Structural and Shear Characteristics of Adsorbed Î²-Casein and

1886

Ind. Eng. Chem. Res. 2006, 45, 1886-1895

MATERIALS AND INTERFACES Structural and Shear Characteristics of Adsorbed β-Casein and Monoglyceride Mixed Monolayers at the Air/Water Interface Juan M. Rodrı´guez Patino,* Marta Cejudo Ferna´ ndez, Cecilio Carrera Sa´ nchez, and Ma. Rosario Rodrı´guez Nin˜ o Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, UniVersidad de SeVilla, C/. Prof. Garcıá Gonza´ lez 1, 41012 SeVille, Spain

In this work, we have analyzed the structural (structure, topography, reflectivity, miscibility, and interactions) and surface shear characteristics of an adsorbed β-casein and monoglyceride (monopalmitin and monoolein) mixed films at the air/water interface. Different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial shear rheology) have been utilized. The structural, topographical, and shear characteristics of the mixed films are dependent on the surface pressure and the composition of the mixed film. The surface shear viscosity (ηs) varies greatly with the surface pressure. Generally, the greater the surface pressure, the greater the values of ηs. At higher surface pressures, collapsed β-casein residues may be displaced from the interface by monoglyceride molecules with important repercussions on the shear characteristics of the mixed films. Shear-induced change in the topography of monoglyceride and β-casein domains, on one hand, and segregation between domains of the film forming components, on the other hand, were also observed. The displacement of the β-casein by the monoglycerides is facilitated under shear conditions, especially for β-casein-monoolein mixed films. Introduction The optimum use of emulsifiers in food dispersions (emulsions and foams) is dependent on knowledge of their interfacial physicochemical characteristicsssurface activity, structure, miscibility, superficial viscosity, etc.sand the kinetics of the film formation at fluid interfaces.1 Emulsifiers used in commercial food formulations typically consist of a mixture of surface-active derivatives, because they can be produced at a relatively lower cost than pure emulsifiers. In addition, in many emulsifier applications, mixtures of different emulsifiers (mainly polar lipids and proteins) often exhibit properties superior to those of the individual emulsifier alone, because of synergistic interactions between emulsifier molecules. The stability and mechanical properties of dispersed food systems (emulsions and foams) are dependent on the way in which the constituent emulsifiers (lipids and proteins) adsorb and interact at fluid interfaces. Interactions between molecules of emulsifiers could affect not only the film thermodynamic properties (structure, topography, miscibility, etc.), but also dynamic phenomena in mixed films.2 Information about these phenomena would be very helpful in the prediction of optimized formulations for food foams and emulsions, based on microscopic and nanoscopic analysis. So far, there are important experimental results available on the structural characteristics of mixed emulsifiers spread at the air/water interface.2-5 However, as far as we know, there have been few studies of the structural characteristics of adsorbed films at the air/water interface,6-9 although, in practice, mixtures of these emulsifiers usually are used to achieve an optimal effect in food formula* To whom all correspondence should be addressed. Tel: +34 95 4556446. Fax: +34 95 4556447. E-mail: [email protected].

tions.3,4 In fact, in contrast with spread monolayers, adsorbed monolayers are more interesting, from a technological point of view. The monolayer technique has been used successfully to study the properties of mixed emulsifiers spread at the air/water interface.2 The study of the dynamic behavior of interfacial films can be described by interfacial rheology. 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.2,5,10-12 In addition, interfacial rheology is a very sensitive technique that is used 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.2 Interfacial rheology can be defined13,14 for both compressional deformation (dilatational rheology) and shearing motion of the interface (shear rheology). While shear viscosity may contribute appreciably to the long-term stability of dispersions, dilatational rheology has an important role in short-term stability.11,15,16 Moreover, the ability of the protein to resist displacement by emulsifiers is closely linked to surface dilatational rheology, whereas the precise form of the displacement is considered to be more closely related to surface shear behavior.4,12,17,18 The objective of this contribution is to analyze the structural (structure, topography, miscibility, and interactions) and surface shear characteristics of mixed monolayers formed by an adsorbed milk protein (β-casein) and a spread monoglyceride (monopalmitin or monoolein). These experiments mimic the behavior of emulsifiers in food emulsions in which an oil-soluble lipid (monopalmitin or monoolein) interacts at the interface with

10.1021/ie051092+ CCC: $33.50 © 2006 American Chemical Society Published on Web 02/21/2006

Ind. Eng. Chem. Res., Vol. 45, No. 6, 2006 1887

a protein film previously adsorbed from the aqueous bulk phase. Thus, these results would be of direct utility for food foams formulations and, assuming the validity of extrapolating from the air/water interface to the oil/water interface, also for emulsions.19 Experimental Section 1. Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90) and 1-mono (cis-9-octadecanoyl) glycerol (monoolein, RYLO MG 19) were supplied by Danisco Ingredients (Brabran, Denmark) with >95%-98% purity. β-Casein (>99% purity) was supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Samples for interfacial characteristics of β-casein adsorbed films were prepared using Milli-Q ultrapure water and were buffered at pH 7. To form the mixed surface film on a previously adsorbed β-casein 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 ((CH2OH)3CNH2/(CH2OH)3CNH3Cl, Sigma, >99.5% purity) was used to achieve pH 7. Ionic strength was 0.05 M in all the experiments. 2. Surface Film Balance. Measurements of the surface pressure-area (π-A) isotherms of β-casein-monoglyceride mixed films at the air/water interface were performed on a fully automated Wilhelmy-type film balance, using a maximum area between the two barriers of 51 cm × 15 cm, as described previously.20 Before each measurement, the film balance was calibrated at 20 °C. For β-casein adsorbed films from water, a β-casein solution at a concentration of 5 × 10-6-7.5 × 10-6% (wt/wt) was left in the trough, and time was allowed for protein adsorption at the interface. These protein concentrations were selected from previous data of the adsorption isotherm.21 At this protein 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. At this point, the monoglyceride was spread at different points on the β-casein film. In preliminary experiments, the spreading solvent was not observed to have any effect on the adsorbed protein monolayer. For pure adsorbed protein films, the maximum protein concentration in the bulk phase should be selected to obtain a reasonable rate of adsorption at the interface, but maintaining the equilibrium surface pressure at zero.7 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 to obtain the complete π-A isotherm of the mixed film, from the moreexpanded monolayer (at the higher areas) to the more-condensed monolayer, at the collapse point (at the lower areas). Mixtures of particular mass ratiossexpressed as the mass fraction of monopalmitin (XMP) or monoolein (XMO) in the mixtureswere studied. The compression rate was 49.5 cm2/min, which is the highest value for which isotherms were determined to be reproducible in preliminary experiments. The π-A isotherm was measured five times. The reproducibility of the results was better than (0.5 mN/m for the surface pressure and (0.05 m2/mg for the area. 3. Brewster Angle Microscopy. Brewster angle microscopy (BAM), using commercial equipment (BAM2, manufactured by NFT (Go¨ttingen, Germany)), was used to study the topography of the monolayer. The BAM system was positioned over the

film balance. Further characteristics of the device and operational conditions have been described elsewhere.22,23 The surface pressure measurements, area, and imaging at specific surface pressures were conducted simultaneously by means of a device that was connected between the film balance and the BAM system. The imaging conditions were adjusted to optimize image quality by increasing the shutter speed as the surface pressure or the protein content increased. 4. Surface Shear Rheometry. To study the shear characteristics of adsorbed films, a homemade canal viscometer was used in this work. It is an analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past each other and is described elsewhere.24 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 composed 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 (L ) 30 mm), and at a constant flow Q (given in terms of m2/s; Q is 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 a simplified equation (eq 1), which is analogous to the Poiseuille equation for the flow of liquids through capillary tubes.25

ηs )

(π2 - π1)Wc3 12QL

(1)

The monolayer is allowed to flow through the 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. Mixtures of a particular mass fraction of monoglyceride in the mixture ranging from X ) 0 to X ) 0.5 were studied, as a function of the surface pressure and monolayer composition. As for pure components,24 an optimization of Wc and Q is necessary to obtain the best flow conditions of the mixed monolayer through the canal. During these experiments, practically all the monolayer (∼90%) flows through the canal; thus, the data presented in this work represent the overall behavior of the monolayer under shear conditions. These data were obtained after a minimum of three measurements and the repeated results prove the reproducibility of the method. Results and Discussion 1. Structural, Topographical, and Surface Shear Characteristics of β-Casein-Monopalmitin Mixed Monolayers at the Air/Water Interface. 1.1. Structural and Topographical Characteristics. Previous results on disordered9 and globular7,8 proteins demonstrate that it is possible to measure reproducible π-A isotherms for adsorbed protein monolayers from low protein concentration in the bulk phase if a long interval of time is allowed for protein adsorption, especially from low protein concentration in solution, as those used in this work. Because the surface concentration is actually unknown for the adsorbed β-casein monolayer, results are presented for surface pressure versus trough area (AAPPARENT). Results derived from the π-A isotherms (Figure 1) in the modified Wilhelmy-type trough are in good agreement with those obtained in the same unmodified

1888

Ind. Eng. Chem. Res., Vol. 45, No. 6, 2006

Figure 1. Surface pressure-area (π-A) isotherms (compression-expansion curves) for adsorbed β-casein-monopalmitin mixed monolayers on buffered water at pH 7 and at 20 °C for various mass fractions of monopalmitin in the mixture (X): (- ‚ ‚ -) X ) 0, (s) X ) 0.2, (- - -) X ) 0.4, and (‚ ‚ ‚) X ) 0.5. The πe value of β-casein is indicated by the arrow.

trough with pure β-casein adsorbed monolayers after 24 h of adsorption time and with adsorbed β-casein-monopalmitin mixed films.9 Briefly, the results of π-A isotherms confirm that β-casein monolayers at the air/water interface adopt two different structures (structures I and II) and the collapse phase.

At low surface pressures, β-casein molecules may exist as trains with all amino acid segments (structure I) located at the interface.26 This structure was observed at surface pressures lower than ∼12 mN/m (see Figure 1). At higher surface pressures, and up to the equilibrium spreading pressure (πe ≈ 21 mN/m)swhich is indicated in Figure 1 by means of an arrowsamino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails (structure II). The equilibrium surface pressure (πe) is the maximum surface pressure to which a spread monolayer may be compressed without the possibility of monolayer collapse. A β-casein adsorbed monolayer collapses at a surface pressure (πβ-casein ) c of ∼21 mN/m (see Figure 1), a value close to the equilibrium surface pressure. Different structures can be deduced for monopalmitin monolayer as a function of surface pressure.22 The liquid-expanded phase (LE) (at π < 5 mN/m), which represents a first-order phase transition between liquidcondensed (LC) and liquid-expanded structures (at 5 mN/m < π < 30 mN/m), the liquid-condensed structure (at π > 30 mN/ m), and, finally, the solid (S) structure near to the monolayer collapse at a surface pressure of ∼53.1 mN/m were all observed.

Figure 2. Visualization of adsorbed β-casein-monopalmitin mixed monolayers by Brewster angle microscopy (BAM) at 20 °C: (A) pure β-casein monolayer ; (C) β-casein-monopalmitin mixed monolayers at π ≈ πβ-casein ; (D and E) at π ≈ 0; (B) β-casein-monopalmitin mixed monolayers at π < πβ-casein c c β-casein ; and (F) β-casein-monopalmitin mixed monolayers at the collapse point of the mixed film. The β-casein-monopalmitin mixed monolayers at π > πc arrows indicate the existence of a shear plane between β-casein and monopalmitin domains (panel C) or between collapsed regions separated by a fracture (panel E) during the flow of the monolayer. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm.


Figure 3. Difference in surface pressure between the limits of the canal during the flow of β-casein adsorbed monolayers at the air/water interface as a function of surface pressure: (A) 5 mN/m, (B) 10 mN/m, (C) 15 mN/m, and (D) 20 mN/m. (The temperature was 20 °C, and pH 7.) The effect of the width of canal (Wc) and β-casein flow through the canal (Q) is also shown: (s) Q ) 6.25 × 10-6 m2/s and Wc ) 1.4 mm, (- - -) Q ) 6.25 × 10-6 m2/s and Wc ) 0.9 mm, (‚ ‚ ‚) Q ) 1.25 × 10-5 m2/s and Wc ) 1.4 mm, and (- ‚ -) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm.

Figure 4. Effect of surface pressure on surface shear viscosity for (b) β-casein adsorbed films at the air/water interface. The surface shear viscosities for pure monopalmitin (]) and (4) monoolein spread monolayers are included for reference. (Temperature ) 20 °C and pH 7.)

From the π-A isotherms for β-casein + monopalmitin mixed films (see Figure 1), it can be seen that there was a monolayer expansion as the monopalmitin concentration in the mixture was

increased, especially at higher surface pressures. That is, the π-A isotherm is displaced toward higher values of A as the concentration of monopalmitin in the mixture increases. At surface pressures higher than that for the β-casein collapse, the π-A isotherm for mixed monolayers was parallel to that of monopalmitin. These data are in good agreement with those deduced for spread β-casein + monopalmitin20 and β-lactoglobulin-monopalmitin27-29 mixed films. These results suggest , a protein displacement by the monoglycthat, at π > πβ-casein c eride from the air/water interface occurs. However, the protein re-enters the mixed monolayer upon expansion and this supports the idea that the protein remains underneath the monoglyceride film,8,20,30 because the π-A isotherms were repetitive upon expansion and further compression (data not shown). However, for adsorbed β-casein-monopalmitin mixed monolayers a degenerated plateau in the π-A isotherm was observed at π ≈ πβ-casein upon monolayer expansion (Figure 1). Interestingly, e this plateau is more evident and more extended at higher concentrations of monopalmitin in the mixture. This result suggests that the readsorption of previously displaced β-casein has a kinetic character,8 which was not evident for spread mixed films.20 Moreover, the readsorption of previously displaced β-casein is hindered as the concentration of monopalmitin in the mixture increases. , both β-casein and monopalmitin coexist at At π < πβ-casein c the interface. From the π-A isotherm, coupled with the application of the additivity rule and thermodynamic treatment, and from reflectivity measurements of the interface, we have concluded that these interactions between adsorbed β-casein and monopalmitin mixed films are attractive, on average.9 The topography of the interface obtained with adsorbed β-casein, and β-casein-monopalmitin mixed films in the

1890


absence of shear (Figure 2) clearly shows, at a microscopic level, the same structural characteristics as that deduced from the π-A isotherms. During the first compression, β-casein adsorbed films are characterized by a “homogeneous” interface (data not shown). However, after successive compressions, a local heterogeneity of the interface at a microscopic level was observed, because of the existence of isolated interfacial regions with folds or aggregations (see Figure 2A) of collapsed β-casein formed at the higher surface pressures in previous compressions. For adsorbed β-casein and monopalmitin mixed films at π < , a mixed monolayer of monopalmitin and β-casein πβ-casein c may exist (see Figure 2B) with small domains of monopalmitin (with a LC structure at π > 5 mN/m) uniformly distributed on the homogeneous β-casein layer. The circular domains of LC monopalmitin were more numerous as the surface pressure increased (data not shown). At the collapse point of β-casein (see Figure 2C), some regions of collapsed β-casein (bright region) separated from monopalmitin domains (dark region) were observed. The existence of this segregation between β-casein and monopalmitin domains defines a shear plane during the flow of the mixed monolayer. At π > πβ-casein , the mixed c monolayers were practically dominated by monopalmitin domains. That is, at higher surface pressures, a squeezing-out phenomenon is observed (see Figure 2D) with small LC domains of monopalmitin (dark circle) floating over a sublayer of collapsed residues of β-casein (bright region). This topography of the mixed film proves the existence of attractive interactions between film-forming components, because the size of monopalmitin LC domains in the mixed film are smaller than that for a pure monopalmitin monolayer. In addition, both regions are not clearly separated, because of the existence of some miscibility between them, which is a phenomenon that was not observed for spread mixed films. Another topographical characteristic of the adsorbed film was the presence of large (Figure 2E) and small (Figure 2F) fractures in the monolayer at the collapse point of the mixed film, which are characteristic of protein-monoglyceride adsorbed films.8,9 1.2. Effect of Surface Pressure on Surface Shear Characteristics of Adsorbed β-Casein. The time evolution of ∆π during the flow of an adsorbed β-casein monolayer is dependent on the β-casein flow through the canal and the surface pressure (Figure 3). An optimization of Wc and Q was necessary to obtain the best flow conditions of a protein monolayer through the canal within any fluctuation in the ∆π-time flow curve. For β-casein adsorbed monolayer high values of Wc (1.4 mm) and low values of Q (1.25 × 10-5-6.25 × 10-6 m2/s) were necessary to obtain more-reproducible data. From the ∆π value at steady state, the surface shear viscosity was determined as a function of surface pressure (Figure 4). Over the overall range of surface pressures, the values of ηs were not dependent on the flow Q (see Figure 4). The ηs values increased with surface pressure, especially as the residues of β-casein adopt the conformation of loops and tails at the air/ water interface. At high surface pressures, as amino acid segments are extended into the underlying aqueous solution adopting the form of loops and tails, an increase in the viscous drag caused by the subphase may explain the higher values of ηs. The surface shear viscosity for pure monopalmitin and monoolein spread monolayers24 is included in Figure 4 as a reference. Over the overall range of surface pressures of an adsorbed β-casein monolayer (up to πβ-casein ≈ 21 mN/m), the c values of ηs were lower for β-casein than for monoglycerides (see Figure 4A). This behavior is different from that observed for spread monolayers.24 One speculation is that β-casein (a disordered protein) may be adsorbed at the interface, maintaining

Figure 5. Surface shear viscosity for adsorbed mixed films of β-casein and monopalmitin at XMP ) 0.2 and (A) 10 mN/m, (B) 20 mN/m, and (C) 30 mN/m. The surface shear viscosity for a pure β-casein monolayer at 25 mN/m (striped region) is included for reference. Symbol legend is as follows: (s) Q ) 3.75 × 10-6 m2/s and Wc ) 1.4 mm; (- - -) Q ) 1.25 × 10-6 m2/s and Wc ) 1.4 mm; (‚ ‚ ‚) Q ) 3.75 × 10-6 m2/s and Wc ) 0.9 mm; and (- ‚ -) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm.

some of its secondary and tertiary structure. However, the same protein may be more unfolded during the spreading at the interface at high molecular areas.23 Thus, the interactions between unfolded β-casein residues must be higher for spread than for adsorbed β-casein films, which is a phenomenon that explains the higher value of ηs for β-casein spread films, as compared with the adsorbed one. The differences observed in ηs values for monopalmitin at π > πβ-casein can be of utility in c the analysis of the shear characteristics of mixed films in the following sections. 1.3. Effect of Surface Pressure on Surface Shear Characteristics of Adsorbed β-Casein-Monopalmitin Mixed Films. The surface shear characteristics of adsorbed β-caseinmonopalmitin mixed monolayers under different operational conditions and at XMP ) 0.2, 0.4, and 0.5 are shown in Figures 5, 6, and 7, respectively. 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). For some flow conditions, the ηstime curve develops a maximum (see Figures 5A, 5C, and 7B) and then ηs decreases with time, before the ηs “steady state” value is attained. These phenomena were also described by other authors in relation to stress-strain curves for proteins using a


Figure 6. Surface shear viscosity for adsorbed mixed films of β-casein and monopalmitin at XMP ) 0.4 and (A) 10 mN/m, (B) 20 mN/m, (C) 30 mN/m, and (D) 40 mN/m. The surface shear viscosities for pure β-casein (striped region) and monopalmitin (checkered region) monolayer are included for reference. Symbol legend is as follows: (s) Q ) 3.75 × 10-6 m2/s and Wc ) 0.9 mm; (- - -) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm; (‚ ‚ ‚) Q ) 1.25 × 10-5 m2/s and Wc ) 1.4 mm; and (- ‚ -) Q ) 3.75 × 10-6 m2/s and Wc ) 2.05 mm.

Figure 7. Surface shear viscosity for an adsorbed mixed film of β-casein and monopalmitin at XMP ) 0.5 and (A) 20 mN/m, (B) 30 mN/m, (C) 40 mN/m, and (D) 45 mN/m (curve 1) and 50 mN/m (curve 2). The surface shear viscosities for a pure monopalmitin (striped region) and β-casein (checkered region) monolayers are included for reference. Symbol legend is as follows: (s) Q ) 1.25 × 10-5 m2/s and Wc ) 1.4 mm; (- - -) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm; (‚ ‚ ‚) Q ) 3.75 × 10-6 m2/s and Wc ) 2.05 mm; (- ‚ -) Q ) 3.75 × 10-6 m2/s and Wc ) 0.9 mm; and (- ‚ ‚ -) Q ) 6.25 × 10-6 m2/s and Wc ) 2.05 mm.

Couette-type interfacial viscometer.31 According to these authors, the maximum can be associated with the strength of the monolayer, whereas the decrease in ηs after the maximum may

represent a structure breakdown of the mixed film under shear, similar to the overshoot observed with thixotropic fluids in bulk rheology. The breaking of LC domains was evident at a

1892


Figure 8. Surface pressure-area (π-A) isotherms (compression-expansion curves) for adsorbed β-casein-monoolein mixed monolayers on buffered water at pH 7 and at 20 °C for various mass fraction of monoolein in the mixture (X): (- ‚ -) X ) 0, (s) X ) 0.2, (- - -) X ) 0.4, and (‚ ‚ ‚) X ) 0.5. The πe value of β-casein is indicated by the arrow.

microscopic level by means of BAM. In fact, the size of the LC domains was lower under shear than with the open canal (data not shown). However, we have also observed that there exists a deformation of the domains and/or a segregation of β-casein and monopalmitin domains at the interface during the flow of the monolayer. That is, the shear may induce heterogeneity in the structure of the mixed monolayer at a microscopic level along the BAM image. The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers32-35 and may be the cause of the ηs peaks observed during the flow of the monolayer. The “steady-state” ηs value can be associated with the existence of a balance between the formation and breaking of monolayer structures or 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 β-casein: both of these phenomena 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 β-casein monolayers are included in Figures 5-7 as reference. However, there were significant differences in ηs during the flow of the mixed film through the canal, , the ηs depending on the film composition. At π < πβ-casein c values of the mixed films are lower than those for a pure monopalmitin monolayer and tend toward that of β-casein, regardless of the composition of the mixed films, at X ) 0.2 (Figure 5A and B), X ) 0.4 (Figure 6A and B), and X ) 0.5 (Figure 7A and B). These results indicate that, in this region, the monolayer flow was dominated by the presence of β-casein but the LC domains of monopalmitin produced a reduction in the friction in the mixed monolayer in relation to that for a pure β-casein monolayer. There are significant differences in the shear characteristics of the mixed film after the β-casein collapse, at surface pressures of 30, 40, and 50 mN/m (see Figures 5-7). In fact, at these surface pressures, the ηs values of the mixed film were similar to those for a pure monopalmitin monolayer, especially at higher contents of monopalmitin in the mixture. 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, the topography was dominated by monopalmitin with a solid structure (see Figure 2D) or a collapsed monopalmitin film with the presence of some fractures of collapsed β-casein (which were not representative of the overall topography of the mixed film) aligned in the direction of the flow as typical features of the mixed film (see Figure 2E and F). However, we have also observed a segregation of β-casein and monopalmitin domains at the interface during the flow of the monolayer. Figure 2C and D shows the frontier between regions of collapsed β-casein domains and domains of monopalmitin (see Figure 2C) and a fracture in the collapsed monolayer (see Figure 2E) separated by a shear plane, which is indicated in the figure by the two arrows. The existence of isolated spots with collapsed β-casein residues was the cause

Figure 9. Visualization of adsorbed β-casein-monoolein mixed monolayers by Brewster angle microscopy (BAM) at 20 °C: (A) pure β-casein monolayer, ; (B) pure β-casein monolayer or β-casein-monoolein mixed pure monoolein monolayer, or β-casein-monoolein mixed monolayers at π < πβ-casein c monolayers at π < πβ-casein ; (C) β-casein-monoolein mixed monolayers at π > πβ-casein ; and (D) β-casein-monoolein mixed monolayers at the collapse c c point of the mixed films. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm.


Figure 10. Surface shear viscosity for adsorbed mixed films of β-casein and monoolein at XMO ) 0.2 and (A) 10 mN/m and (B) 20 mN/m. The surface shear viscosities for pure monoolein (striped region) and β-casein (checkered region) monolayers are included for reference. Symbol legend is as follows: (s) Q ) 1.25 × 10-5 m2/s and Wc ) 1.4 mm; (- - -) Q ) 3.75 × 10-6 m2/s and Wc ) 0.9 mm; (‚ ‚ ‚) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm; and (- ‚ -) Q ) 3.75 × 10-6 m2/s and Wc ) 1.4 mm.

of the lower values ηs in relation to a pure monopalmitin monolayer, even at higher contents of monopalmitin in the mixture (Figures 5-7). 2. Structural, Topographical, and Surface Shear Characteristics of β-Casein-Monoolein Mixed Monolayers at the Air/Water Interface. 2.1. Structural and Topographical Characteristics. The π-A isotherms for adsorbed β-casein and β-casein-monoolein mixed monolayers are shown in Figure 8. The monolayer structure in a β-casein-monoolein mixed monolayer was liquid-expanded-like, as for pure monoolein36 and β-casein (Figure 8) monolayer components. At surface pressures higher than that for β-casein collapse, the π-A isotherms for the mixed monolayer were parallel to that of pure monoolein.36 These results suggest that the composition of the mixed monolayer was very dependent on the surface pressure. At surface pressures lower than that for β-casein collapse, a mixed monolayer of monoolein and β-casein could exist, β-casein molecules adopting different structures in the isotropic liquid-expanded monoolein monolayer,36 depending on the surface pressure, as described previously. However, at surface pressures higher than that for β-casein collapse, collapsed β-casein residues may be displaced from the interface by monoolein molecules.2 BAM images corroborate the following: (i) the presence of homogeneous β-casein or LE monoolein domains (Figure 9A); (ii) the existence of isolated interfacial regions with folds or aggregations (Figure 9B) of collapsed β-casein formed at the higher surface pressures in previous compressions (both features (i) and (ii) observed at π < πβ-casein ); (iii) the squeezing out of β-casein by monoolein at π c (Figure 9C); and, finally (iv) the presence of short > πβ-casein c fractures in the monolayer at the collapse point of the mixed film (Figure 9D). 2.2. Effect of Surface Pressure on Surface Shear Characteristics. The time evolution of ηs during the flow through

Figure 11. Surface shear viscosity for adsorbed mixed films of β-casein and monoolein at XMO ) 0.4 and (A) 10 mN/m, (B) 20 mN/m, and (C) 30 mN/m. The surface shear viscosities for pure monoolein (striped region) and β-casein (checkered region) monolayers are included for reference. Symbols: (s) Q ) 3.75 × 10-6 m2/s and Wc ) 1.4 mm, (- - -) Q ) 1.25 × 10-5 m2/s and Wc ) 1.4 mm, and (‚ ‚ ‚) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm.

the canal of β-casein-monoolein mixed monolayers at XMO ) 0.2, 0.4, and 0.5 is shown in Figures 10, 11, and 12, respectively, as a function of surface pressure. The presence of a maximum in the ηs-time curve or the existence of large fluctuations in ηs value for adsorbed β-casein-monoolein mixed monolayers were only observed at XMO ) 0.2 (Figure 10). This phenomenon can be associated to the segregation between β-casein and monoolein domains at the interface during the flow of the monolayer34 and/or for the use of inappropriate flow conditions. It can be seen that, over the overall range of surface pressures analyzed, the values of ηs for the mixed monolayer were very small and these values practically were not dependent on the surface pressure (see Figures 10-12). Interestingly, the values of ηs were very similar to those for a pure adsorbed β-casein or monoolein monolayer (see Figure 4). These results indicate that the shear characteristics of the mixed monolayer are dependent on the presence of adsorbed β-casein or monoolein at the interface at π < πβ-casein . At π > πβ-casein , and, especially, at c c the higher surface pressures or at the collapse point of the mixed films, the ηs values were lower than that for a pure β-casein adsorbed film (see Figures 11B, 11C, and 12C), which may be

1894


Figure 12. Surface shear viscosity for an adsorbed mixed film of β-casein and monoolein at XMO ) 0.5 and (A) 10 mN/m, (B) 20 mN/m, (C) 40 mN/m, and (D) at the collapse point. The surface shear viscosity for a pure monopalmitin monolayer (striped region) is included for reference. Symbol legend is as follows: (s) Q ) 1.25 × 10-5 m2/s and Wc ) 1.4 mm and (- - -) Q ) 1.25 × 10-5 m2/s and Wc ) 0.9 mm.

associated with the flow of LE monoolein domains through the canal. Surprisingly, at the collapse of the mixed film, the ηs values of β-casein-monoolein mixed monolayers were lower (see Figure 12D) than those for a pure monoolein monolayer (see Figure 4B). This phenomenon can be associated with the presence of small fractures in the collapsed film (see Figure 9D). 3. Relevance of Protein-Monoglyceride Interactions at a Fluid Interface to Product Engineering. The competition and cooperative interactions that occur between emulsifiers (mainly polar lipids and proteins) as they adsorb at a fluid interface presented in this work is essential to understand the control of texture and long-term stability of food emulsions.4,37 For instance, a key step during the manufacture of milk-base emulsions (cream liqueurs) or whippable emulsions (ice cream and whipping cream) is the destabilization of the milk proteinstabilized oil-water emulsion during the whipping or the freezing/aeration of the mix.38,39 The role of mixed interfacial lipid-protein films is not totally clear and there exists some controversy about its implication in the formation and stabilization of food colloids. It was found that protein displacement by the surfactant in model food emulsions was independent of the nature of the protein (disordered or globular), whether the surfactant was oil-soluble or water-soluble, or whether it was added before or after emulsification.40 However, most studies inform that such interactions are sensitive to the emulsifier.41-45 The presence of commercial monoglycerides (saturated and unsaturated monoglycerides) in dairy-type emulsions can affect both the solid fat content of the droplets and the amount of protein adsorbed at the oil/water interface. The competitive adsorption of monoglyceride may weaken or interfere with the formation of protein/protein interactions in the adsorbed layer, destroying the protective viscoelastic properties. Initially adsorbed monoglyceride could cause weaknesses in the protein film. Monoglyceride crystallization can also lead to proteins being displaced from the droplet surface.39 Penetration of the droplet surface by fat crystals inside droplets will become easier when less protein is present at the interface, thus, the facilitation of shear-induced partial coalescence.44,45 Here, we have observed

that the extent of competitive protein displacement is different for monopalmitin and monoolein, which can explain the differing abilities of saturated and unsaturated monoglyceride to induce shear-induced destabilization of emulsions.44,45 This phenomenon is of practical importance in relation to the control of clumping of fat globules in dairy emulsion aeration and ice cream making.38 On the other hand, β-casein-monoglyceride interactions at a fluid interface and/or the protein displacement by monoglycerides, pushing β-casein loops and tails residues further away from the interface, may increase the steric stabilizing capacity of the protein around the emulsion droplets.43 Finally, the protein displacement by monoglyceride from a fluid interface also is dependent on the protein/monoglyceride ratio and, to a great extent, on the interfacial density (or surface pressure) of the emulsifiers. These phenomena can also explain the effect of the amount of added monoglyceride on the orthokinetic stability of milk-protein-based emulsions.44,45 Conclusions In this work, we have used different and complementary interfacial techniques (surface film balance, Brewster angle microscopy, and interfacial shear rheology) to analyze the structural and surface shear characteristics of an adsorbed β-casein and monoglyceride (monopalmitin and monoolein) mixed films at the air/water interface. At π < πβ-casein , a c mixed monolayer of monoglyceride and β-casein may exist at , the mixed monolayers the interface. However, at π > πβ-casein c were practically dominated by monoglyceride domains. The ηs values of adsorbed β-casein-monoglyceride mixed monolayers are directly linked with their structural and topographical characteristics. Lower values of ηs were observed for a liquidlike expanded structure of the adsorbed β-casein-monoolein monolayer, coinciding with a more homogeneous flow of the monolayer through the canal (see Figures 10-12). Over the overall range of surface pressures and monolayer composition analyzed, the values of ηs were higher for mixed films of β-casein and monopalmitin (see Figures 6 and 7) than for monoolein (see Figures 10-12), especially at the higher surface


pressures. These results confirm the extremely sensitive dependence of surface shear characteristics on the monolayer structure, including the shear-induced segregation in the mixed film. Acknowledgment This research was supported by CICYT (through Grant Nos. AGL2001-3843-C02-01 and AGL2004-1306/ALI). The comments and suggestions raised by the referees are acknowledged. Literature Cited (1) Dickinson E. An Introduction to Food Colloids; Oxford University Press: Oxford, U.K., 1992. (2) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, Ma. R.; Carrera, C. Protein-emulsifier interactions at the air-water interface. Curr. Opin. Colloid Interface Sci. 2003, 8, 387-395. (3) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsifiers and Their Applications; Hasenhuettl, G. L., Hartel, R. W., Eds.; Chapman and Hall: New York, 1997; p 95. (4) Wilde, P. J. Interfaces: their role in foam and emulsion behaviour. Curr. Opin. Colloid Interface Sci. 2000, 5, 176-181. (5) Dickinson, E. Milk protein interfacial layers and the relationship to emulsion stability and rheology. Colloids Surf., B 2001, 20, 197-210. (6) Li, J. B.; Zhao, J.; Miller, R. Morphology and thermodynamics of dipalmitoyl-phosphatidyl-choline monolayers penetrated by β-casein and β-lactoglobulin. Nahrung 1998, 42, 234-235. (7) Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M. Effect of the Aqueous Phase Composition on the Adsorption of Bovine Serum Albumin to the Air-Water Interface. Ind. Eng. Chem. Res. 2002, 41, 1489-1495. (8) Rodrı´guez Patino, J. M.; Cejudo, M. Structural and Topographical Characteristics of Adsorbed WPI and Monoglyceride Mixed Monolayers at the Air-Water Interface. Langmuir 2004, 20, 4515-4522. (9) Cejudo Fernańdez, M.; Carrera Sańchez, C.; Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M. The effect of monoglycerides on structural and topographical characteristics of adsorbed β-casein fims at the air-water interface. Biomacromolecules 2006, 7, 507-514. (10) Dickinson, E. Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology. Colloids Surf., B 1999, 15, 161176. (11) Bos, M. A.; van Vliet, T. Interfacial rheological properties of adsorbed protein layers and surfactants: a review. AdV. Colloids Interface Sci. 2001, 91, 437-471. (12) Murray, B. S. Interfacial rheology of food emulsifiers and proteins. Curr. Opin. Colloid Interface Sci. 2002, 7, 426-461. (13) Murray, B. S.; Dickinson, E. Interfacial rheology and the dynamic properties of adsorbed films of food proteins and surfactants. Food Sci. Technol. Inst. 1996, 2, 131-145. (14) Murray, B. S. Interfacial Rheology of Mixed Food Protein and Surfactant Adsorption Layers with respect to Emulsion and Foam Stability. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 179-220. (15) Benjamins, J.; Lucassen-Reynders, E. H. Surface dilational rheology of proteins adsorbed at air/water and oil/water interfaces. In Proteins at Liquid Interface; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; pp 341-384. (16) Lucassen-Reynders, E. H.; Benjamins, J. Dilational Rheology of Proteins Adsorbed at Fluid Interfaces. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1999; pp 195206. (17) Roth, S.; Murray, B. S.; Dickinson. E. Interfacial shear rheology of aged and heat-treated β-lactoglobulin films: displacement by nonionic surfactant. J. Agric. Food Chem. 2000, 48, 1491-1497. (18) Mackie, A. R.; Gunning, P. A.; Pugnaloni, L. A.; Dickinson, E.; Wilde, P. J.; Morris, V. J. The Growth of Surfactant Domains in Protein Films. Langmuir 2003, 19, 6032-6038. (19) Carrera, C.; Rodrı´guez Patino, J. M. Interfacial, foaming and emulsifying characteristics of sodium caseinate as influenced by protein concentration in solution. Food Hydrocolloids 2005, 19, 407-416. (20) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Analysis of β-casein-monopalmitin mixed films at the air-water interface. J. Agric. Food Chem. 1999, 47, 4998-5008. (21) Rodrı´guez Ninõ, Ma. R.; Carrera, C.; Cejudo, M., Rodrı´guez Patino, J. M. Protein and lipid adsorbed and spread films at the air-water interface at the equilibrium. J. Am. Oil Chem. Soc. 2001, 78, 873-879.

(22) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, Ma. R. Morphological and Structural Characteristics of Monoglyceride Monolayers at the Air-Water Interface Observed by Brewster Angle Microscopy. Langmuir 1999, 15, 2484-2492. (23) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, Ma. R. Structural and morphological characteristics of β-casein monolayers at the air-water interface. Food Hydrocolloids 1999, 13, 401-408. (24) Rodrı´guez Patino, J. M.; Carrera, C. Structural, Topographical, and Shear Characteristics of Milk Protein and Monoglyceride Monolayers Spread at the Air-Water Interface. Langmuir 2004, 20, 4530-4539. (25) Harkins, W. D.; Kirkwood, J. G. Theory of the surface slit viscometer. Nature (London) 1938, 141, 38-39. (26) Graham, D. E.; Phillips, M. C. Proteins at liquid interfaces. III. Molecular structures of adsorbed films. J. Colloid Interface Sci. 1979, 70, 427-439. (27) Cornec, M.; Narsimhan, G. Adsorption and Exchange of β-Lactoglobulin onto Spread Monoglyceride Monolayers at the Air-Water Interface. Langmuir 2000, 16, 1216-1225. (28) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C.; Cejudo, M. Whey Protein Isolate-Monoglyceride Mixed Monolayers at the Air-Water Interface. Structure, Morphology, and Interactions. Langmuir 2001, 17, 7545-7553. (29) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Static and Dynamic Properties of a Whey Protein Isolate and Monoglyceride Mixed Films at the Air-Water Interface. Ind. Eng. Chem. Res. 2002, 41, 2652-1661. (30) Li, J. B.; Kra¨gel, J.; Makievski, A. V.; Fainermann, V. B.; Miller, R.; Mo¨hwald, H. A study of mixed phospholipid/β-casein monolayers at the water/air surface. Colloids Surf., A 1998, 142, 355-360. (31) Izmailova, V. N. Structure formation and rheological properties of proteins and surface-active polymers of interfacial adsorption layers. Prog. Surf. Membr. Sci. 1979, 13, 141-209. (32) Murayama, T.; Fuller, G.; Frank, C.; Robertson, C. Flow-induced molecular orientation of a Langmuir film. Science 1996, 274, 233-235. (33) Murayama, T.; Lauger, J.; Fuller, G.; Frank, C.; Robertson, C. Orientation in a Fatty Acid Monolayer: Effect of Flow Type. Langmuir 1998, 14, 1836-1845. (34) Igne´s-Mullol, J.; Schwartz, D. K. Shear-Induced molecular precession in a hexatic Langmuir monolayer. Nature 2001, 410, 348-351. (35) Carrera, C.; Rodrı´guez Patino, J. M. Flow-Induced Molecular Segregation in β-Casein-Monoglyceride Mixed Films Spread at the AirWater Interface. Langmuir 2004, 20, 6327-6334. (36) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M.; Carrera, C.; Cejudo, M.; Navarro, J. M. Physicochemical characteristics of food lipids and proteins at fluid-fluid interfaces. Chem. Eng. Commun. 2003, 190, 1547. (37) Gupta, R.; Muralidhara, H. S. Interfacial challenges in the food industry: a review. Trends Food Sci. Technol. 2001, 12, 382-391. (38) Goff, H. D. Colloidal aspects of ice creamsa review. Int. Dairy J. 1997, 7, 363-373. (39) Krog, N. In Ice Cream: Proceedings of the IDF International Symposium on Ice Cream, Athens, Greece, September 18-19, 1997; Buchheim, W., Ed.; International Dairy Federation: Northfield, IL, 1997; pp 37-44. (40) de Feinjter, J. A.; Benjamins, J.; Tamboer, M. Adsorption displacement of proteins by surfactants in oil-in-water emulsions. Colloids Surf. 1987, 27, 243-266. (41) Cornec, M.; Wilde, P. J.; Gunning, P. A.; Mackie, A. R.; Husband, F. A.; Parker, M. L.; Clark, D. C. emulsion stability as affected by competitive adsorption between an oil-soluble emulsifier and milk proteins at the interfaced. J. Food Sci. 1998, 63, 39-43. (42) Krog, N. In Emulsifiers: Functionality and Applications; Berger, K., Hamilton, R. J., Eds.; Society Chemical Industry (SCI): London, 1998. (43) Dickinson, E.; Narhan, S. K.; Stainsby, G. Stability of cream liqueurs containing low-molecular-weight surfactants. J. Food Sci. 1989, 54, 77-81. (44) Davies, E.; Dickinson, E.; Bee, R. Shear stability of sodium caseinate emulsions containing monoglyceride and diglycerides crystals. Food Hydrocolloids 2000, 14, 145-153. (45) Davies, E.; Dickinson, E.; Bee, R. Orthokinetic destabilization of emulsions by saturated and unsaturated monoglycerides. Int. Dairy J. 2001, 11, 827-836.

ReceiVed for reView September 30, 2005 ReVised manuscript receiVed January 16, 2006 Accepted January 26, 2006 IE051092+

Structural and Shear Characteristics of Adsorbed Î²-Casein and

Recommend Documents