Shear Characteristics, Miscibility, and Topography of Sodium

Biomacromolecules 2004, 5, 2065-2072

2065

Shear Characteristics, Miscibility, and Topography of Sodium Caseinate-Monoglyceride Mixed Films at the Air-Water Interface Juan M. Rodrı´guez Patino* and Cecilio Carrera Sa´ nchez Departamento de Ingenierıá Quı´mica. Facultad de Quı´mica. Universidad de Sevilla. C/. Prof. Garcıá Gonza´ lez 1. 41012-Seville. Spain Received May 24, 2004; Revised Manuscript Received June 24, 2004

In this contribution, we are concerned with the study of structure, topography, and surface rheological characteristics (under shear conditions) of mixed sodium caseinate and monoglycerides (monopalmitin and monoolein) at the air/water interface. Combined surface chemistry (surface film balance and surface shear rheometry) and microscopy (Brewster angle microscopy, BAM) techniques have been applied in this study to mixtures of insoluble lipids and sodium caseinate spread at the air-water interface. At a macroscopic level, sodium caseinate and monoglycerides form an heterogeneous and practically immiscible monolayer at the air-water interface. The images from BAM show segregated protein and monoglyceride domains that have different topography. At surface pressures higher than that for the sodium caseinate collapse, this protein is displaced from the interface by monoglycerides. These results and those derived from interfacial shear rheology (at a macroscopic level) appear to support the idea that immiscibility and heterogeneity of these emulsifiers at the interface have important repercussions on the shear characteristics of the mixed films, with the alternating flow of segregated monoglyceride domains (of low surface shear viscosity, ηs) and protein domains (of high ηs) across the canal. Introduction Interfacial characteristics of spread and adsorbed layers of emulsifiers are important for food colloids (emulsions and foams) because the structural and the mechanical properties of food emulsifiers (proteins and lipids) at fluid interfaces have an influence on the stability and texture of the product.1-6 In addition, interfacial rheology is a very sensitive technique to monitor the interfacial structure, the competitive adsorption, and the magnitude of interactions between different emulsifiers (proteins and lipids) at the interface.3,5,6,7 Interfacial rheology can be defined for both compressional deformation (dilatational rheology) and shearing motion of the interface (shear rheology).7-10 While shear viscosity may contribute appreciably to the long-term stability of dispersions, dilatational rheology plays an important role in shortterm stability.7,11,12 The shear characteristics of the interfacial film are governed by the composition and structure of the adsorbed material. In addition, surface shear viscosity is a very sensitive technique to analyze the competitive adsorption of protein and water-soluble emulsifier at the air-water interface.7-13 In a previous paper,14 we have confirmed the sensitivity of the interfacial shear viscosity (ηs) to the behavior of emulsifier (protein or monoglyceride) films at the air-water interface, as a function of surface pressure. In fact, ηs was higher for proteins than for monoglycerides. Moreover, the ηs value is also sensitive to the monolayer structure, as was observed for monoglycerides with a rich * To whom correspondence should be addressed. Tel: +34 95 4556446. Fax: +34 95 4557134. E-mail: [email protected].

structural polymorphism (i.e., monopalmitin) and to the capacity of the protein to form physical (such as for β-casein) or covalent-disulfide (such as for κ-casein, caseinate, and WPI) interfacial gels, with the higher values of ηs for the latter. The aim of this work, was to analyze the structural, topographical, and shear characteristics of caseinate-monoglyceride mixed monolayers at the air-water interface. The differences observed between monoglycerides and proteins in shear rheology make it possible to use this technique to analyze the interfacial characteristics (structure, interactions, miscibility, squeezing out phenomena, etc.) of proteinmonoglyceride mixed films at the air-water interface. This work is center in the use of a real food protein (caseinate), to see if the shear characteristics of monoglyceride-caseinate mixed films depend on the proper nature of the film forming components, as observed for mixed films with a model protein (β-casein)15 forming part of caseinate. Materials and Methods Materials. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90) and 1-mono(cis-9octadecanoyl)glycerol (monoolein, RYLO MG 19) were supplied by Danisco Ingredients with over 95-98% purity. 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. Caseinate (a mixture of ≈38% β-casein, ≈39%

10.1021/bm049694k CCC: $27.50 © 2004 American Chemical Society Published on Web 08/04/2004

2066

Biomacromolecules, Vol. 5, No. 5, 2004

Rodrı´guez Patino and Sa´ nchez

Rs1-casein, ≈12% κ-casein, and ≈11% Rs2-casein) was supplied and purified from bulk milk from Unilever Research (Colworth, U.K.). Samples for interfacial characteristics of caseinate films were prepared using Milli-Q ultrapure water at pH 7. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution (Sigma, >99.5%) called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) was used to adjust the subphase at pH 7. Ionic strength was 0.05 M in all the experiments. Surface Shear Rheometry. To study the shear characteristics of spread 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 elsewhere14 was used in this work. 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. 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-1, recorded as a variation of the area associated with the movement of the barrier as a function of time). 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 tubes:16 ηs ) ((π2 - π1)Wc3/12QL)

(1)

The monolayer is allowed to flow through a canal without compression 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 value of ∆π ) π2 - π1 was deduced during the flow of the monolayer. The advantages of our instrument in relation to conventional devices have been analyzed previously.14 Mixtures of a particular mass fraction of monoglyceride in the mixture of X ) 0.5 were studied, as a function of the surface pressure and monolayer composition. Aliquots of aqueous solutions of caseinate (1.405 × 10-4-1.490 × 10-4 mg/µL) at pH 7 were spread on the interface by means of a micrometric syringe. 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 caseinate necessary to form the mixed film should be spread before the lipid. Afterward, a monoglyceride solution in a hexane:ethanol mixture (1.35 × 10-4-1.55 × 10-4 mg/µL) was spread at different points on the caseinate film. To allow for spreading and caseinate-monoglyceride interactions, 30 min were 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, 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. As for pure components,14 an optimization of Wc and Q is necessary in order to obtain the best flow conditions of the mixed monolayer through the canal. During these experiments, practically all of the monolayer (c.a. 90%) flows through the canal, thus the data presented in this work represent the overall behavior of the monolayer under 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. Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on the same modified Wilhelmy-type film balance, as described elsewhere.17,18 The measurement of the π-A isotherm was performed before the experiments of surface shear rheology. 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 mean deviation for four independent π-A isotherms was within ( 0.1 mN/m for surface pressure and ( 0.125 × 10-3 m2 per mg for area. Brewster Angle Microscopy. For microscopic observation of the monolayer structure, a Brewster angle microscope, BAM 2 plus (NFT, Germany) was used as described elsewhere.19,20 The BAM was located at the exit of the canal. In all BAM images under the flow of the monolayer, the imaging conditions were adjusted to optimize image quality and not to enable quantitative measurement reflectance (I). Thus, the reflectance of each image was quantified by the gray level in a direction diagonal to the interface. The gray level as a function of distance gives complementary information about the heterogeneity of each image (i.e., the relative film thickness is proportional to the square root of the gray level for the proper monolayer) along the diagonal, but cannot be used to compare the BAM images with each other at different experimental conditions. Some BAM images under shear conditions are accompanied by the evolution of the gray level with the distance in the direction of the diagonal in the image, which is indicated by means of a bar. The application of different devices (Wilhelmy-type film balance, Brewster angle microscopy, and surface shear rheology) to the same monolayer makes possible the characterization of the monolayer under shear conditions and at equilibrium, at a macroscopic and at a microscopic scale. Results Structural and topographical characteristics of caseinate-monoglyceride mixed monolayers at the air-water interface. Caseinate-Monopalmitin Mixed Monolayers. Figure 1A shows the π-A isotherms obtained in the modified Wilhelmy-type trough for caseinate, monopalmitin, and a caseinate-monopalmitin mixed monolayer at pH 7 and at 20 °C. These results are in good agreement with those obtained for

Sodium Caseinate-Monoglyceride Mixed Films

Figure 1. (A) Surface pressure(π)-area (A) isotherm of (;) caseinate, (- - -) monopalmitin, and (O) caseinate-monopalmitin mixed film at XMP ) 0.5. (B) Relative reflectivity as a function of surface pressure for (;) caseinate, (- - -) monopalmitin, and (O) caseinate-monopalmitin mixed film at XMP ) 0.5. Temperature 20 °C. pH 7. The equilibrium surface pressures of monopalmitin (πe MP) and caseinate (πe CS) are indicated by means of arrows.

the same systems in a Langmuir-type trough.21 Briefly, the results of π-A isotherms confirm that caseinate have a liquid-expanded-like structure at every surface pressure. At surface pressures near to the equilibrium surface pressure (πe ≈ 29 mN/m), which is indicated in Figure 1A by means of an arrow, caseinate collapses and amino acid segments are extended into the underlying aqueous solution. The equilibrium surface pressure (πe) is the maximum surface pressure to which a spread monolayer may be compressed without the possibility of monolayer collapse. Different structures can be deduced for monopalmitin monolayer as a function of surface pressure (Figure 1A). The liquidexpanded phase (LE) (at π < 5 mN/m), an intermediate region at the broad plateau due to a degenerated first-order phase transition between liquid-condensed (LC) and liquidexpanded structures (at 5 < π < 30 mN/m), the liquidcondensed structure (at π > 30 mN/m), and finally, the solid (S) structure near to the monolayer collapse at a surface pressure of about 53.1 mN/m were observed. Caseinate-monopalmitin (Figure 1A), at a monopalmitin mass fraction of XMP ) 0.5 (as an example), and at surface pressures lower than that for caseinate collapse (πC = 2829 mN/m), adopts a structural polymorphism, as for pure components. In this region a mixed monolayer of monoglyceride and caseinate may exist. However, at surface pressures higher than that for caseinate collapse, the π-A isotherm for the mixed monolayer at XMP ) 0.5 was parallel to that of monopalmitin, which demonstrates that the arrangement of the monopalmitin hydrocarbon chain in mixed monolayers is practically the same.21 That is, at higher surface pressures, collapsed caseinate residues may be displaced from the interface by monopalmitin molecules.

Biomacromolecules, Vol. 5, No. 5, 2004 2067

Moreover, the mixed film collapses at a surface pressure similar than that for monopalmitin, which is an indication of the immiscibility between film forming components at the highest surface pressures. Results of BAM, in particular the relative reflectivity as a function of surface pressure (Figure 1B) and topography (data not shown) obtained with caseinate, monopalmitin, and a caseinate-monopalmitin mixed film at XMP ) 0.5 in the absence of shear clearly show at a microscopic level the same structural characteristics as those deduced from the π-A isotherms. Briefly, at π < 20 mN/m, the I-π plots were the same as for pure components. In this region, LC domains of monopalmitin existed in an environment of homogeneous LE monopalmitin and LE-like caseinate domains, during the compression. The main difference was observed in the region near and after the caseinate collapse (22 mN/m < π < 42 mN/m), because the relative reflectivity of the mixed film was lower than for pure caseinate. This suggests that monopalmitin is able to displace caseinate residues from the interface toward a sublayer beneath the monopalmitin monolayer.21 The peaks in the relative reflectivity are due to the fact that in this region domains of pure caseinate (with high I) and monopalmitin (with low I) passed alternatively through the spot were this measurement was performed. Near to the collapse of the mixed films (at 42 mN/m < π < 52 mN/m), the peaks in relative reflectivity practically disappeared and the I values decreased to those typical for a pure monopalmitin monolayer. This suggests that in this region monopalmitin predominates at the interface. Caseinate-Monoolein Mixed Monolayers. The π-A isotherms for monoolein, caseinate, and a caseinate-monoolein mixed monolayer at a mass fraction of monoolein in the mixture of XMO ) 0.5 (as an example) are shown in Figure 2A. As expected,19,22 monoolein monolayers had a liquid-expanded structure under all experimental conditions. The collapse of monoolein monolayer occurred at a surface pressure close to the equilibrium surface pressure (πc = 45.7 mN/m). The monolayer structure in a caseinate-monoolein mixed monolayer at XMO ) 0.5 was liquid-expanded-like, as for pure monolayer components. At surface pressures higher than that for caseinate collapse (πC = 28-29 mN/ m), the π-A isotherms for the mixed monolayer were parallel to that of pure monoolein. These results suggest that the composition of the mixed monolayer was very dependent on the surface pressure. At surface pressures lower than that for caseinate collapse a mixed monolayer of monoolein and caseinate could exist, caseinate molecules adopting a LElike structure in the isotropic LE monoolein monolayer. However, at surface pressures higher than that for caseinate collapse, the mixed monolayer was practically dominated by monoolein molecules. That is, at higher surface pressures, collapsed caseinate residues may be displaced from the interface by monoolein molecules.3,21 The I values (Figure 2B) for a caseinate-monoolein mixed film at XMO ) 0.5 were similar to that for caseinate or monoolein as the surface pressures are lower than 10 mN/ m. At higher surface pressures, the I-π plot for the mixed film approached that for pure monoolein monolayer, denoting the displacement of caseinate by monoolein from the air-

2068


Figure 2. (A) Surface pressure(π)-area (A) isotherm of (;) caseinate, (- - -) monoolein, and (O) caseinate-monoolein mixed film at XMO ) 0.5. (B) Relative reflectivity as a function of surface pressure for (;) caseinate, (- - -) monoolein and (O) caseinate-monoolein mixed film at XMO ) 0.5. Temperature 20 °C. pH 7. The equilibrium surface pressures of monoolein (πe MO) and caseinate (πe CS) are indicated by means of arrows.

water interface. Finally, the mixed film collapsed at a surface pressure similar than that for monoolein. These results suggests that caseinate and monoolein are practically immiscible with regions of monoolein or caseinate alternating at the air-water interface, depending on the surface pressure. In summary, at surface pressures lower than that for caseinate collapse, caseinate and monoglyceride (monopalmitin or monoolein) domains are present at the interface in the mixed film but with few interactions between them. However, at surface pressures higher than that for caseinate collapse, the structural and topographical characteristics of the mixed film were dominated by the presence of the monoglyceride at the interface. Effect of Surface Pressure on Surface Shear Characteristics of Caseinate-Monoglyceride Mixed Monolayers at the Air-Water Interface. Caseinate-Monopalmitin Mixed Monolayers. The surface shear characteristics of a caseinate-monopalmitin mixed monolayer at XMP ) 0.5, at surface pressures lower than that for caseinate collapse, at 10 mN/m (Figure 3B) and at 20 mN/m (Figure 4B), and at different operational conditions, at a constant width of the canal (Wc ) 1.5 mm) and different flows of the monolayer (3.75 × 10-6 to 1.25 × 10-5 m2 s-1), have been determined. As can be seen, a steady state ηs value was not reached during the flow of the mixed film as for pure monopalmitin and caseinate monolayers.14 In addition, the fluctuations in ηs values were more significant and extended in time than during the flow of pure components.14 The peaks had ηs values lower than that for a pure caseinate monolayer, at 10 mN/m, ηs ) 0.007-0.008 mPa s m and at 15 mN/m, ηs ) 0.02-0.025 mPa s m, but the lower ηs values were similar than those for a pure mono-


Figure 3. (A1, A2, and A3) Visualization of caseinate-monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of caseinate and monopalmitin at XMP ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal, Q (m2 s-1): (;) 3.75 × 10-6, and (‚‚‚) 1.25 × 10-5. The surface shear viscosity for pure monopalmitin (checked pattern) monolayer is included as reference. Surface pressure 10 mN/m. Temperature 20 °C. pH 7.

Figure 4. (A1 and A2) Visualization of caseinate-monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of caseinate and monopalmitin at XMP ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal Q (m2 s-1): (Timea, ;) 6.25 × 10-6 and (Timeb, ‚‚‚) 1.25 × 10-5. The surface shear viscosity for pure monopalmitin (checked pattern) monolayer is included as reference. Surface pressure 20 mN/m. Temperature 20 °C. pH 7.

palmitin monolayer, which is included in the figures as reference, denoting the importance of monopalmitin on the shear characteristics of the mixed film. The fluctuations in the ηs value may be due to friction between LC monopalmitin domains and/or between segregated regions of monopalmitin and caseinate, both of these phenomena denoting the heterogeneity of the mixed monolayer. The region of LC domains of monopalmitin produced a reduction in the friction


Figure 5. (A1, A2, and A3) Visualization of caseinate-monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film caseinate and monopalmitin at XMP ) 0.5, at Wc ) 2.0 mm and at Q ) 3.75 × 10-6 m2 s-1. The surface shear viscosity for pure monopalmitin (checked pattern) monolayer is included as reference. Surface pressure 30 mN/ m. Temperature 20 °C. pH 7.

(i.e., lower ηs values) in the mixed monolayer in relation to that for a pure caseinate monolayer. The results of BAM corroborate these hypotheses. In fact, at 10 mN/m images A2 and A3 (Figure 3) clearly show the coexistence between circular LC monopalmitin domains with different sizes and homogeneous liquid-expanded (LE) domains of monopalmitin and caseinate (image A1). Unfortunately, by BAM image we are unable to distinguish between liquid-expanded monopalmitin domains and caseinate in the continuous homogeneous phase (image A1). However, a heterogeneity in the structure of the mixed monolayer at a microscopic level was observed by the evolution of the film thickness along the BAM images (data not shown), confirming the coexistence of LE monopalmitin domains and caseinate in the mixed film. The LC monopalmitin domains are more numerous and grow in size at the expense of the LE ones as the surface pressure increased to 20 mN/m (Figure 4, images A1 and A2). There were significant differences in ηs during the flow of the mixed film through the canal at 30 mN/m, near to the collapse of caseinate (Figure 5). At the beginning of the flow, the ηs values for the mixed film were similar to that for a pure monopalmitin monolayer. However, at long-term flow, great fluctuations in ηs values were also observed. These fluctuations in ηs values were more significant and extended in time than during the first period and may be associated with the flow of large regions of collapsed caseinate through the canal instead of film heterogeneities associated with the presence of LC monopalmitin. In fact, at 30 mN/m, image A1 (Figure 5) shows a characteristic squeezing out phenomenon with many LC domains of monopalmitin (the circulardeformed dark regions) floating over a sublayer of collapsed caseinate molecules (characterized by a completely white image with a reflectivity similar to that for pure caseinate). However, we also observed regions of segregated monopalmitin and caseinate domains (Figure 5, image A2) and regions of collapse caseinate (Figure 5, image A3) during


Figure 6. (A1 and A2) Visualization of caseinate-monopalmitin mixed monolayers at XMP ) 0.5 by BAM. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of caseinate and monopalmitin at XMP ) 0.5 and at (;) 40 mN/m and (‚‚‚‚‚) at the monolayer collapse. The surface shear viscosity for a pure monopalmitin monolayer at 40 mN/m (striped pattern) and at the collapse point (checked pattern) is included as reference. Width of canal Wc ) 2.0 mm. Monolayer flow through the canal, Q ) 3.75 × 10-6 m2 s-1. Temperature 20 °C. pH 7.

the flow of the mixed monolayer. These phenomena were an indication that a segregation in the mixed monolayer was produced during the flow with LC monopalmitin deformed domains due to the shear, with low ηs values, alternating with caseinate-monopalmitin mixed monolayer depleted in monopalmitin content and even with regions of collapse caseinate, with high ηs values. There are significant differences in the shear characteristics of the mixed film after the caseinate collapse, at 40 mN/m and at the collapse of the mixed monolayer (Figure 6B). In fact, at these surface pressures, the ηs values of the mixed film were similar to those for a pure monopalmitin monolayer, with the absence of fluctuations in the ηs values. These results suggest that in this region the mixed monolayer was dominated by the presence of monopalmitin at the interface. In fact, at the higher surface pressures (at the collapse point of the mixed film), the topography was dominated for monopalmitin with a closed-packed LC structure (Figure 6, image A1) or collapsed monopalmitin film with the presence of some fractures of collapsed caseinate (which were not representative of the overall topography of the mixed film) aligned in the direction of the flow (Figure 6, image A2) as typical features of the mixed film. This topography under shear conditions is also in good agreement with the absence of reflectivity peaks in the I-π plot and with the decrease in relative reflectivity to a value close to that for a pure monopalmitin monolayer (Figure 1B). Caseinate-Monoolein Mixed Monolayers. The time evolution of ηs during the flow of a caseinate-monoolein mixed monolayer at XMO ) 0.5 through the canal is shown in Figures 7-10 as a function of surface pressure. As for caseinate-monopalmitin monolayers, fluctuations in ηs values were observed for a wide range of surface pressures lower and higher than that to the collapse of caseinate. The lower

2070


Figure 7. (A1 and A2) Visualization of caseinate-monoolein mixed monolayers at XMO ) 0.5 by BAM. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of caseinate and monoolein at XMO ) 0.5. Width of canal Wc ) 1.5 mm. Monolayer flow through the canal, Q (m2 s-1): (; and ‚‚‚) 3.75 × 10-6. The surface shear viscosity for a pure monoolein (checked pattern) monolayer is included as reference. Surface pressure 10 mN/m. Temperature 20 °C. pH 7.

Figure 8. Surface shear viscosity for a mixed film of caseinate and monoolein at XMO ) 0.5, at Q ) 3.75 × 10-6 m2 s-1, and at (;) Wc ) 2.0 mm. The surface shear viscosity for pure monoolein (checked pattern) monolayer is included as reference. Surface pressure 20 mN/m. Temperature 20 °C. pH 7.

values of ηs were very similar to those for a pure monoolein monolayer, which indicates that the shear characteristics of the mixed monolayer depends on the presence of monoolein at the interface. However, numerous peaks in ηs were observed and extended in time, which may be associated with the flow of large regions of caseinate through the canal. The peaks had ηs values lower than that for a pure caseinate monolayer, caseinate-monoolein mixed film at 10 mN/m is an exception, indicating that the monolayer flow was dominated by the presence of caseinate in these regions. However, the LE domains of monoolein produced a reduction in the friction in the mixed monolayer in relation to that for a pure caseinate monolayer, specially at higher surface pressures (π g 20 mN/m). The results of BAM corroborate these hypotheses. In fact, at surface pressures lower than the equilibrium surface pressure of caseinate (πe ≈ 29 mN/m), at 10 (Figure 7) and 20 mN/m (Figure 8), the evolution of film thickness (data not shown) along images A1 and A2 present regions of monoolein and caseinate, both of then with a homogeneous


Figure 9. (A1 and A2) Visualization of caseinate-monoolein mixed monolayers at XMO ) 0.5 by BAM. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. The evolution of the gray level along the interface is included for images A1 and A2. The segregation between collapse caseinate (CS) and monoolein (MO) is indicated in image A2. (B) Surface shear viscosity for a mixed film of caseinate and monoolein at XMO ) 0.5, at (Timea, ;) Q ) 3.75 × 10-6 m2 s-1 and at Wc ) 2.0 mm and at (Timeb, ‚‚‚) Q ) 1.25 × 10-5 m2 s-1 and at Wc ) 1.5 mm. The surface shear viscosity for pure monoolein (checked pattern) monolayer is included as reference. Surface pressure 30 mN/m. Temperature 20 °C. pH 7.

liquid-expanded-like structure, during the flow of the mixed film, specially at higher surface pressures (π g 20 mN/m). The topography of a caseinate-monoolein mixed monolayer at 20 mN/m (data not shown) was similar to that at 10 mN/m (Figure 7). At higher surface pressures, (at 30 mN/m), images A1 and A2 (Figure 9) show regions of monoolein with a LE structure (image A1) and regions of collapsed caseinate (image A2) during the flow of the mixed film. The presence of collapsed caseinate domains was corroborated by the saturation of the camera shown in image A2 (bright image). As a consequence of this topography the monolayer thickness (gray level) presented significant fluctuations along the interface. These phenomena were an indication that a shearinduced segregation was produced in the mixed monolayer, with the flow of homogeneous LE monoolein domains and homogeneous LE-like caseinate or collapsed caseinate domains in the range of surface pressures between 10 and 30 mN/m. As expected,21 after the caseinate collapse, at 40 mN/m and at the collapse point of the mixed film (Figure 10), the ηs values of caseinate-monoolein mixed monolayers were similar to those for a pure monoolein monolayer. These results suggest that in this region the mixed monolayer was dominated by the presence of monoolein at the interface. In fact, at the higher surface pressures, the topography was dominated for monoolein with a LE structure at 40 mN/m



Figure 11. The effect of surface pressure on surface shear viscosity for (b) β-casein, (4) k-casein, (O) caseinate, (]) monopalmitin, and (3) monoolein monolayers at the air-water interface. Temperature 20 °C. pH 7. Figure 10. (A1, A2, and A3) Visualization of caseinate-monoolein mixed monolayers at XMO ) 0.5 by BAM a the collapse point. The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm. (B) Surface shear viscosity for a mixed film of caseinate and monoolein at XMO ) 0.5, at Wc ) 1.5 mm, and at (Timea, ;) Q ) 3.75 × 10-6 m2 s-1 and at (Timeb, ‚‚‚) at Q ) 1.25 × 10-5 m2 s-1. The surface shear viscosity for a pure monoolein monolayer at 40 mN/m is included as reference. Surface pressure (s) 40 mN/m and (‚‚‚) collapse point. Temperature 20 °C. pH 7.

(image not shown) or collapsed monoolein film (Figure 10, images A1, A2, and A3). This topography under shear conditions is also in good agreement with the absence of reflectivity peaks in the I-π plot and with the decrease in relative reflectivity to a value similar to that for a pure monoolein monolayer (Figure 2B). However, we have also observed the coexistence of collapsed domains of monoolein (Figure 10, image A1) and caseinate (Figure 10, image A2) and islands of collapsed caseinate Figure 10, (image A3) in minor regions of the interface, which explain the peaks of ηs in Figure 10B. Discussion In this contribution, we have analyzed the structure, topography, surface rheological characteristics (under shear conditions), and squeezing out phenomena of mixed caseinate and monoglycerides at the air/water interface. Combined surface chemistry (surface film balance and surface shear rheometry) and microscopy (BAM) techniques have been applied in this study to mixtures of insoluble lipids (monopalmitin and monoolein) and sodium caseinate spread at the air-water interface. To study the shear characteristics of spread films, a homemade canal was used. The shear characteristics of pure and caseinate-monoglyceride mixed films were sensitive to the composition of the mixed film and the surface pressure. That is, the ηs values (Figures 3-10) depend on the structural characteristics of the monolayer (Figures 1 and 2). We have observed that, at the same surface pressure, the ηs values were higher for caseinate than for monoglycerides (monopalmitin and monoolein), with the lower ηs values for monoolein (Figure 11). This is due to lateral interactions between the protein due to hydrogen bonding, hydrophobic and covalent bonding, and/or electrostatic interactions.14 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.3,5,23 Therefore, differences between ηs values for various proteins (caseinate, β-casein, κ-casein, etc.) are rather great and reflect differences, among others, in the protein structure and the potential for the formation of disulfide cross-linking and the formation of interfacial aggregates of significant sizes. The values of ηs were higher for proteins that form interfacial gels by cross-linking of disulfide residues and the formation of interfacial aggregates of significant size (κ-casein and caseinate, in this order) as compared with β-casein that only can form physical gels stabilized by intermolecular hydrogen bonds.14 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 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, explains the lower values of ηs in comparison to those for proteins, at the same surface pressure. The differences observed between monoglyceride and caseinate monolayers in shear rheology justify the use of this technique to analyze the interfacial characteristics (structure, interactions, miscibility, squeezing out phenomena, etc.) of caseinate-monoglyceride mixed films at the air-water interface and thus enable a global consistency check on the same monolayer. For a 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 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 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 caseinate-monoolein mixed monolayer

2072


practically did not depend on the surface pressure. 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. Moreover, the ηs value is also sensitive to the displacement of film forming components at the interface. At surface pressures lower than that for caseinate collapse, a shearinduced segregation in the topography of monoglycerides and caseinate domains was observed. At surface pressures higher than that for the caseinate collapse, the abovementioned changes in the topography of the mixed monolayer were accompanied by the squeezing of collapsed caseinate domains by monoglycerides. 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). The heterogeneity of the mixed films (help by the effect of the shear) justifies the fact that we are unable to obtain a representative steady-state ηs value for the proteinmonoglyceride mixed film, as observed for pure components.14 Different proteins and monoglycerides show different interfacial topography, confirming the importance of protein and emulsifier structure in determining the mechanism of interfacial interactions.3,23,24 Comparing the data presented in this paper to those for β-casein-monoglyceride mixed films,15 we conclude that the structural and topographical characteristics, the shear-induced segregation, and squeezing out phenomena of mixed proteins and monoglycerides at the air/water interface observed with protein (either a model β-casein or a real caseinate protein) and monoglyceride mixed films appear to be generic. The reasons for these behaviors must be associated with the immiscibility between protein and monoglyceride at the air-water interface, to the protein displacement by the monoglyceride at surface pressures higher than that for the protein collapse, and to the shear-induced segregation in the mixed films. These phenomena have significant repercussions on surface shear properties. That is, the surface shear characteristics reflect the complex phenomena that take place in protein-monoglyceride mixed films under flow conditions. In fact, at surface pressures lower than those for protein collapse (β-casein or caseinate), the values of ηs peaks, associated to the flow of the protein through the canal, were higher for caseinatemonoglyceride than for β-casein-monoglyceride mixed films, as observed for pure protein monolayers (Figure 11). However, at surface pressures higher than those for protein collapse (β-casein or caseinate), and especially near to the collapse of the mixed films, the values of ηs for caseinateand β-casein-monoglyceride mixed films were practically the same. This fact is in agreement with the opinion that at high surface pressures the mixed films are dominated by the presence of the monoglyceride (monopalmitin or monoolein) in the mixed film. On the other hand, this work has demonstrated that there exist differences in caseinate displacement by monoglycerides (monopalmitin or monoolein) in the absence or in the presence of shear. In the absence of shear, we have observed


that the lower surface activity of monoolein justified the idea that this monoglyceride has a lower capacity than monopalmitin for protein displacement,3 a phenomenon that was not observed in this work under shear conditions. In fact, under shear conditions, the additional segregation of the monolayer facilitates the displacement of the caseinate by the monoglyceride, especially for caseinate-monoolein mixed films. That is, the shear may induce an additional heterogeneity in the structure of the mixed monolayer at a microscopic level as observed by the BAM images. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers25-27 and may be an additional cause of the ηs peaks observed during the flow of the monolayer. Clearly, caseinate displacement by monoolein was facilitated by the monolayer flow. Acknowledgment. The authors acknowledge the support of CICYT thought the grant AGL2001-3843-C02-01. References and Notes (1) Dickinson, E. An Introduction to Food Colloids; Oxford University Press: Oxford, 1992. (2) McClements, D. J. Food Emulsions: Principles, Practice and Techniques; CRC Press: Boca Raton, FL, 1999. (3) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, Ma. R.; Carrera, C. Curr. Opin. Colloid Interface Sci. 2003, 8, 387. (4) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsions and their Applications; Hasenhuette, G. L., Hartel, R. W., Eds.; Chapman and Hall: New York, 1997; p 95. (5) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (6) Dickinson, E. Colloids Surf. B: Biointerfaces 2001, 20, 197. (7) Bos, M. A.; van Vliet, T. AdV. Colloids Interface Sci. 2001, 91, 437. (8) Murray, B. S. Curr. Opinion Colloid Interface Sci. 2002, 7, 426. (9) Murray, B. S.; Dickinson, E. Food Sci. Technol. Inst. 1996, 2, 131. (10) Murray, B. S. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 179. (11) Benjamins, J.; Lucassen-Reynders, E. H. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 341. (12) Lucassen-Reynders, E. H.; Benjamins, J. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, 1999; p 195. (13) Dickinson, E. Colloids Surf. B: Biointerfaces 1999, 15, 161. (14) Rodrı´guez Patino, J. M.; Carrera, C. Langmuir 2004, 20, 4530. (15) Rodrı´guez Patino, J. M.; Carrera, C. Langmuir 2004, 20, 6327. (16) Harkins, W. D.; Kirkwood, J. G. Nature (London) 1938, 141, 38. (17) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, Ma. R.; Cejudo, M. Langmuir 2001, 17, 4003. (18) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez, Ma. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (19) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez, Ma. R. Langmuir 1999, 15, 2484. (20) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez, Ma. R. Food Hydrocolloids 1999, 13, 401. (21) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez, Ma. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 240, 113. (22) Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M.; Carrera, C. Cejudo, M.; Navarro, J. M. Chem. Eng. Com. 2003, 190, 15. (23) Gunning, P. A.; Mackie, A. R.; Gunning, A. P.; Woodward, N. C.; Wilde, P. J.; Morris, V. J. Biomacromolecules 2004, 5, 984. (24) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, Ma. R.; Carrera, C.; Cejudo, M. Langmuir 2001, 17, 7545. (25) Murayama, T.; Fuller, G.; Frank, C.; Robertson, C. Science 1996, 274, 233. (26) Murayama, T.; Lauger, J.; Fuller, G.; Frank, C.; Robertson, C. Langmuir 1998, 14, 1836. (27) Igne´s-Mullol, J.; Schwartz, D. K. Nature 2001, 410, 348.

BM049694K

Shear Characteristics, Miscibility, and Topography of Sodium

Recommend Documents