Structural, Topographical, and Shear Characteristics of Milk Protein

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Structural, Topographical, and Shear Characteristics of Milk Protein and Monoglyceride Monolayers Spread at the Air-Water Interface Juan M. Rodrı´guez Patino* and Cecilio Carrera Sańchez Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcıá Gonza´ lez 1, 41012-Seville, Spain Received December 1, 2003. In Final Form: March 9, 2004 In this contribution we are concerned with the study of structure, topography, and surface rheological characteristics under shear conditions of monoglyceride (monopalmitin and monoolein) and milk protein (β-casein, κ-casein, caseinate, and WPI) spread monolayers 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 pure emulsifiers (proteins and monoglycerides) spread at the air-water interface. To study the shear characteristics of spread films, a homemade canal viscometer was used. The experiments have demonstrated the sensitivity of the surface shear viscosity (ηs) of protein and monoglyceride films at the air-water interface, as a function of surface pressure (or surface density). The surface shear viscosity was higher for proteins than for monoglycerides. In addition, ηs was higher for the globular WPI than for disordered β-casein and caseinate due to the strong forces acting on spread globular proteins. This technique makes it possible to distinguish between β-casein and caseinate spread films, with the higher ηs values for the later due to the presence of κ-casein. The ηs value varies greatly with the surface pressure (or surface density). In general, the greater the surface pressure, the greater the values of ηs. Finally, the ηs value is also sensitive to the monolayer structure, as was observed for monoglycerides with a rich structural polymorphism (i.e., monopalmitin).

Introduction The dynamic behavior of emulsifier (protein, lipid, phospholipid, surfactants, etc.) films is recognized as being of importance to the formation and stability of food colloids in which these emulsifiers are used. The study of such dynamic behavior can be described by interfacial rheology.1-7 Interfacial rheology is important for food colloids (emulsions and foams) because the structural and mechanical properties of food emulsifiers at fluid-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.6,8-11 * To whom all correspondence should be addressed. Tel: +34 95 4556446. Fax: +34 95 4557134. E-mail: [email protected]. (1) Murray, B. S.; Dickinson, E. Food Sci. Technol. Inst. 1996, 2, 131. (2) Murray, B. S. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 179. (3) 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 196. (4) Benjamins, J.; Lucassen-Reynders, E. H. In Proteins at Liquid Interface; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 341. (5) Wasan, D. T. In Emulsions, Foams, and Thin Films; Mittal, K. L., Kumar, P., Eds.; Marcel Dekker: New York, 2000; p 1. (6) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437. (7) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2002, 7, 426. (8) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. Langmuir 2001, 17, 4003. (9) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (10) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Ind. Eng. Chem. Res. 2002, 41, 2652. (11) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. J. Agric. Food Chem. 2003, 51, 112.

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 stability of dispersions, dilatational rheology plays an important role in short-term stability.3-6 In fact, during the formation of food colloids a new interface is continuously formed during the dispersion process, over a time scale typical of dilatational rheology. In addition, the type of deformation that interfaces undergo during emulsification and foaming is expansion and, to a lesser extent, compression. 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.12 In addition, surface shear rheology has an effect on the flow of liquid through the films and Plateau borders and the so-called marginal regeneration in thin liquid films, determining foam stability.13 In this contribution we are concerned with the study of structure, topography, and surface rheological characteristics under shear conditions of monoglycerides (monopalmitin and monoolein) and milk proteins (β-casein, κ-casein, caseinate, and WPI) at the air-water interface. The pH and the ionic strength were maintained constant at 7 and 0.05 M, respectively. Combined surface chemistry (surface film balance and surface shear rheometry) and microscopy (Brewster angle microscopy, BAM) techniques have been applied in this study to pure emulsifiers (proteins and monoglycerides) spread at the air-water interface. In previous studies in our laboratory we have analyzed the structural and dilatational characteristics (12) Miller, R.; Wu¨stneck, R.; Kra¨gel, J.; Kretzsmara, G. Colloids Surf., A 1996, 111, 75. (13) Prins, A.; Bos, M. A.; Boerboom, F. J. G.; van Kalsbeek, H. K. A. I. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 221.

10.1021/la036255i CCC: $27.50 © 2004 American Chemical Society Published on Web 04/20/2004

Milk Protein Monolayers

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of monoglyceride8 and proteins9 spread films at the airwater interface. To study the shear characteristics of spread films, a homemade canal was used. To our best knowledge this method has not been used before in protein monolayers.1,2,5-7 This classical method was applied in this work for the first time to protein monolayers after the optimization of the main experimental parameters (mainly the width of the canal and the flow of the monolayer through the canal). Materials and Methods Materials. 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 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. Pure (99%) β-casein and κ-casein were supplied and purified from bulk milk from the Hannah Research Institute (Ayr, Scotland). Caseinate (a mixture of ≈38% β-casein, ≈39% Rs1-casein, ≈12% κ-casein, and ≈11% Rs2-casein) was supplied and purified from bulk milk from Unilever Research (Colworth, U.K.). Whey protein isolate (WPI), a native protein with a high content of β-lactoglobulin (protein 92 ( 2%, β-lactoglobulin >95%, R-lactalbumin 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. There are many experimental devices for measuring surface shear rheology.1,2,12,14 These methods, which detect stresses in the surface film in the presence of stresses from the underlying subphase, can be classified into indirect and direct. Direct methods determine torsional stress values by the rotational motion of a knife-edge bob, disk, or ring when placed in the plane of the interface. The arrangement is the equivalent of a typical Couette viscometer. Indirect methods (that measure velocity profiles) such as canal and deep-channel surface viscometers require measurements of fluid flow using easily visible inert particles from which surface viscosities can be determined. Indirect methods have been restricted to the airwater interface,1,2,15 although some modifications of this technique enable it to be used at the oil-water interface.16 To study the shear characteristics of spread films, a homemade canal viscometersan analogue of the conventional Ostwald viscometer, where there is no area change but where the different interfacial elements slip past one anotherswas used in this work (Figure 1). The instrument is a modification of a previous one based on the same principle.17-21 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, (14) Warburton, B. Curr. Opin. Colloid Interface Sci. 1996, 1, 481. (15) Prins, A. In New Physicochemical Techniques for the Characterization of Complex Food Systems; Dickinson, E., Ed.; Chapman and Hall: London, 1995; p 214. (16) Nagaranjan, R.; Wasan, D. T. Rev. Sci. Instrum. 1994, 65, 2675. (17) Harvey, N.; Rose, P.; Porter, N. A.; Huff, J. B.; Arnett, E. M. J. Am. Chem. Soc. 1988, 110, 4395. (18) Harvey, N. G.; Rose, P. L.; Mirajovsky, D.; Arnett, E. M. J. Am. Chem. Soc. 1990, 112, 3547. (19) Lucassen, J.; Akamatsu, S.; Rondelez, F. J. Colloid Interface Sci. 1991, 144, 434. (20) Heath, J. G.; Arnett, E. M. J. Am. Chem. Soc. 1992, 114, 4500. (21) Rolandi, R.; Dante, S.; Gussoni, A.; Leporatti, S.; Maga, L.; Tundo, P. Langmuir 1995, 11, 3119.

Figure 1. Detail of the Wilhelmy film balance modified for measurement of surface shear viscosity. The monolayer is allowed to flow 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 optimization of the canal width and monolayer flow is included as a function of the monolayer composition (for details see the text). 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 was calculated from the rate of film flow Q by an simplified equation (eq 1), which is analogous to the Poiseuille equation for the flow of liquids through capillary tubes.22 Because we are only interested in the comparison of surface shear viscosities for monoglycerides and proteins, the classical correction17,18,20,21 for the drag of the underlying viscous liquid (Wcη0/π) was not included in the viscosity calculations (η0 is the viscosity of the subphase liquid).

ηs )

(π2 - π1)Wc3 12QL

(1)

This equation only is valid if the canal is narrow, if L . Wc, if the walls are smooth and parallel with no slip of the film along them, and if the flow is Newtonian (independent of the shear rate) and the viscosity is constant within the canal. 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 under steady-state conditions as ∆π tends to a plateau during the flow of the monolayer. The time required to achieve a constant ∆π value depended on the monolayer as will be discussed later. An advantage of our instrument as compared with other devices17,18,20,21 is that it is possible to obtain steady-state conditions and a constant value of ∆π during the flow of the monolayer through the canal within the time of the experiment, due to the movement of two barriers in the same direction. Before each experiment 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. The experiments have demon(22) Harkins, W. D.; Kirkwood, J. G. Nature (London) 1938, 141, 38.

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strated the sensitivity of the interfacial shear viscosity (ηs) of monoglyceride and protein films at the air-water interface, as a function of surface pressure. In fact, ηs was higher for proteins than for monoglycerides. The high values of ηs for protein films introduce important difficulties in the experiments. Thus, an optimization of Wc and Q is necessary in order to obtain the flow of the protein film through the canal. Thus, in contrast to the opinion of other authors,1,2 the flow originated by the movement of two barriers in the same direction makes it possible to apply our instrument to the analysis of protein monolayers. This point will be discussed more deeply in the next section. The data presented in this work were obtained after a minimum of three measurements, and the repeated results prove the reproducibility of the method. A problem here is that the surface shear viscosity values given in various references1,2,6,13 are difficult to compare with each other because emulsifier films show different behavior as a function of the applied shear stress. Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on the same fully automated Wilhelmy-type film balance, as described elsewhere.8,9 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 mean deviation was within (0.1 mN/m for surface pressure and (0.125 × 10-3 m2/mg for area. The subphase temperature was controlled 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. Brewster Angle Microscopy. For microscopic observation of the monolayer structure, a Brewster angle microscope, BAM 2 plus (NFT, Germany), was used as described elsewhere.23,24 The BAM was located at the exit of the canal. To measure the relative thickness of the film, I ) Cδ2, where C is a constant and δ is the film thickness, a previous camera calibration is necessary in order to determine the relationship between the gray level (GL) and the relative reflectivity (I), according to a procedure described previously.23,24 Finally, another important advantage of our instrument is that it allows the application of different devices (Wilhelmy-type film balance, Brewster angle microscopy, and surface dilatational rheology) to the same monolayer without the use of any probe that can introduce contamination in the monolayer and makes possible the characterization the monolayer under shear conditions and at equilibrium, at a macroscopic and at a microscopic scale. The flow visualization using BAM has been utilized recently by other authors.25-28

Results and Discussion Structural, Topographical, and Surface Shear Characteristics of Monopalmitin Monolayers at the Air-Water Interface. At a macroscopic level, results derived from π-A isotherms (data not shown) are in good agreement with those obtained in the same unmodified Wilhelmy-type trough8 and in the Langmuir-type trough with the same monoglyceride.29 Briefly, different structures can be deduced for a monopalmitin monolayer as a function of surface pressure. Liquid expanded (LE) phase (at π < 5 mN/m), liquid-condensed (LC) structure (at 5 < π < 32 mN/m), solid (S) structure (at 32 < π < 53 mN/m), and, finally, collapse at a surface pressure of about 53.1 mN/m were observed. (23) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 2484. (24) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Food Hydrocolloids 1999, 13, 401. (25) Ivanova, A.; Kurnaz, M. I.; Schwartz, D. K. Langmuir 1999, 15, 4622. (26) Ivanova, A.; Schwartz, D. K. Langmuir 2000, 16, 9433. (27) Igne´s-Mullol, J.; Schwartz, D. K. Phys. Rev. Lett. 2000, 85, 1476. (28) Igne´s-Mullol, J.; Schwartz, D. K. Nature 2001, 410, 348. (29) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 2484.

Rodrı´guez Patino and Carrera Sa´ nchez

At a microscopic level, BAM images (Figure 2) reveal that monopalmitin monolayers present a rich polymorphism as a function of surface pressure. In fact, a homogeneous LE phase is present during compression at π < 5 mN/m (data not shown). The morphology of the monopalmitin monolayer at π > 5 mN/m (Figure 2Aa) shows circular LC domains from the homogeneous ambient phase with an LE structure. The LC domains grow in size, and the monolayer is covered with LC domains as the surface pressure is increased (Figure 2Ab). At the highest surface pressure, the LC domains are so closely packed that they occupy the entire field of view and the contrast vanishes suddenlysa typical morphology of a solid structure (Figure 2Ac). Finally, at the collapse point the presence of monolayer fractures can be observed (Figure 2Ad). The evolution with the monolayer compression of the relative thickness (δ) gives complementary information about the topographical and structural characteristics of monopalmitin monolayers during compression and flow (Figure 2B). It can be seen that δ increases as the monolayer is compressed, passes through a maximum at the end of the LC structure, and then decreases at the collapse point. The δ peaks are observed as the circular monopalmitin domains with LC structure pass through the spot where this measurement is performed denoting a heterogeneity in the monolayer structure at a microscopic level. The δ peaks increased in frequency during the compression of the monolayer with LC domains, especially at the beginning of the LE f LC transition as the LC are smaller and more numerous. Finally, the δ peaks vanish at higher surface pressures as the monolayer adopts an isotropic solid structure. Surface shear characteristics of monopalmitin monolayers have been determined at different operational conditions, including the effect of the width of the canal (Wc) and the flow of the monolayer (Q), as a function of the surface pressure (π). That is, an optimization of Wc and Q was necessary in order to obtain the best flow conditions of the monolayer through the canal. At π < 20 mN/m (Figure 2C1) we have observed that the monolayer flows through the canal even at the higher values of Q (3.75 × 10-5 m2‚s-1). However, at higher surface pressures (at π > 30 mN/m) the flow of the monolayer requires (Figure 2C) the use of a higher width of the canal (Wc ) 2 mm) and lower values of Q (3.75 × 10-6 to 6.25 × 10-6 m2‚s-1). The values of ηs did not depend on the flow Q within the experimental error ((0.002 mPa‚s‚m), which is an indication that monopalmitin monolayers display a Newtonian behavior. The time evolution of ∆π during the flow of a monopalmitin monolayer through the canal is shown in Figure 3. As can be seen, the ∆π-time curves depend on the monopalmitin flow through the canal (Q) and the surface pressure (that is, on the monolayer structure). At higher values of Q and surface pressure, the ∆π-time curves develop a maximum, then ∆π decreases with time before the ∆π steady-state value is attained. The maximum was higher and the decrease of ∆π after the maximum was steeper as the value of Q increased. The maximum disappears as both the flow and the surface pressure decrease. These phenomena were also described by other authors in relation to stress-strain curves for proteins using a Couette-type interfacial viscometer.30,31 According to these authors the maximum can be associated to the (30) Izmailova, V. N. Prog. Surf. Membr. Sci. 1979, 13, 141. (31) Martin, A.; Bos, M.; Cohen Stuart, M.; van Vliet, T. Langmuir 2002, 18, 1238.


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Figure 2. (A) Visualization of monopalmitin monolayers by BAM: (a) coexistence of LE (dark regions) and LC (bright region) domains at π < 30 mN/m, (b) LC domains at π < monolayer collapse, (c) solid domains at π = monolayer collapse, and (d) fractures in a collapsed monolayer. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm. (B) Relative thickness of monopalmitin monolayers as a function of surface pressure. (C) The surface pressure dependence of surface shear viscosity for monolayers of monopalmitin spread at the air-water interface at 20 °C and at pH 7. To optimize the sensibility of the method in figures C and insert C1 and C2 different width of canal (Wc, mm) and monopalmitin flow through the canal are used. Q (m2‚s-1): ()) 3.75 × 10-6, (0) 6.25 × 10-6, (O) 1.25 × 10-5, (4) 2.5 × 10-5, and (3) 3.75 × 10-5. The lines are drawn to help the reading (for details see the text).

strength of the monolayer whereas the decrease in ∆π after the maximum may represent a structure breakdown of the monopalmitin monolayer under shear, similar to the overshoot observed with tixotropic fluids in bulk rheology. The breaking of LC domains was evident at a microscopic level by means of BAM (Figure 2A). In fact, the size of the LC domains was lower under shear than with the open canal (data not shown). But, we have also observed that there exists a deformation of the domains during the shear or even a deformation and alignment of fractures

of the monopalmitin collapsed monolayer (Figure 2Ad). The flow-induced orientational alignment has recently been the topic of abundant research in a variety of spread monolayers.25-28,32,33 The intensity of flow (Figure 3A) and the surface pressure (Figure 3B) did increase the breaking and deformation of LC monopalmitin domains, and thus the overshoot in the ∆π-time curves was more evident. (32) Murayama, T.; Fuller, G.; Frank, C.; Robertson, C. Science 1996, 274, 233. (33) Murayama, T.; Lauger, J.; Fuller, G.; Frank, C.; Robertson, C. Langmuir 1998, 14, 1836.

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Figure 3. Difference in surface pressure between the limits of the canal during the flow of monoglyceride monolayers spread at the air-water interface, at 20 °C and at pH 7. (A) Effect of monopalmitin flow through the canal. Q (m2‚s-1): (s) 6.25 × 10-6, (- - -) 1.25 × 10-5, (‚ ‚ ‚) 2.5 × 10-5, and (- ‚ ‚ -) 3.75 × 10-5 at π ) 20 mN/m and Wc ) 1.5 mm. (B) Effect of monopalmitin surface pressure: (s) 10 mN/m, Q ) 2.5 × 10-5 m2‚s-1, and Wc ) 1.5 mm, (- - -) 20 mN/m, Q ) 2.5 × 10-5 m2‚s-1, and Wc ) 1.5 mm, and (‚ ‚ ‚) 30 mN/m, Q ) 1.25 × 10-5 m2‚s-1, and Wc ) 2.0 mm. (C) Effect of monoolein flow through the canal at 20 mN/m and at Wc ) 1.5 mm, Q (m2‚s-1) (- ‚ - ) 3.75 × 10-6, (s) 6.25 × 10-6, (- - -) 1.25 × 10-5, (‚ ‚ ‚) 2.5 × 10-5, and (- ‚ ‚ -) 3.75 × 10-5.

Interestingly, at very slow monolayer flow or as the size of the LC domains of monopalmitin became smaller (at low surface pressures), the maximum disappeared (Figure 3A,B). 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 monopalmitin monolayer domains along the canal. The fluctuations in the steady-state ∆π value may be due to friction between LC domains or between regions of fractured monopalmitin at the higher surface pressures, both of these phenomena denoting the heterogeneity of the monolayer.


For monopalmitin (Figure 3A,B) the steady state in ∆π was reached relatively quickly. From the ∆π at steady state, the surface shear viscosity (ηs) was determined by means of eq 1. In Figure 2C ηs is given for monopalmitin monolayers as a function of surface pressure. There is a general tendency (with exceptions) for ηs to increase as a function of surface pressure, as might be expected on the basis of greater intermolecular interactions. There are significant differences between different monolayer structures as a function of surface pressure. At surface pressure lower than 30 mN/m, as the monolayer is in the transition between LE and LC structures, we observed a very small value of ηs. At higher surface pressures a steep increase in ηs was observed which is consistent with a more condensed and thicker monolayer with a developed LC structure. The plateau in the surface pressure evolution of ηs coincided with the monopalmitin solid structure near to the collapse point. Clearly, the surface shear characteristics of a monopalmitin monolayer are directly linked with their structural and topographical characteristics. The results in Figure 2 also show the extremely sensitive dependence of the surface shear viscosity on the monolayer structure. In fact, the value of ηs was 17.5 times higher for a solid than for a LC monopalmitin monolayer structure. In addition the value of ηs was more than seven times higher at the beginning than at the end of the LE to LC transition. That is, as the interactions between monopalmitin molecules at the air-water interface are at a maximum, with a solid structure in the monolayer, the value of ηs is higher than that with the minimum interactions between monopalmitin molecules, with a liquid-expanded structure in the monolayer. The same behavior has been observed for other surfactants.20,34,35 The effect of temperature on ηs is shown in Figure 4A. In these experiments the highest width of the canal (Wc ) 2 mm) for a constant flow (Q ) 6.25 × 10-6 m2‚s-1) was used at the higher surface pressures and lower temperatures. As expected, a significant decrease in ηs was observed at the higher temperatures, especially at the higher surface pressures. As the temperature increases, the interactions between the molecules at the interface decrease and a more expanded structure is observed in the monopalmitin monolayer,36 which explains the lower values of ηs. Structural, Topographical, and Surface Shear Characteristics of Monoolein Monolayers at the Air-Water Interface. Monoolein monolayer (Figure 5) presents only the liquid expanded structure and collapses at the equilibrium surface pressure (πe ≈ 45.7 mN/m). BAM images corroborate that only the homogeneous LE phase is present during the compression of a monoolein monolayer (Figure 5Aa) up to the collapse point (Figure 5Ab). From the observation with BAM along the film balance, no fractures were visualized after the monoolein collapse, which demonstrates that the collapse of monopalmitin and monoolein monolayers is quite different and these differences are associated with the homogeneous LE structures of monoolein at the surface pressures corresponding to πe.36 The evolution of the relative thickness (δ) for monoolein monolayers with monolayer compression (Figure 5B) also shows important differences from monopalmitin monolayers (Figure 2B). It can be seen that δ increases monotonically with monolayer compression, but in con(34) Peng, J. B.; Barnes, G. T.; Abraham, B. M. Langmuir 1993, 9, 3574. (35) Sacchetti, M. Yu, H. Zografi, G. Langmuir 1993, 9, 2168. (36) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M.; Carrera, C.; Cejudo, M.; Garcıá, J. M. Chem. Eng. Commun. 2003, 190, 15.


Figure 4. The temperature dependence of surface shear viscosity for monolayers of (A) monopalmitin and (B) monoolein spread at the air-water interface at 20 °C, at pH 7, and different surface pressures (mN/m): (0, 9) 10, (O, b) 20, (4, 2) 30, and (3, 1) 40. Monoglyceride flow through the canal 6.25 × 10-6 m2‚s-1. Width of canal 1.5 mm (open symbols) and 2.0 mm (full symbols). The lines are drawn to help the reader (for details see the text).

trast to monopalmitin monolayers no discontinuity was observed which corroborates the fact that during the compression a denser monoolein film is formed but without any change in its structure. The time evolution of ∆π during the flow of a monoolein monolayer through the canal is shown in Figure 3C. As can be seen the ∆π-time curves depend on the monoolein flow through the canal (Q) and the surface pressure (data not shown). However, in contrast with monopalmitin monolayers, (i) the ∆π-time curves did not develop any maximum, (ii) the steady state in ∆π was reached more quickly for monoolein than for monopalmitin monolayers, and (iii) at the steady state the fluctuations in ∆π value observed for monopalmitin (Figure 3A,B) were absent in monoolein monolayers (Figure 3C). These results corroborate the opinion that both the friction between LC domains and the heterogeneity of the monolayer are the main causes of the evolution of ∆π with time during the flow of a monoglyceride monolayer through the canal. From the ∆π at steady state, the surface shear viscosity of a monoolein monolayer was determined (Figure 5C). Over the overall range of surface pressures analyzed, the values of ηs did not depend on the flow Q within the experimental error ((0.002 mPa‚s‚m), which is an indication that monoolein monolayers display a Newtonian behavior. However, the values of ηs were lower at the lower width of the canal, especially at the higher surface pressures (data not shown). The same behavior was observed for other surfactants37 and was attributed to the “meniscus effect” in narrower canals. For a narrow canal (37) Hu¨hnerfuss, H. J. Colloid Interface Sci. 1985, 107, 84.

Langmuir, Vol. 20, No. 11, 2004 4535

Figure 5. (A) Visualization of monoolein monolayers by BAM: (a) homogeneous LE structure, (b) monolayer collapse. (B) Relative thickness of monoolein monolayers as a function of surface pressure. (C) The surface pressure dependence of surface shear viscosity for monolayers of monoolein spread at the airwater interface at 20 °C and at pH 7. Width of canal Wc ) 1.5 mm. Monoolein flow through the canal, Q (m2‚s-1): ()) 3.75 × 10-6, (0) 6.25 × 10-6, (O) 1.25 × 10-5, (∆) 2.5 × 10-5, and (∇) 3.75 × 10-5. The lines are drawn to help the reader (for details see the text).

the profile of a meniscus approximates a semicircle and, as a consequence, the effective canal width available for transportation of the monolayer through the canal is given by Weff ) (π/2)Wc instead of Wc. If this Weff is included in eq 1, the values of ηs at Wc ) 1 mm and at Wc ) 1.5 mm are practically the same within the experimental error. Figure 5C shows the surface pressure dependence of ηs for monoolein monolayers. It can be seen that over the overall range of surface pressures analyzed, the values of ηs were very small and that this value practically did not depend on the surface pressure. The low values of ηs measured for a monoolein monolayer with the canal viscometer deserve mentioning because these measurements cannot be performed with direct methods. Interestingly, these values of ηs were very similar to those for monopalmitin monolayers in the LE-LC phase transition at π < 20 mN/m (Figure 2C1). These results confirm the extremely sensitive dependence of surface shear characteristics on the monolayer structure. That is, the ηs values of a monoglyceride (monopalmitin or monoolein) monolayer are directly linked with their structural and topographical characteristics (Figures 2 and 5). The lower values of ηs were observed for a liquid-expanded structure of the monolayer, coinciding with a more homogeneous flow of the monolayer through the canal even at the higher values of Q (Figure 3). The temperature dependence of ηs for monoglyceride monolayers (Figure 4) strengthens this hypothesis. In fact,

4536


for a monoolein monolayer an increase in temperature produces a small expansion of the monolayer but with a constant LE structure,36 which explains the lack of dependence of ηs on temperature within the experimental error ((0.002 mPa‚s‚m) (Figure 4B). This strengthens the hypothesis that there exists a correlation between the surface shear rheology and the generalized phase diagram.36 In addition, the evolution of ηs with temperature is different in the various monolayer structures. They vary with temperature in the LC structure (Figure 4A) but are constant in the LE structure (Figure 4B). Structural, Topographical, and Surface Shear Characteristics of β-Casein Monolayers at the AirWater Interface. The results of π-A isotherms (data not shown) confirm that a β-casein monolayer at the airwater interface adopts two different structures and the collapse phase.36,38 Results of the relative film thickness as a function of surface pressure (Figure 7B) confirm that at low surface pressures (π < 12-14 mN/m) β-casein molecules exist with a tail-train structure with amino acid segments located at the interface. At higher surface pressures, and up to the collapse point, amino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails. The residues of β-casein molecules at the air-water interface appeared to be of uniform thickness (Figure 7B) and isotropy (Figure 7 Aa). The relative thickness (Figure 7B) increased with the surface pressure and is a maximum at the collapse point, at the higher surface pressures. At the collapse point we can observe the shear effect on the topography of residues of collapsed β-casein. In fact, the folds (Figure 7 Ab) or collapsed protein (Figure 7 Ac) appeared aligned in the direction of the flow due to the effect of the shear on β-casein monolayer. The time evolution of ∆π during the flow of a β-casein monolayer through the canal (Figure 6A) shows similar characteristics to that observed for a monopalmitin monolayer (Figure 3). Briefly, the ∆π-time curves depend on the β-casein flow through the canal and the surface pressure. At higher surface pressure the ∆π-time curves develop a maximum, then ∆π decreases with time before the ∆π steady-state value is attained. Thus, the same reasoning as above can be applied here. After the overshoot in the ∆π-time curves, fluctuations in the steady-state ∆π value were also observed. However, for a β-casein monolayer these fluctuations were more extended in time (Figure 6A) than those for monopalmitin (Figure 3) and may be associated with the flow of large folds or regions of collapsed proteins through the canal instead of the presence of film heterogeneitiessassociated with the presence of LC domains or interfacial fractures, as observed for collapsed monopalmitin monolayers at the higher surface pressures (Figure 2A). An optimization of Wc and Q was necessary in order to obtain the best flow conditions of a protein monolayer through the canal. In fact for β-casein monolayer higher values of Wc (1.5 mm) and lower values of Q (1.25 × 10-5 to 6.25 × 10-6 m2‚s-1) were necessary in order to obtain more reproducible data (Figure 7C). Over the overall range of surface pressures, the values of ηs did not depend on the flow Q within the experimental error ((0.002 mPa‚s‚m), especially for a canal width of 1.5 mm. In this regard, after an optimization of the method we did not detect the experimental difficulties observed by other authors39-42 as the shear characteristics of protein adsorbed films were determined by means of Couette-type interfacial rheometers. (38) Horne, D.; Rodrı´guez Patino, J. M. In Biopolymers at Interfaces, 2nd ed.; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; p 857.


Figure 6. Difference in surface pressure between the limits of the canal during the flow of (A) β-casein and (B and C) WPI monolayers spread at the air-water interface, at 20 °C and at pH 7. (A) Effect of surface pressure on β-casein flow through the canal: (s) 5 mN/m, Q 3.75 × 10-6 m2‚s-1, and Wc 0.9 mm; (- - -) 10 mN/m, Q 6.25 × 10-6 m2‚s-1, and Wc 0.9 mm; (‚ ‚ ‚) 15 mN/m, Q 6.25 × 10-6 m2‚s-1, and Wc 1.5 mm, and (- ‚ ‚ -) 20 mN/m, Q 3.75 × 10-6 m2‚s-1, and Wc 1.5 mm. (B) Flow of WPI through the canal at 10 mN/m: (s) Q 3.75 × 10-6 m2‚s-1 and Wc 1.5 mm; (- - -) Q 6.25 × 10-6 m2‚s-1, and Wc 1.5 mm. (C) Flow of WPI through the canal at 20 mN/m: (- - -) Q 1.25 × 10-6 m2‚s-1 and Wc 2.0 mm; (‚ ‚ ‚) Q 1.25 × 10-6 m2‚s-1 and Wc 2.5 mm.

The time required to obtain steady-state values in ∆π was significantly higher for β-casein (Figure 6) than for (39) Castle, J.; Dickinson, E.; Murray, B. S. In Proteins at Interfaces; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series, American Chemical Society: Washington, DC, 1987. (40) Dickinson, E.; Murray, A.; Murray, B. S.; Stainsby, G. In Food Emulsions and Foams, Dickinson, E., Ed.; Royal Society of Chemistry: London, 1987.


Langmuir, Vol. 20, No. 11, 2004 4537

Figure 8. (A) Relative thickness of a caseinate monolayer as a function of surface pressure. (B) The surface pressure dependence of surface shear viscosity for monolayers of κ-casein (full symbols) and caseinate (open symbol) spread at the airwater interface at 20 °C and at pH 7. Flow through the canal: (0, 9) 3.75 × 10-6 m2‚s-1; (3, 1) 6.25 × 10-6 m2‚s-1. The lines are drawn to help the reader. Figure 7. (A) Visualization of β-casein monolayers by BAM: (a) homogeneous LE structure at π lower than that for monolayer collapse; (b and c) monolayer collapse. (B) Relative thickness of a β-casein monolayer as a function of surface pressure. (C) The surface pressure dependence of surface shear viscosity for monolayers of β-casein spread at the air-water interface at 20 °C and at pH 7. Symbols (open, Wc 1.5 mm; full, Wc 0.9 mm): (0, 9) 3.75 × 10-6 m2‚s-1; (O, b) 6.25 × 10-6 m2‚s-1; (4, 2) 1.25 × 10-5 m2‚s-1. The line is drawn to help the reader.

monoglycerides (Figure 3). From the ∆π at steady state, the surface shear viscosity was determined as a function of surface pressure (Figure 7C). The values of ηs increased with surface pressure, especially as the residues of β-casein adopt the conformation of loops and tails at the air-water interface. At surface pressures higher than about 10 mN/m, the charged section of the β-casein molecule (approximately 50 residues from N-terminal end) is displaced from the interface and extends into the subphase. This may cause an increase in the viscous drag caused by the subphase. The data presented here highlight the sensitivity of shear rheology to intermolecular interactions, something that dilatation and topography are not very sensitive to. Over the overall range of surface pressures analyzed, the values of ηs were higher for β-casein than for monoglycerides. We speculate that the higher interactions and probably the flow of large regions (unfortunately we cannot observe the morphology of homogeneous β-casein domains by BAM) of β-casein with loops and tails conformation may have an effect on the higher values of ηs in relation to those observed for monopalmitin monolayers with crystalline LC or solid structure (Figure 2).

Interestingly, the ηs value continues to rise, even as the monolayer is saturated by the protein at the collapse point. The same behavior was observed by Dickinson et al.41,43 for globular proteins adsorbed at the oil-water interface and was attributed to interfacial polymerization via disulfide linkage. As β-casein has no potential for covalent cross-linking, the phenomenon observed here may be related to the formation of a physical gel stabilized by intermolecular hydrogen bonds, leading to the formation of aggregated β-sheets.38,44 Structural, Topographical, and Surface Shear Characteristics of Caseinate and K-Casein Monolayers at the Air-Water Interface. The structural, topographical, and flow conditions (∆π-time curves) of caseinate are essentially the same as those observed for β-casein monolayers. In fact, caseinate monolayers at low surface pressures (π < 15 mN/m) adopt the conformation of tails and trains with amino acid segments located at the interface. At higher surface pressures, and up to the monolayer collapse, the amino acid segments are extended into the underlying aqueous solution and adopt the form of loops and tails. The regions of caseinate at the air-water interface appeared to be of uniform thickness (Figure 8A) and isotropy (data not shown), except at the collapse point where folds and collapsed protein appeared aligned in the direction of the flow as for β-casein monolayers (Figure 7A). The relative thickness (Figure 8A) increased with the surface pressure and is a maximum at the collapse point. However, for the flow of a caseinate monolayer through the canal, a higher value of Wc and lower values of Q were necessary (data not shown) in

(41) Dickinson, E. ACS Symp. Ser. 1991, 448, 114. (42) Benjamins, J.; van Voorst Vader, F. Colloids Surf. 1992, 65, 161.

(43) Dickinson, E.; Rolfe, S. E.; Dalgleish, D. G. Int. J. Biol. Macromol. 1990, 12, 189. (44) Bantchev, G. B.; Schawartz, D. K. Langmuir 2003, 19, 2673.

4538


comparison with those used for the flow of β-casein monolayers (Figure 6A). The values of ηs for caseinate monolayers at the airwater interfacesdeduced from the ∆π at steady states increased with the surface pressure (Figure 8B). The value of ηs of caseinate is somewhat higher than that for β-casein under the same conditions. This result was expected but at the same time surprising. In fact, the same behavior was described by other authors.2,6,45,46 However, the higher value of ηs for caseinate as compared with β-casein does not correlate well with the similar monolayer structure of the two proteins at the air-water interface. That is, the significant differences in ηs values between caseinate and β-casein were not observed for other properties of the film such as monolayer structure, topography, dilatational characteristics, and long-term relaxation phenomena.36,38 According to other authors1,2,6 this is probably mainly attributed to the presence of κ-casein in sodium caseinate. To corroborate this explanation, experiments on shear characteristics were performed with pure κ-casein monolayers spread at the air-water interface. The results included in Figure 8B demonstrate that the values of ηs were higher for κ-casein than for caseinate and β-casein, especially at the higher surface pressures as the protein forms typical two-dimensional gels. Due to the cysteine residues (which are not present in the structure of β-casein), κ-casein has the ability to form interfacial protein gel stabilized by covalent disulfide cross-linked networks. The formation of this interfacial gel may be the cause of the higher values of ηs for κ-casein and caseinate monolayers at the higher surface pressures (or surface densities) in comparison to those for β-casein. Thus, the higher values of ηs for caseinate monolayers may be associated with the presence of κ-casein and Rs2-casein, both proteins with cysteine residues in the molecule and the potential for interfacial gelation by cross-linking of disulfide residues and the formation of interfacial aggregates of significant size. The latter phenomenon can explain the lower flow conditions of caseinate and κ-casein (data not shown) in relation to β-casein (Figure 6A). Structural, Topographical, and Surface Shear Characteristics of WPI Monolayers at the Air-Water Interface. The differences in solution structure between β-casein, a flexible protein lacking secondary structure, and β-lactoglobulin (the main component of WPI), a globular protein with a compact conformation, are reflected in their adsorption behavior.38 Upon adsorption or spreading, WPI retains elements of the native structure, not fully unfolded at the interface. Thus, most amino acid residues in WPI adopt loop conformation at the air-water interface. But the loop conformation is more condensed at higher surface pressures and is displaced toward the bulk phase at the collapse point. These data are in agreement with those deduced for globular proteins.47 Results of BAM (topography and relative thickness) as a function of surface pressure (Figure 9A) obtained with WPI monolayers show the same structural characteristics as those deduced from the π-A isotherms (data not shown). The residues of WPI molecules at the air-water interface appeared to be of uniform reflectivity, suggesting homogeneity in thickness and film isotropy. The relative thickness (Figure 9A) increases with the surface pressure and is a maximum at the collapse point, at the highest surface pressure. (45) Dickinson, E.; Murray, B. S.; Stainsby, G.; J. Colloid Interface Sci. 1985, 106, 259. (46) Dickinson, E.; Rolfe, S. E.; Dalgleish, D. G. Food Hydrocolloids 1989, 3, 193. (47) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427.


Figure 9. (A) Relative thickness of a WPI monolayer as a function of surface pressure. (B) The surface pressure dependence of surface shear viscosity for monolayers of WPI spread at the air-water interface at 20 °C and at pH 7. Flow through the canal: (0) 3.75 × 10-6 m2‚s-1; (O) 6.25 × 10-6 m2‚s-1; (4) 1.25 × 10-5 m2‚s-1. The line is drawn to help the reader.

The flow of a WPI monolayer through the canal is even more difficult than for disordered proteins (β-casein, κ-casein, and caseinate). Thus, for the flow of a WPI monolayer through the canal the higher values of Wc and lower values of Q were necessary (Figure 6B,C). In addition, the time to attain the maximum value of ∆π, the extent of the overshoot, and the level of the fluctuations in ∆π after the overshoot were higher for WPI than for monoglycerides (Figure 2) and disordered proteins (Figure 6A). The value of ηs for WPI is somewhat higher than those for disordered proteins (β-casein, κ-casein, and caseinate) under the same experimental conditions. The same behavior was reported by other authors.39,48,49 The high values of ηs for WPI correlate well with the capacity of WPI for interfacial gelation. Even though the phenomenon is not well understood,50-53 the interfacial gelation of globular proteins may possess great technological importance. The interfacial gelation by cross-linking of disulfide residues in WPI films and the formation of interfacial aggregates of significant sizes may be the causes of the high values of ηs (Figure 9B) and the fluctuations in ∆π during the flow of the monolayer though the canal (Figure 6B,C). That is, in contrast with individual molecules, interfacial WPI gels present a high shear (48) Dickinson, E.; Matsumura, Y. Int. J. Biol. Macromol. 1991, 13, 26. (49) Benjamins, J. Static and Dynamic Properties of Proteins Adsorbed at Liquid Interfaces. PhD Dissertation, Wageningen University: Wageningen, The Netherlands, 2000. (50) Ball, A.; Jones, R. A. L. Langmuir 1995, 11, 3542. (51) Green, R. G.; Hopkinson, I.; Jones, R. A. L. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; The Royal Society of Chemistry: Cambridge, 1999; p 285. (52) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. J. Agric Food Chem. 1999, 47, 3640. (53) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C.; Navarro, J. M.; Rodrı´guez, G.; Cejudo, M. Colloids Surf., B 2001, 21, 87.


resistance during their flow through the canal.54 The reversible gels formed upon compression and flow of WPI monolayers deserves mentioning. As the WPI monolayer flows repeatedly through the canal, its structural, topographical, and shear characteristics were the same under the same experimental conditions. This trend was different from that observed for thermal WPI gels at the air-water interface.52,53 In summary, the surface shear viscosities of proteins are higher than those of monoglycerides. This is due to lateral interactions between the protein due to hydrogen bonding, hydrophobic and covalent bonding, and/or electrostatic interactions. These interactions between adsorbed protein molecules may vary strongly. Moreover, for a fully packed adsorbed layer (at the higher surface pressures) the deformability of the protein molecules may be an important factor. Therefore, differences between surface shear viscosity data for the various proteins 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. In general, the greater the internal cohesion and structuring of a protein molecule (such as for WPI), the greater the value of ηs. The values of ηs were also higher for proteins that form interfacial gels by cross-linking of disulfide residues and the formation of interfacial aggregates of significant size (WPI, κ-casein, and caseinate, in this order) as compared with β-casein that only can form physical gels stabilized by intermolecular hydrogen bonds. 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. In every monolayer (those of proteins or monoglycerides), due to their sensitivity to interactions between the adsorbed monolayer molecules and the possible structural changes involved, ηs values change as a function of the surface pressure.

Langmuir, Vol. 20, No. 11, 2004 4539

Conclusions

shear conditions of monoglyceride (monopalmitin and monoolein) and milk protein (β-casein, κ-casein, caseinate, and WPI) spread monolayers at the air-water. Combined surface chemistry (surface film balance and surface shear rheometry) and microscopy (Brewster angle microscopy, BAM) techniques have been applied in this study to pure emulsifiers (proteins and monoglycerides) spread at the air-water interface. Combined π-A isotherms, and especially BAM measurements (monolayer topography and thickness), on the same monolayer under shear conditions provide complementary information on the monolayer flow, and thus enable a global consistency check. To study the shear characteristics of spread films, a canal viscometersan analogue of the conventional Ostwald viscometerswas used. An optimization of the width of the canal (Wc) and flow of the monolayer through the canal (Q) were necessary in order to obtain the best conditions of flow of the monolayer (Figure 1). The experiments have demonstrated the sensitivity of the interfacial shear viscosity (ηs) to 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. This technique makes it possible to distinguish between β-casein, κ-casein, and caseinate spread films, with the higher ηs values for κ-casein, and the lower ηs values for β-casein. The higher ηs values were observed for WPI monolayers. The ηs varies greatly with the surface pressure (or surface density) of the emulsifier (monoglyceride or protein) at the interface. In general, the greater the surface pressure (i.e., at the higher surface density), the greater the values of ηs. Finally, the ηs value is also sensitive to the monolayer structure, as was observed for monoglycerides with a rich 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 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 protein-monoglyceride mixed films at the air-water interface. This work is under way at present and will be presented in a future paper.

In this contribution we have studied the structure, topography, and surface rheological characteristics under

Acknowledgment. The authors acknowledge the support of CICYT thought Grant AGL2001-3843-C02-01.

(54) Wijmans, C. M.; Dickinson, E. Langmuir 1998, 14, 7278.

LA036255I

Structural, Topographical, and Shear Characteristics of Milk Protein

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