin, (B) β-casein and monopalmitin domains, (C) domains of monopalmitin, (D) frontier between β-casein and monopalmitin domains, and (E) monopalmitin over a sublayer of β-casein.

Figure 4. The time evolution of (solid line) surface pressure and (O) relative reflectivity (at a shutter speed of 1/250 s) during the compression-expansion cycle for (A) β-casein-monopalmitin and (B) β-casein-monoolein mixed monolayers on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monoglyceride in the mixture) ) 0.5. Key: 1(A), collapse of the mixed film; 2(A), equilibrium surface pressure of monopalmitin; 3(A), squeezing out of β-casein by monopalmitin; 4(A), β-casein collapse; 5(A), main transition in the pure monopalmitin monolayer; 1(B), monoolein collapse; 2(B), equilibrium surface pressure of monoolein; 3(B), squeezing out of β-casein by monoolein; 4(B), β-casein collapse.

figure and BAM images (data not shown) show that as the relaxation progresses, domains of monopalmitin (region A), domains of β-casein and monopalmitin (region B), new domains of monopalmitin (region C), a frontier between β-casein and monopalmitin domains (region D), and the squeezing out of β-casein by monopalmitin with monopalmitin over a sublayer of β-casein (region E) appear alternatively on the air-water interface. These results also corroborate the idea that during the relaxation of a mixed film of β-casein and monopalmitin at a constant surface pressure (at 20 mN/m) both components are present at the interface but with minor interactions between them. Thus, the relaxation phenomena observed in these systems under these experimental conditions may be associated essentially with organization/reorganization changes in the β-casein structure after the compression at 20 mN/m, but mediated by the presence of monopalmitin in the mixture. Relaxation Phenomena at Constant Surface Area (at the Collapse Point). β-Casein-monopalmitin mixed films were also tested under the most adverse conditions, at the maximum superficial density at constant collapse area. (46) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C.; Cejudo, M. J. Colloid Interface Sci. 2001, 240, 113.

Figure 6. Relaxation at constant area (at the collapse point) for β-casein-monopalmitin mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): (O) 0, (solid line) 0.2, (dashed line) 0.4, (dotted line) 0.6, (dashed-dotted line) 0.8, and (4) 1. The equilibrium surface pressures for monopalmitin (πe MP) and β-casein (πe BC) are indicated by arrows.

Figure 6 shows the relaxation in surface pressure at constant surface area (at the collapse point) for β-caseinmonopalmitin mixed films. Monopalmitin and β-caseinmonopalmitin mixed films relaxed toward the equilibrium surface pressure of pure monopalmitin,23 which is indicated in Figure 6 by an arrow. That is, at higher relaxation time, the surface pressure tends to a plateau, which is practically coincident with the value of πe for monopalmitin. This indicates that the relaxation phenomena in mixed films are similar to those observed for the pure monopalmitin monolayer.23 The (π/π0) relaxation has a logarithmic time dependence instead of an exponential dependency. In fact, processing the experimental data according to the Prout-Tompkins equation (eq 4) showed that it was possible to obtain a linear fit (Table 2). This indicates that the relaxation phenomena under these conditions may be due to nucleation and growth of critical nuclei of monopalmitin. Again, the reason for this behavior must be associated with the immiscibility between both components at the air-water interface, as was deduced for these systems at the collapse point.31 In fact, at surface pressures higher than that for the β-casein collapse, a squeezing out of

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Table 2. Characteristic Parameters [Equation 4] for Relaxation of β-Casein-Monopalmitin Mixed Films at Constant Area (at the Collapse Point) and at 20 °C XMP 0 0.2 0.4 0.6 0.8 1

C1 (LR)

C1′ (LR)

(π/π0)60

0.38 (0.999)

0.14 (0.992) 0.14 (0.999) 0.17 (0.998) 0.15 (0.984) 0.19 (0.996)

0.90 0.87 0.94 0.90 0.89 0.85

β-casein by monopalmitin is produced (Figure 4A shows an example of this behavior). The displacement of β-casein by monopalmitin, as can be observed in β-caseinmonopalmitin mixed films at any surface composition,31 progresses with surface pressure up to the collapse point. Thus, at the collapse point the mixed film is dominated by the presence of monopalmitin. The time evolution of surface pressure and relative reflectivity (Figure 7) of a β-casein-monopalmitin mixed monolayer on water at pH 7 and at a mass fraction of monopalmitin in the mixture of 0.5 (as an example) during the relaxation at constant area (at the collapse point) supports this hypothesis. It can be seen that the reflectivity of the mixed films is practically constant during the relaxation of surface pressure at the collapse point with some peaks of high reflectivity as domains of collapsed β-casein or fractures of monopalmitin pass through the spot where these measurements are made (Figure 7). Over the overall relaxation process, the mixed film presents a reflectivity lower than that for pure monopalmitin at the equilibrium surface pressure, indicated in Figure 7 by the lined area, except as regions of monopalmitin fractures or islands of collapsed β-casein pass through the spot where these measurements are performed, indicated in Figure 7 by arrows. These results corroborate that (i) the original mixed film is dominated by the presence of monopalmitin, (ii) the displacement of β-casein by monopalmitin is not quantitative, and (iii) the mixed films are immiscible with domains of monopalmitin (in a large extension) and β-casein (in a lower extension) on the interface. Finally, the displacement of β-casein by monopalmitin at the air-water interface is a dynamic and reversible process, as can be deduced from data presented in Figures 3 and 4. It can be seen (Figure 4) that the evolution of the surface pressure and, especially, of the reflectivity during the compression-expansion cycle is not symmetrical. The presence of numerous and more intense reflectivity peaks during the expansion than during the compression, due to the transformation of the 3-D collapsed monopalmitin phase into the liquid-expanded and finally in the liquidexpanded phases, on one hand, and the readsorption of the previously displaced β-casein, on the other hand, as the expansion progresses involve some time. The reversibility of these processes can be deduced from the repetitiveness in the π-A isotherms after continuous compression-expansion cycles (Figure 3). Relaxation Phenomena in β-Casein-Monoolein Mixed Films. Relaxation Phenomena at Constant Surface Pressure. Figure 8 shows the relaxation in relative molecular area at 20 °C and at 20 mN/m for β-caseinmonoolein mixed films. For the pure monoolein monolayer, the long-term relaxation phenomena can be quantified by a desorption mechanism in two steps:23 dissolution (eq 1, A1 ) 7.0 × 10-3 min-0.5, LR ) 0.997) and diffusion into the aqueous phase (eq 2, A2 ) 2.8 × 10-4 min-0.5, LR ) 0.989). For β-casein and β-casein-monoolein mixed films, the (A/A0) relaxation has an exponential time dependence as

Figure 7. The time evolution of (O) surface pressure and (4) relative reflectivity (at a shutter speed of 1/250 s) during the relaxation at constant area (at the collapse point) for a β-caseinmonopalmitin mixed monolayer on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture) ) 0.5. The lined area represents the relative reflectivity of the pure monopalmitin monolayer at the equilibrium surface pressure (πe MP).

Figure 8. Relaxation at constant surface pressure (π ) 20 mN/m) for β-casein-monoolein mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monoolein in the mixture): (O) 0, (solid line) 0.2, (dashed line) 0.4, (dotted line) 0.6, (dashed-dotted line) 0.8, and (4) 1.

expected for conformation/reorganization changes within the spread molecules. The best fits of the experimental data were obtained by means of eq 5 with two relaxation times. In Table 1, the relaxation times and the value of relative area at 60 min of relaxation time, (A/A0)60, are shown. It can be seen that the amplitude of the (A/A0)60 relaxation is lower for pure β-casein than for β-caseinmonoolein mixed films. As for the β-casein-monopalmitin mixed monolayer, the relaxation phenomena at 20 mN/m for β-casein-monoolein mixed films are reversible processes, as can be deduced from the repetitiveness in π-A isotherms after continuous compression-expansion cycles (data not shown). The relaxation phenomena observed for β-caseinmonoolein and β-casein-monopalmitin mixed films after the compression up to 20 mN/m are essentially similar. Thus, the same reasoning used previously for the latter would be utilized for β-casein-monoolein mixed films. Relaxation Phenomena at Constant Surface Area (at the Collapse Point). β-Casein-monoolein mixed films were also tested at the maximum superficial density at constant collapse area. Figure 9 shows the relaxation in surface pressure at constant surface area (at the collapse point) for β-casein-monoolein mixed films. β-Casein-monoolein monolayers behave differently than β-casein-monopalm-


Figure 9. Relaxation at constant area (at the collapse point) for β-casein-monoolein mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monoolein in the mixture): (O) 0, (solid line) 0.2, (dashed line) 0.4, (dotted line) 0.6, (dashed-dotted line) 0.8, and (4) 1. The equilibrium surface pressures for monoolein (πe MO) and β-casein (πe BC) are indicated by arrows.

Langmuir, Vol. 18, No. 22, 2002 8461

Figure 10. Relaxation at constant surface pressure (π ) 20 mN/m) for β-casein-monolaurin mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monolaurin in the mixture): (O) 0, (solid line) 0.2, (dashed line) 0.4, (dotted line) 0.6, (dashed-dotted line) 0.8, and (4) 1.

Table 3. Characteristic Parameters [Equation 6] for Relaxation of β-Casein-Monoolein Mixed Films at Constant Area (at the Collapse Point) and at 20 °C XMO

τ1

τ2

(π/π0)60

0 0.2 0.4 0.6 0.8 1

46.3 10.2 8.85 8.9 6.75

15.8 12.3 18.0 7.58 3.43

0.90 0.84 0.82 0.85 0.82 0.62

Table 4. Characteristic Parameters [Equations 1 and 2] for Relaxation of β-Casein-Monolaurin Mixed Films at Constant Surface Pressure (π ) 20 mN/m) and at 20 °C XML

A1 × 103 (LR)

A2 × 105 (LR)

0.2 0.4 0.6 0.8 1.0

10.7 (0.991) 19 (0.992) 31.7 (0.998) 42.6 (0.999) 90 (0.999)

19 (0.938) 29 (0.991) 35 (0.970) 96 (0.096) 243 (0.938)

itin monolayers. It can be seen (Figure 9) that in experiments at constant collapse area, the surface pressure relaxes from the collapse value, which is close to πe (see arrow in Figure 9), toward lower π values at longer times. Constant area relaxation studies are difficult to interpret due to the interference of different relaxation processes, such as desorption, organization/reorganization changes, and monolayer collapse, and to the fact that the monolayer is continuously passing through the various monolayer states during the course of the experiment. In fact, during the monolayer compression-expansion cycle up to the collapse point different events occur in the filmforming components (Figure 4B): (i) monoolein adopts a liquid-expanded structure up to the collapse point at a surface pressure close to πe; (ii) the absence of any defined structure reduces the reflectivity peaks of the mixed film under both monolayer compression and expansion, a consequence of the monolayer isotropy as deduced from BAM images;32 (iii) a squeezing out of β-casein by monoolein is detected by BAM after the β-casein collapse; (iv) we observed some peaks of higher intensity, especially during the monolayer expansion, when a domain of collapsed β-casein molecules passed through the spot where this measurement was performed; and (v) the I-π plot during the compression-expansion cycle is not symmetrical, indicating that the molecular reorganization and readsorption of β-casein molecules at the interface upon expansion requires some time, as a consequence of

Figure 11. The time evolution of (O) relative area and (4) relative reflectivity (at a shutter speed of 1/50 s) during the relaxation at constant surface pressure (π ) 20 mN/m) for a β-casein-monolaurin mixed monolayer on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monolaurin in the mixture) ) 0.5. The lined area represents the relative reflectivity of the pure monolaurin monolayer at 20 mN/m. Key: (A) β-casein and (B) monolaurin.

the dynamic behavior of the monolayer. However, the structural changes produced during the compressionexpansion cycle for β-casein-monoolein are lower than for β-casein-monopalmitin mixed films due to the facts that monopalmitin has a rich structural polymorphism and monoolein collapses by formation of lenses,23 a typical behavior of liquid surfactants. As the surface pressure value is lower than πe at a higher relaxation time, the relaxation characteristics of monoolein and β-casein-monoolein mixed monolayers at the collapse area could be assigned to different phenomena, such as desorption and collapse occurring concurrently. The possibility of a concurrence of these effects considerably complicates the interpretation of the overall kinetics. The concurrence of different phenomena, such as desorption and collapse, in the relaxation of pure monoolein monolayers at the collapse point has been quantified recently with a satisfactory result.23 On the basis of both the immiscibility between the filmforming components and the practical displacement of β-casein by monoolein at the collapse area, the relaxation phenomena in β-casein-monoolein mixed films under these conditions may be associated with the concurrence of different relaxation phenomena, including the desorption, reorganization, and readsorption of β-casein molecules, and collapse. This hypothesis can be supported by

8462


Figure 12. Relaxation at constant area (at the collapse point) for β-casein-monolaurin mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monoolein in the mixture): (O) 0, (solid line) 0.2, (dashed line) 0.4, (dotted line) 0.6, and (dashed-dotted line) 0.8.

the quantification of the (π/π0) relaxation by means of an exponential dependency (eq 5), with two relaxation times (Table 3). On the other hand, the magnitude of relaxation in surface pressures is practically the same for all mixed films. Relaxation Phenomena in β-Casein-Monolaurin Mixed Films. Relaxation Phenomena at Constant Surface Pressure. Figure 10 shows the relaxation in relative molecular area at 20 °C and at 20 mN/m for β-caseinmonolaurin mixed films. The relaxation phenomena are more pronounced for these systems than for β-caseinmonopalmitin (Figure 2) and for β-casein-monoolein (Figure 8) mixed films. Unlike monopalmitin- and monoolein-β-casein mixed films, a mechanism with two steps in accord with eqs 1 and 2 fits the data better. Thus the phenomena of monolayer relaxation may be due to a desorption mechanism by dissolution and diffusion. The characteristic parameters for destabilization of β-caseinmonolaurin monolayers are included in Table 4. The


significant monolaurin monolayer molecular loss that is observed in these experiments (Figure 10) has important consequences not only from a practical point of view but also for the characteristics of the β-casein-monolaurin film at the air-water interface, as will be discussed later. The relaxation phenomena in β-casein-monolaurin mixed films at 20 mN/m are a consequence of the immiscibility between the film-forming components and may be associated with the instability of monolaurin in the mixture by desorption into the bulk aqueous phase. However, we do not reject the concurrence of the phenomenon of looping of the amino acid segments of β-casein into the underlying aqueous phase, as discussed in previous sections. In fact, from the evolution of the reflectivity of a mixed monolayer of β-casein + monolaurin at a mass fraction of monolaurin in the mixture of 0.5 (as an example) during the relaxation at 20 mN/m (Figure 11), the mixed film presents the existence of some heterogeneity. In this experiment, we have selected a shutter speed of 1/50 s in order to increase the sensitivity in the observation of monolaurin (see in Figure 11 the characteristic reflectivity peaks of liquid-condensed monolaurin domains), although for this shutter speed the domains of β-casein with high reflectivity saturate the camera. Figure 11 shows that as the relaxation progresses, regions of β-casein-rich and monolaurin-rich domains passed alternatively through the spot where this measurement was performed, the reflectivity of the mixed film being higher than that for pure monolaurin, which is indicated in Figure 11 by the lined area. Relaxation Phenomena at Constant Surface Area (at the Collapse Point). Figure 12 shows the relaxation in surface pressure at constant surface area (at the collapse point) for β-casein-monolaurin mixed films. It can be seen that in experiments at constant collapse area, the surface pressure relaxes from the collapse value to very low surface pressures. However, important differences with monopalmitin- and monoolein-β-casein mixed films can be

Figure 13. Compression-expansion cycles for β-casein-monolaurin mixed monolayers on water at pH 7. Temperature ) 20 °C. Monolayer composition (mass fraction of monolaurin in the mixture): (A) 0.2, (B) 0.4, (C) 0.6, and (D) 0.8. The first cycle was at 30 min after spreading, at the maximum area. The second cycle was after 30 min at the maximum area, after the relaxation experiment at constant surface pressure (π ) 20 mN/m).


Langmuir, Vol. 18, No. 22, 2002 8463

of high reflectivity as domains of collapsed β-casein and domains of monolaurin with a liquid-condensed structure pass through the spot where these measurements were made. However, as visualized by BAM images (data not shown), the frequency of the β-casein domains increases at the expense of monolaurin domains as the relaxation progresses. Interestingly, the surface pressure relaxes toward the πe value of the pure β-casein monolayer, which is indicated in the figure by an arrow. That is, from the relaxation experiments at the collapse area it can be concluded that monolaurin molecules are depleted from the mixed films due to their instability by desorption into the aqueous bulk phase. Figure 14. The time evolution of (4) surface pressure and (O) relative reflectivity (at a shutter speed of 1/125 s) during the relaxation at constant area (at the collapse point) for a β-caseinmonolaurin mixed monolayer on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monolaurin in the mixture) ) 0.5. The lined area represents the relative reflectivity of the pure β-casein monolayer at 20 mN/m (just before the collapse point). The equilibrium surface pressures for monolaurin (πe ML) and β-casein (πe BC) are indicated by arrows. Key: (A) monolaurin domains, (B) β-casein domains, and (C) β-casein with minor monolaurin domains.

observed. First, the magnitude of the relaxation in surface pressure is higher for monolaurin- than for monopalmitin- and monoolein-β-casein mixed films. Second, the (π/π0) relaxation has a logarithmic time dependence as for β-casein-monopalmitin mixed films, but this dependence cannot be associated with a mechanism of collapse by nucleation and growth of nuclei of monolaurin because the surface pressure decreases to values lower than πe for monolaurin. Clearly, the desorption of monolaurin, which is practically instantaneous for pure films at the collapse point,23 has an important role in the relaxation phenomena observed in the mixed films (Figure 12). The instability of the mixed films can be also deduced from the plots of Figure 13. The displacement toward the π-axis of the π-A isotherm after continuous compressionexpansion cycles is a consequence of a monolayer molecular loss, because the molecular area A is calculated by assuming that all the spread monolaurin molecules remain in the monolayer. The hysteresis observed in π-A isotherms (compression curves) after continuous compression-expansion cycles is due to an irreversible process of monolayer molecular loss. In fact, the isotherm does not return to its original state on recompression after a waiting period of 24 h at the maximum area (π ) 0). On the other hand, the hysteresis in the π-A isotherm during a compression-expansion cycle is higher as the content of monolaurin in the mixture increases, a phenomenon opposite to that observed for monopalmitin- (Figure 3) and monoolein-β-casein (data not shown) mixed films. These results also support evidence on the importance of the monolaurin molecular loss, as compared with the structural changes that take place in the β-casein molecules present in the mixed films during the relaxation process. The time evolution of surface pressure and relative reflectivity (Figure 14) for a β-casein-monolaurin mixed monolayer at a mass fraction of monolaurin in the mixture of 0.5 (as an example) during the relaxation at constant area (at the collapse point) supports the above hypothesis. It can be seen that the mean reflectivity of the mixed films is practically constant during the relaxation of surface pressure at the collapse point with numerous peaks

Conclusions This article presents experimental studies of long-term relaxation phenomena in β-casein-monoglyceride mixed films at the air-water interface. Data have been analyzed according to models for desorption, collapse, and organization/reorganization changes, after the monolayer compression to a surface pressure of 20 mN/m or at the collapse area of the mixed film. At a constant surface pressure (at 20 mN/m), just below the β-casein collapse, the organization/reorganization changes of β-casein molecules in monopalmitin- and monoolein-β-casein mixed films are the mechanism that controls the relaxation process. At constant surface area (at the collapse point of the mixed film), the relaxation phenomena in mixed films may be due either to nucleation and growth of critical nuclei of monoglyceride, such as was deduced for β-casein-monopalmitin mixed films, or to a complex mechanism including competition between collapse and monolayer desorption, such as was deduced for β-casein-monoolein mixed films. For β-casein-monolaurin mixed films at every surface pressure, the relaxation phenomena are mainly due to the irreversible loss of monolaurin molecules by desorption into the bulk aqueous phase. The reasons for these behaviors must be associated with the immiscibility between β-casein and monoglyceride at the air-water interface and to the β-casein displacement by the monoglyceride at surface pressures higher than that for β-casein collapse. Recent studies47-49 have shown that the displacement of milk proteins from the air-water interface involves a novel orogenic mechanism. Such a model allows an explanation of the displacement of protein by monoglycerides. The orogenic displacement mechanism is a consequence of the existence of few interactions between proteins and monoglycerides at the air-water interface.50 The long-term relaxation phenomena also serve to analyze the surface dilatational characteristics of proteinmonoglyceride mixed films.51 Acknowledgment. This research was supported in part by EU through Grant FAIR-CT96-1216 and by DGICYT through Grant PB97-0734. LA0202071 (47) Mackie, A. R.; Gunning A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (48) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. Langmuir 2000, 16, 2242. (49) Mackie, A. R.; Gunning A. P.; Ridout, M. J.; Wilde, P. J.; Rodrı´guez Patino, J. M. Biomacromolecules 2001, 2, 1001. (50) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C.; Cejudo, M. Langmuir 2000, 17, 7545. (51) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Ind. Eng. Chem. Res. 2002, 41, 2652.

Dynamic Properties of Î²-CaseinâMonoglyceride Mixed Films at the Air

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