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Determination of Fracture Stresses of Adsorbed Protein Layers at Air-Water Interfaces M. A. Bos,†,‡ K. Grolle,† W. Kloek,†,§ and T. van Vliet*,† Wageningen Centre for Food Sciences, P.O. Box 557, 6700 AN Wageningen, The Netherlands, and TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received July 29, 2002 Proteins are widely used for the physical stabilization of emulsions and foams. Stabilization strongly depends on the interfacial properties of the proteins. In many cases likely a kind of interfacial gel layer is formed. This paper deals with fracture properties of such interfacial gelled protein layers as studied by the overflowing cylinder technique. In the first part of the paper, equations are presented for the calculation of the fracture stress of these protein layers from experimental data. This is illustrated in the second part of this paper for ovalbumin, glycinin, β-casein, and β-lactoglobulin. Obtained fracture stresses are on the order of 2 × 105 Pa and dependent on the type of protein. Protein properties such as conformation seem to be important aspects, determining the height of the fracture stress. Although interfacial protein gels are fracturable, relaxation phenomena within the gelled protein layers are also important.
Introduction The occurrence of stagnant surfaces, surfaces which do not move irrespective of flow of the liquid below, is a wellknown phenomenon in a lot of practical systems such as natural water, milk, and beer. When a liquid is flowing through an open canal and the surface meets an obstacle, often a stagnant layer can be observed upstream of it. It is unclear whether such a layer is caused by surface tension gradients or by a kind of skin at the surface, which has rheological properties that can stop the surface movement. Proteins are known to form stagnant surface layers, which is presumably due to their ability to form network structures at the interface.1,2 Stagnant surface layers have been studied using the canal method1 and with the overflowing cylinder technique.3-5 Elastic and viscoelastic phenomena of these stagnant layers have been measured. Prins et al.3 and Prins5 attributed the occurrence of these stagnant layers to lateral interactions between adsorbed molecules. They also concluded that interfacial gel layer formation was promoted by compression of the interface. The latter probably relates to the irreversible character of protein adsorption. In the case of adsorbed proteins, a twodimensional network of protein molecules, often called protein gel layer or protein skin, may be formed. According to Prins,5 random coil proteins are less able to form interfacial gel layers than globular proteins. Stagnant surface behavior has consequences for foaming and emulsifying behavior.3,4,6 Stagnant bubble surfaces * Corresponding author. † Wageningen Centre for Food Sciences. ‡ TNO Nutrition and Food Research Institute. § Present affiliation: DMV International, P.O. Box 13, 5460 BA, Veghel, The Netherlands. (1) Prins, A.; Jochems, A. M. P.; van Kalsbeek, H. A. I. K.; Boerboom, J. F. G., Wijnen, M. E.; Williams, A. Colloid Polym. Sci. 1996, 100, 321. (2) van Aken, G. A.; Merks, M. T. E. Colloids Surf., A: Physicochem. Eng. Aspects 1996, 114, 221. (3) Prins, A.; Boerboom, F. J. G.; van Kalsbeek, H. K. A. I. Colloids Surf., A: Physicochem. Eng. Aspects 1998, 143, 395. (4) Prins, A.; Bos, M. A.; Boerboom, F. J. G.; van Kalsbeek, H. K. A. I. In Proteins at interfaces; Miller R. Mo¨bius, D., Ed.; Elsevier Science B. V.: Amsterdam, 1998; Chapter 5. (5) Prins, A. Colloids Surf., A: Physicochem. Eng. Aspects 1999, 149, 647. (6) Williams, A.; Prins, A. Colloids Surf., A: Physicochem. Eng. Aspects 1996, 114, 267.
slow the drainage of liquid out of foam. The viscous resistance of the liquid that moves downward through thin films will be at maximum when the surfaces of the bubbles are motionless.4 Williams and Prins6 and Williams et al.7 found for an aqueous droplet in oil that break up of the droplet in shear flow took place more efficiently when the surface of the droplets was more stagnant, i.e., had a higher surface dilational modulus. As a result, droplets with a smaller average droplet diameter were formed, which will influence the long-term stability of the emulsion. Furthermore, it is expected that stagnant surface layers might play a role in the coalescence process of oil droplets, either spontaneous coalescence due to Brownian motion or coalescence induced by shear.8 In the present work, a method to determine the fracture stress of the stagnant layers by using the overflowing cylinder is described. Results for ovalbumin, glycinin, β-casein, and β-lactoglobulin are discussed. Theoretical The overflowing cylinder is an apparatus in which a liquid is subjected to continuous radial expansion at an interface. The contained liquid is pumped around, causing it to flow over the rim of the inner cylinder (see Figure 1). The flow of the liquid along the surface, which is in radial direction and laminar, and the stresses in the surface are coupled. Two situations can be distinguished: (1) the liquid drives the surface and (2) the surface accelerates the liquid just below it. Both situations are illustrated in Figure 1. Mechanical equilibrium is ensured, which means that the shear stress excerted by the viscous liquid on the surface is fully compensated by the surface tension gradient acting along the surface
( )
dvx dγ )η dx dz
z)0
(1)
where γ represents the surface tension (N m-1), η the viscosity of the liquid (Pas), and vx the liquid velocity in (7) Williams, A.; Janssen, J. J. M.; Prins, A. Colloids Surf., A: Physicochem. Eng. Aspects 1997, 125, 189. (8) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91/3, 437-471.
10.1021/la020675a CCC: $25.00 © 2003 American Chemical Society Published on Web 02/14/2003
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Figure 1. Schematic vertical cross section of the overflowing cylinder apparatus showing the direction of the flow. The top view shows the horizontal expanding surface of the overflowing cylinder and the direction of flow. L is the length of the falling film. The enlargement on the right-hand side of the figure illustrates two distinct situations that may occur. (A) The surface opposes the radial expansion speed of the liquid just below and (B) the surface enhances the radial expansion speed.
Figure 2. Expansion rate of the surface [d(ln A)/dt] of an overflowing cylinder as a function of the length of the falling film (L) for water, an aqueous “soap” solution, and an aqueous protein solution (typical examples; data from ref 4).
the x-direction (m s-1). The x-direction points in the same direction as the liquid velocity, and the z-coordinate points in the direction perpendicular to the top surface, which is situated at z ) 0. In Figure 2 typical figures for the expansion rate of the surface [d(ln A)/dt] as a function of the length L of the falling film outside the cylinder is given for water, for a low molecular weight surfactant like Teepol, and for a macromolecular surfactant like a protein. The expansion rate depends not on L for pure water, but it does in the presence of surface-active substances. d(ln A)/dt values smaller than for pure water indicate that the surface tension gradient opposes the liquid flow (Figure 1, situation A), whereas d(ln A)/dt data higher than for pure water indicate that the surface tension gradient drives the liquid (Figure 1, situation B). From Figure 2 it appears that low molecular weight surfactants are better at increasing the surface expansion speed than proteins and thus can generate higher surface tension gradients under the conditions studied.9 On the other hand, protein (9) Boerboom, F. J. G. Ph.D. thesis; Proteins and protein/surfactant mixtures at interfaces in motion; Wageningen University, Wageningen, 2000; p 213.
Figure 3. Schematic representation of the forces acting on a protein gel at the liquid-air surface in the overflowing cylinder.
solutions are much better to slow the surface expansion rate than the low molecular weight surfactants. Proteins (or the adsorbed protein layer) may cause the surface to stay almost motionless as a shear stress is excerted on them (it), indicating that they can withstand a stress at the surface generated by the stress excerted by the liquid moving along that surface. Dependent on the magnitude of the stress, a homogeneous three-dimensional protein network (gel) is formed at the surface. One can imagine that by increasing the shear stress (by increasing the flux Q) it is possible to overcome the coherence of the adsorbed layer. This implies that the protein network at the surface (also called protein surface gel) will fracture. The yield stress/fracture stress of the surface protein network can be calculated from the shear stress exerted. To do so, the balance of forces acting on a protein layer on top of the overflowing cylinder has to be considered. Figure 3 gives a schematic representation of the forces and with that the stresses acting on a protein layer at the liquid-air surface in the overflowing cylinder. Three stresses have to be taken into account: first, the shear stress by the flowing liquid on top of the cylinder (σshear); second, the stress generated by the liquid flowing downward along the inner cylinder, assuming Poiseuille flow between two flat plates (inner cylinder and stagnant
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protein gel layer) (σfilm); and, finally, stresses due to interfacial tension gradients (dγ1/dx and dγ2/dz). Assuming that the protein layer is homogeneous, stresses due to surface tension gradients may be neglected. Boerboom calculated these stresses and found them to be on the order of 1-2 Pa.9 Then the forces acting on the protein film are given by
σgel ) σnetwork ) σshear(Q) + σfilm(L)
(2)
Shear Stress on Top of the Cylinder. The shear stress acting on top of the overflowing cylinder caused by the flowing liquid is equal to the shear stress in the liquid directly beneath it, which in turn depend on the velocity gradients in the liquid. The latter, in turn, depends on the flow profile below the surface and the relative rate of expansion of the surface in the case it is either accelerated or slowed-down with respect to a pure liquid. In a first approximation, the flow profile under the surface is supposed to be linear (simple shear) and to depend only on the flux Q, assuming no acceleration or deceleration of the surface. This assumption has been supported by results found for CTAB solutions by Bain et al.10 They found a velocity profile that was almost linear between the top surface and a depth of 1 mm. By changing the flux in the overflowing cylinder, one can vary the tangential shear stress and thus the shear stress acting on the protein film on top of the overflowing cylinder. The shear stress is than given by9,11
σshear ) η
( ) dvx dz
z)0
)η
( ) vx(Q) hdyn
(3)
in which vx(Q) is the radial expansion speed depending on the flux Q, hdyn the hydrodynamic height on the overflowing cylinder (see Figure 3), and η the viscosity of the liquid. Here it is assumed that in the steady-state situation dvx/ dz is constant and that the liquid flow below the surface has a thickness equal to hdyn. The hydrodynamic height hdyn is assumed to be independent of Q. In practice, this is true for pure water, but for surfactant systems it is not true.9 Bergink-Martens11 determined hdyn for water and for a surfactant solution as a function of Q. For our calculations, we will use an average value of 4 × 10-3 m for hdyn. Bain et al.10 showed that the thickness of the liquid flow below the surface was on the order of 3 mm for water and CTAB solutions. Therefore, our assumption that the liquid flow below the surface has a thickness equal to hdyn is justified. Bergink-Martens11 has derived an equation for the radial expansion speed vx for pure water. It was found that vx increases linearly with the flux in the overflowing cylinder used (see Figure 5b)
r r vx(Q) ) [d(ln A)/dt] ) [(2.1 × 104)Q + 0.19] (4) 2 2 where r is the radial distance from the center of the cylinder. The slope of the d(ln A)/dt vs Q curve is for water equal to 2.1 × 104 (m-3) and the intercept is 0.19 s-1. In case of a protein solution, the relation between d(ln A)/dt and Q is not always linear, but in most cases it is.11 (10) Bain, C. J.; Manning-Benson, S.; Darton, R. C. J. Colloid. Interface Sci. 2000, 229, 247-256. (11) Bergink-Martens, D. J. M. Ph.D. thesis; Interface Dilation. The Overflowing Cylinder Technique; Wageningen Agricultural University, Wageningen, 1993; p 151.
Figure 4. (A) View on top of the overflowing cylinder covered by a motionless protein film. (B) The same view after the flux has been changed to a higher value, inducing fracture of the protein film.
(See the curve for caseinate in Figure 4b.) In this study, we found for ovalbumin a slope of 4.2 × 104 (m-3) and an intercept of 0.5 s-1, assuming a linear dependency (Figure 5b). The most right-hand side of eq 4 gives the relation for the overflowing cylinder used in this study. Substituting this equation into eq 3 gives for the shear stress
σshear )
( )
rη [d(ln A)/dt) ) 2hdyn rη [(2.1 × 104)Q + 0.19] (5) 2hdyn
( )
Liquid Flowing through the Falling Film. When a protein network is present on top of the overflowing cylinder, then also at the outside of the falling film a protein layer is present that can be regarded as a fixed wall. Therefore, a Poiseuille flow profile will develop in the gap between the cylinder wall and the protein network. This will cause a tangial stress acting on the stagnant protein layer, which can be estimated in the following way. First it is assumed that the liquid in the falling film is flowing between two motionless (flat) plates. The assumption of flat plates is valid because h, the thickness of the falling film, is much smaller than the radius R of the inner cylinder.
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Figure 6. Lstill as function of the flux Q for various proteins (the protein concentration was 0.01 g/L): (b) β-lactoglobulin, (2) glycinin pH 3, ([) ovalbumin, (9) glycinin pH 6.7, and (O) β-casein.
is the thickness of the protein film on top of the overflowing cylinder. This results in a film stress of
1 L ) σwall σfilm ) σwall2πRL 2πR∆x ∆x
Figure 5. (A) Surface expansion rate [d(ln A)/dt] as function of the falling film L for an ovalbumin solution of 0.1 g/L for different values of the flux Q: (b) Q ) 6.7 cm3 s-1, (2) Q ) 13.4 cm3 s-1; ([) Q ) 33.6 cm3 s-1; (9) Q ) 54.6 cm3 s-1. For comparison, the same dependency is given for water for Q ) 33.6 cm3 s-1 (*). (B) Surface expansion rate as function of Q for L ) 4 cm for a 0.1 g/L ovalbumin solution (b), water ([), and a 2% caseinate solution (9). Data for water and caseinate are taken from ref 10.
For stationary flow between two parallel plates neglecting gravity, the strain rate at the wall γ˘ wall is12
γ˘ wall )
( )
dvz h ∆p h 12η h 12η Q ) ) ) 〈v 〉 ) dx η ∆z η h2 z η h2 2πRh 6 Q (6) h2 πR
where h is the thickness of the falling film, ∆p the pressure difference over the length of the parallel plates z, and 〈vz〉 the average velocity. Note that h is not hdyn. ∆p over ∆z is Fg, where F is the density of the liquid and g acceleration due to gravity. It gives for the stress σwall on the protein layer
6η Q 6η 1 2Fgh3πR ) Fgh (7) ) 2 σwall ) η γ˘ ) 2 h πR h πR 12η
( )
Next we have to translate this shear stress on the protein layer covering the falling film into a tensile stress in the protein film on top of the overflowing cylinder. Thereto σwall is multiplied by the area Awall (Awall ) 2πRL) it is acting on and divided by the cross section, 2πR∆x, of the protein at the rim of the overflowing cylinder, where ∆x (12) Lyklema, J. Fundamentals of Interface and Colloid Science. Volume I: Fundamentals; Academic Press: San Diego, 1991; Chapter 6, p 6.40.
(8)
Inserting eqs 5 and 8 in eq 2 will give the following for the stress acting on the protein gel layer:
ηr L L [d(ln A)/dt] σgel ) σwall + σshear ) Fgh + ∆x ∆x 2hdyn (9) In Figure 2, the relative expansion rate is given for an adsorbed protein film for a given Q. Below a certain length L (Lstill), the interface does not expand [d(ln A)/dt ) 0]. For values of L higher than Lstill, the surface is continuously expanding radially. Therefore, Lstill corresponds to the point were the protein layer fractures/yields. Different values of Q will result in different Lstill values. In Figure 6 (see results) Lstill is given as function of Q for various proteins. Below the drawn lines, a protein gel layer is present on top of the overflowing cylinder, whereas above this line no gel layer is present on top of the cylinder, because the hydrodynamic forces are too high to form such a layer. This dependence of Lstill on Q does not have to be linear. Along these lines, the strength of the gelled protein layer, i.e., its fracture/yield stress, is equal to the sum of the stresses acting on the layer
σfracture ) ηr h [d(ln A)/dt] σgel ) FgLstill + ∆x 2hdyn
(10)
ηr h ) FgLstill + [(2.1 × 104)Q + 0.19] ∆x 2hdyn for the overflowing cylinder used. The fracture stress can now be calculated as a function of the ratio h/∆x, which can be assessed either by assuming a certain thickness for the adsorbed protein gel layer or by measurement of the thickness, e.g., by ellipsometry. By substitution of η ) 0.001 m2 s-1, F ) 1000 kg m-3, g ) 9.81 m2 s-1, Lstill ) 0.02 m, r ) 0.03 m, hdyn ) 0.004 m, and Q ) 33.4 cm3 s-1 and assuming that h/∆x is >1, it can be concluded that the second term on the right-hand side of eq 10 can be neglected with respect to the first term (0.0033 vs 100h/ ∆x). In other words, the stress acting on top of the
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Table 1. Some Properties of the Proteins Used
b
protein
ability to form intermolecular S-S bridges
pI
Mw (kDa)
ovalbumin glycinin, pH 3a glycinin, pH 6.7a β-casein β-lactoglobulin gliadinb
yes yes yes no yes yes
4.5-6.7 4.8 4.8 4.5 5.2 6.5-8.1
45 43 350 24 18 30-40
a pH 3, 3S form; pH 6.7, 11S form; ionic strength 30 mM. Contains approximately 25% puroindolines.
overflowing cylinder caused by the liquid flow beneath it can be neglected in the determination of the fracture stress of a protein film. It would imply that the fracture stress does not depend on Q, unless h/∆x depends on Q, which is not unrealistic. ∆x for adsorbed monolayers as obtained by ellipsometry is for the proteins used in this study on the order of 10 nm. Assuming that h is on the order of 1 mm, the fracture stress for adsorbed protein films is expected to be on the order of 107 Pa. However, in the present study, adsorbed layers are build up while they are continuously compressed. Continuous compression experiments showed that it was possible to draw threads of protein from the compressed surface just by touching the surface with a pencil. It is thus expected that the thickness of the adsorbed protein gel layer is larger than the 10 nm found for a molecular layer (see Results and Discussion), leading to much lower calculated fracture stresses. Materials and Methods Proteins. Proteins used were ovalbumin, (wheat) gliadin, soy glycinin, β-casein, and β-lactoglobulin. Some typical features of the proteins are given in Table 1. Ovalbumin grade V was purchased from Sigma (catalog no. A5503). Soy glycinin (WCFS code PR004) was isolated from soybeans according to the fractionation scheme given by Thanh and Shibasaki,13 and the wheat gliadin was isolated from an acidified gluten extract. The glycinin and gliadin-enriched fractions were dissolved in a 10 mM phosphate buffer pH 7.8 at 4 °C in the presence of 10 mM 2-mercaptoethanol and 20% glycerol and stored at -40 °C. After defrosting, glycinin and gliadin were dialyzed prior to use to remove glycerol. Dialysis tubings were boiled at least 10 min in distilled water in the presence of approximately 5 mM EDTA and rinsed afterward with demineralized water. Defrosted material was then dialyzed eight times against 50 times excess buffer under gently stirring: once 30 min at 20 °C, then 60 min at 20 °C, five times 60 min at 4 °C, and finally overnight at 4 °C. Large amounts of buffer were required and, therefore, the first four buffers were made using tap water. Gliadin appeared gray and untransparent after dialysis. Glycinin changed into a liquid with white aggregates, which changed into a grayish untransparent solution after warming up from 4 to 22 °C. To reduce aggregation, glycinin was diluted 1:10 with buffer prior to dialysis. It was assumed that the aggregation did not affect the efficiency of dialysis. β-casein (WCFS code PR208) and β-lactoglobulin (WCFS code PR210) were obtained from the Wageningen Centre for Food Sciences. Both proteins were isolated from milk by first removing the fat by centrifuation and filtering. Subsequent acidification of the skimmed milk gave a precipitated caseinate fraction and a whey protein fraction (supernatant). The caseinate and the whey protein fractions were further purified to obtain β-casein and β-lactoglobulin.14 Both proteins, supplied as freeze-dried powder, were dissolved in buffer by stirring with a magnetic stirrer the day before and kept overnight at 4 °C. If not dissolved (13) Tanh, V. H.; Shibasaki, K. J. Agric. Food. Chem. 1976, 24, 1117. (14) de Jongh, H. H. J.; Gro¨neveld, T.; de Groot, J. J. Dairy Sci. 2001, 84, 562-571.
completely, stirring was continued overnight. On the day of the measurement, the solution was allowed to equilibrate to room temperature for 1 h with slow stirring. Protein solutions were diluted with buffer to the appropriate concentration. The final protein concentration of the solutions was determined by the Biureet method. A phosphate buffer (Merck Germany) was used for pH 6.7. The final pH was obtained by adding small amounts of 2 M HCl or NaOH. The ionic strength of the phosphate buffer was 30 mM. Stirring of protein solutions was always done at sufficiently low speeds to prevent foam formation. Overflowing Cylinder. The overflowing cylinder technique used in this study has been described by Bergink-Martens et al.11,15 This technique is often used to obtain the interfacial dilational viscosity ηd ()∆γ/[d(ln A)/dt]), in which ∆γ is the difference between the equilibrium surface tension and the dynamic surface tension and d(ln A)/dt the relative surface expansion rate of the surface on top of the cylinder. The latter is determined using laser Doppler Anemometry.15 The values of the radial expansion speeds presented in this study are averages of 10 individual measurements. Protein solutions (4 °C) were slowly adjusted to 22 °C and poured into the cylinder. The flux was varied between 0 and 70 cm3/s, the diameter of the cylinder was 6 cm, and the total volume was 5 L. The temperature during the measurements was 22 °C. At the beginning of the experiment, the top surface and the surface in the gutter of the overflowing cylinder were cleaned by suction. Directly after cleaning of the surfaces, gel formation on the surface in the gutter was observed due to irreversible protein adsorption and continuous compression of the protein layer. Slowly this compressed gel “moves” up in the direction of the rim of the overflowing cylinder, and after a while, the protein layer (gel layer) is all over the top surface, and radial expansion was not observed any more. The time span to fully cover the top surface with a protein gel layer varied with the type of protein and protein concentration. Therefore, fracture stresses were determined at a constant time (5 min) after cleaning of the surfaces.
Results and Discussion Figure 4 illustrates the fracture behavior of protein films on top of the overflowing cylinder. Talcum powder particles were homogeneously distributed on top of the protein gel by sprinkling. No movement of the talcum powder particles was observed (Figure 4a). After increasing the force by increasing the flux Q, the talcum powder particles moved instantaneously radially and collected in a ring around the center of the overflowing cylinder (Figure 4b). Disturbance of the protein skin by touching it with the back of a pencil resulted in the same behavior of the talcum particles. In this case, even fracture lines in the protein gel could be observed on top of the cylinder. Talcum powder rings at the outside of the falling film move slowly downward with time, showing relaxation phenomena of the gelled protein layers. The protein layer fractures on top of the overflowing cylinder. Theoretically, according to eq 10, fracture of the protein should take place at r ) rmax. At rmax, σ is the highest. However, as discussed above, the second term on the right-hand side of eq 10 is very small compared with the first term on the right-hand side of eq 10, indicating that the effect of r on the exact point of fracture is negligible. In practice, fracture occurs always on top of the overflowing cylinder and not at random somewhere in the surface. This indicates that the gel layer is stronger at rmax than in the center of the overflowing cylinder. Going from the center to the rim, the protein gel layer is older and the protein molecules have more time to interact with each other and/or to form a thicker protein gel layer. (15) Bergink-Martens, D. J. M.; Bos, H. J.; Prins, A.; Schulte, B. C. J. Colloid Interface Sci. 1990, 138, 1.
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The typical dependence of d(ln A)/dt on L of the overflowing cylinder has been discussed for a protein solution in the theoretical section. As an example, in Figure 5a d(ln A)/dt as function of the length of the falling film L is shown for various Q for a 0.1 g/L ovalbumin solution. At high L d(ln A)/dt increased with Q. In Figure 5b, d(ln A)/dt as function of Q is given for L ) 4 cm. Larger Q resulted in higher d(ln A)/dt. For comparison, data for pure water and 2% w/w caseinate solution are also given.11 For small values of the flow rate a rather steep increase in d(ln A)/dt was measured, whereas for higher values of the flow rate the slope of the curves is comparable with that measured for pure water. The first steep increase is caused by an increase in the surface tension gradient between the center and the rim of the inner cylinder. At a certain flow rate, the surface tension gradient has reached its maximum. This flow rate differs between different solutions and depends on the transport properties of the surface-active molecules. Above this flow rate, the slope of {d[d(ln A)/dt]/dt}/dQ is about the same as obtained for pure water and is determined solely by hydrodynamic effects. With increasing flux Lstill decreases. This is in agreement with the theoretical expression for Lstill. A theoretical expression for Lstill can be derived from eq 10 and is given by
[
] [
]
∆xσfracture 0.19ηr∆x (2.1 × 104)ηr∆x + Lstill ) Q Fgh Fgh2hdyn Fgh2hdyn (11)
Figure 7. Lstill as function of the flux Q for ovalbumin solutions with different concentrations: (b) 0.01 g/L, (2) 0.1 g/L, (9) 1.0 g/L. Table 2. Measured Values for Lstill and Surface Dilational Modulus (E) and Calculated Yield Stress for Various Interfacial Protein Gels When h/∆x Is 5 × 103 (See Eq 10) type of protein ovalbumin β-casein β-lactoglobulin
This equation indeed shows that Lstill decreases with increasing Q. However, as described after eq 10, the Q-containing term may be neglected compared to the first term. So the effect of Q on Lstill must be indirect. This is likely by affecting ∆x/h. The same dependency of Lstill on Q shown in Figure 5a was observed for various other proteins (Figure 6). In all cases the protein concentration was 0.01 g/L in a 30 mM phosphate buffer. At this protein concentration, β-lactoglobulin and glycinin at pH 3 have relatively high Lstill values as function of Q (Lstill between 3 and 2 cm), and β-casein, ovalbumin, and glycinin at pH 6.7 exhibit relatively low Lstill as a function of Q (between 1.5 and 0.7 cm). For all proteins investigated the slope of the Lstill vs Q plot is more or less the same, indicating that ∆x/h varies more or less in the same way for all proteins with Q (first term of eq 11 and assuming σfracture to be independent of Q). This can be understood if one realize the main function of the adsorbed protein in this respect is to make the surface motionless. Protein specific properties may express themselves in the height of the curve via ∆x and σy. Since the protein gel layer is a compressed protein layer that expands from the gutter outside the cylinder to cover the top of the overflowing cylinder, it is not unrealistic that the gel layer thickness of the proteins are similar. The results of glycinin at pH 6.7 and 3.0 show that the conformation in which the protein is present may have a big effect on the yield/fracture stress. At pH 3.0 glycinin is present is the 7S and 3S form and at pH 6.7 in the 11S form.16-18 The molecular weight is 175, 45, and 350 kDa, respectively. The smaller protein molecules at pH 3 are more effective in forming a stronger network at the (16) Wolf, W. J.; Briggs, D. R. Arch. Biochem. Biophys. 1958, 76, 377. (17) Peng, I. C.; Quass, D. W.; Dayton, W. R.; Allen, C. E. Cereal Chem. 1984, 61, 480. (18) Wolf, W. J.; Nelsen, T. C. J. Agric. Food Sci. 1996, 44, 785.
glycinin, pH 6.7 glycinin, pH 3 gliadin
protein concn
Lstill (cm)
σyield (kPa)
0.01 0.1 1.0 0.01 0.1 0.01 0.03 0.1 0.01 0.1 0.3 0.01 0.1 0.1
1.0 2.4 2.4 1.2 1.8 2 2.25 2.25 1.3 2.0 2.0 2.3 2.5 1.9
490 1170 1170 589 883 981 1104 1104 638 981 981 1128 1226 932
E (mN/m) 77 15 20 68 82 76 40 48 60 58
interface. This is probably due to faster adsorption at the interface and more hydrophobic interactions (more denatured protein). Moreover, differences in capabilities to form SS bridges may play a role. The higher force needed to break a glycinin gel layer at pH 3 than at pH 6.7 corresponds with the higher dilational modulus found at pH 3, 60 mN/m vs 40 mN/m at pH 6.7.19 In Figure 7 the effect of protein concentration on the dependence of Lstill on Q for ovalbumin is given for 5-minold protein films. Higher protein concentration resulted in larger Lstill vs Q. Likely, at low concentrations the adsorbed amount is not high enough at the prevailing conditions (waiting time, etc.) to form a strong cohesive interfacial layer in the available time. Also for glycinin at pH 6.7 and β-casein, an increase in Lstill values as function of Q was found when the protein concentration was increased from 0.01 to 0.1 g/L, but the increase was less sharp. However, an increase in concentration from 0.01 to 0.1 g/L did not result in a higher Lstill (Q) for glycinin at pH 3 and for β-lactoglobulin pH 6.7 (Table 2). From the measured Lstill values, yield stresses have been calculated for the proteins using for flux 33.4 cm3 s-1, assuming that the thickness of the protein layer on top of the overflowing cylinder is about 1/5000 of the thickness of the falling film (liquid plus protein layer). In practice, the thickness of the falling film is about 1-2 × 10-3 m. (19) Bos, M. A.; Martin, A.; Bikker, J.; van Vliet, T. In Food Colloids 2002. Fundamentals of Formulation; Miller, R., Dickinson, E., Eds.; Royal Chemical Society, London, 2002; p 223-232.
Fracture Stresses of Adsorbed Protein Layers
Langmuir, Vol. 19, No. 6, 2003 2187
This gives an estimated thickness of the protein gel layer of about 2 µm, which is not unrealistic regarding the protein threads that can be obtained. We have no indications for strong variations in the thickness of the adsorbed strong compressed protein layers. More research is required before a more definitive conclusion can be drawn. Furthermore, for adsorbed surface layers of ovalbumin that have been compressed by a factor of 2, thicknesses of 100-500 nm (depending on time) have been found using the IRRAS technique.20 Measured Lstill values and calculated yield stresses are given in Table 2. The latter are on the order of 105-106 Pa. Since the yield stress is linearly proportional with Lstill, differences in Lstill are reflected in differences in yield stresses. For a h/∆x ratio of 5 × 103 the yield stresses are on the order of (5-10) × 105 Pa. Yield stresses of three-dimensional ovalbumin and soybean protein gels are on the order of 103-104 Pa at concentrations of 20%.21 Therefore, the calculated yield stresses for gel layers at an air/water interface could be realistic and support the assumption made that the adsorbed protein gel layer behaves as a first approximation like a thin three-dimensional gel. The latter argument was also strengthened by van Vliet et al.,22 who observed clear phenomenological similarities between bulk and
interfacial gelling behavior with changes in the molecular structure of glycinin. All measurements were performed 5 min after cleaning of the air/water interface. In practice, it will take time for a protein solution to build up a protein gel layer. The rate will depend on protein concentration and on the nature (viscoelasticity) of the formed adsorbed protein layer. At identical concentrations, one protein will much better be able to form a gel layer than another protein in 5 min. Besides, relaxation processes in the adsorbed protein layer play a role. This time dependency to built up the protein film and the effects due to relaxation processes in such films are currently under investigation. In conclusion, we have shown that, making use of the overflowing cylinder technique, it is possible to measure fracture properties of interfacial gelled protein layers. Equations have been presented for the calculation of fracture stress of adsorbed protein layers. Obtained fracture stresses are on the order of 2 × 105 Pa and depend on the type of protein. Protein properties such as conformation seem to be important aspects, determining the height of the fracture stress.
(20) Kundryashova, E. V.; Meinders, M. B. J.; Visser, A. J. W. G.; de Jongh, H. H. J. Submitted to Biophys. J., 2001. (21) van Kleef, F. S. M. Biopolymers 1986, 25, 31.
(22) van Vliet, T.; Martin, A.; Renkema, J. M. S.; Bos, M. A. Proceedings of the 2nd workshop on Plant Biopolymer Science: Food and non Food Applications, 2001, Nantes, France.
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