Relaxation Phenomena in Whey Protein Isolate and Monoglyceride

monolaurin, and monoolein) mixed films at the air-water interface were studied ... relaxation phenomena are mainly due to the irreversible loss of mon...
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Ind. Eng. Chem. Res. 2002, 41, 3169-3178

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Relaxation Phenomena in Whey Protein Isolate and Monoglyceride Mixed Films at the Air-Water Interface Juan M. Rodrı´guez Patino,* Ma. Rosario Rodrı´guez Nin ˜ o, and Cecilio Carrera Sa´ nchez Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/Prof. Garcı´a Gonza´ lez, s/nu´ m, 41012 Seville, Spain

Relaxation phenomena in a whey protein isolate (WPI) and monoglycerides (monopalmitin, monolaurin, and monoolein) mixed films at the air-water interface were studied using surface film balance and Brewster angle microscopy (BAM). Relaxation in surface area at a constant surface pressure (at 20 mN/m) or at constant area at the collapse point have been analyzed according to models for desorption, collapse, and/or organization/reorganization changes. At a constant surface pressure, the organization/reorganization change of WPI molecules in monopalmitin- and monoolein-WPI 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 WPI-monolaurin mixed films the relaxation phenomena are mainly due to the irreversible loss of monolaurin molecules by desorption into the bulk aqueous phase. Introduction Food dispersions (emulsions and foams) are complicated multicomponent systems containing many emulsifiers that may show surface activity by themselves (proteins and low-molecular-weight emulsifiers) or by association with each other.1-3 To control the production, stability, and other organoleptic and textural properties of food dispersions, detailed knowledge of the role of emulsifiers at fluid interfaces is required. Food proteins are distinguished by their good foaming and emulsifying properties and for these reasons are widely used in food formulations.4,5 Proteins show a strong tendency to adsorb to fluid interfaces (air-water and oil-water), and thus they find an important use in the manufacture of stable emulsions (i.e., ice cream, cream liqueurs, whipped toppings, coffee whiteners, products for infant nutrition, etc.), where long-term emulsion stability is essential.6 On the other hand, in many food formulations, proteins are not the only emulsifiers present because small molecule emulsifiers (monoglycerides, phospholipids, surfactants, etc.) are also incorporated in the formulation2,7,8. The small molecule emulsifiers can cover the interface that proteins do not, resulting in an emulsion with smaller particles, leading to greater stability. However, more important in some products is the effect of the small molecules in destabilizing the emulsion.9 Thus, an important effect of the small molecule emulsifiers is to promote the displacement of proteins from the interface.10 In particular, knowledge is required about the interactions between different emulsifiers at fluid interfaces because when a mixture of proteins and lowmolecular-weight emulsifiers exists at the interface a reduction in stability is often observed.11 This is believed to be because low-molecular-weight emulsifiers and proteins stabilize interfaces by different mechanisms.11,12 * To whom correspondence should be addressed. Tel.: +34 95 4557183. Fax: +34 95 4557134. E-mail: [email protected].

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.6,10,13 An understanding of the structure and dynamic properties of the monolayer is essential for the prediction of the properties of food colloids stabilized by emulsifiers. This is because the stability of foams and emulsions are governed to a large extend by the dynamic characteristics of the film around the bubbles or the droplets.5 The aim of this work is the study of the relaxation phenomena in WPI 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). In previous works we have analyzed the relaxation phenomena in pure monoglycerides14 and proteins15 at the air-water interface. Whey proteins and monoglycerides are widely used in the food industry as emulsifiers and stabilizers in foams and emulsions.16 Nonequilibrium processes occurring in systems containing fluid-fluid interfaces with a surfactant present are of great practical significance.17-21 Experimental Section Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90), 1-mono(cis-9octadecanoyl)glycerol (monoolein, RYLO MG 19), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMODAN ML 90) were supplied by Danisco Ingredients with over 95-98% of purity. Whey protein isolate (WPI), a native protein with a high content of β-lactoglobulin (protein 92 ( 2%, β-lactoglobulin > 95%, R-lactalbumin < 5%) obtained by fractionation, was supplied by Danisco Ingredients (Brabran, Denmark). The sample was stored below 0 °C and all work was done without further purification. Samples for interfacial characteristics of

10.1021/ie010868d CCC: $22.00 © 2002 American Chemical Society Published on Web 05/31/2002

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WPI films were prepared using Milli-Q ultrapure water and were buffered at pH 7. To adjust the subphase at pH 7, a commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) was used. 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 a Millipore filtration device (Milli-Q). All these products were supplied by Sigma (>99.5%). The 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 WPI-monoglyceride mixed films at the air-water interface were performed on a commercial fully automated Langmuir-type film balance (Lauda, Germany). The method has been described previously for pure14,15 and mixed components.22,23 Before each measurement, the film balance was calibrated at 20 °C. Mixtures of particular mass ratiossranging between 0 and 1, expressed as the mass fraction of monoglyceride in the mixture, Xswere studied. To allow the quantitative adsorption of the protein on the interface, the monolayer was not under any surface pressure during the spreading process. Thus, the WPI necessary to form the mixed film should be spread before the monoglyceride. Aliquots of aqueous solutions of WPI (1.636 × 10-4 mg/µL) at pH 7 were spread on the interfacewith a micrometric syringe. To allow for spreading, adsorption, and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. The spreading method adopted in these experiments ensured the quantitative spreading of the protein on the interface as was discussed in a previous paper.24 Afterward, a monoglyceride solution in a hexane:ethanol mixture was spread at different points on the WPI film. The monoglyceride solutions were spread on the subphase with a micrometric syringe at 20 °C. To allow for spreading and WPI-monoglyceride interactions, 30 min was allowed to elapse before compression was performed. To ensure interactions and homogeneity, the mixed film was compressed near the collapse point of the mixture and then expanded immediately to avoid the collapse. After 30 min at the maximum area, a new π-A isotherm was performed. The compression rate was 3.3 cm‚min-1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. 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. Two kinds of experiment were used for the analysis of relaxation in WPI-monoglyceride mixed monolayers. First, the surface pressure (π) is kept constant, and the area A is measured as a function of time. This relaxation experiment is the usual, preferred method and is capable of being interpreted kinetically.25 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.

Various relaxation mechanisms can be fitted to the results derived from the above-mentioned experiments. Relaxation mechanisms other than desorption and collapse are difficult to quantify in typical experimental relaxation experiments such as those used in this work. Desorption of a spread monolayer at any constant surface pressure involves two stages.26 The first is dissolution into the bulk aqueous phase to form a saturated aqueous layer. During the initial non-steadystate 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 due to either a macroscopic film fracture or a process of nucleation and the growth of bulk surfactant fragments, whenever a characteristic surface pressure is exceeded.27 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 by applying the Prout-Tompkins28 equation to the data derived from relaxation experiments at constant surface collapse area:

Log

π0-π ) C1 Log θ + C2 π

(4)

where π and π0 are the surface pressures at time θ and at the initial moment, respectively, and C1 and C2 are coefficients that depend on the experimental conditions. For proteins, various relaxation mechanisms ascribed to desorption or collapse of spread monolayer were applied to the results derived from relaxation data.15 However, the best fit of the results was obtained by 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 conformationorganization change and hydrophilic group hydration,15 with repercussions in surface dilatational properties.29

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.

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Figure 1. Relaxation at constant surface pressure (π ) 20 mN/ m) of WPI-monopalmitin mixed monolayers on water at pH 7. Temperature 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): (O) 0, (s) 0.2, (- - -) 0.4, (‚‚‚) 0.6, (- ‚ -) 0.8, and (4) 1.

Brewster Angle Microscopy. 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. A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the morphology and reflectivity of the monolayer. 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 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.30,31 The relative film reflectivity can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light through the method described elsewhere.30,31 In the experiments presented here, the reflectivity for the protein film was relatively high and that for monoglyceride films relatively low. Thus, 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 at Constant Surface Pressure. Relaxation Phenomena in WPI-Monopalmitin and WPI-Monoolein Mixed Films. Figures 1 and 2 show the relaxation in relative molecular area at 20 °C and at 20 mN/m for WPI-monopalmitin and WPImonoolein mixed films, respectively, at pH 7 as an example. For pure monopalmitin and monoolein monolayers the relaxation phenomena can be quantified by a desorption mechanism in two steps:14 dissolution (eq 1) and diffusion into the aqueous phase (eq 2). For WPI, WPI-monopalmitin, and WPI-monoolein 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 eq 5 with two relaxation times, which means that the relaxation of WPI, WPI-monopalmitin, and WPI-monoolein mixed films is not a simple process. In Table 1 the relaxation

Figure 2. Relaxation at constant surface pressure (π ) 20 mN/ m) for WPI-monoolein mixed monolayers on water at pH 7. Temperature 20 °C. Monolayer composition (mass fraction of monoolein in the mixture): (O) 0, (s) 0.2, (- - -) 0.4, (‚‚‚) 0.6, (- ‚ -) 0.8, and (4) 1. Table 1. Characteristic Parameters (Equation 5) for Relaxation of WPI-Monopalmitin and WPI-Monoolein Mixed Films at Constant Surface Pressure (π ) 20 mN/m) and at 20 °C system

X

τ1

τ2

(A/A0)60

WPI-monopalmitin

0 0.2 0.4 0.6 0.8 1.0

5.3 6.0 5.9 6.4 5.7

79.6 62.9 47.9 85.7 96.3

0.91 0.92 0.91 0.91 0.91 0.88

WPI-monoolein

0 0.2 0.4 0.6 0.8 1.0

5.3 6.2 1.82 2.15 3.21

67.9 65.7 70.1 106 145

0.91 0.95 0.91 0.94 0.95 0.88

Table 2. Characteristic Parameters for Desorption (Equations 1 and 2) of Monoglycerides and a WPI-Monolaurin Mixed Film at XML ) 0.5, at Constant Surface Pressure (π ) 20 mN/m) and at 20 °C monopalmitin monoolein monolaurin WPI-monolaurin XML ) 0.5

A1 × 103 (LR)

A2 × 105 (LR)

7.0 (0.996) 7.0 (0.997) 90 (0.999) 22.8 (0.997)

16 (0.983) 28 (0.989) 243 (0.938) 32 (0.973)

times and the value of relative area at 60 min of relaxation time, (A/A0)60, are shown. The data presented in Figures 1 and 2 and Table 1 may be associated with the immiscibility between WPI and monoglycerides (monopalmitin and monoolein) in the mixtures. In fact, pure monopalmitin and monoolein monolayers at 20 mN/m are unstable because of a mechanism of desorption in two steps (Table 2). However, WPI and mixed monolayers show exponential relaxation phenomena typical of pure proteins monolayers.14 Presumably, these phenomena may be due, among other things, to reorganization changes between WPI native molecules and WPI molecules in various degrees of unfolding after the compression up to 20 mN/ m. In fact, WPI is a globular protein that retains some elements of structure from the native state at low surface pressures. Most amino acid residues in WPI adopt loop conformation at the air-water interface.32 But the loop conformation is more condensed at higher surfaces pressures and is displaced toward the bulk phase at the collapse point. From the π-A isotherm of mixed monolayers,24 including the application of the additivity rule on

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Figure 3. Compression-expansion cycles for WPI-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. (O) First cycle at 30 min after spreading, at the maximum area. (s) Second cycle at 30 min after the first one, at the maximum area. (4) Third cycle at 30 min after the relaxation experiment at constant surface pressure (π ) 20 mN/m), at the maximum area.

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 WPImonoglyceride mixed monolayers form a mixed monolayer at the air-water interface with few interactions between film-forming components, at surface pressures lower than that for the WPI collapse (π ≈ 31 mN/m). However, the data in Table 1 also show that the presence of monoglyceride (monopalmitin or monoolein) 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. The relaxation phenomena at 20 mN/m for pure WPI or WPI-monoglyceride mixed films are reversible processes, as can be deduced from data presented in Figure 3 for WPI-monopalmitin mixed films (as an example). 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 WPI and WPI-monoglyceride 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 and to the effect of the viscoelastic characteristics of the protein on this process.29 In fact, the hysteresis in π-A isotherms during the compression-expansion cycle decreased with the WPI proportion in the mixture. This is an indication of the importance of the WPI content in the mixture in the dynamic relaxation phenomena. The higher the WPI/monopalmitin ratio in the mixture, the higher the hysteresis becomes because of the fact that pure protein monolayers have higher viscoelastic characteristics15,29 and lower superficial diffusion and mobility33 than monoglycerides. At the higher content of monopalmitin in the mixture (Figure 3D) the hys-

Figure 4. The time evolution of (s) surface pressure and (O) relative reflectivity (at a shutter speed of 1/250 s) during a compression-expansion cycle for (A) WPI-monopalmitin and (B) WPI-monoolein mixed monolayers on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monoglyceride in the mixture): 0.5. The collapse of the mixed films (πc WPI-MP) and (πc WPI-MO), and WPI (πc WPI), monopalmitin (πc MP), and monoolein (πc MO) pure films, the equilibrium surface pressures, monopalmitin (πe MP), and monoolein (πe MO), and the squeezing out phenomena of WPI by monopalmitin and monoolein are indicated.

teresis in the π-A isotherm was the same as that for pure monopalmitin monolayer. Further information about the mechanism or mechanisms that control the relaxation phenomena in WPImonoglyceride 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 for a WPI-monopalmitin (Figure 4A) or a WPI-monoolein (Figure 4B) mixed monolayer at a mass fraction of monoglyceride in the mixture of 0.5 (as an example). It can be seen that, at 20 mN/m, monopalmitin in the mixture (Figure 4A) has a liquid-condensed structureswhich 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 WPI in the mixture can be also deduced because some peaks have a reflectivity similar to that for pure WPI. From these results some differences can be deduced as compared to those recorded for monoolein-WPI mixed monolayers (Figure 4B). Namely, the I-π plots for WPI-monoolein mixed films were practically the same as those for pure monoolein,30 over the overall range of surface pressures, which strengthens the conclusion that monoolein, with the lower relative thickness, predominates at the interface. However, at higher surface pressures, near to and after the WPI collapse, the relative intensity of some spots was the same as that for pure WPI. These data also support the hypothesis that WPI and monoglyceride form a

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Figure 5. Time evolution of (O) relative area 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 WPI-monopalmitin mixed monolayer on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): 0.5. The crossed area represents the relative reflectivity of pure WPI or monopalmitin monolayer at 20 mN/m. Key (see also Figure 6): (A) WPI and monopalmitin domains, (B) domains of WPI, (C) domains of monopalmitin, and (D) squeezing out of WPI by monopalmitin.

practically immiscible monolayer at the air-water interface. In Figures 5 and 6 we observe some interesting features of the evolution of the reflectivity and the morphology, respectively, for a mixed monolayer of WPI + 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 shows the existence of some heterogeneity, which is typical of monoglycerideprotein mixed films at the air-water interface.22,23,30 These figures show that as the relaxation progresses, the presence of domains of WPI and monopalmitin (region A in Figure 5 and Figure 6A), domains of WPI (region B in Figure 5 and Figure 6B), domains of monopalmitin (region C in Figure 5 and Figure 6C), and the squeezing out of WPI by monopalmitin with monopalmitin over a sublayer of WPI (region D in Figure 5 and Figure 6D) appear alternatively on the air-water interface. These results also corroborate the idea that during the relaxation of a mixed film of WPI and monopalmitin (or monoolein) 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 WPI structure after the compression at 20 mN/m, but mediated by the presence of monoglyceride in the mixture. Relaxation Phenomena in WPI-Monolaurin Mixed Films. Figure 7 shows the relaxation in relative molecular area at 20 °C and at 20 mN/m (Figure 7B) for WPImonolaurin mixed films at a mass fraction of monolaurin in the mixture of 0.5 (as an example). The relaxation phenomena are more pronounced for these systems than for WPI-monopalmitin (Figure 1) and for WPI-monoolein (Figure 2) mixed films. Unlike monopalmitinand monoolein-WPI 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 WPI-monolaurin monolayer are included in

Table 2. It can be seen that the magnitude of the rates of dissolution (coefficient A1) and diffusion (coefficient A2) is higher for pure monolaurin than for the mixed film. That is, the presence of WPI in the mixture produces a reduction of the magnitude of the relaxation phenomena in the mixed film. The significant monolaurin monolayer molecular loss that is observed in these experiments (Figure 7) has important consequences not only from a practical point of view but also for the characteristics of WPI-monolaurin film at the air-water interface. The instability of the mixed films can also be deduced from the plots of Figure 7A. The displacement toward the π-axis of the π-A isotherm after continuous compression-expansion 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 upon 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 compressionexpansion cycle is higher as the content of monolaurin in the mixture increases (data not shown), a phenomenon opposite to that observed for monopalmitin(Figure 3) and monoolein-WPI (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 WPI molecules present in the mixed films during the relaxation process. The relaxation phenomena in WPI-monolaurin mixed films at 20 mN/m are also 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 organization/rearrangement of the amino acid segments of WPI into the underlying aqueous phase, as discussed in previous sections. Relaxation Phenomena at Constant Surface Area (at the Collapse Point). Relaxation Phenomena in WPI-Monopalmitin Mixed Films. WPI-monopalmitin mixed films were also tested under the most adverse conditions, at the maximum superficial density at constant collapse area. Figure 8 shows the relaxation in surface pressure at constant surface area (at the collapse point) for WPI-monopalmitin mixed films. It can be seen that monopalmitin and WPI-monopalmitin mixed films relaxed toward the equilibrium surface pressure of pure monopalmitin,14 which is indicated in Figure 8B 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 pure monopalmitin monolayer.14 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 3). This indicates that the relaxation phenomena under these conditions may be due to nucleation and growth of critical nuclei of monopalmitin. On the other hand, the

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Figure 6. Visualization of WPI-monopalmitin mixed film on water at pH 7 during the relaxation (π ) 20 mN/m). Monolayer composition (mass fraction of monopalmitin in the mixture): 0.5. Temperature 20 °C. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm. Key: (A) WPI and monopalmitin domains (π ) 20 mN/m), (B) domains of WPI (π ) 20 mN/m), (C) domains of monopalmitin (π ) 20 mN/m), (D) squeezing out of WPI by monopalmitin (π ) 20 mN/m), (E) island of collapsed WPI (at the collapse point), (F) monopalmitin domains with a solid structure (at the collapse point), (G) monopalmitin domains over a layer of collapse WPI (at the collapse point), and (H) fracture of a collapsed monopalmitin monolayer (at the collapse point).

magnitude of relaxation in surface pressure is practically the same for all mixed films. WPI-monopalmitin mixed films behave in a different way as they relax under different experimental conditions, either at constant surface pressure (at 20 mN/m)

or at constant surface area (at the collapse point). Again, the reason for this behavior must be associated with the immiscibility between both components at the airwater interface, as was deduced for these systems at the collapse point.24 In fact, at surface pressures higher

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Figure 8. Relaxation at constant area (at the collapse point) for WPI-monopalmitin mixed monolayers on water at pH 7. Temperature 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): (O) 0, (s) 0.2, (- - -) 0.4, (‚‚‚) 0.6, (- ‚ -) 0.8, and (4) 1. The equilibrium surface pressures for monopalmitin (πe MP) and WPI (πe WPI) are indicated by arrows. Figure 7. (A) π-A isotherms (compression curves): (O) first compression at 30 min after spreading, at the maximum area, (s) second compression after 30 min after the first one, and (4) third compression after the relaxation experiment at constant surface pressure (π ) 20 mN/m). (B) Relaxation at constant surface pressure (π ) 20 mN/m) and (C) relaxation at constant surface area (at the collapse point) for WPI-monolaurin mixed monolayers on water at pH 7. Temperature 20 °C. Monolayer composition (mass fraction of monolaurin in the mixture): 0.5. The equilibrium surface pressure for monolaurin (πe ML) is indicated by an arrow.

than that for the WPI collapse a squeezing out of WPI by monopalmitin is produced (the results in Figure 4 are an example of this behavior). The displacement of WPI by monopalmitin, which can be observed in WPImonopalmitin mixed films at any surface composition,24 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 9) of a WPI-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), on one hand, and the visualization of the mixed film (Figure 6), on the other hand, 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 WPI (region E in Figure 9 and Figure 6E) or fractures of monopalmitin (region H in Figure 9 and Figure 6H) pass through the spot where these measurements are made. Figure 6F shows the crystalline nuclei of collapsed monopalmitin (bright points), in a homogeneous solid environment of monopalmitin. As the relaxation progresses, the existence of monopalmitin domains with a liquid-

Figure 9. 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 WPI-monopalmitin mixed monolayer on water at pH 7 and at 20 °C. Monolayer composition (mass fraction of monopalmitin in the mixture): 0.5. The crossed area represents the relative reflectivity of pure monopalmitin monolayer at the equilibrium surface pressure (πe MP). Key (see also Figure 6): (E) domains of collapsed WPI, (F) crystalline nuclei of collapsed monopalmitin (bright points), in a homogeneous solid environment of monopalmitin, (G) monopalmitin domains with a liquid-condensed structure over a sublayer of collapsed WPI, and (H) fractures of collapsed monopalmitin. Table 3. Characteristic Parameters (Equation 4) for Relaxation of WPI-Monopalmitin Mixed Films at Constant Area (at the Collapse Point) and at 20 °C XMP 0 0.2 0.4

C1 (LR)

(π/π0)60

XMP

C1 (LR)

(π/π0)60

0.10 (0.980) 0.13 (0.998)

0.88 0.90 0.91

0.6 0.8 1

0.17 (0.997) 0.19 (0.998) 0.19 (0.996)

0.91 0.91 0.85

condensed structure over a sublayer of collapsed WPI can be clearly distinguished (Figure 6G). Over the overall relaxation process, the mixed film presents a reflectivity similar to that for pure mono-

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Figure 10. Relaxation at constant area (at the collapse point) for WPI-monoolein mixed monolayers on water at pH 7. Temperature 20 °C. Monolayer composition (mass fraction of monoolein in the mixture): (O) 0, (s) 0.2, (- - -) 0.4, (‚‚‚) 0.6, (- ‚ -) 0.8, and (4) 1. The equilibrium surface pressures for monoolein (πe MO) and WPI (πe WPI) are indicated by arrows.

palmitin at the equilibrium surface pressure, indicated in Figure 9 by the crossed area, except as regions of monopalmitin fractures or islands of collapsed WPI pass through the spot where these measurements are performed, indicated in Figure 9 by arrows. These results corroborate that (i) the original mixed film is dominated by the presence of monopalmitin, (ii) the displacement of WPI by monopalmitin is not quantitative, and (iii) the mixed films are immiscible with domains of monopalmitin (to a large extent) and WPI (to a lower extent) on the interface. Finally, the displacement of WPI 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 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 compression than during the expansion because of the transformation of the 3-D collapsed monopalmitin phase into the liquid-condensed phase and finally into the liquid-expanded phase, on one hand, and the re-adsorption of the previously displaced WPI, on the other hand, as the expansion progresses involves some time. The reversibility of these processes can be deduced from the repetitivity in the π-A isotherms after continuous compression-expansion cycles (Figure 3). Relaxation Phenomena in WPI-Monoolein Mixed Films. WPI-monoolein mixed films were also tested at the maximum superficial density at constant collapse area. Figure 10 shows the relaxation in surface pressure at constant surface area (at the collapse point) for WPImonoolein mixed films. WPI-monoolein monolayers behave differently from WPI-monopalmitin monolayers. It can be seen (Figure 10) that, in experiments at

constant collapse area, the surface pressure relaxes from the collapse value, which is close to πe (see arrow in Figure 10), toward lower π values at longer times. Constant area relaxation studies are difficult to interpret because of the interference of different relaxation processesssuch as desorption, organization/ reorganization changes, and monolayer collapsesand 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 film-forming components (Figure 4B): (i) monoolein adopts a liquid-expanded structure up to the collapse point at a surface pressure close to πe, (ii) the presence of the reflectivity peaks under both monolayer compression and expansion is a consequence of the presence of WPI in the mixed film, (iii) a squeezing out of WPI by monoolein is detected by BAM after the WPI collapse, (iv) we observed some peaks of higher intensity, especially during the monolayer compression, when a domain of collapsed WPI 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 re-adsorption of WPI molecules at the interface upon expansion requires some time, as a consequence of the dynamic behavior of the monolayer. In addition, the structural changes produced during the compression-expansion cycle for WPI-monoolein are lower than those for WPImonopalmitin mixed films because of the fact that monopalmitin has a rich structural polymorphism and monoolein collapses by formation of lenses,14 a typical behavior of liquid surfactants. If in experiments at constant surface pressure there is an equilibrium between the monolayer and the subphase, desorption is not possible. This is the case for slightly soluble monolayers, such as monopalmitin and WPI-monopalmitin monolayers, at π < 20 mN/m (Figure 1). Thus, the relaxation phenomena observed with WPI-monopalmitin mixed monolayers at the collapse area could be assigned to the mechanism of monolayer collapse by nucleation and growth of the critical nuclei. However, as the surface pressure value is lower than πe at higher relaxation time, the relaxation characteristics of monoolein and WPI-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.14 On the basis of both the immiscibility between the film-forming components and the practical displacement of WPI by monoolein at the collapse area, the relaxation phenomena in WPI-monoolein mixed films under these conditions may be associated with the concurrence of different relaxation phenomena, including the desorption, reorganization, and re-adsorption of WPI molecules, and collapse. This hypothesis can be supported by the quantification of the (π/π0) relaxation by means of an exponential dependency (eq 6), with two relaxation times (Table 4). On the other hand, the magnitude of relaxation in surface pressures is practically the same for all mixed films. Relaxation Phenomena in WPI-Monolaurin Mixed Films. Figure 7C shows the relaxation in surface pressure at constant surface area (at the collapse point)

Ind. Eng. Chem. Res., Vol. 41, No. 13, 2002 3177 Table 4. Characteristic Parameters (Equation 6) for Relaxation of WPI-Monoolein Mixed Films at Constant Area (at the Collapse Point) and at 20 °C XMO

τ1

τ2

(π/π0)60

XMO

τ1

τ2

(π/π0)60

0 0.2 0.4

0.41 4.34 3.86

23.5 68.4 91.5

0.82 0.85 0.76

0.6 0.8 1

9.05 5.46

245 102

0.79 0.80 0.62

for WPI-monolaurin mixed films at a mass fraction of monolaurin in the mixture of 0.5, as an example. 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-WPI mixed films can be observed. First, the magnitude of the relaxation in surface pressure is higher for monolaurinthan for monopalmitin- and monoolein-WPI mixed films. Second, the (π/π0) relaxation has a logarithmic time dependence like that for WPI-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,14 has an important role in the relaxation phenomena observed in the mixed films (Figure 7C). Conclusions This article presents experimental studies of relaxation phenomena in WPI-monoglyceride mixed films at the air-water interface. Data have been analyzed according to models for desorption, collapse, and/or 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), below the WPI collapse, the organization/reorganization changes of WPI molecules in monopalmitin- and monoolein-WPI mixed films are the mechanisms that control 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 monoglyceridessuch as that deduced for WPI-monopalmitin mixed filmssor to a complex mechanism including competition between collapse and monolayer desorptionssuch as that deduced for WPImonoolein mixed films. For WPI-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 WPI and monoglyceride at the air-water interface and to the WPI displacement by the monoglyceride at surface pressures higher than that for WPI collapse. These relaxation phenomena have significant repercussion in surface dilatational properties.36 Thus, as in many food formulations, proteins and monoglycerides are incorporated together; the dynamic behavior of mixed films (including relaxation phenomena and surface dilatational properties) are important for controlling the formation and stability of emulsions and foams in the food industry. To relate the dynamic properties of mixed emulsifiers layers to texture, storage, and processing behavior of real food dispersions (emulsions and foams), emphasis on the systematic study of sys-

tems containing mixtures of emulsifiers are required. Therefore, the main objectives for future research are to understand how molecular structure, component interactions, and surface composition of mixed emulsifiers at fluid-fluid interfaces influence and control the interfacial properties that are important for foam and emulsion behavior. Acknowledgment This research was supported in part by EU through Grant FAIR-CT96-1216 and by DGICYT through Grant PB97-0734. Literature Cited (1) Dickinson E. An Introduction to Food Colloids; Oxford University Press: Oxford, 1992. (2) Friberg, S. E.; Larsson, K. Food Emulsion, 3rd ed.; Marcel Dekker: New York, 1997. (3) Sjo¨blom, J. Emulsions and Emulsion Stability; Marcel Dekker: New York, 1996. (4) Dickinson, E. Proteins at Interfaces and in Emulsion: Stability, Rheology and Interactions. J. Chem. Soc., Faraday Trans. 1998, 94, 1657. (5) Halling, P. J. Protein-Stabilized Foams and Emulsions. Crit. Rev. Food Sci. Nutr. 1981, 13, 155. (6) Dickinson, E. Adsorption Protein Layers at Fluid Interfaces: Interactions, Structure and Surface Rheology. Colloids Surf. B 1999, 15, 161. (7) Gunstone, F. D.; Padley, F. N. Lipid Technologies and Applications; Marcel Dekker: New York, 1997. (8) Hartel, R.; Hasenhuette, G. R. Food Emulsifiers and Their Applications; Chapman and Hall: New York, 1997. (9) Goff, H. D.; Jordan, W. K. Action of Emulsifiers in Promoting Fat Destabilization During the Manufacture of Ice Cream. J. Dairy Sci. 1989, 72, 18. (10) Wilde, P. J. Interfaces: Their Role in Foam and Emulsion Behaviour. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (11) 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. (12) Coke, M.; Wilde, P. J.; Russell, E. J.; Clark, D. C. The Influence of Surface Composition and Molecular Diffusion on the Stability of Foams Formed from Proteins-Detergent Mixtures. J. Colloid Interface Sci. 1990, 138, 489. (13) Dickinson, E. Milk Protein Interfacial Layers and the Relationship to Emulsion Stability and Rheology. Colloid Surf. B 2001, 20, 197. (14) Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; Rodrı´guez Patino, J. M. Relaxation Phenomena in Monoglyceride Films at the AirWater Interface. Colloids Surf. B 1999, 12, 175. (15) Rodrı´guez Nin˜o, Ma. R.; Carrera, C.; Rodrı´guez Patino, J. M. Interfacial Characteristics of β-Casein Films at the Air-Water Interface. Colloids Surf. B 1999, 12, 161. (16) Cayot, P.; Lorient, D. In Food Proteins and Their Applications; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997. (17) Baszkin, A.; Norde, W. Physical Chemistry of Biological Interfaces; Marcel Dekker: New York, 2000. (18) Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1985. (19) Dukhin, S. S.; Kretzschmar, G.; Miller, R. Dynamics of Adsorption at Liquid Interfaces; Elsevier: Amsterdam, 1995. (20) Malhotra, A. K.; Wasan, D. T. Interfacial Rheological Properties of Adsorbed Surfactant Films with Applications to Emulsion and Foam Stability. Surfactant Sci. Ser. (Thin Solid Films) 1988, 29, 829. (21) Mittal, K. L.; Kumar, P. Emulsions, Foams, and Thin Films; Marcel Dekker: New York, 2000. (22) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Analysis of β-Casein-Monopalmitin Mixed Films at the AirWater Interface. J. Agric. Food Chem. 1999, 47, 4998.

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(23) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Is Brewster Angle Microscopy a Useful Technique To Distinguish between Isotropic Domains in β-Casein-Monoolein Mixed Monolayers at the Air-Water Interface? Langmuir 1999, 15, 4777. (24) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R.; Carrera, C.; Cejudo, M. Whey Protein Isolate-Monoglyceride Mixed Monolayers at the Air-Water Interface. Morphology, Structure, and Interactions. Langmuir 2001, 17, 7545. (25) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interface; Wiley: New York, 1966. (26) Ter Minassian-Saraga, L. Recent Work on Spread Monolayers, Adsorption and Desorption. J. Colloid Interface Sci. 1956, 11, 398. (27) Smith, R. D.; Berg, J. C. The Collapse of Surfactant Monolayers at the Air-Water Interface. J. Colloid Interface Sci. 1980, 74, 273. (28) Prout, E. G.; Tompkins, F. C. The Thermal Decomposition of Potassium Permanganate. Trans. Faraday Soc. 1944, 40, 488. (29) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; Cejudo, M. Structural and Dynamic Properties of Milk Proteins Spread at the Air-Water Interface. J. Colloid Interface Sci. 2001, 242, 141. (30) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Morphological and Structural Characteristics of Monoglyceride Monolayers at the Air-Water Interface Observed by Brewster Angle Microscopy. Langmuir 1999, 15, 2484. (31) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Nin˜o, Ma. R. Structural and Morphological Characteristics of β-Casein

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Received for review October 23, 2001 Revised manuscript received April 17, 2002 Accepted April 23, 2002 IE010868D