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Structural and shear characteristics of adsorbed sodium caseinate and monoglyceride mixed monolayers at the air–water interface
Journal of Colloid and Interface Science 313 (2007) 141–151 www.elsevier.com/locate/jcis
Structural and shear characteristics of adsorbed sodium caseinate and monoglyceride mixed monolayers at the air–water interface Juan M. Rodríguez Patino ∗ , Marta Cejudo Fernández, Cecilio Carrera Sánchez, Ma . Rosario Rodríguez Niño Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, C/. Prof. García González 1, 41012-Seville, Spain Received 20 February 2007; accepted 12 April 2007 Available online 18 April 2007
Abstract The structural and shear characteristics of mixed monolayers formed by an adsorbed Na-caseinate film and a spread monoglyceride (monopalmitin or monoolein) on the previously adsorbed protein film have been analyzed. Measurements of the surface pressure (π )–area (A) isotherm and surface shear viscosity (ηs ) were obtained at 20 ◦ C and at pH 7 in a modified Wilhelmy-type film balance. The structural and shear characteristics of the mixed films depend on the surface pressure and on the composition of the mixed film. At surface pressures lower than the equilibrium surface pressure of Na-caseinate (at π < πeCS ), both Na-caseinate and monoglyceride coexist at the interface, with a structural polymorphism or a liquid expanded structure due to the presence of monopalmitin or monoolein in the mixture, respectively. At higher surface pressures, collapsed Na-caseinate residues may be displaced from the interface by monoglyceride molecules. For a Na-caseinate–monopalmitin mixed film the ηs value varies greatly with the surface pressure (or surface density) of the mixed monolayer at the interface. In general, the greater the surface pressure, the greater are the values of ηs . However, the values of ηs for a Na-caseinate–monoolein mixed monolayer are very low and practically do not depend on the surface pressure. The collapsed Na-caseinate residues displaced from the interface by monoglyceride molecules at π > πeCS have important repercussions on the shear characteristics of the mixed films. © 2007 Elsevier Inc. All rights reserved. Keywords: Food emulsifiers; Food dispersions; Protein–monoglyceride mixed films; Interfacial shear viscosity; Adsorbed films; Monolayer structure; Interfacial rheology; Air–water interface
1. Introduction The stability, textural, and mechanical properties of dispersed food systems (emulsions and foams) depend on the way in which the constituent emulsifiers—low-molecular weight emulsifiers (lipids, phospholipids, and surfactants) and biopolymers (proteins and some polysaccharides)—adsorb and interact at fluid interfaces [1,2]. The optimum use of these emulsifiers depends on knowledge of their interfacial physico-chemical characteristics (such as surface activity, structure, miscibility, superficial viscosity, etc.) and the kinetics of the film formation at fluid interfaces. The distribution of lipids and proteins at fluid interfaces is determined by competitive or co-operative adsorp* Corresponding author. Fax: +34 95 4557134.
E-mail address: [email protected] (J.M. Rodríguez Patino). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.04.025
tion between the two types of emulsifiers, which in turn depend on interactions between emulsifier molecules [3,4]. Interactions between molecules of emulsifiers could affect not only the film structure, topography and mechanical properties, but also dynamic phenomena in mixed films [4–7]. Interfacial rheology is important for food colloids (emulsions and foams) because the structural and mechanical properties of food emulsifiers at fluid interfaces have an influence on the stability and texture of the product. In addition, interfacial rheology is a very sensitive technique to monitor the interfacial structure and concentration of single emulsifiers at the interface or the relative concentration, the competitive adsorption, and the magnitude of interactions between different emulsifiers at the interface [4,5,8–11]. Interfacial rheology can be defined for both compressional deformation (dilatational rheology) and shearing motion of the interface (shear rheology). While shear viscosity may contribute appreciably to the long-term stabil-
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ity of dispersions, dilatational rheology plays an important role in short-term stability [5,12–19]. Interfacial shear rheology is most useful for polymer and mixed polymer surfactant adsorption layers and insoluble monolayers and gives access to interaction forces in two dimensional layers [20,21]. In addition, the shear may induce heterogeneity and segregation between protein and lipid domains at the interface during the flow of the monolayer [22,23]. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers [22–26]. In this contribution we are concerned with the analysis of structural and shear characteristics of mixed monolayers formed by an adsorbed milk protein (Na-caseinate) and a spread monoglyceride (monopalmitin or monoolein). Na-caseinate is distinguished by its good foaming and emulsifying properties and for this reason is widely used in food formulations in combination with monoglycerides [1,2,27]. Monolayer technique has been used successfully for studying the properties of mixed emulsifiers spread [4] and adsorbed and/or penetrated [28] at the air–water interface. This work is an extension of previous studies of structural, topographical and shear characteristics of spread mixed films at the air–water interface [29,30]. 2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90) and 1-mono (cis-9-octadecanoyl) glycerol (monoolein, RYLOTM MG 19) were kindly supplied by Danisco Ingredients (Brabran, Denmark) with over 95–98% of purity. Na-caseinate (a mixture of ≈38% β-casein, ≈39% αs1 -casein, ≈12% κ-casein, and ≈11% αs2 -casein) was kindly supplied and purified from bulk milk from Unilever Research (Colworth, UK). Samples for interfacial characteristics of Nacaseinate adsorbed films were prepared using Milli-Q ultra pure water and were buffered at pH 7. To form the mixed surface film on a previously adsorbed Na-caseinate 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 subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution called trizma ((CH2 OH)3 CNH2 /(CH2 OH)3 CNH3 Cl, Sigma, >99.5%) was used to achieve pH 7. Ionic strength was 0.05 M in all the experiments. 2.2. Surface film balance Measurements of the surface pressure (π ) versus average trough area (A) were performed on a modified Wilhelmy-type film balance, which integrates surface shear rheometry, as described elsewhere [31]. Before each measurement, the film balance was calibrated at 20 ◦ C. For Na-caseinate adsorbed films from water a Na-caseinate solution at 7.5 × 10−6 −1 × 10−5 % (wt) was left in the trough and time allowed for protein adsorption at the interface. These protein concentrations were selected
from previous data of the adsorption isotherm [32]. At this Na-caseinate concentration in solution the surface pressure at equilibrium is zero. In fact, after 24 h the surface pressure (π ) at the maximum area of the trough was practically zero and then the monoglyceride (≈1.5 × 10−4 mg/µl) was spread at different points on the previously adsorbed Na-caseinate film. For pure adsorbed protein films the maximum protein concentration in the bulk phase should be selected in order to obtain a reasonable rate of adsorption at the interface, but maintaining as zero the equilibrium surface pressure. On the other hand, for mixed films, at low protein concentrations in the aqueous phase we cannot observe the collapse point, especially for low monoglyceride concentrations. Thus, in these experiments we have selected optimum conditions in order to obtain the complete π–A isotherm of the mixed film, from the more expanded monolayer (at the higher areas) to the more condensed monolayer, at the collapse point (at the lower areas). Mixtures of particular mass fractions—expressed as the mass fraction of monoglyceride in the mixture, X—were studied. The compression rate was 3.3 cm/min, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. To allow for Na-caseinate–monoglyceride interactions, 30 min were allowed to elapse before compression was performed. After 30 min at the maximum area, measurements of compression–expansion cycles were performed with 30 min of waiting time between each expansion–compression cycle. The measurement of the π–A isotherm was performed before the surface shear rheology experiments. This isotherm was recorded with the maximum width of the canal at which the surface pressure at both sides of the canal was the same (i.e. π was zero during the monolayer compression). The initial surface pressure of the mixed film at the maximum area before compression was zero. The reproducibility of the results was better than ±0.5 mN/m for surface pressure and ±0.05 m2 /mg. 2.3. Surface shear rheometry To study the shear characteristics of adsorbed films a homemade canal viscometer—an analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past one another—as described elsewhere was used in this work [31]. Briefly, surface shear viscosity measurements were taken on a modified Wilhelmy-type film balance, with two surface pressure sensors located at both sides of the canal in the center of the trough (Fig. 1). The canal was constructed of two Teflon bars to render them hydrophobic and was mounted on the trough. The separation between the two bars can be varied and adjusted by means of a precision screw. The monolayer is allowed to flow through a canal of a width Wc and fixed length, L = 30 mm, and at a constant flow (Q, m2 /s, recorded as a variation of the area associated with the movement of the barrier as a function of time). For a constant rate R (m/s) of translation of the two barriers, the flow Q (m2 /s) is Q = R · T ; were T (m) is the width of the trough. The surface shear viscosity (ηs ) was calculated from the rate of film flow Q by Eq. (1), which is analogous to the Poiseuille equation for the flow of liquids through capillary
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Fig. 1. Wilhelmy-type film balance modified for measurement of surface shear viscosity. The monolayer is allowed to flow in the direction of the arrow through a canal of width Wc and length L by the movement in the same direction of two barriers from a region of higher surface pressure (π2 ) to a region of lower surface pressure (π1 ). The width of the canal can be modified by means of the precision screw. For details see the text.
Fig. 2. Surface pressure–area isotherms (compression–expansion curves) for adsorbed Na-caseinate–monopalmitin mixed monolayers on buffered water at pH 7 and at 20 ◦ C. Mass fraction of monopalmitin in the mixture (XMP ): (!) 0, (—) 0.2, (- - -) 0.4, and (· · ·) 0.5. The equilibrium surface pressure of Na-caseinate (πeCS ) and the collapse pressure of monopalmitin (πcMP ) are indicated by means of arrows.
tubes [33]
The results of the π−A isotherms (Fig. 2) confirm that a Nacaseinate monolayers at the air–water interface adopt a liquid expanded-like structure at every surface pressure. The monolayer collapses and amino acid segments are extended into the underlying aqueous solution at a surface pressure a little higher than the equilibrium surface pressure (πeCS ≈ 29 mN/m), which is indicated in Fig. 2 by means of an arrow [32]. The equilibrium surface pressure is the maximum surface pressure at which a spread monolayer can be compressed just before the monolayer collapse. Mixtures of particular mass fractions—ranging between 0 and 0.5, expressed as the mass fraction of monoglyceride in the mixture, X—were studied. The amount of spread monoglyceride was calculated on the basis of the mass of previously adsorbed Na-caseinate, which was calculated form the adsorbed π–A isotherm [34,35]. It must be emphasized that, due to this assumption, in Fig. 2 the area in x-axis is not the true area per unit mass of mixed film but the trough (apparent) area (AAPPARENT ). On the other hand, as opposed to spread monolayers [29], for adsorbed monolayers the mixtures with mass fractions higher than X = 0.5 saturate the interface, under the experimental conditions used in this work. From the π–A isotherms for Na-caseinate + monopalmitin mixed films (Fig. 2) it can be seen that there was a monolayer expansion as the monopalmitin concentration in the mixture was increased, especially at higher surface pressures (at π > πeCS ). That is, the π–A isotherm is displaced towards higher areas as the concentration of monopalmitin in the mixture increases. At surface pressures lower than the equilibrium surface pressure of Na-caseinate (at π < πeCS ), both Na-caseinate and monopalmitin coexist at the interface. In fact, we can see the main transition between liquid expanded (LE) and liquid condensed (LC) phases, which is typical of monopalmitin monolayers at low surface pressures (at π ≈ 5 mN/m) [36]. In a previous paper [29] we deduced by means of the excess area and excess free energy that spread Na-caseinate and monopalmitin
ηs =
(π2 − π1 ) · Wc3 . 12 · Q · L
(1)
The monolayer was compressed to the desired surface pressure and then the flow of the monolayer through the canal was facilitated by the movement of the two barriers in the same direction, from a region of higher surface pressure (π2 ) to a region of lower surface pressure (π1 ). The value of π = π2 − π1 was deduced during the flow of the monolayer. As for pure components, an optimization of Wc and Q is necessary in order to obtain the best flow conditions of the mixed monolayer through the canal [31]. During these experiments practically all the monolayer (ca. 90%) flows through the canal, thus the data presented in this work represent the overall behavior of the monolayer under pure shear conditions. The subphase temperature was controlled at 20 ◦ C by water circulation from a thermostat, within an error range of ±0.3 ◦ C. The temperature was measured by a thermocouple located just below the air– water interface. Data were obtained after a minimum of three measurements and the repeated results prove the reproducibility of the method. 3. Results 3.1. Structural and shear characteristics of Na-caseinate– monopalmitin mixed monolayers adsorbed at the air–water interface 3.1.1. Structural characteristics The π–AAPPARENT isotherm for adsorbed Na-caseinate monolayer was obtained as a control at the beginning of each experiment (Fig. 2). This isotherm is practically the same as that obtained directly by spreading [29,31]. Thus, the structures of the monolayers formed in the two different ways must be identical, at least for adsorption from low bulk protein concentrations.
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Fig. 3. Difference in surface pressure between the limits of the canal during the flow of Na-caseinate adsorbed monolayers at the air–water interface as a function of surface pressure (mN/m): (A) 5, (B) 10, (C) 15, and (D) 20. Temperature 20 ◦ C and pH 7. Effect of width of canal (Wc , mm) and Na-caseinate flow through the canal (Q, m2 /s): (—) Q = 3.75 × 10−6 and Wc = 1.4, (- - -) Q = 1.25 × 10−5 and Wc = 1.4, (· · ·) Q = 3.75 × 10−6 and Wc = 0.9, (- · -) Q = 6.25 × 10−6 and Wc = 0.9, and (- · · -) Q = 1.25 × 10−5 and Wc = 0.9.
form a mixed monolayer at the air–water interface with few repulsive interactions between film forming components (even phase separation could occur) at low monopalmitin concentrations in the mixed film (at XMP < 0.8) and at surface pressures lower than that for the Na-caseinate collapse (at π < πeCS ). Results of BAM (topography and reflectivity) also show that at a microscopic level domains of monopalmitin and Na-caseinate coexist at the air–water interface in spread [30] and adsorbed [29] mixed films. At surface pressures higher than that for Na-caseinate collapse (at π > πeCS ), the π–A isotherms for mixed monolayers were parallel to that of monopalmitin [36]. These results suggest that at the higher surface pressures the arrangement of the monopalmitin hydrocarbon chain in mixed monolayers is practically the same in the entire Na-caseinate/monopalmitin fraction. That is, at higher surface pressures, collapsed Na-caseinate residues may be displaced from the interface by monopalmitin molecules. At the highest surface pressures (at π > πeCS ), at the
collapse point of the mixed film, immiscibility between monolayer forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monopalmitin monolayer (Fig. 2). The squeezing out of Na-caseinate by monopalmitin and the clear evidence that the mixed monolayer is practically dominated by the presence of monopalmitin at the interface both in spread and adsorbed was also corroborates by BAM [29,30]. The fact that upon expansion and further compression, the π–A isotherms were repetitive (data not shown) suggests that the protein displaced by monopalmitin during the compression (at π > πeCS ) reenters the mixed monolayer upon expansion and supports the idea that the protein remains underneath the monoglyceride film either through hydrophobic interactions between protein and lipid or by local anchoring through the monoglyceride layer [37,38]. However, for adsorbed Na-caseinate– monopalmitin mixed monolayers a first order-like phase transition was observed upon the monolayer expansion (Fig. 2) at
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surface pressure values close to the equilibrium surface pressure of Na-caseinate (at π ≈ πeCS )—with an atypical plateau in the π–A isotherm. These results suggest that the re-adsorption of previously displaced Na-caseinate has a kinetic character, which was not evident for spread mixed films [34,39]. 3.1.2. Shear characteristics The time evolution of π during the flow of an adsorbed Nacaseinate monolayer depends on the Na-caseinate flow through the canal and the surface pressure (Fig. 3). An optimization of Wc and Q was necessary in order to obtain the best flow conditions of a protein monolayer through the canal, with few fluctuation in the π –time flow curve, and more reproducible data. The steady state in π was reached relatively quickly during the flow of the Na-caseinate monolayer at low surface pressures, but more time is necessary at high surface pressure. From the π the surface shear viscosity (ηs ) was determined by means of Eq. (1). The steady state π value can be associated with the equilibrium shear viscosity in stationary flow. At this point there exists a balance between the formation and breaking of monolayer structure or a constant friction within the Nacaseinate monolayer domains along the canal. The fluctuations in the π value may be due to friction between Na-caseinate domains at the higher surface pressures, a phenomenon that denotes the heterogeneity of the monolayer. From the π at steady state, the surface shear viscosity was determined as a function of surface pressure (Fig. 4). Over the overall range of surface pressures the values of ηs did not depend on the flow Q (Fig. 4A). The values of ηs increased with surface pressure, especially as the residues of Na-caseinate adopt the more condensed conformation at the air–water interface. The values of ηs are higher for spread [30] than for adsorbed Na-caseinate films (Fig. 4C). Thus, even the π–A isotherms for adsorbed Na-caseinate monolayer are practically the same as those obtained directly by spreading, the surface shear viscosity is different. One speculation is that Na-caseinate may be spread at the interface maintaining a more unfolded structure as compared with adsorbed films. Thus, the interactions between unfolded Na-caseinate residues must be higher for spread than for adsorbed Na-caseinate films, a phenomenon that explains the higher value of ηs for Na-caseinate spread films as compared with adsorbed one. The values of ηs for Nacaseinate are also higher (Fig. 4C) than those for β-casein [26] especially at the higher surface pressure and under the same experimental conditions, as proteins have the possibility to form two-dimensional gels at the air–water interface. The same behavior was reported for spread films [30] and was attributed to the presence of κ-casein in sodium Na-caseinate, which has the possibility to form interfacial gel stabilized by covalent disulfide cross-linked networks (which are not possible for β-casein films). The surface shear viscosity for pure monopalmitin and monoolein spread monolayers is included in Figs. 4A and 4B as a reference [31]. At surface pressures lower than 10 mN/m the values of ηs were similar for Na-caseinate and for monoglycerides (Fig. 4A), because at these surface pressures Nacaseinate and monoglycerides adopt a similar liquid expanded-
Fig. 4. (A) The effect of surface pressure on surface shear viscosity for Na-caseinate adsorbed films at the air–water interface. Symbols: (!, ") Q = 3.75 × 10−6 and (P, Q) Q = 1.25 × 10−5 , open symbols Wc = 1.4 mm and closed symbols Wc = 0.9 mm. The surface shear viscosity for pure (E) monopalmitin and (e) monoolein monolayers are included as reference. (B) The surface shear viscosity for pure (E) monopalmitin and (e) monoolein spread monolayers. (C) The effect of surface pressure on surface shear viscosity for (") adsorbed and ( ) spread Na-caseinate adsorbed films at the air–water interface. The surface shear viscosity of (P) β-casein monolayers is included as reference. Temperature 20 ◦ C and pH 7.
like structure at the interface. The differences observed in ηs values for Na-caseinate and monopalmitin at π > 15 mN/m (Fig. 4A), and especially after the Na-caseinate collapse at
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As can be observed, the ηs -time curves are dependent on the monolayer flow through the canal (Q) and the width of the canal (Wc ). The “steady-state” ηs value can be associated with the existence of a balance between the formation and breaking of monolayer structures or with a constant friction within the mixed monolayer domains along the canal. The fluctuations in the “steady-state” ηs value may be due to friction between LC monopalmitin domains or between segregated regions of monopalmitin and Na-caseinate, these both phenomena would denote the heterogeneity of the mixed monolayer. There was a general tendency for ηs to increase as the surface pressure increased, as might be expected on the basis of greater intermolecular interactions. The “steady-state” ηs values for pure monopalmitin and Na-caseinate monolayers are included in Figs. 5–7 as reference. There were significant differences in ηs during the flow of the mixed film through the canal, depending on the film composition. At π < πeCS , the ηs values of the mixed films are lower than those for a pure component (monopalmitin and Na-caseinate) monolayer, regardless of the composition of the mixed films, at XMP = 0.2 (Figs. 5A and 5B), XMP = 0.4 (Fig. 6A), and XMP = 0.5 (Fig. 7B). These results indicate that, in this region, Na-caseinate and liquidcondensed (LC) domains of monopalmitin flow through the canal, but with few interactions between them, producing a reduction in the friction in the mixed monolayer in relation to that for pure component monolayers and fluctuations in ηs values. After the Na-caseinate collapse, the ηs values of the mixed film were similar to those for a pure monopalmitin monolayer (Figs. 5C, 6B, 6D, and 7D) or are between those of pure monopalmitin and Na-caseinate monolayers (Figs. 5C, 6C, and 7C), with few exceptions (see Fig. 7B). These exceptions may have coincided with less than optimum conditions for the flow of the monolayer, because the values of Q and Wc were not the most appropriate. These results suggest that, in this region, the mixed monolayer was dominated by the presence of monopalmitin at the interface. The segregation of Na-caseinate and monopalmitin domains at the interface during the flow of the monolayer can explain the fact that the ηs values of the mixed film were coincident with those in the lower limit for a pure monopalmitin monolayer or even a little lower (Fig. 7B).
Fig. 5. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monopalmitin at XMP = 0.2 and at (A) 10, (B) 20, and (C) 30 mN/m. The surface shear viscosity for a pure monopalmitin monolayer at 25 mN/m (striped pattern) is included as reference. Symbols: (A and B) (—) Q = 1.25 × 10−5 and Wc = 1.4, (- - -) Q = 1.25 × 10−5 and Wc = 0.9, (· · ·) Q = 3.75 × 10−6 and Wc = 0.9, and (- · -) Q = 6.25 × 10−6 and Wc = 0.9; (C) (—) Q = 6.25 × 10−6 and Wc = 2.05, (- - -) Q = 3.75 × 10−6 and Wc = 1.4, and (· · ·) Q = 3.75 × 10−6 and Wc = 0.9. Temperature 20 ◦ C and pH 7.
π > πeCS (Fig. 4B), can be of utility in the analysis of the shear characteristics of adsorbed mixed films. The effect of surface pressure on surface shear characteristics of adsorbed Na-caseinate–monopalmitin mixed monolayers under different operational conditions and at XMP = 0.2, 0.4, and 0,5 are shown in Figs. 5, 6, and 7, respectively.
3.2. Structural and shear characteristics of Na-caseinate– monoolein mixed monolayers adsorbed at the air–water interface 3.2.1. Structural characteristics The structural characteristics of adsorbed Na-caseinate– monoolein mixed monolayers were essentially different to those of monopalmitin in the mixture, as deduced from π–A isotherms (Fig. 8). Briefly, as expected [26,39], Na-caseinate– monoolein mixed films at surface pressures lower than that for Na-caseinate collapse (at π < πeCS ) adopt a liquid-likeexpanded structure, as for pure components. There was a monolayer expansion due to the presence of monoolein in the mixture. In a previous paper [29] we deduced by means of the excess area and excess free energy that spread Na-caseinate and
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Fig. 6. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monopalmitin at XMP = 0.4 and at (A) 10, (B) 20, and (C) 30mN/m, and (D) (1) 40 mN/m and (2) at the collapse point. The surface shear viscosity for a pure monopalmitin monolayer (striped pattern) and Na-caseinate is included as reference. Symbols: (A and B) (—) Q = 1.25 × 10−5 and Wc = 1.4, (- - -) Q = 1.25 × 10−5 and Wc = 0.9, (· · ·) Q = 3.75 × 10−6 and Wc = 0.9, and (- · -) Q = 6.25 × 10−6 and Wc = 0.9; (C and D) (—) Q = 3.75 × 10−6 and Wc = 2.05, (- - -) Q = 6.25 × 10−6 and Wc = 2.05, (· · ·) Q = 6.25 × 10−6 and Wc = 1.4, and (- · -) Q = 3.75 × 10−6 and Wc = 2.05. Temperature 20 ◦ C and pH 7.
monoolein form a mixed monolayer at the air–water interface with few repulsive interactions between them. This phenomenon occurs at XMO < 0.8 and at π < πeCS , as the film–forming components adopt a similar liquid-like structure. Results of BAM (topography and reflectivity) also show that at a microscopic level domains of monoolein and Na-caseinate coexist at the air–water interface in spread [29] and adsorbed [30] mixed films. However, in this region both components and the mixed monolayer form an isotropic film without any difference in the domain morphology. At surface pressures higher than that for Na-caseinate collapse (at π > πeCS ), the π–A isotherm for mixed monolayers was practically parallel to that of monoolein [36]. At the highest surface pressures, at the collapse point of the mixed film, immiscibility between monolayer forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of a pure monoolein monolayer (Fig. 8). BAM images and the reflectivity of the mixed film also prove the squeezing out of Na-caseinate by monoolein and the presence of monoolein at the interface both in spread and adsorbed films [29,30]. The re-adsorption of previously dis-
placed Na-caseinate upon expansion is easier for monoolein than for monopalmitin because the hysteresis in the π–A isotherms during the compression–expansion cycles is lower for Na-caseinate–monoolein (Fig. 8) than for Na-caseinate– monopalmitin (Fig. 2) adsorbed films. 3.2.2. Shear characteristics The time evolution of ηs during the flow through the canal of Na-caseinate–monoolein mixed monolayers at XMO = 0.2, 0.4, and 0.5, is shown in Figs. 9, 10, and 11, respectively, as a function of surface pressure. The existence of large fluctuations in ηs value are absent for adsorbed Na-caseinate–monoolein mixed monolayers. This phenomenon can be associated with the absence of segregation between Na-caseinate and monoolein domains at the interface during the flow of the monolayer because the film homogeneity. Over the overall range of surface pressures analyzed, the values of ηs for the mixed monolayer are very small and these values are practically independent of the surface pressure (see Figs. 9–11). From these results we concluded that: (i) At π < πeCS , the values of ηs are very similar to those for
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Fig. 7. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monopalmitin at XMP = 0.5 and at (A) 20, (B) 30, and (C) 40 mN/m, and (D) (1) 45 and (2) 50 mN/m. The surface shear viscosity for a pure monopalmitin monolayer (striped pattern) and Na-caseinate is included as reference. Symbols: (A and B) (—) Q = 6.25 × 10−6 and Wc = 0.9, (- - -) Q = 1.25 × 10−5 and Wc = 1.4, (· · ·) Q = 1.25 × 10−5 and Wc = 0.9, (- · -) Q = 6.25 × 10−6 and Wc = 1.4, and (- · · -) Q = 6.25 × 10−6 and Wc = 0.9; (C and D) (—) Q = 3.75 × 10−6 and Wc = 2.05. Temperature 20 ◦ C and pH 7.
a pure monoolein monolayer. These results indicate that the shear characteristics of the mixed monolayer are dependent on the presence of monoolein at the interface at π < πeCS . (ii) At π > πeCS , and, especially, at the higher surface pressures or at the collapse point of the mixed films, the ηs values are lower than that for a pure Na-caseinate adsorbed film, especially at XMO > 0.2 (see Figs. 9D and 11A), which may be associated with the flow of LE monoolein domains through the canal. These data corroborate the hypothesis that Na-caseinate is displaced from the interface by monoolein at π > πeCS . (iii) For some systems, at the collapse of the mixed film, the ηs values of Na-caseinate–monoolein mixed monolayers are at the lower limit or a little lower than the ηs values for a pure monoolein monolayer. These phenomena demonstrated that the mixed film is dominated by the presence of monoolein. Fig. 8. Surface pressure–area isotherms (compression–expansion curves) for adsorbed Na-caseinate–monoolein mixed monolayers on buffered water at pH 7 and at 20 ◦ C. Mass fraction of monoolein in the mixture (XMO ): (!) 0, (—) 0.2, (- - -) 0.4, and (· · ·) 0.5. The equilibrium surface pressure of Na-caseinate (πeCS ) and the collapse pressure of monoolein (πcMO ) are indicated by means of arrows.
4. Discussion Different proteins and monoglycerides show different interfacial structure, confirming the importance of protein and emulsifier molecules in determining the mechanism of inter-
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Fig. 9. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monoolein at XMO = 0.2 and at (A) 10, (B) 20, (C) 30, and (D) 40 mN/m. The surface shear viscosity for a pure monoolein monolayer (striped pattern) is included as reference. Symbols: (—) Q = 1.25 × 10−5 and Wc = 0.9 and (- - -) Q = 1.25 × 10−5 and Wc = 1.4. Temperature 20 ◦ C and pH 7.
facial interactions in spread [4] and adsorbed [28] mixed films. The comparison between spread and adsorbed mixed films is important because the concentrations of the film forming components are well known in the former. Thus, spread mixed films can be used for fundamental studies as reference for the analysis of adsorbed mixed films, because in the latter the concentrations of the film forming components are unknown in experiment involving film balance [28]. Comparing the data presented in this paper to those for Na-caseinate–monoglyceride spread mixed films [29,30] we conclude that the structural and topographical characteristics, the shear-induced segregation, and squeezing out of proteins by monoglycerides appear to be generic phenomena. The reasons for these behaviors must be associated with (i) the immiscibility between protein and monoglyceride at the air–water interface, (ii) to the protein displacement by the monoglyceride at surface pressures higher than that for the protein collapse, and (iii) to the shear-induced segregation in the mixed films. These phenomena have significant repercussion on surface shear properties of spread [4] and adsorbed [28] mixed films. That is, the surface shear viscosity reflects the complex phenomena that take place in protein–monoglyceride
mixed films under flow conditions, as analyzed in preceding sections. The shear characteristics of pure Na-caseinate (Fig. 4) and Na-caseinate–monoglyceride mixed films (Figs. 5–7 and 9–11) were sensitive to the composition of the mixed film and the surface pressure. That is, the ηs values depend on the structural characteristics of the monolayer (Figs. 2 and 8). We have observed that, at the same surface pressure, the ηs values were higher for Na-caseinate than for monoglycerides (monopalmitin and monoolein), with the lower ηs values for monoolein (Fig. 4). This is due to lateral interactions between the protein due to hydrogen bonding, hydrophobic and covalent bonding and/or electrostatic interactions [31]. These interactions between adsorbed protein molecules may vary strongly. Moreover, for a fully packed adsorbed layer (at the higher surface pressures) the deformability (i.e., the mechanical properties) of the protein molecules may be an important factor [4,40,41]. Therefore, differences between ηs values for Nacaseinate and β-casein, (Fig. 4) are rather great and reflect differences, among others, in the protein structure and the potential for the formation of disulfide cross-linking and the
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Fig. 10. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monoolein at XMO = 0.4 and at (A) 20, (B) 30, and (C) 40 mN/m, and (D) at the collapse point. The surface shear viscosity for a pure monoolein monolayer (striped pattern) is included as reference. Symbols: (—) Q = 1.25 × 10−5 and Wc = 1.4 and (- - -) Q = 1.25 × 10−5 and Wc = 0.9. Temperature 20 ◦ C and pH 7.
Fig. 11. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monoolein at XMO = 0.5 and at (A) (—) 30 and (B) (- - -) 40 mN/m, and (· · ·) at the collapse point. The surface shear viscosity for a pure monopalmitin monolayer (striped pattern) is included as reference. Q = 1.25 × 10−5 m2 /s and Wc = 0.9 mm. Temperature 20 ◦ C and pH 7.
formation of interfacial aggregates of significant sizes. The values of ηs were higher for Na-caseinate that form interfacial gels by cross-linking of disulfide residues and the formation of interfacial aggregates of significant size as compared with
β-casein that only can form physical gels stabilized by intermolecular hydrogen bonds [31]. On the other hand, the absence of any interfacial domains—such as for monopalmitin at low surface pressures (with a LE structure) or monoolein with a
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LE expanded structure at every surface pressure—or the presence of small interfacial crystalline-like domains—such as for monopalmitin monolayers with LC or solid structures, at higher surface pressures—(Fig. 4) explains the lower values of ηs for monoglycerides in comparison to those for proteins, at the same surface pressure [36]. For a Na-caseinate–monopalmitin mixed film the ηs value varies greatly with the surface pressure (or surface density) of the mixed monolayer at the interface. In general, the greater the surface pressure (i.e., at the higher surface density), the greater were the values of ηs , as might be expected on the basis of greater intermolecular interactions. That is, as the interactions in the mixed monolayer between monopalmitin molecules— with a LC or solid structure in the monolayer—or between collapsed Na-caseinate are at a maximum, the value of ηs are higher than that with the minimum interactions between monopalmitin molecules—with a LE structure in the monolayer. The lower values of ηs were observed for a LE structure of the monoglyceride (monopalmitin or monoolein) in the mixed monolayer, coinciding with a more homogeneous flow of the monolayer through the canal. In addition, it was observed that over the overall range of surface pressures analyzed, the values of ηs for a Na-caseinate–monoolein mixed monolayer practically did not depend on the surface pressure. For this system film forming components adopt a LE-like structure at every surface pressure (Fig. 8). Moreover, the ηs value is also sensitive to the displacement of film forming components at the interface in spread [29,30] and adsorbed (Figs. 5–7 and 9–11) mixed films. At surface pressures lower than that for Na-caseinate collapse a shear-induced segregation in the topography of monoglycerides and Na-caseinate domains was deduced. At surface pressures higher than that for the Na-caseinate collapse the above mentioned changes in the topography of the mixed monolayer were accompanied by the squeezing of collapsed Na-caseinate domains by monoglycerides [29,30]. Near to the collapse point the mixed film was dominated by the presence of the monoglyceride and the ηs values were practically similar to those for pure monoglyceride monolayers (monopalmitin or monoolein). These results also confirm the extremely sensitive dependence of surface shear characteristics on the immiscibility between film forming components, including the shear-induced segregation in the mixed film. Acknowledgment This research was supported by CICYT through Grant AGL2004-01306/ALI. References [1] E. Dickinson, An Introduction to Food Colloids, Oxford Univ. Press, Oxford, 1992. [2] D.J. McClements, Food Emulsions: Principles, Practice and Techniques, second ed., CRC Press, Boca Raton, FL, 2005. [3] M. Bos, T. Nylander, T. Arnebrant, D.C. Clark, in: G.L. Hasenhuette, R.W. Hartel (Eds.), Food Emulsions and Their Applications, Chapman & Hall, New York, 1997, p. 95.
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[4] J.M. Rodríguez Patino, M.R. Rodríguez Niño, C. Carrera, Curr. Opin. Colloid Interface Sci. 8 (2003) 387. [5] M.A. Bos, T. van Vliet, Adv. Colloid Interface Sci. 91 (2001) 437. [6] P.J. Wilde, A.R. Mackie, F. Husband, P. Gunning, V. Morris, Adv. Colloid Interface Sci. 108–109 (2004) 63. [7] L.A. Pugnaloni, E. Dickinson, R. Ettelaie, A.R. Mackie, P.J. Wilde, Adv. Colloid Interface Sci. 107 (2004) 27. [8] J.M. Rodríguez Patino, C. Carrera, M.R. Rodríguez Niño, M. Cejudo, Langmuir 17 (2001) 4003. [9] J.M. Rodríguez Patino, C. Carrera, M.R. Rodríguez Niño, M. Cejudo, J. Colloid Interface Sci. 242 (2001) 141. [10] J.M. Rodríguez Patino, M.R. Rodríguez Niño, C. Carrera, Ind. Eng. Chem. Res. 41 (2002) 2652. [11] J.M. Rodríguez Patino, M.R. Rodríguez Niño, C. Carrera, J. Agric. Food Chem. 51 (2003) 112–119. [12] E.H. Lucassen-Reynders, J. Benjamins, in: E. Dickinson, J.M. Rodríguez Patino (Eds.), Food Emulsions and Foams: Interfaces, Interactions and Stability, Royal Soc. Chem., Cambridge, 1999, p. 196. [13] J. Benjamins, E.H. Lucassen-Reynders, in: D. Möbius, R. Miller (Eds.), Proteins at Liquid Interface, Elsevier, Amsterdam, 1998, p. 341. [14] A.H. Martin, K. Grollem, M.A. Bos, M.A. Cohen Stuart, T. van Vliet, J. Colloid Interface Sci. 254 (2002) 175. [15] D.T. Wasan, in: K.L. Mittal, P. Kumar (Eds.), Emulsions, Foams, and Thin Films, Marcel Dekker, New York, 2000, p. 1. [16] B.S. Murray, E. Dickinson, Food Sci. Technol. Inst. 2 (1996) 131. [17] B.S. Murray, in: D. Möbius, R. Miller (Eds.), Proteins at Liquid Interfaces, Elsevier, Amsterdam, 1998, p. 179. [18] B.S. Murray, Curr. Opin. Colloid Interface Sci. 7 (2002) 426. [19] A. Prins, M.A. Bos, F.J.G. Boerboom, H.K.A.I. van Kalsbeek, in: D. Möbius, R. Miller (Eds.), Proteins at Liquid Interfaces, Elsevier, Amsterdam, 1998, p. 221. [20] R. Miller, R. Wüstneck, J. Krägel, G. Kretzsmara, Colloids Surf. A Physicochem. Eng. Aspects 111 (2006) 75. [21] R. Miller, V.B. Fainermann, A.V. Makievski, J. Krägel, D.O. Grigoriev, V.N. Kazakov, O.V. Sinyachenco, Adv. Colloid Interface Sci. 86 (2000) 39. [22] J. Ignés-Mullol, D.K. Schwartz, Nature 410 (2001) 348. [23] C. Carrera, J.M. Rodríguez Patino, Langmuir 20 (2004) 6327. [24] T. Murayama, G. Fuller, C. Frank, C. Robertson, Science 274 (1996) 233. [25] T. Murayama, J. Lauger, G. Fuller, C. Frank, C. Robertson, Langmuir 14 (1998) 1836. [26] J.M. Rodríguez Patino, M. Cejudo, M.R. Rodríguez Niño, C. Carrera, Ind. Eng. Chem. Res. 45 (2006) 1886. [27] S. Damodaran, A. Paraf, Food Proteins and their Applications, Marcel Dekker, New York, 1997. [28] M. Cejudo, C. Carrera, M.R. Rodríguez Niño, J.M. Rodríguez Patino, Food Hydrocolloids 21 (2007) 906. [29] J.M. Rodríguez Patino, M.R. Rodríguez Niño, C. Carrera, M. Cejudo, J. Colloid Interface Sci. 240 (2001) 113. [30] J.M. Rodríguez Patino, C. Carrera, Biomacromolecules 5 (2004) 2065. [31] J.M. Rodríguez Patino, C. Carrera, Langmuir 20 (2004) 4530. [32] M.R. Rodríguez Niño, C. Carrera, M. Cejudo, J.M. Rodríguez Patino, J. Am. Oil Chem. Soc. 78 (2001) 873. [33] W.D. Harkins, J.G. Kirkwood, Nature (London) 141 (1938) 38. [34] J.M. Rodríguez Patino, M. Cejudo, Langmuir 20 (2004) 4515. [35] M. Cejudo, C. Carrera, M.R. Rodríguez Niño, J.M. Rodríguez Patino, Biomacromolecules 7 (2006) 507. [36] J.M. Rodríguez Patino, C. Carrera, M.R. Rodríguez Niño, Langmuir 15 (1999) 2484. [37] J.B. Li, J. Krägel, A.V. Makievski, V.B. Fainermann, R. Miller, H.A. Möhwald, Colloids Surf. A Phys. Eng. Aspects 142 (1998) 355. [38] J.M. Rodríguez Patino, M. Cejudo, Langmuir 20 (2004) 4515. [39] J.M. Rodríguez Patino, M.R. Rodríguez Niño, C. Carrera, M. Cejudo, Langmuir 17 (2001) 7545. [40] P.J. Wilde, Curr. Opin. Colloid Interface Sci. 5 (2000) 176. [41] P.A. Gunning, A.R. Mackie, A.P. Gunning, N.C. Woodward, P.J. Wilde, V.F. Morris, Biomacromolecules 5 (2004) 984.
Structural and shear characteristics of adsorbed sodium caseinate and monoglyceride mixed monolayers at the air–water interface Juan M. Rodríguez Patino ∗ , Marta Cejudo Fernández, Cecilio Carrera Sánchez, Ma . Rosario Rodríguez Niño Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, C/. Prof. García González 1, 41012-Seville, Spain Received 20 February 2007; accepted 12 April 2007 Available online 18 April 2007
Abstract The structural and shear characteristics of mixed monolayers formed by an adsorbed Na-caseinate film and a spread monoglyceride (monopalmitin or monoolein) on the previously adsorbed protein film have been analyzed. Measurements of the surface pressure (π )–area (A) isotherm and surface shear viscosity (ηs ) were obtained at 20 ◦ C and at pH 7 in a modified Wilhelmy-type film balance. The structural and shear characteristics of the mixed films depend on the surface pressure and on the composition of the mixed film. At surface pressures lower than the equilibrium surface pressure of Na-caseinate (at π < πeCS ), both Na-caseinate and monoglyceride coexist at the interface, with a structural polymorphism or a liquid expanded structure due to the presence of monopalmitin or monoolein in the mixture, respectively. At higher surface pressures, collapsed Na-caseinate residues may be displaced from the interface by monoglyceride molecules. For a Na-caseinate–monopalmitin mixed film the ηs value varies greatly with the surface pressure (or surface density) of the mixed monolayer at the interface. In general, the greater the surface pressure, the greater are the values of ηs . However, the values of ηs for a Na-caseinate–monoolein mixed monolayer are very low and practically do not depend on the surface pressure. The collapsed Na-caseinate residues displaced from the interface by monoglyceride molecules at π > πeCS have important repercussions on the shear characteristics of the mixed films. © 2007 Elsevier Inc. All rights reserved. Keywords: Food emulsifiers; Food dispersions; Protein–monoglyceride mixed films; Interfacial shear viscosity; Adsorbed films; Monolayer structure; Interfacial rheology; Air–water interface
1. Introduction The stability, textural, and mechanical properties of dispersed food systems (emulsions and foams) depend on the way in which the constituent emulsifiers—low-molecular weight emulsifiers (lipids, phospholipids, and surfactants) and biopolymers (proteins and some polysaccharides)—adsorb and interact at fluid interfaces [1,2]. The optimum use of these emulsifiers depends on knowledge of their interfacial physico-chemical characteristics (such as surface activity, structure, miscibility, superficial viscosity, etc.) and the kinetics of the film formation at fluid interfaces. The distribution of lipids and proteins at fluid interfaces is determined by competitive or co-operative adsorp* Corresponding author. Fax: +34 95 4557134.
E-mail address: [email protected] (J.M. Rodríguez Patino). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.04.025
tion between the two types of emulsifiers, which in turn depend on interactions between emulsifier molecules [3,4]. Interactions between molecules of emulsifiers could affect not only the film structure, topography and mechanical properties, but also dynamic phenomena in mixed films [4–7]. Interfacial rheology is important for food colloids (emulsions and foams) because the structural and mechanical properties of food emulsifiers at fluid interfaces have an influence on the stability and texture of the product. In addition, interfacial rheology is a very sensitive technique to monitor the interfacial structure and concentration of single emulsifiers at the interface or the relative concentration, the competitive adsorption, and the magnitude of interactions between different emulsifiers at the interface [4,5,8–11]. Interfacial rheology can be defined for both compressional deformation (dilatational rheology) and shearing motion of the interface (shear rheology). While shear viscosity may contribute appreciably to the long-term stabil-
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ity of dispersions, dilatational rheology plays an important role in short-term stability [5,12–19]. Interfacial shear rheology is most useful for polymer and mixed polymer surfactant adsorption layers and insoluble monolayers and gives access to interaction forces in two dimensional layers [20,21]. In addition, the shear may induce heterogeneity and segregation between protein and lipid domains at the interface during the flow of the monolayer [22,23]. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers [22–26]. In this contribution we are concerned with the analysis of structural and shear characteristics of mixed monolayers formed by an adsorbed milk protein (Na-caseinate) and a spread monoglyceride (monopalmitin or monoolein). Na-caseinate is distinguished by its good foaming and emulsifying properties and for this reason is widely used in food formulations in combination with monoglycerides [1,2,27]. Monolayer technique has been used successfully for studying the properties of mixed emulsifiers spread [4] and adsorbed and/or penetrated [28] at the air–water interface. This work is an extension of previous studies of structural, topographical and shear characteristics of spread mixed films at the air–water interface [29,30]. 2. Experimental 2.1. Chemicals Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90) and 1-mono (cis-9-octadecanoyl) glycerol (monoolein, RYLOTM MG 19) were kindly supplied by Danisco Ingredients (Brabran, Denmark) with over 95–98% of purity. Na-caseinate (a mixture of ≈38% β-casein, ≈39% αs1 -casein, ≈12% κ-casein, and ≈11% αs2 -casein) was kindly supplied and purified from bulk milk from Unilever Research (Colworth, UK). Samples for interfacial characteristics of Nacaseinate adsorbed films were prepared using Milli-Q ultra pure water and were buffered at pH 7. To form the mixed surface film on a previously adsorbed Na-caseinate 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 subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution called trizma ((CH2 OH)3 CNH2 /(CH2 OH)3 CNH3 Cl, Sigma, >99.5%) was used to achieve pH 7. Ionic strength was 0.05 M in all the experiments. 2.2. Surface film balance Measurements of the surface pressure (π ) versus average trough area (A) were performed on a modified Wilhelmy-type film balance, which integrates surface shear rheometry, as described elsewhere [31]. Before each measurement, the film balance was calibrated at 20 ◦ C. For Na-caseinate adsorbed films from water a Na-caseinate solution at 7.5 × 10−6 −1 × 10−5 % (wt) was left in the trough and time allowed for protein adsorption at the interface. These protein concentrations were selected
from previous data of the adsorption isotherm [32]. At this Na-caseinate concentration in solution the surface pressure at equilibrium is zero. In fact, after 24 h the surface pressure (π ) at the maximum area of the trough was practically zero and then the monoglyceride (≈1.5 × 10−4 mg/µl) was spread at different points on the previously adsorbed Na-caseinate film. For pure adsorbed protein films the maximum protein concentration in the bulk phase should be selected in order to obtain a reasonable rate of adsorption at the interface, but maintaining as zero the equilibrium surface pressure. On the other hand, for mixed films, at low protein concentrations in the aqueous phase we cannot observe the collapse point, especially for low monoglyceride concentrations. Thus, in these experiments we have selected optimum conditions in order to obtain the complete π–A isotherm of the mixed film, from the more expanded monolayer (at the higher areas) to the more condensed monolayer, at the collapse point (at the lower areas). Mixtures of particular mass fractions—expressed as the mass fraction of monoglyceride in the mixture, X—were studied. The compression rate was 3.3 cm/min, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. To allow for Na-caseinate–monoglyceride interactions, 30 min were allowed to elapse before compression was performed. After 30 min at the maximum area, measurements of compression–expansion cycles were performed with 30 min of waiting time between each expansion–compression cycle. The measurement of the π–A isotherm was performed before the surface shear rheology experiments. This isotherm was recorded with the maximum width of the canal at which the surface pressure at both sides of the canal was the same (i.e. π was zero during the monolayer compression). The initial surface pressure of the mixed film at the maximum area before compression was zero. The reproducibility of the results was better than ±0.5 mN/m for surface pressure and ±0.05 m2 /mg. 2.3. Surface shear rheometry To study the shear characteristics of adsorbed films a homemade canal viscometer—an analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past one another—as described elsewhere was used in this work [31]. Briefly, surface shear viscosity measurements were taken on a modified Wilhelmy-type film balance, with two surface pressure sensors located at both sides of the canal in the center of the trough (Fig. 1). The canal was constructed of two Teflon bars to render them hydrophobic and was mounted on the trough. The separation between the two bars can be varied and adjusted by means of a precision screw. The monolayer is allowed to flow through a canal of a width Wc and fixed length, L = 30 mm, and at a constant flow (Q, m2 /s, recorded as a variation of the area associated with the movement of the barrier as a function of time). For a constant rate R (m/s) of translation of the two barriers, the flow Q (m2 /s) is Q = R · T ; were T (m) is the width of the trough. The surface shear viscosity (ηs ) was calculated from the rate of film flow Q by Eq. (1), which is analogous to the Poiseuille equation for the flow of liquids through capillary
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Fig. 1. Wilhelmy-type film balance modified for measurement of surface shear viscosity. The monolayer is allowed to flow in the direction of the arrow through a canal of width Wc and length L by the movement in the same direction of two barriers from a region of higher surface pressure (π2 ) to a region of lower surface pressure (π1 ). The width of the canal can be modified by means of the precision screw. For details see the text.
Fig. 2. Surface pressure–area isotherms (compression–expansion curves) for adsorbed Na-caseinate–monopalmitin mixed monolayers on buffered water at pH 7 and at 20 ◦ C. Mass fraction of monopalmitin in the mixture (XMP ): (!) 0, (—) 0.2, (- - -) 0.4, and (· · ·) 0.5. The equilibrium surface pressure of Na-caseinate (πeCS ) and the collapse pressure of monopalmitin (πcMP ) are indicated by means of arrows.
tubes [33]
The results of the π−A isotherms (Fig. 2) confirm that a Nacaseinate monolayers at the air–water interface adopt a liquid expanded-like structure at every surface pressure. The monolayer collapses and amino acid segments are extended into the underlying aqueous solution at a surface pressure a little higher than the equilibrium surface pressure (πeCS ≈ 29 mN/m), which is indicated in Fig. 2 by means of an arrow [32]. The equilibrium surface pressure is the maximum surface pressure at which a spread monolayer can be compressed just before the monolayer collapse. Mixtures of particular mass fractions—ranging between 0 and 0.5, expressed as the mass fraction of monoglyceride in the mixture, X—were studied. The amount of spread monoglyceride was calculated on the basis of the mass of previously adsorbed Na-caseinate, which was calculated form the adsorbed π–A isotherm [34,35]. It must be emphasized that, due to this assumption, in Fig. 2 the area in x-axis is not the true area per unit mass of mixed film but the trough (apparent) area (AAPPARENT ). On the other hand, as opposed to spread monolayers [29], for adsorbed monolayers the mixtures with mass fractions higher than X = 0.5 saturate the interface, under the experimental conditions used in this work. From the π–A isotherms for Na-caseinate + monopalmitin mixed films (Fig. 2) it can be seen that there was a monolayer expansion as the monopalmitin concentration in the mixture was increased, especially at higher surface pressures (at π > πeCS ). That is, the π–A isotherm is displaced towards higher areas as the concentration of monopalmitin in the mixture increases. At surface pressures lower than the equilibrium surface pressure of Na-caseinate (at π < πeCS ), both Na-caseinate and monopalmitin coexist at the interface. In fact, we can see the main transition between liquid expanded (LE) and liquid condensed (LC) phases, which is typical of monopalmitin monolayers at low surface pressures (at π ≈ 5 mN/m) [36]. In a previous paper [29] we deduced by means of the excess area and excess free energy that spread Na-caseinate and monopalmitin
ηs =
(π2 − π1 ) · Wc3 . 12 · Q · L
(1)
The monolayer was compressed to the desired surface pressure and then the flow of the monolayer through the canal was facilitated by the movement of the two barriers in the same direction, from a region of higher surface pressure (π2 ) to a region of lower surface pressure (π1 ). The value of π = π2 − π1 was deduced during the flow of the monolayer. As for pure components, an optimization of Wc and Q is necessary in order to obtain the best flow conditions of the mixed monolayer through the canal [31]. During these experiments practically all the monolayer (ca. 90%) flows through the canal, thus the data presented in this work represent the overall behavior of the monolayer under pure shear conditions. The subphase temperature was controlled at 20 ◦ C by water circulation from a thermostat, within an error range of ±0.3 ◦ C. The temperature was measured by a thermocouple located just below the air– water interface. Data were obtained after a minimum of three measurements and the repeated results prove the reproducibility of the method. 3. Results 3.1. Structural and shear characteristics of Na-caseinate– monopalmitin mixed monolayers adsorbed at the air–water interface 3.1.1. Structural characteristics The π–AAPPARENT isotherm for adsorbed Na-caseinate monolayer was obtained as a control at the beginning of each experiment (Fig. 2). This isotherm is practically the same as that obtained directly by spreading [29,31]. Thus, the structures of the monolayers formed in the two different ways must be identical, at least for adsorption from low bulk protein concentrations.
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Fig. 3. Difference in surface pressure between the limits of the canal during the flow of Na-caseinate adsorbed monolayers at the air–water interface as a function of surface pressure (mN/m): (A) 5, (B) 10, (C) 15, and (D) 20. Temperature 20 ◦ C and pH 7. Effect of width of canal (Wc , mm) and Na-caseinate flow through the canal (Q, m2 /s): (—) Q = 3.75 × 10−6 and Wc = 1.4, (- - -) Q = 1.25 × 10−5 and Wc = 1.4, (· · ·) Q = 3.75 × 10−6 and Wc = 0.9, (- · -) Q = 6.25 × 10−6 and Wc = 0.9, and (- · · -) Q = 1.25 × 10−5 and Wc = 0.9.
form a mixed monolayer at the air–water interface with few repulsive interactions between film forming components (even phase separation could occur) at low monopalmitin concentrations in the mixed film (at XMP < 0.8) and at surface pressures lower than that for the Na-caseinate collapse (at π < πeCS ). Results of BAM (topography and reflectivity) also show that at a microscopic level domains of monopalmitin and Na-caseinate coexist at the air–water interface in spread [30] and adsorbed [29] mixed films. At surface pressures higher than that for Na-caseinate collapse (at π > πeCS ), the π–A isotherms for mixed monolayers were parallel to that of monopalmitin [36]. These results suggest that at the higher surface pressures the arrangement of the monopalmitin hydrocarbon chain in mixed monolayers is practically the same in the entire Na-caseinate/monopalmitin fraction. That is, at higher surface pressures, collapsed Na-caseinate residues may be displaced from the interface by monopalmitin molecules. At the highest surface pressures (at π > πeCS ), at the
collapse point of the mixed film, immiscibility between monolayer forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monopalmitin monolayer (Fig. 2). The squeezing out of Na-caseinate by monopalmitin and the clear evidence that the mixed monolayer is practically dominated by the presence of monopalmitin at the interface both in spread and adsorbed was also corroborates by BAM [29,30]. The fact that upon expansion and further compression, the π–A isotherms were repetitive (data not shown) suggests that the protein displaced by monopalmitin during the compression (at π > πeCS ) reenters the mixed monolayer upon expansion and supports the idea that the protein remains underneath the monoglyceride film either through hydrophobic interactions between protein and lipid or by local anchoring through the monoglyceride layer [37,38]. However, for adsorbed Na-caseinate– monopalmitin mixed monolayers a first order-like phase transition was observed upon the monolayer expansion (Fig. 2) at
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surface pressure values close to the equilibrium surface pressure of Na-caseinate (at π ≈ πeCS )—with an atypical plateau in the π–A isotherm. These results suggest that the re-adsorption of previously displaced Na-caseinate has a kinetic character, which was not evident for spread mixed films [34,39]. 3.1.2. Shear characteristics The time evolution of π during the flow of an adsorbed Nacaseinate monolayer depends on the Na-caseinate flow through the canal and the surface pressure (Fig. 3). An optimization of Wc and Q was necessary in order to obtain the best flow conditions of a protein monolayer through the canal, with few fluctuation in the π –time flow curve, and more reproducible data. The steady state in π was reached relatively quickly during the flow of the Na-caseinate monolayer at low surface pressures, but more time is necessary at high surface pressure. From the π the surface shear viscosity (ηs ) was determined by means of Eq. (1). The steady state π value can be associated with the equilibrium shear viscosity in stationary flow. At this point there exists a balance between the formation and breaking of monolayer structure or a constant friction within the Nacaseinate monolayer domains along the canal. The fluctuations in the π value may be due to friction between Na-caseinate domains at the higher surface pressures, a phenomenon that denotes the heterogeneity of the monolayer. From the π at steady state, the surface shear viscosity was determined as a function of surface pressure (Fig. 4). Over the overall range of surface pressures the values of ηs did not depend on the flow Q (Fig. 4A). The values of ηs increased with surface pressure, especially as the residues of Na-caseinate adopt the more condensed conformation at the air–water interface. The values of ηs are higher for spread [30] than for adsorbed Na-caseinate films (Fig. 4C). Thus, even the π–A isotherms for adsorbed Na-caseinate monolayer are practically the same as those obtained directly by spreading, the surface shear viscosity is different. One speculation is that Na-caseinate may be spread at the interface maintaining a more unfolded structure as compared with adsorbed films. Thus, the interactions between unfolded Na-caseinate residues must be higher for spread than for adsorbed Na-caseinate films, a phenomenon that explains the higher value of ηs for Na-caseinate spread films as compared with adsorbed one. The values of ηs for Nacaseinate are also higher (Fig. 4C) than those for β-casein [26] especially at the higher surface pressure and under the same experimental conditions, as proteins have the possibility to form two-dimensional gels at the air–water interface. The same behavior was reported for spread films [30] and was attributed to the presence of κ-casein in sodium Na-caseinate, which has the possibility to form interfacial gel stabilized by covalent disulfide cross-linked networks (which are not possible for β-casein films). The surface shear viscosity for pure monopalmitin and monoolein spread monolayers is included in Figs. 4A and 4B as a reference [31]. At surface pressures lower than 10 mN/m the values of ηs were similar for Na-caseinate and for monoglycerides (Fig. 4A), because at these surface pressures Nacaseinate and monoglycerides adopt a similar liquid expanded-
Fig. 4. (A) The effect of surface pressure on surface shear viscosity for Na-caseinate adsorbed films at the air–water interface. Symbols: (!, ") Q = 3.75 × 10−6 and (P, Q) Q = 1.25 × 10−5 , open symbols Wc = 1.4 mm and closed symbols Wc = 0.9 mm. The surface shear viscosity for pure (E) monopalmitin and (e) monoolein monolayers are included as reference. (B) The surface shear viscosity for pure (E) monopalmitin and (e) monoolein spread monolayers. (C) The effect of surface pressure on surface shear viscosity for (") adsorbed and ( ) spread Na-caseinate adsorbed films at the air–water interface. The surface shear viscosity of (P) β-casein monolayers is included as reference. Temperature 20 ◦ C and pH 7.
like structure at the interface. The differences observed in ηs values for Na-caseinate and monopalmitin at π > 15 mN/m (Fig. 4A), and especially after the Na-caseinate collapse at
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As can be observed, the ηs -time curves are dependent on the monolayer flow through the canal (Q) and the width of the canal (Wc ). The “steady-state” ηs value can be associated with the existence of a balance between the formation and breaking of monolayer structures or with a constant friction within the mixed monolayer domains along the canal. The fluctuations in the “steady-state” ηs value may be due to friction between LC monopalmitin domains or between segregated regions of monopalmitin and Na-caseinate, these both phenomena would denote the heterogeneity of the mixed monolayer. There was a general tendency for ηs to increase as the surface pressure increased, as might be expected on the basis of greater intermolecular interactions. The “steady-state” ηs values for pure monopalmitin and Na-caseinate monolayers are included in Figs. 5–7 as reference. There were significant differences in ηs during the flow of the mixed film through the canal, depending on the film composition. At π < πeCS , the ηs values of the mixed films are lower than those for a pure component (monopalmitin and Na-caseinate) monolayer, regardless of the composition of the mixed films, at XMP = 0.2 (Figs. 5A and 5B), XMP = 0.4 (Fig. 6A), and XMP = 0.5 (Fig. 7B). These results indicate that, in this region, Na-caseinate and liquidcondensed (LC) domains of monopalmitin flow through the canal, but with few interactions between them, producing a reduction in the friction in the mixed monolayer in relation to that for pure component monolayers and fluctuations in ηs values. After the Na-caseinate collapse, the ηs values of the mixed film were similar to those for a pure monopalmitin monolayer (Figs. 5C, 6B, 6D, and 7D) or are between those of pure monopalmitin and Na-caseinate monolayers (Figs. 5C, 6C, and 7C), with few exceptions (see Fig. 7B). These exceptions may have coincided with less than optimum conditions for the flow of the monolayer, because the values of Q and Wc were not the most appropriate. These results suggest that, in this region, the mixed monolayer was dominated by the presence of monopalmitin at the interface. The segregation of Na-caseinate and monopalmitin domains at the interface during the flow of the monolayer can explain the fact that the ηs values of the mixed film were coincident with those in the lower limit for a pure monopalmitin monolayer or even a little lower (Fig. 7B).
Fig. 5. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monopalmitin at XMP = 0.2 and at (A) 10, (B) 20, and (C) 30 mN/m. The surface shear viscosity for a pure monopalmitin monolayer at 25 mN/m (striped pattern) is included as reference. Symbols: (A and B) (—) Q = 1.25 × 10−5 and Wc = 1.4, (- - -) Q = 1.25 × 10−5 and Wc = 0.9, (· · ·) Q = 3.75 × 10−6 and Wc = 0.9, and (- · -) Q = 6.25 × 10−6 and Wc = 0.9; (C) (—) Q = 6.25 × 10−6 and Wc = 2.05, (- - -) Q = 3.75 × 10−6 and Wc = 1.4, and (· · ·) Q = 3.75 × 10−6 and Wc = 0.9. Temperature 20 ◦ C and pH 7.
π > πeCS (Fig. 4B), can be of utility in the analysis of the shear characteristics of adsorbed mixed films. The effect of surface pressure on surface shear characteristics of adsorbed Na-caseinate–monopalmitin mixed monolayers under different operational conditions and at XMP = 0.2, 0.4, and 0,5 are shown in Figs. 5, 6, and 7, respectively.
3.2. Structural and shear characteristics of Na-caseinate– monoolein mixed monolayers adsorbed at the air–water interface 3.2.1. Structural characteristics The structural characteristics of adsorbed Na-caseinate– monoolein mixed monolayers were essentially different to those of monopalmitin in the mixture, as deduced from π–A isotherms (Fig. 8). Briefly, as expected [26,39], Na-caseinate– monoolein mixed films at surface pressures lower than that for Na-caseinate collapse (at π < πeCS ) adopt a liquid-likeexpanded structure, as for pure components. There was a monolayer expansion due to the presence of monoolein in the mixture. In a previous paper [29] we deduced by means of the excess area and excess free energy that spread Na-caseinate and
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Fig. 6. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monopalmitin at XMP = 0.4 and at (A) 10, (B) 20, and (C) 30mN/m, and (D) (1) 40 mN/m and (2) at the collapse point. The surface shear viscosity for a pure monopalmitin monolayer (striped pattern) and Na-caseinate is included as reference. Symbols: (A and B) (—) Q = 1.25 × 10−5 and Wc = 1.4, (- - -) Q = 1.25 × 10−5 and Wc = 0.9, (· · ·) Q = 3.75 × 10−6 and Wc = 0.9, and (- · -) Q = 6.25 × 10−6 and Wc = 0.9; (C and D) (—) Q = 3.75 × 10−6 and Wc = 2.05, (- - -) Q = 6.25 × 10−6 and Wc = 2.05, (· · ·) Q = 6.25 × 10−6 and Wc = 1.4, and (- · -) Q = 3.75 × 10−6 and Wc = 2.05. Temperature 20 ◦ C and pH 7.
monoolein form a mixed monolayer at the air–water interface with few repulsive interactions between them. This phenomenon occurs at XMO < 0.8 and at π < πeCS , as the film–forming components adopt a similar liquid-like structure. Results of BAM (topography and reflectivity) also show that at a microscopic level domains of monoolein and Na-caseinate coexist at the air–water interface in spread [29] and adsorbed [30] mixed films. However, in this region both components and the mixed monolayer form an isotropic film without any difference in the domain morphology. At surface pressures higher than that for Na-caseinate collapse (at π > πeCS ), the π–A isotherm for mixed monolayers was practically parallel to that of monoolein [36]. At the highest surface pressures, at the collapse point of the mixed film, immiscibility between monolayer forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of a pure monoolein monolayer (Fig. 8). BAM images and the reflectivity of the mixed film also prove the squeezing out of Na-caseinate by monoolein and the presence of monoolein at the interface both in spread and adsorbed films [29,30]. The re-adsorption of previously dis-
placed Na-caseinate upon expansion is easier for monoolein than for monopalmitin because the hysteresis in the π–A isotherms during the compression–expansion cycles is lower for Na-caseinate–monoolein (Fig. 8) than for Na-caseinate– monopalmitin (Fig. 2) adsorbed films. 3.2.2. Shear characteristics The time evolution of ηs during the flow through the canal of Na-caseinate–monoolein mixed monolayers at XMO = 0.2, 0.4, and 0.5, is shown in Figs. 9, 10, and 11, respectively, as a function of surface pressure. The existence of large fluctuations in ηs value are absent for adsorbed Na-caseinate–monoolein mixed monolayers. This phenomenon can be associated with the absence of segregation between Na-caseinate and monoolein domains at the interface during the flow of the monolayer because the film homogeneity. Over the overall range of surface pressures analyzed, the values of ηs for the mixed monolayer are very small and these values are practically independent of the surface pressure (see Figs. 9–11). From these results we concluded that: (i) At π < πeCS , the values of ηs are very similar to those for
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Fig. 7. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monopalmitin at XMP = 0.5 and at (A) 20, (B) 30, and (C) 40 mN/m, and (D) (1) 45 and (2) 50 mN/m. The surface shear viscosity for a pure monopalmitin monolayer (striped pattern) and Na-caseinate is included as reference. Symbols: (A and B) (—) Q = 6.25 × 10−6 and Wc = 0.9, (- - -) Q = 1.25 × 10−5 and Wc = 1.4, (· · ·) Q = 1.25 × 10−5 and Wc = 0.9, (- · -) Q = 6.25 × 10−6 and Wc = 1.4, and (- · · -) Q = 6.25 × 10−6 and Wc = 0.9; (C and D) (—) Q = 3.75 × 10−6 and Wc = 2.05. Temperature 20 ◦ C and pH 7.
a pure monoolein monolayer. These results indicate that the shear characteristics of the mixed monolayer are dependent on the presence of monoolein at the interface at π < πeCS . (ii) At π > πeCS , and, especially, at the higher surface pressures or at the collapse point of the mixed films, the ηs values are lower than that for a pure Na-caseinate adsorbed film, especially at XMO > 0.2 (see Figs. 9D and 11A), which may be associated with the flow of LE monoolein domains through the canal. These data corroborate the hypothesis that Na-caseinate is displaced from the interface by monoolein at π > πeCS . (iii) For some systems, at the collapse of the mixed film, the ηs values of Na-caseinate–monoolein mixed monolayers are at the lower limit or a little lower than the ηs values for a pure monoolein monolayer. These phenomena demonstrated that the mixed film is dominated by the presence of monoolein. Fig. 8. Surface pressure–area isotherms (compression–expansion curves) for adsorbed Na-caseinate–monoolein mixed monolayers on buffered water at pH 7 and at 20 ◦ C. Mass fraction of monoolein in the mixture (XMO ): (!) 0, (—) 0.2, (- - -) 0.4, and (· · ·) 0.5. The equilibrium surface pressure of Na-caseinate (πeCS ) and the collapse pressure of monoolein (πcMO ) are indicated by means of arrows.
4. Discussion Different proteins and monoglycerides show different interfacial structure, confirming the importance of protein and emulsifier molecules in determining the mechanism of inter-
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Fig. 9. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monoolein at XMO = 0.2 and at (A) 10, (B) 20, (C) 30, and (D) 40 mN/m. The surface shear viscosity for a pure monoolein monolayer (striped pattern) is included as reference. Symbols: (—) Q = 1.25 × 10−5 and Wc = 0.9 and (- - -) Q = 1.25 × 10−5 and Wc = 1.4. Temperature 20 ◦ C and pH 7.
facial interactions in spread [4] and adsorbed [28] mixed films. The comparison between spread and adsorbed mixed films is important because the concentrations of the film forming components are well known in the former. Thus, spread mixed films can be used for fundamental studies as reference for the analysis of adsorbed mixed films, because in the latter the concentrations of the film forming components are unknown in experiment involving film balance [28]. Comparing the data presented in this paper to those for Na-caseinate–monoglyceride spread mixed films [29,30] we conclude that the structural and topographical characteristics, the shear-induced segregation, and squeezing out of proteins by monoglycerides appear to be generic phenomena. The reasons for these behaviors must be associated with (i) the immiscibility between protein and monoglyceride at the air–water interface, (ii) to the protein displacement by the monoglyceride at surface pressures higher than that for the protein collapse, and (iii) to the shear-induced segregation in the mixed films. These phenomena have significant repercussion on surface shear properties of spread [4] and adsorbed [28] mixed films. That is, the surface shear viscosity reflects the complex phenomena that take place in protein–monoglyceride
mixed films under flow conditions, as analyzed in preceding sections. The shear characteristics of pure Na-caseinate (Fig. 4) and Na-caseinate–monoglyceride mixed films (Figs. 5–7 and 9–11) were sensitive to the composition of the mixed film and the surface pressure. That is, the ηs values depend on the structural characteristics of the monolayer (Figs. 2 and 8). We have observed that, at the same surface pressure, the ηs values were higher for Na-caseinate than for monoglycerides (monopalmitin and monoolein), with the lower ηs values for monoolein (Fig. 4). This is due to lateral interactions between the protein due to hydrogen bonding, hydrophobic and covalent bonding and/or electrostatic interactions [31]. These interactions between adsorbed protein molecules may vary strongly. Moreover, for a fully packed adsorbed layer (at the higher surface pressures) the deformability (i.e., the mechanical properties) of the protein molecules may be an important factor [4,40,41]. Therefore, differences between ηs values for Nacaseinate and β-casein, (Fig. 4) are rather great and reflect differences, among others, in the protein structure and the potential for the formation of disulfide cross-linking and the
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Fig. 10. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monoolein at XMO = 0.4 and at (A) 20, (B) 30, and (C) 40 mN/m, and (D) at the collapse point. The surface shear viscosity for a pure monoolein monolayer (striped pattern) is included as reference. Symbols: (—) Q = 1.25 × 10−5 and Wc = 1.4 and (- - -) Q = 1.25 × 10−5 and Wc = 0.9. Temperature 20 ◦ C and pH 7.
Fig. 11. Surface shear viscosity for an adsorbed mixed film of Na-caseinate and monoolein at XMO = 0.5 and at (A) (—) 30 and (B) (- - -) 40 mN/m, and (· · ·) at the collapse point. The surface shear viscosity for a pure monopalmitin monolayer (striped pattern) is included as reference. Q = 1.25 × 10−5 m2 /s and Wc = 0.9 mm. Temperature 20 ◦ C and pH 7.
formation of interfacial aggregates of significant sizes. The values of ηs were higher for Na-caseinate that form interfacial gels by cross-linking of disulfide residues and the formation of interfacial aggregates of significant size as compared with
β-casein that only can form physical gels stabilized by intermolecular hydrogen bonds [31]. On the other hand, the absence of any interfacial domains—such as for monopalmitin at low surface pressures (with a LE structure) or monoolein with a
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LE expanded structure at every surface pressure—or the presence of small interfacial crystalline-like domains—such as for monopalmitin monolayers with LC or solid structures, at higher surface pressures—(Fig. 4) explains the lower values of ηs for monoglycerides in comparison to those for proteins, at the same surface pressure [36]. For a Na-caseinate–monopalmitin mixed film the ηs value varies greatly with the surface pressure (or surface density) of the mixed monolayer at the interface. In general, the greater the surface pressure (i.e., at the higher surface density), the greater were the values of ηs , as might be expected on the basis of greater intermolecular interactions. That is, as the interactions in the mixed monolayer between monopalmitin molecules— with a LC or solid structure in the monolayer—or between collapsed Na-caseinate are at a maximum, the value of ηs are higher than that with the minimum interactions between monopalmitin molecules—with a LE structure in the monolayer. The lower values of ηs were observed for a LE structure of the monoglyceride (monopalmitin or monoolein) in the mixed monolayer, coinciding with a more homogeneous flow of the monolayer through the canal. In addition, it was observed that over the overall range of surface pressures analyzed, the values of ηs for a Na-caseinate–monoolein mixed monolayer practically did not depend on the surface pressure. For this system film forming components adopt a LE-like structure at every surface pressure (Fig. 8). Moreover, the ηs value is also sensitive to the displacement of film forming components at the interface in spread [29,30] and adsorbed (Figs. 5–7 and 9–11) mixed films. At surface pressures lower than that for Na-caseinate collapse a shear-induced segregation in the topography of monoglycerides and Na-caseinate domains was deduced. At surface pressures higher than that for the Na-caseinate collapse the above mentioned changes in the topography of the mixed monolayer were accompanied by the squeezing of collapsed Na-caseinate domains by monoglycerides [29,30]. Near to the collapse point the mixed film was dominated by the presence of the monoglyceride and the ηs values were practically similar to those for pure monoglyceride monolayers (monopalmitin or monoolein). These results also confirm the extremely sensitive dependence of surface shear characteristics on the immiscibility between film forming components, including the shear-induced segregation in the mixed film. Acknowledgment This research was supported by CICYT through Grant AGL2004-01306/ALI. References [1] E. Dickinson, An Introduction to Food Colloids, Oxford Univ. Press, Oxford, 1992. [2] D.J. McClements, Food Emulsions: Principles, Practice and Techniques, second ed., CRC Press, Boca Raton, FL, 2005. [3] M. Bos, T. Nylander, T. Arnebrant, D.C. Clark, in: G.L. Hasenhuette, R.W. Hartel (Eds.), Food Emulsions and Their Applications, Chapman & Hall, New York, 1997, p. 95.
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