Thermodynamic and Dynamic Characteristics of Monoglyceride

Langmuir 2006, 22, 4215-4224

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Thermodynamic and Dynamic Characteristics of Monoglyceride Monolayers Penetrated by β-Casein Cecilio Carrera Sańchez, Marta Cejudo Fernańdez, Ma. Rosario Rodrı´guez Ninõ, and Juan M. Rodrı´guez Patino* Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, UniVersidad de SeVilla, Calle Prof. Garcıá Gonza´ lez, 1, E-4012 SeVille, Spain ReceiVed December 28, 2005. In Final Form: February 22, 2006 In this work, we have analyzed the dynamics of the penetration of β-casein into monoglyceride monolayers (monopalmitin and monoolein) and the structural, dilatational, and topographical characteristics of mixed films formed by monoglyceride penetrated by β-casein. Different complementary experimental techniques [dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology] have been used, maintaining the temperature constant at 20 °C and the pH at 7. The surface pressure of the monoglyceride monolayer at the beginning of the penetration process (at πiMP and πiMO for monopalmitin and monoolein, respectively) was the variable studied. β-Casein can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of β-casein into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, is a complex process that is facilitated by monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of the monoglyceride penetrated by β-casein mixed monolayers are essentially dominated by the presence of the monoglyceride (either monopalmitin or monoolein) in the mixed film.

Introduction In many emulsifier applications, such as dispersed food systems (emulsions and foams), mixtures of different emulsifiers (mainly polar lipids and proteins) often exhibit properties superior to those of the individual emulsifier alone because of synergistic interactions between emulsifier molecules. The optimum use of emulsifiers (process function) depends upon the knowledge of their interfacial physicochemical characteristics (property function), such as surface activity, structure, miscibility, superficial viscosity, etc., and the kinetics of film formation at fluid interfaces.1,2 In previous works with mixtures of monoglycerides and milk proteins, we have observed that when molecules of both emulsifiers are spread at the air-water interface some form of association between film-forming components can exist.3 Interactions between molecules of emulsifiers could affect not only the film structure and topography but also dynamic phenomena in mixed films.3-6 Information about these phenomena would be very helpful in the prediction of optimized formulations (product engineering) for food foams and emulsions.2,7 Thus far, there are important experimental results available on structural characteristics of mixed emulsifiers spread at the air-water interface.3,7-9 However, as far as we know, there have been few studies of the structural and dynamic * To whom correspondence should be addressed. Telephone: +34-954556446. Fax: +34-95-4556447. E-mail: [email protected]. (1) Dickinson E. An Introduction to Food Colloids; Oxford University Press: Oxford, U.K., 1992. (2) Van Aken, G. A. In Food Emulsions, 4th ed.; Friberg, S., Larsson, K., Sjoblom, J., Eds.; Marcel Dekker: New York, 2004; Chapter 8. (3) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Curr. Opin. Colloid Interface Sci. 2003, 8, 387-395. (4) Bos, M. A.; van Vliet, T. AdV. Colloid Interface Sci. 2001, 91, 437-471. (5) Pugnaloni, L. A.; Dickinson, E.; Ettelaie, R.; Mackie, A. R.; Wilde, P. J. AdV. Colloid Interface Sci. 2004, 107, 27-49. (6) Miller, R.; Fainerman. V. B.; Lesser, M. W.; Michel, M. Curr. Opin. Colloid Interface Sci. 2004, 9, 350-356. (7) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176-181. (8) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsions and Their Applications; Hasenhuette, G. L., Hartel, R. W., Eds.; Chapman and Hall: New York, 1997; pp 95-146. (9) Dickinson, E. Colloids Surf., B 2001, 20, 197-210.

characteristics of adsorbed10,11 or penetrated8,12-16 films at the air-water interface, although in practice, mixtures of these emulsifiers are usually used to achieve an optimal effect in food formulations.7-9,17 When water-soluble amphiphiles (surfactants or proteins) are injected into the subphase beneath an insoluble monolayer, different types of interactions are possible. One form of interaction is termed penetration and involves the adsorption of the soluble amphiphilic molecules at the interface between the monolayer molecules.18 Monolayer penetration provides a useful model for many biological processes19,20 and is relevant to numerous industrial and food processing operations.21,22 The penetration of phospholipids by proteins has been studied in numerous publications because they serve as models for biomembranes and because of their important biotechnological and biomedical applications.19,20,23-27 For food dispersion formulations (10) Li, J. B.; Kra¨gel, J.; Makievski, A. V.; Fainermann, V. B.; Miller, R.; Mo¨hwald, H. Colloids Surf., A 1998, 142, 355-360. (11) Li, J. B.; Zhao, J.; Miller, R. Nahrung 1998, 42, 234-235. (12) Bos, M. A.; Nylander, T. Langmuir 1996, 12, 2798-2801. (13) Zhao, J.; Vollhardt, D.; Brezesinski, G.; Siegel, S.; Wu, J.; Li, J. B.; Miller, R. Colloids Surf., A 2000, 171, 175-184. (14) Ferri, J. K.; Miller, R.; Makievski, A. V. Colloids Surf., A 2005, 261, 39-48. (15) Lucero, A. Propiedades Interfaciales de Emulsionantes Alimentarios: Fosfolı´pidos y Proteıńas. Ph.D. Thesis, University of Seville, Spain, 2005. (16) Lucero, A.; Rodrı´guez Ninõ, M. R.; Carrera, C.; Rodrı´guez Patino, J. M.; Gunning, A. P.; Mackie, A. R. In Food Colloids: Interactions, Microstructure and Processing; Dickinson, E., Ed.; Royal Society of Chemistry: Cambridge, U.K., 2005; pp 160-175. (17) Friberg, S.; Larsson, K.; Sjoblom, J. Food Emulsions, 4th ed.; Marcel Dekker: New York, 2004. (18) Barnes, G. T. Colloids Surf., A 2001, 190, 145-151. (19) Notter, R. H. Lung Surfactants: Basic Science and Clinical Applications; Marcel Dekker: New York, 2000. (20) Nag, K. DeVelopments in Lung Surfactant (Dys)Function; Marcel Dekker: New York, 2003. (21) Goddard, E. D., Ananthapadmanabha, K. P., Eds.; Interactions of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, FL, 1993. (22) Brockman, H. Curr. Opin. Struct. Biol. 1999, 9, 438-443. (23) Vollhardt, D.; Fainerman, V. B. AdV. Colloid Interface Sci. 2000, 86, 103-151. (24) Brezesinski, G.; Mo¨hwald, H. AdV. Colloid Interface Sci. 2003, 100102, 563-584.

10.1021/la053506+ CCC: $33.50 © 2006 American Chemical Society Published on Web 03/23/2006

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(emulsions and foams), the penetration of a protein into a lipid or phospholipid monolayer is of special interest. In recent years, new methods, such as fluorescence,28,29 Brewster angle microscopy (BAM),30,31 ellipsometric microscopy,32 atomic force microscopy,5,15,16,33 FITR spectroscopy,34 etc., along with traditional Langmuir monolayers,35,36 have contributed to considerable progress relating thermodynamics and structures of penetrated monolayers at microscopic and nanoscopic length scales. To relate penetration concepts (property function) to industrial processes (process function), the so-called product engineering or formulation engineering, it is essential to determine the thermodynamic and dynamic characteristics of monolayers penetrated by amphiphilic molecules, a field of research that requires further investigation.37 Product engineering, which is concerned with physical or physicochemical principles, may improve the quality and performance of products with added value by the adequate correlation between property function and process function.38,39 In this study, we are concerned with both the analysis of the dynamics of penetration of a model milk protein (β-casein) into a spread monoglyceride (monopalmitin or monoolein) monolayer and with the structural and surface dilatational characteristics of mixed monolayers formed after long-term penetration. As far as we know, these systems have not been analyzed thus far. These results may have direct relevance for foam formation and stabilization and, assuming the validity of extrapolating from the air-water interface to the oil-water interface, for emulsions. These experiments also mimic the behavior of emulsifiers in food emulsions during the storage of the product in which an oil-soluble lipid (monopalmitin or monoolein) is quickly adsorbed at the fluid interface and then interacts with a protein film slowly adsorbed from the aqueous bulk phase. The monolayer technique has been used successfully for studying the properties of mixed emulsifiers spread at the air-water interface. Although these experiments have demonstrated the relevance of spread monolayers for fundamental studies,3,40,41 penetrated monolayers are more interesting from a technological point of view. The penetration of β-casein into a spread monoglyceride monolayer at the air-water interface was studied by different complementary techniques, such as dynamic tensiometry, surface pressure (π)area (A) isotherm (monolayer structure), BAM, and interfacial dilatational rheology. (25) Santos Magalhaes, N. S.; de Oliveira, H. M.; Baszkin, A. Colloids Surf., A 1996, 118, 63-73. (26) Baszkin, A.; Norde, W. Physical Chemistry of Biological Interfaces; Marcel Dekker: New York, 2000. (27) Ronzon, F.; Desbat, B.; Chauvet, J.-P.; Roux, B. Colloids Surf., B 2002, 23, 365-373. (28) Mo¨hwald, H. Annu. ReV. Phys. Chem. 1990, 41, 441-476. (29) McConnell, H. M. Annu. ReV. Phys. Chem. 1991, 42, 171-195. (30) Heńon, S.; Meunier, J. ReV. Sci. Instrum. 1991, 62, 936. (31) Ho¨ning, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590-4592. (32) Reiter, R.; Motschmann, H.; Orendel, H.; Nemetz, A.; Knoll, W. Langmuir 1992, 8, 1784-1788. (33) Wilde, P. J.; Mackie, A. R.; Husband, F.; Gunning, P.; Morris, V. AdV. Colloid Interface Sci. 2004, 108-109, 63-71. (34) Dluhy, R. G.; Cornell, D. G. J. Phys. Chem. 1985, 89, 3196. (35) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interface; Interscience: New York, 1966. (36) Davies, J. T.; Rideal, E. K. Interfacial Phenomena, 2nd ed.; Academic Press: London, U.K., 1963. (37) Rider, K. B.; Hwang, K. S.; Salmeron, M. Phys. ReV. Lett. 2001, 86, 4330-4333. (38) Schubert, H.; Ax, K.; Behrend, O. Trends Food Sci. Technol. 2003, 14, 9-16. (39) He, L.; Dexter, A. F.; Middelberg, A. P. J. Chem. Eng. Sci. 2006, 61, 689-1003. (40) Horne, D.; Rodrı´guez Patino, J. M. In Biopolymers at Interfaces, 2nd ed.; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; pp 857-900. (41) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M.; Carrera, C.; Cejudo, M.; Garcıá, J. M. Chem. Eng. Commun. 2003, 190, 15-47.

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Experimental Section 1. Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90) and 1-mono(cis-9-octadecanoyl)glycerol (monoolein, RYLO MG 19) were supplied by Danisco Ingredients (Brabran, Denmark) with over 95-98% purity. β-Casein (>99%) was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Samples for the penetration of β-casein solutions were prepared using Milli-Q ultrapure water and were buffered at pH 7. To form the monolayer, 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. The water used as a subphase was purified by 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 of the experiments. 2. Dynamic Surface Pressure Measurements. The study of the dynamics of penetration of β-casein into a monoglyceride monolayer spread at the air-water interface was performed on a fully automated Langmuir-type film balance, by following the time evolution of the surface pressure after the injection of a β-casein solution underneath the monoglyceride monolayer. Before each measurement, the film balance was calibrated at 20 °C, as described previously.42,43 The monoglyceride solutions in hexane/ethanol (9:1, v/v) were spread on the subphase by a micrometric syringe at 20 °C. Aliquots of 300-350 µL (from 1.5 × 10-4 to 2 × 10-4 mg/µL) were spread in each experiment. The same precautions as in previous works were taken to allow for the evaporation of the spreading solvent (15 min was allowed to elapse before beginning the isotherm recording) and for the choice of compression rate (0.062 nm2 molecule-1 min-1).42,43 Afterward, a surface pressure (π)-area (A) isotherm for the monoglyceride was recorded and used as a control. Some experiments were repeated (at least twice). In these cases, the mean deviation was within (0.3 mN/m for the surface pressure and (0.05 m2/mg for the area. Then, the monoglyceride monolayer was compressed up to the desired surface pressure (at 10 mN/m, 20 mN/m, or the collapse point) before experiments for β-casein penetration were carried out. For β-casein-adsorbed films from water, a protein solution from 5 × 10-6 to 7.5 × 10-6 wt % was left in the trough and time was followed for protein penetration at the interface. These β-casein concentrations ware selected from previous data of the adsorption isotherm .44 At this β-casein concentration in solution, the surface pressure at equilibrium is 0. In fact, in control experiments, we have observed that for β-casein solutions at 5 × 10-6 and 7 × 10-6 wt % the surface pressure (π) at the maximum area of the trough was practically 0 after 24 h of adsorption on a clean air-water interface. For the dynamics of penetration experiments, the β-casein solution was added to the aqueous subphase, being injected beyond the barrier, underneath the monolayer. The injection method used for penetration experiments has the disadvantage that the kinetics were limited by the diffusion of the protein in the subphase after injection, and this could result in poor reproducibility or artifacts. To avoid this problem, we built a device that allows fast mixing of the β-casein solution in the subphase. This device consists of two parallel lines with multiple points for the injection of the protein solution and the suction of the same amount of subphase. These lines are situated along the film balance and connected to a peristaltic pump. Thus, the volume of the subphase is maintained constant at 750 mL during the experiment. Measurements of surface relaxation in monoglyceride monolayers at the air-water interface were also performed as a control on the same Langmuir-type film balance. The method has been described previously.45 The monoglyceride solutions were spread on the subphase by a micrometric syringe at 20 °C. In these experiments, the area is kept constant (at the collapse point of each monoglyceride) (42) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 2484-2492. (43) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Food Hydrocolloids 1999, 13, 401-408. (44) Rodrı´guez Ninõ, M. R.; Carrera, C.; Cejudo, M.; Rodrı´guez Patino, J. M. J. Am. Oil Chem. Soc. 2001, 78, 873-879. (45) Carrera, C.; Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 175-192.

Penetration of β-Casein into Monoglyceride Monolayers and the surface pressure decreases. This decrease is measured as a function of time. 3. Surface Film Balance. Measurements of surface pressure (π)area (A) isotherms of the monoglyceride and monoglyceride penetrated by β-casein mixed films at the air-water interface were performed on the same Langmuir-type film balance as described previously.42,43 These experiments were performed after the surface pressure relaxed to a steady-state value during previous penetration experiments. The π-A isotherm was measured at least 5 times. The reproducibility of the results was better than (0.5 mN/m for the surface pressure and (0.05 m2/mg for the area. 4. BAM. A commercial Brewster angle microscope, BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography (morphology and reflectivity) of the monolayer. The Brewster angle microscope was positioned over the film balance. Further characteristics of the device and operational conditions have been described elsewhere.42,43 The surface pressure measurements, area, and gray level as a function of time were carried out simultaneously by a device connected between the film balance and Brewster angle microscope. To measure the relative reflectivity (I) of the film, a previous camera calibration is necessary.42,43 The imaging conditions were adjusted to optimize both the image quality and quantitative measurement of reflectivity. Thus, generally, as the surface pressure or the protein content increased, the shutter speed was also increased. The reflected beam passes through a focal lens, into an analyzer at a known angle of incident polarization, and finally to a CCD camera. Rotation of the analyzer allows the image contrast to be adjusted by varying the reflected polarization that is passing to the camera. If the emulsifier domain does not have a uniform reflectivity, this reflectivity changes with the analyzer angle. Thus, the optical anisotropy, which is typical for liquid-condensed (LC) structures because of crystalline-like domains being formed at the air-water interface42, can be visualized for different positions of the analyzer relative to the plane of incidence. Some images were recorded in the absence of the automatic gain control (AGC) to distinguish between protein (with high reflectivity, which saturate the camera with an image completely white) and monoglyceride (with low reflectivity) domains in a continuous homogeneous phase. 5. Surface Dilatational Rheology. To obtain surface rheological parameters, such as surface dilatational modulus, its elastic, Ed, and viscous, Ev, components, and the loss angle tangent, tan θ, a modified Wilhelmy-type film balance (KSV 3000) was used as described elsewhere.46,47 In this method, the surface is subjected to small periodic sinusoidal compressions and expansions by two oscillating barriers at a given frequency (ω) and amplitude (∆A/A) and the response of the surface pressure is monitored. Surface pressure was directly measured by two roughened platinum plates situated on the surface between the two barriers. The surface dilatational modulus derived from the change in the surface pressure resulting from a small change in the surface area may be described by the equation48 E ) -(δπ/δ ln A) ) Ed + iEv The dilatational modulus is a complex quantity and is composed of real and imaginary parts. The real part of the dilatational modulus or storage component is the dilatational elasticity, Ed ) |E|cos θ. The imaginary part of the dilatational modulus or loss component is the surface dilatational viscous modulus, Ev ) |E|sin θ. The loss angle tangent can be defined by tan θ ) Ev/Ed. Thus, for a perfectly elastic monolayer tan θ is 0. Measurements were performed at least 3 times. The reproducibility of these results was better than 5%.

Results and Discussion 1. Dynamic of the Penetration of β-Casein into Monoglyceride Monolayers. The penetration of β-casein into an insoluble monoglyceride monolayer was followed by measurements of (46) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. Langmuir 2001, 17, 4003-4013. (47) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141-151. (48) Lucassen, J.; van den Tempel, M. Chem. Eng. Sci. 1972, 27, 1283-1291.

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Figure 1. Time dependence of surface pressure during the penetration of β-casein into a spread monopalmitin monolayer as a function of the initial surface pressure (πiMP): (+) 10 mN/m, (- - -) 20 mN/m, and (‚‚‚) at the collapse point of monopalmitin. Temperature at 20 °C and pH 7. The concentration of β-casein in the bulk phase is 5 × 10-6 wt %. The πe of β-casein is indicated by an arrow. The long-term relaxation of a pure monopalmitin monolayer (s) is included as a reference. (Inset) Time evolution of the surface pressure of the penetration of β-casein into a spread monopalmitin monolayer.

Figure 2. Time dependence of the surface pressure during the penetration of β-casein into a monoolein monolayer as a function of the initial surface pressure (πiMO): (+) 10 mN/m, (- - -) 20 mN/ m, and (‚‚‚) at the collapse point of monoolein, at pH 7 and 20 °C. The concentration of β-casein in the bulk phase is 5 × 10-6 wt %. The πe of β-casein is indicated by an arrow. The long-term relaxation of a pure monoolein monolayer (s) is included as a reference. (Inset) Time evolution of the surface pressure of the penetration of β-casein into a spread monoolein monolayer.

the time evolution of the surface pressure at a constant surface area. β-Casein was injected into the aqueous bulk phase, underneath the monopalmitin (Figure 1) or monoolein (Figure 2) monolayer, at different initial surface pressures (at πiMP or πiMO, respectively). It can be observed that the dynamics of surface pressure evolution after the injection of β-casein into the aqueous bulk phase, underneath the monoglyceride monolayer, depend upon the level of initial surface pressure. Significant differences can be observed if the initial surface pressure is lower or higher than the equilibrium surface pressure of β-casein (πeβ-casein). The equilibrium surface pressure (πe) is the maximum surface pressure to which a spread monolayer may be compressed without the possibility of monolayer collapse. β-Casein and monoolein collapse at πe; however, monopalmitin collapses at πc > πe. Thus, a monopalmitin monolayer is in a metastable state at π > πe.42,43 1.1. Penetration at Surface Pressures Lower Than the Equilibrium Surface Pressure of β-Casein (πeβ-casein). At initial surface pressures (πiMP or πiMO) lower than the equilibrium

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Table 1. Effect of the Initial Surface Pressure of the Monoglyceride Monolayer (πimonoglyceride) on the Final Surface Pressure after 1200 min of β-Casein Penetration into the Monopalmitin (πMP1200) or Monoolein (πMO1200) Monolayer πimonoglyceride (mN m)

πMP1200 (mN m)

πMO1200 (mN m)

10 20 collapse

18.9 20.7 24.3

19.2 21.3 25.3

surface pressure of β-casein (πeβ-casein ) 20.9 mN/m),44 the evolution of the surface pressure with time after the injection of β-casein corresponds to an adsorption mechanism with two kinetic steps: (i) initially the surface pressure was constant (see insets in Figures 1 and 2) and, finally, (ii) π increased progressively with the penetration time up to a maximum plateau value. The initial plateau in π could be associated with the penetration of β-casein from underneath the monoglyceride monolayer, which produces an accumulation of the protein, which finally destabilizes the monoglyceride monolayer and promotes a progressive penetration of β-casein into the monoglyceride monolayer at long-term penetration. As a consequence of the β-casein penetration into the monoglyceride monolayer, the surface pressure increases up to the final maximum plateau value (Table 1), which is close to the πeβ-casein value. The differences observed in the π-time plot as a function of both monoglyceride (monopalmitin or monoolein) and the initial surface pressure of the monoglyceride monolayer suggest that the penetration of β-casein is sensitive to the structure of the monoglyceride monolayer (Figures 1 and 2). From the insets in Figures 1 and 2, it can be seen that for the penetration of β-casein into monopalmitin monolayers (Figure 1) the initial plateau in the π-time curve at πiMP ) 10 mN/m disappears at a higher surface pressure (at πiMP ) 20 mN/m) but the opposite is observed for monoolein monolayers (Figure 2). Interestingly, the maximum surface pressure attained at longer times is close to the equilibrium surface pressure for a pure β-casein monolayer, which is indicated in Figures 1 and 2 by an arrow. For the same amount of β-casein injected into the subphase, the surface pressure at equilibrium is 0 after the adsorption to a clean interface. Thus, the monoglyceride monolayer (either monopalmitin or monoolein) acts as a promoter of access of β-casein at the air-water interface. However, the accessibility of β-casein at the air-water interface is hindered in monoolein monolayers because the extent of the plateau at πι ) 10 mN/m is higher for monoolein (Figure 2) than for monopalmitin (Figure 1). We must reject the possibility that a change in the monoglyceride monolayer structure may be produced as a consequence of the penetration of β-casein at the interface, as observed for phospholipids at low surface pressures,13,23,49 because in the range of surface pressures attained (Figures 1 and 2), the monopalmitin monolayer is in the region of LC-liquid-expanded (LE) transition and monoolein adopts a LE structure.42 However, we must not reject the possibility that a change in the monoglyceride monolayer condensation may be produced, maintaining the monolayer structure constant, as a consequence of the penetration of β-casein at the interface. The topographical characteristics of the mixed monolayers will be analyzed later. If the plot of the maximum surface pressure increases (∆πmax), attained after the β-casein injection into a monoglyceride monolayer as a function of the initial surface pressure (πimonoglyceride) yields a straight line of negative slope,16,22 the initial surface pressure of a monoglyceride monolayer for which the (49) Wang, X.; Zhang, Y.; Wu, J.; Wang, M.; Cui, G.; Li, J.; Brezesinski, G. Colloids Surf., B 2002, 23, 339.

addition of β-casein does not induce a surface pressure increase defines the exclusion surface pressure (πex).22 The πex value obtained by extrapolation of the linear ∆πmax-πi curves is ≈21 mN/m for monopalmitin or monoolein monolayer. This πex value is practically the same as the πeβ-casein value at the same pH. These results corroborate the idea that the penetration of β-casein into an insoluble monoglyceride (monopalmitin or monoolein) monolayer is facilitated at surface pressures lower than the πeβ-casein value. 1.2. Penetration at Surface Pressures Higher Than the Equilibrium Surface Pressure of β-Casein (πeβ-casein). At an initial surface pressure higher than the equilibrium surface pressure of the monoglyceride (at the collapse point of the monoglyceride monolayer),44 the evolution of the surface pressure with time was unexpected, but the same results were observed after repeated experiments with monopalmitin (Figure 1) or monoolein (Figure 2). Similar results were also observed for the same monoglycerides with different proteins (unpublished results). It can be seen that starting the injection of β-casein in the aqueous subphase at the initial surface pressures of monopalmitin or monoolein collapse (at ≈53 and ≈46 mN/m for monopalmitin and monoolein, respectively),42 the evolution of the surface pressure with time was the opposite to that discussed at π < πeβ-casein in the preceding section. In fact, it can be seen that, because the molecules in the monopalmitin or monoolein monolayer are in a state involving the strongest intermolecular interactions, with maximum density packing, with a solid monopalmitin monolayer or a LE monoolein monolayer at the maximum condensation,42 the surface pressure decreases progressively with the penetration time and tends to a minimum value (Table 1), which is a little higher than the πeβ-casein value at the same pH. The results shown in Figures 1 and 2 at an initial surface pressure higher than the equilibrium surface pressure of the monopalmitin and monoolein monolayer (πemonoglyceride) may be explained by the concurrence of a complex competition between the instability of the monoglyceride monolayer, which causes a monolayer molecular loss by collapse and/or desorption45 and facilitates the penetration of the protein into the interface; that is, monoglyceride molecules that desorb from the interface increase the available area so that the β-casein molecules will have a greater access to penetrate at the interface. A prerequisite for this penetration competition for the interface is the absence of interactions between monoglyceride and β-casein molecules, as was confirmed for spread50,51 and adsorbed52 β-caseinmonoglyceride mixed films. To gain some insight into these complex dynamic phenomena, we have included in Figures 1 and 2 the results of long-term relaxation for pure monopalmitin and monoolein monolayers, respectively. It was observed that at surface pressures higher than πemonoglyceride, the relaxation phenomena in these experimental conditions are different for monopalmitin than for monoolein monolayers. For monopalmitin monolayers, the relaxation phenomena may be due to either monolayer collapse by nucleation or growth of critical nuclei until the πe value of monopalmitin is attained (πeMP ≈ 49 mN/m),45 followed by a monolayer molecular loss by desorption at a long-term relaxation time (Figure 1). For monoolein monolayers and after a short relaxation time, at which the relaxation data can be quantified by monolayer collapse, the monolayer molecular loss by desorption controls (50) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. M.; J. Agric. Food Chem. 1999, 47, 4998-5008. (51) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. M. Langmuir 1999, 15, 4777-4788. (52) Cejudo, M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M. Biomacromolecules 2006, 7, 507-514.

Penetration of β-Casein into Monoglyceride Monolayers

the monolayer instability at long-term relaxation.45 Clearly, the collapse and desorption of monopalmitin and monoolein monolayers explain the penetration of β-casein into the spread monoglyceride monolayer at the highest state of condensation, at the collapse point of the spread monolayer. The penetration of β-casein into the monoglyceride monolayer can explain the fact that at the beginning of the penetration process the surface pressure decreases faster for the monoglyceride monolayer penetrated by β-casein than for a pure monoglyceride monolayer (Figures 1 and 2). On the other hand, the surface pressure decreases with the penetration time faster for monoolein (Figure 2) than for monopalmitin (Figure 1). A speculation is that the penetration of β-casein at the collapse point of the monoglyceride monolayer may be easier for monoolein (with a more expanded structure) than for monopalmitin (with a more condensed structure). Interestingly, the penetration of β-casein into the monoolein monolayer hindered the instability of the monolayer by desorption, because the relaxation in a pure monoolein monolayer was faster than for the monolayer penetrated by β-casein (Figure 2). However, the opposite was observed for a monopalmitin monolayer penetrated by β-casein (Figure 1). 2. Topography of Monoglyceride Monolayers Penetrated by β-Casein under Dynamic Conditions. BAM images and the evolution of reflectivity (I) evolutions during the penetration of β-casein into a monoglyceride monolayer as a function of the initial surface pressure of the monoglyceride (Figures 3-6) give support to the conclusions derived from the dynamics of surface pressure evolution with time (Figures 1 and 2). 2.1. Topography of Monoglyceride Monolayers at Surface Pressures Lower Than the Equilibrium Surface Pressure of β-Casein (πeβ-casein) Penetrated by β-Casein under Dynamic Conditions. For a monopalmitin monolayer at πiMP ) 10 mN/m penetrated by β-casein, the monolayer reflectivity increases with the surface pressure as the penetration process takes place (Figure 3B). The monolayer reflectivity is a maximum at the highest surface pressure at the plateau, which is close to the πeβ-casein value. The increase in I with π and especially near the β-casein monolayer collapse suggests that an increase in the monolayer thickness from a more expanded to a more condensed film structure takes place. Similar results are observed for the penetration of β-casein into a monopalmitin monolayer at πiMP ) 20 mN/m (data not shown). Interestingly, the reflectivity of the mixed film at πiMP < πeβ-casein is higher than that for pure components, which are included in Figure 3 as a reference. These results strengthen the hypothesis that for mixed films of adsorbed monopalmitin penetrated by β-casein some degree of attractive interactions between film-forming components does exist at a microscopic level, giving a mixed film with greater thickness as compared with those of pure components. The same phenomenon was observed for β-casein-monopalmitin adsorbed mixed films.52 At a microscopic level, the penetration of β-casein into a monopalmitin monolayer gives mixed films with characteristic features (Figure 3A). Briefly, the results reported here suggest that, in β-casein-monopalmitin mixed films, islands of protein and monoglyceride do exist at the air-water interface. In contrast with the typical circular LC domains of monopalmitin at 5 < π < 30 mN/m uniformly distributed on the homogeneous LE phase (panel a in Figure 3A), a mixed film of monopalmitin penetrated by β-casein may exist at πiMP < πeβ-casein with large (panel b in Figure 3A) and deformed (panel c in Figure 3A) domains of monopalmitin with a LC structure. Another topographical characteristic of monopalmitin monolayers penetrated by β-casein (not observed in spread mixed film)50 is the presence of numerous

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Figure 3. (A) Visualization by BAM and (B) reflectivity of monopalmitin monolayers penetrated by β-casein as a function of the penetration time and at 20 °C. The initial surface pressure of a spread monopalmitin monolayer is 10 mN/m. The concentration of β-casein in the bulk phase is 5 × 10-6 wt %. Key of images in A: (a) monopalmitin monolayer at 5 < π < 30 mN/m and (b-i) monopalmitin monolayers penetrated by β-casein at representative surface pressures (mN/m): (b and c) 9, (d) 14, and (e-i) 16 (further information is provided in the text). The horizontal direction of the image corresponds to 630 µm, and the vertical direction of the image corresponds to 470 µm. Symbols in B: (O) monopalmitin monolayers penetrated by β-casein, (4) monopalmitin, and (*) β-casein pure films.

small holes in the interior of the LC monopalmitin domains. These holes are the nuclei of β-casein domains penetrating into monopalmitin LC domains and may be the cause of the large size of LC monopalmitin domains in the mixed film. However, we do not reject the possibility that penetration of β-casein takes place into the homogeneous region with the LE monopalmitin structure. However, the homogeneous region of both β-casein and monopalmitin domains cannot be distinguished by BAM.51 These topographical characteristics prove the existence of few interactions between film-forming components. In fact, as the penetration process progresses, at higher surface pressures, a frontier between monopalmitin with small and large LC domains and a homogeneous phase of β-casein is present at the interface (panel d in Figure 3A). The fact that the reflectivity of the small domains changes with the analyzer angle (image not shown) proves that they are due to regions of monopalmitin with a anisotropic topography. The presence of small LC monopalmitin domains is associated with the existence of a homogeneous phase of β-casein. After a further increase of π, at higher penetration time, the holes of β-casein domains increase in size and the LC monopalmitin domains are so deformed that they no longer have

4220 Langmuir, Vol. 22, No. 9, 2006

Figure 4. Visualization by BAM of monopalmitin monolayers penetrated by β-casein at the end of the penetration (after 1200 min of β-casein penetration) during the compression. Temperature at 20 °C. The initial surface pressure of a spread monopalmitin monolayer is 10 mN/m. The concentration of β-casein in the bulk phase is 5 × 10-6 wt %. Key: (a-c) 38 mN/m and (d) 51 mN/m (further information is provided in the text). The horizontal direction of the image corresponds to 630 µm, and the vertical direction of the image corresponds to 470 µm.

a circular shape (panel e in Figure 3A). The presence of monopalmitin in the morphology of this region can be confirmed by changes in the reflectivity with the analyzer angle (data not shown) because of the anisotropy of this phase. However, the presence of β-casein can also be confirmed by the presence of regions of high brightness because the image is registered in the absence of AGC (panel f in Figure 3A). The image shown in panel g in Figure 3A also proves the existence of a large region of β-casein in the mixed film, because the morphology does not change with the analyzer angle. At the end of the penetration process, near the collapse point of β-casein, the morphology of the interface shows the presence of regions of monopalmitin penetrated by β-casein with large extensions and aligned in the direction of the compression (panel h in Figure 3A) and a characteristic morphology52 of adsorbed aggregates of β-casein (panel i in Figure 3A). The reflectivity peaks (Figure 3B) are deeper for penetrated than for pure components and prove the heterogeneity of the film. Moreover, the penetration of β-casein into a monopalmitin monolayer can also be visualized at a microscopic level by the reduction of mobility in LC monopalmitin domains, which correlates with the higher shear viscosity of β-casein and monopalmitin mixed films at the higher surface pressures53,54 as the penetration process progresses. The topography of the mixed film of monopalmitin monolayers penetrated by β-casein at the end of the penetration (after 1200 min of β-casein penetration) and during the compression (Figure 4) also demonstrates the existence of some domains of collapsed protein in the mixed film. In fact, during the compression at π > πeβ-casein, the presence of large regions of collapsed β-casein can be observed (Figure 4a) in addition to regions of collapsed β-casein with large holes of LC domains of monopalmitin (Figure 4b) and regions of LC domains of monopalmitin with holes of collapsed β-casein (Figure 4c). At the collapse point of the mixed film, monopalmitin domains with a solid structure occupy the (53) Carrera, C.; Rodrı´guez Patino, J. M. Langmuir 2004, 20, 627-6334. (54) Rodrı´guez Patino, J. M.; Cejudo, M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Ind. Eng. Chem. Res. 2006, in press.

Sa´ nchez et al.

Figure 5. (A) Visualization by BAM and (B) reflectivity of monoolein monolayers penetrated by β-casein as a function of the penetration time and at 20 °C. The initial surface pressure of a spread monoolein monolayer is 10 mN/m. The concentration of β-casein in the bulk phase is 5 × 10-6 wt %. Key of images in A: (a and b) 13.7 mN/m and (c) 18 mN/m. The horizontal direction of the image corresponds to 630 µm, and the vertical direction of the image corresponds to 470 µm. Symbols in B: (O) monoolein monolayers penetrated by β-casein, (4) monoolein, and (*) β-casein pure films.

overall morphology of the interface, with the exception of the presence of large fractures of collapse β-casein (Figure 4d). The topography of monoolein monolayers at πiMO < πeβ-casein penetrated by β-casein (Figure 5) is essentially different to that of monopalmitin in the mixture (Figure 3). Briefly, for a monoolein monolayer at πiMO ) 10 mN/m (similar results are observed at πiMO ) 20 mN/m, data not shown) penetrated by β-casein, the monolayer reflectivity increases with the surface pressure as the penetration process takes place (Figure 5B). However, the reflectivity of the mixed films is between those of pure components, which are included in Figure 5B as a reference. The increase in I with π and especially near the β-casein monolayer collapse suggests that an increase in the monolayer thickness from a more expanded to a more condensed film takes place but maintaining the same LE-like structure of the filmforming components. In fact, as expected,51 the morphology of the interface with pure components and the mixed film is practically identical because in this region both components and the mixed film form an isotropic (homogeneous) monolayer without any difference in the domain topography (panels a and b of Figure 5A). The presence of β-casein can be confirmed by the existence of small regions of high brightness as the image is registered in the absence of AGC (panel c in Figure 5A). At surface pressures near β-casein collapse (at the end of the penetration process), BAM images (panel c in Figure 5A) demonstrated that monoolein and β-casein molecules adopt an isotropic structure in the mixed film with some white regions, which correspond to the presence of a thicker β-casein collapsed monolayer. The reduction of reflectivity peaks (Figure 5B) also proves the homogeneous topography of mixed penetrated films. 2.2. Topography of Monoglyceride Monolayers at Surface Pressures Higher Than the Equilibrium Surface Pressure of β-Casein (πeβ-casein) Penetrated by β-Casein under Dynamic Conditions. The topography of a monopalmitin monolayer at an


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Figure 7. Surface pressure-area isotherms (compression curve) for monolayers of monopalmitin penetrated by β-casein at 5 × 10-6 wt % in buffered water at pH 7 and 20 °C. The initial surface pressure of a monopalmitin monolayer before the injection of β-casein into the aqueous phase (πiMP): (- - -) 10 mN/m, (s) 20 mN/m, and (‚‚‚) at the collapse point. The π-A isotherms for pure (O) adsorbed β-casein and (4) spread monopalmitin monolayers are included as a reference. The πe of monopalmitin and β-casein is indicated by arrows.

Figure 6. (A) Visualization by BAM and (B) reflectivity (O) of monopalmitin monolayers penetrated by β-casein as a function of the penetration time and at 20 °C. The initial surface pressure of a spread monopalmitin monolayer coincides with the collapse surface pressure. The concentration of β-casein in the bulk phase is 5 × 10-6 wt %. Key of images in A: (a) 44.5 mN/m, (b and c) 39 mN/m, and (d-f) 26.5 mN/m (further information is provided in the text). The horizontal direction of the image corresponds to 630 µm, and the vertical direction of the image corresponds to 470 µm. The reflectivity of monopalmitin and β-casein at the collapse points (at 50 and 25 mN/m, respectively) is included as a reference.

initial surface pressure higher than the equilibrium surface pressure of the monoglyceride (at the collapse point) penetrated by β-casein (Figure 6) confirms the conclusions derived from the evolution of π with time (Figure 1). In the same figure, the reflectivity of pure components at the respective collapse points is included. Similar results are observed for a monoolein monolayer penetrated by β-casein (data not shown). It can be seen that, when the injection of β-casein is started in the aqueous subphase, at the initial surface pressure of monopalmitin collapse (at πiMP ≈ 53 mN/m), the reflectivity of the interface is similar to that of a pure monopalmitin monolayer but increases as the surface pressure decreases because of the penetration of β-casein into the interface. The results shown in Figure 6B also prove that the penetration of β-casein into the monopalmitin monolayer does not take place with a collapsed structure because the reflectivity peaks of the mixed film are lower than those for a collapsed β-casein monolayer. In fact, it can be seen that as the penetration of β-casein into the monopalmitin takes place the reflectivity of the interface is a little higher than that for pure monopalmitin monolayer but much lower than the reflectivity of a pure β-casein collapsed film adsorbed at the interface, although the surface pressure at the end of the penetration process is higher than πcβ-casein. The morphology of the interface at the beginning of the penetration process shows a high isotropy (panel a in Figure 6A), which is characteristic of a pure monopalmitin monolayer with a solid structure.42 The presence of monopalmitin in large regions of the film at lower surface pressures (at 39 mN/m) was confirmed by the change in the isotropy of the image in the absence of the analyzer (panel b in Figure 6A) and with the

analyzer at an angle of 50° (panel c in Figure 6A). However, as the penetration process progresses (at lower surface pressures, at 26.5 mN/m) small nuclei of β-casein can be well-distinguished in the absence of the analyzer (panel d in Figure 6A), with the analyzer at an angle of 60° (panel e in Figure 6A), or in the absence of AGC (panel f in Figure 6A). From the data presented in the preceding sections, certain questions emerge: What are the consequences of the β-casein penetration into a monoglyceride monolayer at πimonoglyceride < πeβ-casein or the combination of β-casein penetration and monoglyceride monolayer molecular loss at πimonoglyceride > πeβ-casein on the structural and rheological characteristics of the mixed films of the monoglyceride penetrated by β-casein? The answers to these questions will be given in the next sections. 3. Structural Characteristics of Monoglyceride Monolayers Penetrated by β-Casein. The π-A isotherms of monoglyceride monolayers penetrated by β-casein were registered after the penetration experiments. At the end of each penetration experiment, the monolayer was expanded at the maximum area (at π ≈ 0 mN/m) and a waiting time of 60 min was allowed before the monolayer compression. The π-A isotherms for monopalmitin and monoolein mixed monolayers penetrated by β-casein are shown in Figures 7 and 8, respectively, as a function of the initial surface pressure of the monoglyceride monolayer before the β-casein penetration (at πiMP or πiMO). From these results, significant differences between structural characteristics of mixed films depending upon the monoglyceride (either monopalmitin or monoolein) can be observed. 3.1. Structural Characteristics of Monopalmitin Monolayers Penetrated by β-Casein. The π-A isotherms for monopalmitin monolayers penetrated by β-casein on the basis that only monopalmitin was present at the interface (A, m2/ mgmonopalmitin) at different πiMP and at 20 °C are shown in Figure 7. In the same figure, we have included the π-A isotherms for a spread monopalmitin monolayer registered at the beginning of each experiment and for an adsorbed β-casein monolayer, which can be used as a control and for a comparison, respectively. The π-A isotherms for pure spread monopalmitin and adsorbed β-casein monolayers are in good agreement with data in the literature.42,52 The results of the π-A isotherms (Figure 7) with the help of the compressional coefficient (data not shown) deduced from the slope of the π-A isotherm (κ ) -dπ/dA) indicate that

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Figure 8. Surface pressure-area isotherms (compression curve) for monolayers of monoolein penetrated by β-casein at 5 × 10-6 wt % in buffered water at pH 7 and 20 °C. Initial surface pressure of a monoolein monolayer before the injection of β-casein into the aqueous phase (πiMO): (- - -) 10 mN/m, (s) 20 mN/m, and (‚‚‚) at the collapse point. The π-A isotherms for pure (O) adsorbed β-casein and (4) spread monoolein monolayers are included as a reference. The πe of monopalmitin and β-casein is indicated by arrows.

adsorbed β-casein monolayers at the air-water interface adopt two different structures or condensation states and the collapse phase.55 The surface pressure at the transition of an adsorbed β-casein monolayer is πta ≈ 12.1 mN/m. At low surface pressures (at π < 12.1 mN/m), adsorbed β-casein monolayers exist as trains with all amino acid segments located at the interface (structure 1). At higher surface pressures (at π > 12.1 mN/m) and up to the equilibrium surface pressure (πeβ-casein), amino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails (structure 2). The monolayer can be compressed at a surface pressure higher than πeβ-casein, which is indicated in Figure 7 by an arrow. Spread monopalmitin monolayers show a rich structural polymorphism as a function of the surface pressure (Figure 7). The LE phase (at π < 5 mN/m), an intermediate region at the broad plateau because of a degenerated first-order phase transition between LC and LE structures (at 5 < π < 30 mN/m), the LC structure (at π > 30 mN/m), and finally, the solid (S) structure near the monolayer collapse at a surface pressure of about 53.1 mN/m were observed; that is, monopalmitin monolayers are in a metastable state at π > πeMP ≈ 49 mN/m. For spread monopalmitin monolayers penetrated by β-casein at different πiMP, it can be seen (Figure 7) that, on the basis that only monopalmitin was present at the interface, the π-A isotherms for monopalmitin and for β-casein-monopalmitin mixed monolayers are practically coincident; that is, the structures of the mixed monolayers are practically dominated by the presence of monopalmitin. In fact, the first-order transition between LC and LE structures can be distinguished, which is typical of a pure monopalmitin monolayer. In addition, the mixed film collapsed at the collapse pressure of a pure monopalmitin monolayer. These results suggest that at π > πcβ-casein a protein displacement by the monoglyceride from the air-water interface takes place. At π < πcβ-casein, both β-casein and monopalmitin may coexist at the interface but the structural characteristics of the mixed monolayers are dominated by the presence of monopalmitin. β-Casein-monopalmitin mixed monolayers at πiMP ) 10 mN/m are an exception. For this system and at π < πcβ-casein, the π-A isotherms for β-casein-monopalmitin mixed monolayers are different from those of pure components. Thus, under these conditions, the presence of both β-casein and monopalmitin has an effect on the structural characteristics of the mixed monolayer. (55) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427.

Sa´ nchez et al.

BAM images and the evolution of the reflectivity of the interface with the surface pressure (data not shown) corroborate at a microscopic level for these conclusions. In summary, although β-casein molecules have the capacity to penetrate in a spread monopalmitin monolayer, when a steadystate surface pressure close to πcβ-casein is reached, monopalmitin molecules have the capacity to re-enter the monolayer after the expansion and recompression of the mixed monolayer. As monopalmitin re-enters the air-water interface, the structure of β-casein-monopalmitin mixed monolayers is practically dominated by the presence of monopalmitin. These results also suggest that the monopalmitin molecular loss by collapse and/or desorption (deduced in the previous section from dynamic surface pressure measurements) is reversible. We speculate that, during the β-casein penetration into a spread monopalmitin monolayer at the collapse point, monopalmitin molecules are displaced from the interface toward the subphase region and that an irreversible molecular loss into the bulk phase does not take place. 3.2. Structural Characteristics of Monoolein Monolayers Penetrated by β-Casein. The π-A isotherms for monoolein monolayers penetrated by β-casein on the basis that only monoolein was present at the interface (A, m2/mgmonoolein) at different πiMO and at 20 °C are shown in Figure 8. In the same figure, we have included the π-A isotherms for a spread monoolein monolayer registered at the beginning of each experiment and for an adsorbed β-casein monolayer. The π-A isotherm for a pure spread monoolein monolayer is in good agreement with data in the literature.42 In contrast to monopalmitin, the monoolein monolayer presents only the LE structure and collapses at the equilibrium surface pressure (πe ≈ 45.7 mN/m). For spread monoolein monolayers penetrated by β-casein at different πiMO (Figure 8), it can be seen that, on the basis that only monoolein was present at the interface, the π-A isotherms for pure monoolein and β-casein monolayers are different from those for β-casein-monoolein mixed monolayers; that is, although pure and mixed monolayers present the same LE-like structure, the π-A isotherms for β-casein-monoolein mixed monolayers are displaced in relation to pure monolayer components. Interestingly, at π > πcβ-casein, the π-A isotherms for β-casein-monoolein mixed monolayers are parallel but displaced toward lower monolayer mass areas. These results suggest that the monoolein monolayer molecular loss because of collapse and/or desorption that takes place during the preceding penetration experiments is irreversible. In fact, the displacement of π-A isotherms for β-casein-monoolein mixed monolayers toward lower monolayer mass areas is more intense as πiMO increases, especially at the collapse point, in agreement with long-term relaxation data for a pure monoolein monolayer.45 In summary, at π > πcβ-casein, a protein displacement by monoolein from the air-water interface, preceded by a previous monoolein molecular loss, takes place. At π < πcβ-casein, both β-casein and monoolein may coexist at the interface. The evolution of the reflectivity of the interface with the surface pressure (data not shown) corroborates at a microscopic level for these conclusions. 4. Surface Dilatational Characteristics of Monoglyceride Monolayers Penetrated by β-Casein. Changes in surface dilatational modulus (E) and loss angle tangent (tan θ) for spread monopalmitin and monoolein monolayers penetrated by β-casein at different monoglyceride initial surface pressures (at πiMP and πiMO), at a amplitude of deformation of 5% and at 20 °C are shown in Figures 9 and 10, respectively. The percentage area change was determined in preliminary experiments to be in the linear region. In the same figures, we have included the evolution


Figure 9. Frequency dependence of (A) surface dilatational modulus and (B) loss angle tangent for monolayers of monopalmitin penetrated by β-casein at an amplitude of deformation of 5% at pH 7 and 20 °C. The initial surface pressure of a monopalmitin monolayer before the injection of β-casein into the aqueous phase at 5 × 10-6 %, wt (πiMP): (]) 10 mN/m, (O) 20 mN/m, and (4) at the collapse point of monopalmitin. The surface pressure dependence of surface dilatational modulus for pure (*) monopalmitin and (- - -) β-casein monolayers at a frequency of 50 mHz is included as a reference.

of E with the surface pressure for pure monopalmitin and monoolein monolayers at the same amplitude and at a frequency of 50 mHz.46 It can be seen that E and tan θ increased with frequency and progressed toward a plateau at the higher frequencies (Figures 9 and 10). This behavior corroborates the conclusion that the surface dilatational characteristics of mixed monolayers of the monoglyceride penetrated by β-casein are practically viscoelastic. The frequency dependence of the surface dilatational properties may be associated with the effect of the rate of deformation on the structure and relaxation phenomena in the mixed monolayers. The viscoelastic behavior observed for mixed monolayers in the range of frequencies studied may be associated with the organization/reorganization of the monolayer structure and with the formation/destruction of protein multilayers and/or monoglyceride collapse, especially at high πiMP and πiMO. The reason for this behavior must be associated with the immiscibility between monolayer components at the air-water interface, as was observed for β-casein-monoglyceride spread mixed monolayers.56 Surprisingly, although the surface pressure at the end of the penetration process (at πMP1200 or πMO1200), which coincides with the beginning of the surface dilatational experiments, is the same no matter what the monolayer composition or the initial surface pressure of a spread monoglyceride monolayer is (Table 1), the values of E are higher for monopalmitin (Figure 9A) than for monoolein (Figure 10A) monolayers penetrated by β-casein. In addition, the values of E also increase with πiMP and πiMO, for monopalmitin and monoolein monolayers penetrated by β-casein, respectively. In addition, at the same frequency (at 50 mHz), the (56) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. J. Agric. Food Chem. 2003, 51, 112-119.

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Figure 10. Frequency dependence of (A) surface dilatational modulus and (B) loss angle tangent for monolayers of monoolein penetrated by β-casein at 5 × 10-6 wt % into the aqueous phase at an amplitude of deformation of 5% at pH 7 and 20 °C. The initial surface pressure of a monoolein monolayer before the injection of β-casein into the aqueous phase (πiMO): (]) 10 mN/m, (O) 20 mN/ m, and (4) at the collapse point. The surface pressure dependence of surface dilatational modulus for pure (*) monoolein and (- - -) β-casein monolayers at a frequency of 50 mHz is included as a reference.

values of E for mixed monolayers of monopalmitin penetrated by β-casein at πiMP of 10 and 20 mN/m are similar to those for a pure monopalmitin monolayer. For a monopalmitin monolayer at the collapse point (Figure 9A) or monoolein monolayers at every πiMO (Figure 10A) penetrated by β-casein, the values of E are close to but a little lower than those for a pure monoglyceride monolayer. At the values of πMP1200 and πMO1200 (at π ≈ 19-25 mN/m, Table 1), the value of E for a pure β-casein film is ≈17 mN/m.46 From these results, it can be deduced that (i) although the monoglyceride monolayer is penetrated by β-casein, the presence of the monoglyceride (monopalmitin or monoolein) determines the value of E of the mixed film, even at the end of the penetration process because πiMP or πiMO is ≈πeβ-casein (at ≈21 mN/m). These results support the hypothesis that the monoglyceride molecules that are displaced during the preceding penetration experiments may be located near the interface, in agreement with the conclusions derived from the structural characteristics of the mixed films in the preceding section. (ii) The lower values of E for the mixed films in relation to those for a pure monopalmitin monolayer at the collapse point or monoolein monolayers at every πiMO are due to a monolayer molecular loss, as deduced from experiments on the dynamics of the penetration (dynamic surface pressure) of β-casein into monoglyceride monolayers and BAM (dynamic topographical characteristics) in preceding sections. (iii) The fact that the E values are higher for monopalmitin than for monoolein monolayers penetrated by β-casein also confirms that both the dilatational characteristics of the mixed monolayers are dominated by the monoglyceride and the immiscibility between monolayerforming components, because the same behavior was observed

4224 Langmuir, Vol. 22, No. 9, 2006

for pure monoglyceride46 and for spread mixed56 monolayers at the air-water interface.

Conclusions In this work, dynamic tensiometry, surface film balance, BAM, and surface dilatational rheology have been used to analyze the dynamics of the penetration of β-casein into monoglyceride monolayers (monopalmitin and monoolein) and the structural, dilatational, and topographical characteristics of mixed films formed by monoglyceride after the penetration of β-casein. The surface pressure of the monoglyceride monolayer at the beginning of the penetration process (at πiMP and πiMO) was the variable studied, maintaining the temperature constant at 20 °C and the pH at 7. From the time dependence of surface pressure, BAM images, and film reflectivity, one may concluded that β-casein can penetrate into monoglyceride monolayers at every surface pressure. However, the penetration of β-casein at the collapse point of the monoglyceride (with the most condensed structure) is facilitated by monoglyceride molecular loss by collapse and/

Sa´ nchez et al.

or desorption. The structural, topographical, and dilatational characteristics of the monoglyceride penetrated by β-casein mixed monolayers corroborate the conclusion that at surface pressures lower than the equilibrium surface pressure of β-casein both β-casein and monoglyceride may coexist at the interface. At higher surface pressures, β-casein can be squeezed out and the interfacial characteristics of the mixed film are essentially dominated by the presence of the monoglyceride (either monopalmitin or monoolein). However, monoglyceride molecules that are displaced during the penetration experiments or β-casein molecules that are squeezed out during the compression of the mixed film may be located near the interface and can re-enter into the monolayer. The interactions between monolayer-forming components are quite weak. Acknowledgment. This research was supported by CICYT through Grants AGL2001-3843-C02-01 and AGL2004-1306/ALI. LA053506+

Thermodynamic and Dynamic Characteristics of Monoglyceride

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