Simultaneous Control of pH and Ionic Strength ... - ACS Publications

Article pubs.acs.org/Langmuir

Simultaneous Control of pH and Ionic Strength during Interfacial Rheology of β‑Lactoglobulin Fibrils Adsorbed at Liquid/Liquid Interfaces Patrick A. Rühs,* Nathalie Scheuble, Erich J. Windhab, Raffaele Mezzenga, and Peter Fischer Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland ABSTRACT: Proteins can aggregate as amyloid fibrils under denaturing and destabilizing conditions such as low pH (2) and high temperature (90 °C). Fibrils of β-lactoglobulin are surface active and form adsorption layers at fluid−fluid interfaces. In this study, βlactoglobulin fibrils were adsorbed at the oil−water interface at pH 2. A shear rheometer with a bicone geometry set up was modified to allow subphase exchange without disrupting the interface, enabling the investigation of rheological properties after adsorption of the fibrils, as a function of time, different pH, and ionic strength conditions. It is shown that an increase in pH (2 to 6) leads to an increase of both the interfacial storage and loss moduli. At the isoelectric point (pH 5−6) of β-lactoglobulin fibrils, the maximum storage and loss moduli are reached. Beyond the isoelectric point, by further increasing the pH, a decrease in viscoelastic properties can be observed. Amplitude sweeps at different pH reveal a weak strain overshoot around the isoelectric point. With increasing ionic strength, the moduli increase without a strain overshoot. The method developed in this study allows in situ subphase exchange during interfacial rheological measurements and the investigation of interfacial ordering. have shown the potential as encapsulation systems.22 The aggregation and formation kinetics of peptides into βlactoglobulin fibrils has been studied extensively.23−26 βLactoglobulin fibrils show isotropic−nematic transitions in bulk at remarkably low concentrations, and they exhibit a liquid-crystalline behavior at the interface.27 Furthermore, βlactoglobulin fibrils have been studied for their interfacial adsorption behavior and rheology at the interface with different morphologies; however, the response to pH changes and ionic strength changes after adsorption has not been studied.28 By modification of the ionic strength and pH conditions, the isotropic nematic transition concentration can be modified and the fibril properties tuned.29,30 Through the positive charge (20e) of fibrils at pH 2, a double layer of opposite charges is formed. Through an ionic strength increase, the twisting periodicity of β-lactoglobulin fibrils can be increased.31 An increase in ionic strength diminishes the Debye length and thus the effective diameter.29,32 With pH modifications, the effective charge of the β-lactoglobulin fibrils can be changed, thus leading to aggregation and destabilization of the fibrils. A pH increase from 2 to 12 led to a complete degradation in the bulk of the fibrils formed by lysozyme.33 In this study, β-lactoglobulin fibrils are used as a model system for semiflexible particles at the interface capable to undergo an isotropic−nematic transition.27 To study their

I. INTRODUCTION Interfacial shear rheology is important for the characterization of complex soft fluid systems such as emulsions and foams. Several methods are able to measure interfacial shear rheology including the magnetic rod stress rheometer,1 the double wall ring geometry with the du Noüy ring,2 and the bicone geometry mounted on a rotational rheometer.3 Shear rheology is used to study the viscoelasticity of interfaces by applying shear forces. The resulting rheological data are used to fundamentally understand the mechanisms behind interface stabilization.4 A great variety of different material can have a stabilizing effect including proteins, surfactants, and particles.5 Interfaces that are stabilized by proteins, in contrast to surfactants, may form gellike interfacial layers.6,7 Proteins, being amphiphilic, are sensitive to pH and ionic strength changes. Physiological conditions influence their internal structure and thus their charge. To address the influence of pH and ionic strength, interfacial rheology and adsorption of proteins at different pH values have been measured. Some of these measurements modify the physicochemical parameters prior to adsorption and the actual measurements.8−10 Several studies have performed bulk exchange in dilatational rheology11−18 and in shear rheology8,19 to change the conditions after adsorption of proteins and asphaltenes. Their shortcomings are that none have performed controlled physicochemical modifications of the subphase after complete adsorption of proteins. Distinct morphologies of proteins from spherical aggregates to fibrils can be formed through denaturation processes.20,21 One aggregate form of interest is β-lactoglobulin fibrils, which © 2012 American Chemical Society

Received: July 3, 2012 Revised: July 31, 2012 Published: August 2, 2012 12536

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

R)), the interfacial flow is considered to be decoupled from the bulk phase flow, and so the setup can be treated as a 2D Couette device. In this case, given a defined angular velocity Ω of the rotating biconical disk, the interfacial shear viscosity η*i is calculated as follows:3

adsorption at the interface, we present a new rheometrical setup, which enables in line subphase exchanges during shear rheological experiments at the interface and simultaneous monitoring of the pH value. This was modified, because proteins can not be adsorbed at the interface at their isoeletrical point, due to aggregation and the resulting subsequent sedimentation. The ionic strength of the subphase was also changed to observe effects caused by the decrease of the Debye length. Dynamic changes occurring in systems after creation of an interface can thus be studied and understood.

8

|η*i | =

M − 3 R 23(η(1) + η(2))Ω 4πR 22 Ω

(1)

where M is the torque, R is the characteristic distance of the geometry, R2 is the bob radius of the biconical disk, and η(1), η(2) are the viscosities of the two bulk phases. Viscoelastic properties of the interface are extracted by sinusoidal oscillation of the biconical disk. By applying a defined strain γ(t) = γ0·cos(ωt), a periodic stress response τ(t) = τ0·sin(ωt + δ) with a phase shift δ is caused, and the dynamic complex interfacial shear modulus G*i(ω) can be calculated according to the following equation:

II. MATERIALS AND METHODS A. Materials. The β-lactoglobulin fibrils were produced according to the protocol published in detail elsewhere.20,28,32 Therefore, only a brief summary is given. The BioPURE β-lactoglobulin powder (Davisco Foods International, Le Sueur, MN) was dissolved in Milli-Q water to obtain a 10 w/w% solution. Given that ions can greatly affect the fibrillation, the solution was dialyzed through a dialysis membrane with a MWCO of 6000−8000 Da (Spectra/Por) first against pH 2 water and then against Milli-Q water. After dialysis, the pH was adjusted to pH 2. The solution (2 w/w%) was boiled at (90 °C) during 5 h at pH 2 to form the fibrils. The β-lactoglobulin fibrils solution was dialyzed against pH 2 Milli-Q water to remove monomers and peptides. The fibrillation was checked by polarization filters and by atomic force microscopy (see Figure 1) in tapping mode

G*i (ω) = τ0eiδ /γ0 = |G*i |(cos δ + i sin δ) = G′i (ω) + iG″i (ω) (2) The dynamic elastic properties of the interfacial layer are described by the interfacial storage modulus G′i and the dynamic viscous properties by the interfacial loss modulus G″i. The measuring protocol was as follows: Interfacial rheological measurements at the water/MCT interface were performed with a βlactoglobulin fibril solution at pH 2 and a concentration of 0.01 w/w% in bulk. To monitor the transient buildup of the interfacial layer, time sweeps at constant strain (γ = 0.1%) and frequency (ω = 1 s−1) were performed for 15−16 h. After this equilibration period, the pH was adjusted by adding 0.05 M NaOH, and the time sweep was continued. Amplitude sweeps at different pH values and NaCl concentrations are performed to identify the linear viscoelastic region. During amplitude sweeps, the angular frequency is constant with increasing shear strain. To check the influence of the high strains (through the amplitude sweeps) on the time sweep, those measurements were performed with amplitude sweeps and without amplitude sweeps to ensure that no additional effects were introduced. All measurements were repeated to ensure reproducibility of the observed effects. C. Setup of Subphase Exchange System. To change the subphase conditions during measurements, the measuring cell of the existing interfacial rheology setup was modified to achieve different pH conditions (see Figure 2, design A) and changed ionic strength conditions (see Figure 2, design B). A schematic overview on the experimental setup can be seen in Figure 3 for pH and ionic strength changes. The pH measuring cell has an integrated pH electrode (1 mm minimum immersion depth, Metrohm, Switzerland) enabling online pH measurements directly comparable to the shear rheological data. To optimize the exchange process, the fluid amount was reduced by

Figure 1. Atomic force microscopy (AFM) image of 0.01 w/w% βlactoglobulin fibril solution at pH 2 adsorbed on a MICA surface. Inset: The β-lactoglobulin fibrils contour length distribution is depicted. with a Nanoscope V Multimode scanning force microscope (Veeco Inc., U.S.). A detailed methodology can be found in refs 23,31. The formed fibrils have a contour length distribution (see inset of Figure 1) as reported in the literature.31 The rheological properties of the formed β-lactoglobulin fibrils were tested at the water−oil interface. As the oil phase, food grade MCT oil (Delios GmbH) was used. To change the physicochemical conditions inside the measuring cup, a 0.05 M solution of NaOH (Sigma-Aldrich, Switzerland) and a 1 M NaCl at pH 2 (Sigma-Aldrich, Switzerland) solution were used. B. Methods: Interfacial Rheology. A direct shear rheometer (Physica MCR501, Anton Paar) with a biconical disk geometry was used. With the Boussinesq number Bo ≫ 1 (Bo = ηi*/((η(1) + η(2))

Figure 2. Modified measuring cell for pH (design A) and ionic strength (design B) change during interfacial rheology measurements. The circles highlight the holes inside the modified cells. In total, the measuring cells possess seven holes (A) and three holes (B) to ensure optimal flow behavior inside the measuring cell. In design A, a pH electrode is placed directly under the biconical disk. 12537

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

Figure 3. Schematic view of the modified measuring cell (A) for ionic strength and pH changes with connected syringe pumps (B and F) and the peristaltic pump (C) is presented. The biconical disk (D) is placed at the fluid−fluid interface. A flow is created inside the cup with a peristaltic pump. The pH is increased by using a syringe pump with NaOH solution. To increase the ionic strength, the syringe pump was used with a NaCl solution. The pH electrode (E) is measuring inside the subphase and is directly connected to a pH meter. The pH electrode was omitted when measuring the ionic strength increase. The excess volume is expelled from the measuring cell by pumping contrary with a second syringe pump (F).

Figure 4. (A) Interfacial loss G″i and storage G′i moduli of 0.01 w/w% β-lactoglobulin fibrils against time t at different pH adsorbed at the water− MCT oil interface (ionic strength = 0.01 M at pH 2, T = 20 °C). (B) Scheme of β-lactoglobulin fibrils at pH 2, pH 6, and pH 11. (C) Close-up of the first 15 h of the measurement. The Boussinesq number (Bo) at different pH values is: Bo = 5 (pH 2), Bo = 250 (pH 6), and Bo = 10 (pH 10). one-half of the original design3 and with several inlets and outlets in the measuring cell (see Figure 2, design A). The syringe pumps (Aladdin-1010, World Precision Instruments, U.S.) and the peristaltic pump (CA4 ISM 721, Ismatec, Switzerland) were connected through flexible tubing to the measuring cell (see Figure 3). The pH electrode was placed exactly in the middle under the bicone to avoid any flow field disturbances. With injection of the NaOH solution, the subphase

was removed through the outlet to keep the volume in the cup constant during the experiment. This was necessary to keep the interface pinned at the bicone edge. With the peristaltic pump, a continuous flow was forced in the cell. Through the continuous mixing, a constant flow was created across the pH electrode to ensure a correct pH measurement. After each measurement, the pH electrode 12538

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

Figure 5. Interfacial loss G″i and storage G′i moduli as a function of strain γ at 1 rad/s, different pH, and layer ages (A−H) of 0.01 w/w% βlactoglobulin fibrils adsorbed at the water−MCT oil interface (ionic strength = 0.01 M at pH 2, T = 20 °C). The circles highlight the weak strain overshoot. where tr describes the residence time of the NaCl in the measuring cell and C0 the incoming NaCl concentration of the syringe. It can be calculated with the measuring cell volume VD and the volumetric flow rate RE, tr = VD/RE. We assume for these calculations that the liquid in the measuring cell is perfectly mixed.16

was cleaned of protein solution by submerging it into a pepsin/HCl solution to avoid erroneous measurements. The ionic strength measuring cell was designed to optimally change the NaCl conditions in the cup. To avoid NaCl concentration gradients at the interface, an inlet was placed directly under the biconical disk in the middle of the measuring device. The outlet was placed as far away as possible from the inlet (see Figure 2, design B). The third hole in combination with the first one was used to create a flow inside the measuring cell via a peristaltic pump to generate a subtle flow to enhance the mixing of the subphase. With injection of the NaCl solution, the same amount of subphase fluid was removed through the outlet to keep the volume in the cup constant. The syringe pumps, pumping contrary to each other, enabled a smooth exchange of the bulk phase. The transient concentration C(t) of NaCl and thus the ionic strength can be calculated as follows: C(t ) = C0(1 − exp(− t /tr))

III. RESULTS AND DISCUSSION A. pH Influence on the Interfacial Storage and Loss Moduli G′i and G″i over Time and Strain. With the 0.01 w/ w% β-lactoglobulin fibril solution at the water−MCT oil interface, a viscoelastic layer is formed already at the beginning of the measurement. The viscoelastic moduli of the βlactoglobulin fibril solution interface increases until a constant value of G′i and G″i is reached as depicted in the inset of Figure

(3) 12539

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

Figure 6. (A) Interfacial loss G″i and storage G′i moduli of 0.01 w/w% β-lactoglobulin fibrils against time t at different calculated ionic strength adsorbed at the water−MCT oil interface (pH 2, T = 20 °C). (B) Scheme of β-lactoglobulin fibrils at low and high ionic strengths. The Boussinesq number (Bo) at different ionic strength is: Bo = 5 (I = 0.01 M) and Bo = 36 (0.45 M).

Figure 7. Interfacial loss G″i and storage G′i moduli as a function of strain γ at different ionic strengths and layer ages with an angular frequency of 1 s−1 of 0.01 w/w% β-lactoglobulin fibrils at the water−MCT oil interface (pH 2, T = 20 °C). (A) 0.01 M ionic strength (I) /12 h, (B) 0.21 mol/L I/ 20 h, (C) 0.49 mol/L I/30 h, (D) 0.79 mol/L of I/81 h.

lactoglobulin fibrils aggregate and build a strong network. At this pH, the amount and strength of network points between the fibrils are at its maximum, and therefore the interfacial elasticity reaches it highest value. The difference observed in this experiment between the isoelectric point and the maximum elasticity in this network can be explained by transient effects. Additionally, the adsorption of the fibrils at the interface might cause a shift of the isoelectric point to higher values. As reported in literature, the isoelectric point of proteins adsorbed on solid surfaces can shift after adsorption and differ from the

4. The fibrils are highly charged at pH 2, and electrostatic repulsion is strong, which leads to a viscoelastic interaction. After 15 h, the pH of the β-lactoglobulin solution was changed with a 0.05 M NaOH solution, which was injected with the syringe pump as described previously (design A). The time sweep and a scheme of β-lactoglobulin fibrils at different pH values are shown in Figure 4A. A strong correlation between the increase of G′i and G″i can be observed up to a pH of 6. At the isoelectric point of the β-lactoglobulin fibrils (5.13),34 the attracting forces are dominant and the β12540

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

Figure 8. (A) The nonlinear onset (NLO) and the corresponding strain (γ) pH dependency are depicted. The dotted line displays the pH after which the NL onset decreases. (B) The NLO and the corresponding strain (γ) ionic strength dependency are depicted. The lines are to guide the eye.

bulk phase.35 This pH increase from 2 to 6 caused the moduli to increase a 80-fold as compared to the initial value at pH 2. If the pH is further increased, the network weakens as G′i and G″i are decreasing. At a critical pH 8, the network breaks down and the fibrils are now negatively charged. At higher pH, the contour length of the fibrils is also decreased.32 In theory, at a pH higher than 10, the conformation is similar to that of pH 2 (see Figure 4B). Because of the long residence time at the isoeletric point, it is unlikely that the fibrils retain their pristine fibrillar structure. The final value (0.03 Pa m) of G′i and G″i is higher than the starting value. The formed aggregates do not untangle at higher pH; however, the connecting points are disrupted by the negative charges. Furthermore, the addition of NaOH increases the ionic strength, which could lead to the increased moduli. As seen in the next section, an increase in ionic strength causes the fibrils to form stronger networks. To investigate the network properties of the interfacial adsorption layers at the water−oil interface at different pH’s, amplitude sweeps were performed (see Figure 5). At pH 2, the formed layer can resist strains up to 10% (see Figure 5A). This is supported by the theory that at low pH the fibrils do not directly interact with each other due to their positive charge. This is confirmed by fibrils made from lysozyme, which are stable at pH 2−4.33 Increasing the pH to 3−4 causes a shift of G′i and G″i (Figure 5B and C) to higher values as confirmed by the time sweep. Amplitude sweeps in the region of the isoelectric point (pH 5.13) not only show a further shift in G′i and G″i, but a decrease of the linear viscoelastic region (see Figure 5D and E). The network starts to form a soft gel and breaks at lower strains. The layer resists against deformation up to strains where G″i increases (highlighted with circles). At a critical strain, the deformation leads to a network breakdown (Figure 5F and G). At this strain, G″i increases. This is known as a so-called weak strain overshoot, one of four classifications often used in large amplitude oscillatory shear (LAOS) rheology (the other three being strain thinning, strain thickening, and strong overshoot).36 The pH determines the amount and the strength of network points, which are the driving force of interfacial elasticity.8 Finally, at pH 8 (Figure

5H), the network breaks down completely as it can not resist the strains. At this point, the fibrils are negatively charged and repel each other. To further elucidate structure changes, nonlinear interfacial rheology might provide insight into the network formation behavior at different pH values.37 B. Ionic Strength Influence on the Interfacial Storage and Loss Moduli G′i and G″i over Time and Strain. The modified cup as seen in Figure 2 (design B) was used to investigate the influence of the ionic strength on the interfacial shear rheological properties of the β-lactoglobulin fibril solution at the MCT oil interface after an equilibration period of 15 h. A time sweep was performed during alteration of the subphase by injection of a 1 M NaCl solution. In Figure 6, the resulting values of G″i and G′i are depicted. At pH 2, the high charge of β-lactoglobulin fibrils is not shielded by ions. Therefore, after the increase of ionic strength, the Debye length decreases and the fibrils pack closer to each other.29 This leads to interactions between them, thus making the layer more elastic. With increasing ionic strength, the interfacial viscoelastic moduli increase. Results by Maldonado-Valderrama et al.18 on native βlactoglobulin at interfaces confirm that an increase in the ionic strength conditions caused an increase of the dilatational elasticity. In Figure 7, amplitude sweeps at different layer age and ionic strength are shown. The interfacial moduli increases with time and ionic strength. The four amplitude sweeps look similar in shape. There is an overall increase of G′i and G″i (from Figure 7A to D), but there is a decrease of the linear viscoelastic region at higher ionic strength and age of the layer. In comparison to the network formation during different pH’s, there was no weak strain overshoot, which indicates that different interfacial structures must be present. The network properties caused by changes in ionic strength and pH are different (Figures 5 and 7). This is summarized in Figure 8. The nonlinear onset (NLO) is defined as the value of G′i where the linear viscoelastic regime is abandoned at the corresponding strain. The pH and ionic strength changes led to a decrease of the linear viscoelastic regime and thus the onset of the nonlinear regime. In Figure 8A, the influence of the pH on the 12541

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

(2) Vandebril, S.; Franck, A.; Fuller, G.; Moldenaers, P.; Vermant, J. A double wall-ring geometry for interfacial shear rheometry. Rheol. Acta 2010, 49, 131−144. (3) Erni, P.; Fischer, P.; Windhab, E. J. Stress- and strain-controlled measurements of interfacial shear viscosity and viscoelasticity at liquidliquid and gas-liquid interfaces. Rev. Sci. Instrum. 2003, 74, 4916−4924. (4) Erni, P.; Windhab, E. J.; Fischer, P. Emulsion drops with complex interfaces: Globular versus flexible proteins. Macromol. Mater. Eng. 2011, 296, 249−262. (5) Sagis, L. M. C. Dynamic properties of interfaces in soft matter: Experiments and theory. Rev. Mod. Phys. 2011, 83, 1367−1403. (6) Fischer, P.; Erni, P. Emulsion drops in external flow fields − the role of liquid interfaces. Curr. Opin. Colloid Interface Sci. 2007, 12, 196−205. (7) Bos, M. A.; van Vliet, T. Interfacial rheological properties of adsorbed protein layers and surfactants: a review. Adv. Colloid Interface Sci. 2001, 91, 437−471. (8) 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. (9) Pezennec, S.; Gauthier, F.; Alonso, C.; Graner, F.; Croguennec, T.; Brulé, G.; Renault, A. The protein net electric charge determines the surface rheological properties of ovalbumin adsorbed at the airwater interface. Food Hydrocolloids 2000, 14, 463−472. (10) Roberts, S. A.; Kellaway, I. W.; Taylor, K. M. G.; Warburton, B.; Peters, K. Combined surface pressure interfacial shear rheology study of the effect of pH on the adsorption of proteins at the air water interface. Langmuir 2005, 21, 7342−7348. (11) Kotsmár, C.; Grigoriev, D.; Makievski, A.; Ferri, J.; Krägel, J.; Miller, R.; Möhwald, H. Drop profile analysis tensiometry with drop bulk exchange to study the sequential and simultaneous adsorption of a mixed β-casein /c12dmpo system. Colloid Polym. Sci. 2008, 286, 1071−1077. (12) Svitova, T.; Wetherbee, M.; Radke, C. Dynamics of surfactant sorption at the air/water interface: continuous-flow tensiometry. J. Colloid Interface Sci. 2003, 261, 170−179. (13) Fainerman, V. B.; Leser, M. E.; Michel, M.; Lucassen-Reynders, E. H.; Miller, R. Kinetics of the desorption of surfactants and proteins from adsorption layers at the solution/air interface. J. Phys. Chem. B 2005, 109, 9672−9677. (14) Romoscanu, A. I.; Mezzenga, R. Cross linking and rheological characterization of adsorbed protein layers at the oil-water interface. Langmuir 2005, 21, 9689−9697. (15) Freer, E. M.; Yim, K. S.; Fuller, G. G.; Radke, C. J. Interfacial rheology of globular and flexible proteins at the hexadecane/water interface: comparison of shear and dilatation deformation. J. Phys. Chem. 2003, 108, 3835−3844. (16) Ferri, J. K.; Kotsmar, C.; Miller, R. From surfactants adsorption kinetics to asymmetric nanomembrane mechanics: Pendant drop experiments with subphase exchange. Adv. Colloid Interface Sci. 2010, 161, 29−47. (17) Miller, R.; Grigoriev, D.; Krägel, J.; Makievski, A.; MaldonadoValderrama, J.; Leser, M.; Michel, M.; Fainerman, V. Experimental studies on the desorption of adsorbed proteins from liquid interfaces. Food Hydrocolloids 2005, 19, 479−483. (18) Maldonado-Valderrama, J.; Miller, R.; Fainerman, V. B.; Wilde, P. J.; Morris, V. J. Effect of gastric conditions on β-lactoglobulin interfacial networks: Influence of the oil phase on protein structure. Langmuir 2010, 26, 15901−15908. (19) Spiecker, P. M.; Kilpatrick, P. K. Interfacial rheology of petroleum asphaltenes at the oil-water interface. Langmuir 2004, 20, 4022−4032. (20) Jung, J.-M.; Savin, G.; Pouzot, M.; Schmitt, C.; Mezzenga, R. Structure of heat-induced β-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate. Biomacromolecules 2008, 9, 2477−2486. (21) Krebs, M. R.; Devlin, G. L.; Donald, A. M. Amyloid fibril-like structure underlies the aggregate structure across the pH range for βlactoglobulin. Biophys. J. 2009, 96, 5013−5019.

strain and NLO is depicted. With increasing pH from 2 to 6.5, G′i is shifted from 0.001 to 0.1 Pa m, whereas at the same time, the strain necessary for the nonlinear onset decreases. After pH 7, the NLO decreases sharply as a consequence of charge inversion and the restoring of an increasing linear charge density. Although the NLO remains higher than at the beginning, the strain necessary for this onset is lower. In Figure 8B, the influence of the ionic strength is depicted. With increasing ionic strength, the NLO is steadily increasing, while the strain decreases monotonically. This shows that the adsorbed layer becomes stronger, but more brittle. To summarize, the effects caused by ionic strength and pH are similar up to a certain pH. After the isoelectric point of the protein, the systems reacts differently and inverts the evolution of the NLO.

IV. CONCLUSION We measured the transient influence of pH and ionic strength on the interfacial viscoelasticity of β-lactoglobulin fibrils at the water−oil interface. To perform these measurements, the measuring cell was modified to simultaneously control the physicochemical conditions of the bulk phase during interfacial rheological measurements. The interfacial storage G′i and loss G″i moduli are influenced by both the pH and the ionic strength. Both factors led to an increase of G′i and G″i. The maximum of G′i observed during the ionic strength change was 1 order of magnitude smaller than with pH change. A weak strain overshoot was observed in the amplitude sweeps at pH ≈ 6, whereas no weak strain overshoot occurred during the increase of the ionic strength. The increase of ionic strength leads to a decrease of the Debye length, which leads to a thicker layer. The pH from 2 to 6 led to aggregation of the βlactoglobulin fibrils at the interface, whereas a further increase of pH from 6 to 11 led to a change in charge of the fibrils, which then led to a decrease in viscoelastic properties. Our experiments show the simultaneous in-line pH and ionic strength to rheological values dependency. This enables us to observe the rheological response at the isoeletric point of proteins after adsorption. Additionally, time effects can be taken into account due to the simultaneous control of rheological and physiological parameters. Dynamic processes occurring in emulsions (such as pH) can be directly compared to this method, as the pH can be changed after adsorption.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We would like to express our thanks to Jan Corsano for construction of the subphase exchange cell. Sophia Jordens and Lucio Isa are thanked for their fruitful discussions. P.R. acknowledges financial support by ETH Zurich (Project ETHIIRA TH 32-1 “Amyloid Protein Fibers at Surfaces and Interfaces”).

■

REFERENCES

(1) Brooks, C. F.; Fuller, G. G.; Frank, C. W.; Robertson, C. R. An interfacial stress rheometer to study rheological transitions in monolayers at the air water interface. Langmuir 1999, 15, 2450−2459. 12542

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543

Langmuir

Article

(22) Humblet-Hua, K. N. P.; Scheltens, G.; van der Linden, E.; Sagis, L. Encapsulation systems based on ovalbumin fibrils and high methoxyl pectin. Food Hydrocolloids 2011, 25, 569−576. (23) Adamcik, J.; Jung, J. M.; Flakowski, J.; De Los Rios, P.; Dietler, G.; Mezzenga, R. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat. Nanotechnol. 2010, 5, 423−428. (24) Akkermans, C.; van der Goot, A. J.; Venema, P.; van der Linden, E.; Boom, R. M. Formation of fibrillar whey protein aggregates: Influence of heat and shear treatment, and resulting rheology. Food Hydrocolloids 2008, 22, 1315−1325. (25) Akkermans, C.; Venema, P.; van der Goot, A. J.; Gruppen, H.; Bakx, E. J.; Boom, R. M.; van der Linden, E. Peptides are building blocks of heat-induced fibrillar protein aggregates of β-lactoglobulin formed at pH 2. Biomacromolecules 2008, 9, 1474−1479. (26) Kroes-Nijboer, A.; Venema, P.; Bouman, J.; van der Linden, E. The critical aggregation concentration of β-lactoglobulin-based fibril formation. Food Biophys. 2009, 4, 59−63. (27) Isa, L.; Jung, J.-M.; Mezzenga, R. Unravelling adsorption and alignment of amyloid fibrils at interfaces by probe particle tracking. Soft Matter 2011, 7, 8127−8134. (28) Jung, J.-M.; Gunes, D. Z.; Mezzenga, R. Interfacial activity and interfacial shear rheology of native β-lactoglobulin monomers and their heat-induced fibers. Langmuir 2010, 26, 15366−15375. (29) Mezzenga, R.; Jung, J.-M.; Adamcik, J. Effects of charge double layer and colloidal aggregation on the isotropic-nematic transition of protein fibers in water. Langmuir 2010, 26, 10401−10405. (30) Loveday, S.; Wang, X.; Rao, M.; Anema, S.; Creamer, L.; Singh, H. Tuning the properties of β-lactoglobulin nanofibrils with pH, NaCl and CaCl2. Int. Dairy J. 2010, 20, 571−579. (31) Adamcik, J.; Mezzenga, R. Adjustable twisting periodic pitch of amyloid fibrils. Soft Matter 2011, 7, 5437−5443. (32) Jung, J.-M.; Mezzenga, R. Liquid crystalline phase behavior of protein fibres in water: Experiments versus theory. Langmuir 2009, 26, 504−514. (33) Shammas, S.; Knowles, T. P. J.; Baldwin, A. J.; MacPhee, C. E.; Welland, M. E.; Dobson, C. M.; Devlin, G. L. Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions. Biophys. J. 2011, 100, 2783−2791. (34) Jones, O. G.; Handschin, S.; Adamcik, J.; Harnau, L.; Bolisetty, S.; Mezzenga, R. Complexation of β-lactoglobulin fibrils and sulfated polysaccharides. Biomacromolecules 2011, 12, 3056−3065. (35) Maheshwari, R.; Bhavani, R.; Dhathathreyan, A. Solid−liquid interfacial energy as a tool to estimate shifts in isoelectric points of adsorbed proteins on solid surfaces. J. Colloid Interface Sci. 2006, 293, 500−504. (36) Hyun, K.; Kim, S. H.; Ahn, K.; Lee, S. Large amplitude oscillatory shear as away to classify the complex fluid. J. Non-Newtonian Fluid Mech. 2002, 107, 51−65. (37) Erni, P.; Parker, A. Nonlinear viscoelasticity and shear localization at complex fluid interfaces. Langmuir 2012, 28, 7757− 7767.

12543

dx.doi.org/10.1021/la3026705 | Langmuir 2012, 28, 12536−12543