Langmuir 2004, 20, 4881-4884
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Depletion-Flocculation in Oil-in-Water Emulsions Using Fibrillar Protein Assemblies Theo B. J. Blijdenstein, Cecile Veerman, and Erik van der Linden* Food Physics Laboratory, Department of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands Received January 29, 2004. In Final Form: March 23, 2004 This paper shows that low concentrations of β-lactoglobulin fibrils can induce depletion-flocculation in β-lactoglobulin-stabilized oil-in-water emulsions. The minimum required fibril concentration for flocculation was determined experimentally for fibril lengths of about 3 and 0.1 µm. The minimum fibril concentration for flocculation is two orders of magnitude higher for the short fibrils than for the long ones. These experimental results correspond well with a theoretical prediction based on a model of spinodal decomposition. In addition, rheological measurements were performed, verifying that flocculation was induced by a depletion mechanism. The results of this study show that the minimum concentration required for depletion-flocculation can be tuned by varying the length of the fibrils.
1. Introduction The stability and mechanical properties of oil-in-water emulsions can be tuned by addition of polymers.1 Above a minimum concentration, polymers can induce rapid depletion-flocculation of the emulsion droplets, leading to the formation of a weak reversible droplet network.2,3 The stability of such networks depends on the polymer concentration. At low polymer concentrations, depletionflocculated emulsion gels may separate serum as a result of the gravity-induced collapse of the droplet network. However, at larger polymer concentrations, collapse is preceded by a delay time, which may be of the order of days or even months.4 Flexible polymers such as polysaccharides3-7 and compact molecular assemblies such as surfactant or casein micelles are often used to induce depletion-flocculation.8 The minimum concentration for depletion-flocculation roughly depends on the size of the depletion layer thickness, which is related to the size of the polymer molecules. For flexible polymers, which have a compact structure, relatively high concentrations of polymer are required to induce flocculation. To reduce the polymer concentration required for flocculation, stiff or semiflexible polymers may be used, because they infer a larger excluded volume.9,10 Good candidates would be fibrillar protein assemblies composed of, for example, actin or fibrinogen.11,12 Fibrillar assemblies of various food proteins would also be excellent candidates for that matter because they * Corresponding author. Fax: + 31 317 483669. E-mail address:
[email protected]. (1) Dickinson, E. Food Hydrocolloids 2003, 17, 25-39. (2) Poon, W. C. K.; Haw, M. D. Adv. Colloid Interface Sci. 1997, 73, 71-126. (3) Blijdenstein, T. B. J.; Hendriks, W. P. G.; van der Linden, E.; van Vliet, T.; van Aken, G. A. Langmuir 2003, 19, 6657-6663. (4) Manoj, P.; Fillery-Travis, A.; Watson, A. D.; Hibberd, D.; Robins, M. M. J. Colloid Interface Sci. 1998, 207, 283-293. (5) Demetriades, K.; McClements, D. J. J. Agric. Food Chem. 1998, 46, 3929-3935. (6) Demetriades, K.; McClements, D. J. J. Food Sci. 1999, 64, 206210. (7) Tuinier, R.; de Kruif, C. G. J. Colloid Interface Sci. 1999, 218, 201-210. (8) Dickinson, E.; Ritzoulis, C. J. Colloid Interface Sci. 2000, 224, 148-154. (9) Asakura, S.; Oosawa, F. J. Chem. Phys. 1954, 22, 1255-1256. (10) Vrij, A. Pure Appl. Chem. 1976, 48, 471-483. (11) Shah, J. V.; Janmey, P. A. Rheol. Acta 1997, 36, 262-268.
also can form (sub)micrometer fibrils.13,14 For instance, the milk protein β-lactoglobulin (β-lg) can form semiflexible fibrils upon heating for 10 h at 80 °C at pH 2 and low ionic strength. For this condition, 60-70% of the monomeric β-lg is converted into fibrils.15 The length of these fibrils varies between 10-7 and 10-5 m depending on the ionic strength.15 In addition, β-lg is a good emulsifier. The objective of this paper is to determine at what concentrations β-lg fibrils can induce depletion-flocculation in β-lg-stabilized oil-in-water emulsions. The minimum required fibril concentration for flocculation was determined experimentally for fibril lengths of about 3 and 0.1 µm, which were obtained by heating β-lg at 0.01 and 0.09 M, respectively. These experimental results were compared with a theoretical prediction based on a model of spinodal decomposition. In addition, rheological measurements were performed to verify if flocculation was induced by a depletion mechanism. 2. Depletion-Flocculation An estimation of the onset of depletion-flocculation can be predicted using a model of spinodal decomposition.3,7 Spinodal demixing occurs when the osmotic compressibility δΠe/δφe due to interactions between the emulsion droplets becomes negative. The osmotic pressure due to the interacting emulsion droplets, Πe, can be approached by a second-order virial expansion (Πe ∼ φe + B2φe2) up to a droplet volume fraction, φe, of 0.2. Here, B2 is the second virial coefficient. According to this model, spinodal demixing occurs if the volume fraction of emulsion droplets exceeds16
φe ) -
1 ) 2B2
-
∫0
4π
[
∞
(
Ve
)]
UT(h) 1 - exp (de + h) d(de + h) kBT
(1)
where Ve is the volume of an emulsion droplet, UT is the (12) Janmey, P. A.; Shah, J. V.; Janssen, K. P.; Schliwa, M. Subcell. Biochem. 1998, 31, 381-397. (13) Veerman, C.; Sagis, L. M. C.; van der Linden, E. Macromolecular Bioscience 2003, 3, 243-247.
10.1021/la0497447 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/12/2004
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total interaction potential, kB is the Boltzmann constant, T is the absolute temperature, de is the diameter of the emulsion droplet, and h is the distance between the surfaces of the oil droplets. For protein-stabilized emulsion droplets, UT consists of terms for van der Waals attraction, electrostatic repulsion, and depletion attraction. The depletion potential is given by10
Udep(h) ) -
[
cfRT 3(de + h) π(de + 2∆)3 1 + 6M 2(de + 2∆) (de + h)3 2(de + 2∆)3
]
for 0 e h e ∆ (2)
where cf is the concentration of fibrils, R is the gas constant, M is the molecular mass of the protein fibril, and ∆ is the depletion layer thickness inferred by the fibrils. This depletion layer thickness can be calculated from the radius of gyration of the fibril.17 The radius of gyration in turn can be determined from the contour length, Lc, and persistence length, Lp, of the fibril.17
Figure 1. Stability diagram of 10% (w/w) oil-in-water emulsions at varying concentrations of β-lg fibrils and ionic strengths. (2) Creaming; (b) serum separation; ([) delayed serum separation; (9) no serum separation during the time of the experiment; value predicted by theory for the minimum concentration for depletion-flocculation (cff; +).
3. Materials and Methods 3.1. Sample Preparation. β-lg was obtained from Sigma (L0130) and is a mixture of the genetic variants A and B. The protein was dissolved in a HCl solution of pH 2. To remove traces of calcium ions from the β-lg and to obtain a protein solution with the same pH and ionic strength as those of the solvent, the protein was diluted repeatedly with a HCl solution at pH 2 and filtered through a 3K filter in an Omegacell membrane cell (Filtron) at 4 °C and a maximum pressure of 3 bar. The procedure was stopped when the pH and conductivity of the eluted solution and the solvent were the same. The β-lg solution was centrifuged at 22 600g for 30 min. To remove any traces of undissolved protein, the supernatant was filtered through a protein filter (FP 030/2, 0.45 µm, Schleicher & Schuell). A UV spectrophotometer was used to determine the exact β-lg concentration at a wavelength of 278 nm. The obtained stock protein solution of pH 2 (i.e., ionic strength equal to 0.01 M) was used for all measurements. Ionic strengths higher than 0.01 M were prepared by adding NaCl to this stock solution. A 40% oil-in-water stock emulsion, stabilized by native β-lg at pH 2, was prepared by mixing oil into the protein solution using an Ultra Turrax (Polytron, Switzerland). Subsequently, this pre-emulsion was homogenized by 10 passes through a labscale homogenizer (Delta Instruments, Drachten, The Netherlands) operating at a pressure of 50 bar. Stock emulsions had typical d32 values of 1.2 µm as determined from laser diffraction. Protein fibrils were obtained by heating a solution of native β-lg, at pH 2 and ionic strengths of 0.01 or 0.09 M, at 80 °C for 10 h in a water bath. After cooling on ice, varying amounts of β-lg fibrils were added to the stock emulsion. The final concentration of emulsion droplets was 10 wt %, and the final ionic strengths were 0.01 and 0.07 M. Mixing of the emulsion droplets and β-lg fibrils was performed by gently shaking for 30 s. All measurements were carried out at room temperature. 3.2. Demixing Experiments. Demixing of various emulsion samples was followed in time by turbidimetry, using a Turbiscan MA 2000 apparatus (Ramonville St. Agne, France). The initial height of the emulsion samples was 21 ( 1 mm. The interpretation of the backscattering profiles is described in detail elsewhere.3 Shortly, the appearance of a gradient in the backscattering intensity along the height of the emulsion was typical for creaming. The appearance of a sharp boundary between a concentrated emulsion and a serum layer is indicative for serum separation. (14) Sagis, L. M. C.; Veerman, C.; van der Linden, E. Langmuir 2004, 20, 924-927. (15) Veerman, C.; Ruis, H.; Sagis, L. M. C.; van der Linden, E. Biomacromolecules 2002, 3, 869-873. (16) de Hek, H.; Vrij, A. J. Colloid Interface Sci. 1981, 84, 409-422. (17) Tuinier, R. Eur. Phys. J. B 2003, 10, 123-128.
3.3. Viscosity Measurements. Steady shear measurements were carried out using a Bohlin VOR rheometer (Huntingdon, U.K.) with a concentric cylinder geometry (C14). The apparent viscosity was determined as a function of increasing and decreasing shear rate, respectively.
4. Results 4.1. Demixing Experiments. Backscattering profiles of 10 wt % oil-in-water emulsions were measured at varying fibril concentrations and ionic strengths. Note that this concentration denotes the concentration of β-lg fibrils after correction for the conversion of β-lg monomers into fibrils.15 From the backscattering profiles, we can distinguish creaming from serum separation, where serum separation is due to droplet network formation.18 Serum separation may occur instantaneously (serum separation) or after a delay time (delayed serum separation), smaller or larger than the duration of the experiment. The results were collected in a stability diagram (Figure 1). This diagram shows a transition from creaming to serum separation at increasing fibril concentrations. At an ionic strength of 0.01 M, this transition occurs between 0 and 0.14 wt % β-lg fibrils. For 0.07 M, this transition takes place between 0.14 and 0.35 wt % β-lg fibrils. Further increase in fibril concentration leads to delayed serum separation and ultimately to a stable network of emulsion droplets during the time span of our experiments (5 days). This trend was observed in previous studies for polysaccharide-induced depletion-flocculation in emulsions5,19 and can be explained by the increasing droplet-droplet attraction due to increasing osmotic pressure of the fibrils.9,10 The delay at higher concentrations can be explained by a higher viscosity in the aqueous phase and a stronger depletion interaction resulting in stronger droplet-droplet bonds. The trend observed here suggests, therefore, that the droplets flocculate as a result of a depletion mechanism. 4.2. Viscosity Measurements. To confirm that flocculation occurs by a depletion mechanism, we performed steady shear viscosity measurements. Figure 2 shows that the 10 wt % emulsion without added fibrils has a (18) Blijdenstein, T. B. J.; van Vliet, T.; van der Linden, E.; van Aken, G. A. Food Hydrocolloids 2003, 17, 661-669. (19) Cao, Y. H.; Dickinson, E.; Wedlock, D. J. Food Hydrocolloids 1990, 4, 185-195.
Depletion-Flocculation in Oil-in-Water Emulsions
Langmuir, Vol. 20, No. 12, 2004 4883 Table 1. Parameters of Emulsion Droplets and β-lg Fibrils at the Ionic Strengths Used and the Resulting Values of cffa I (M)
Lc (µm)
Lp (µm)
Mfibril (MDa)
rg (µm)
∆ (µm)
cff (wt %)
0.01 0.07
3.0 0.1
1.6 0.04
14 0.46
1 0.03
0.4 0.02
0.003 0.4
a
) 6.9 × 10-10 C V-1 m-1, A ) 4 × 10-21 J, de ) 1 µm.
Figure 2. Apparent viscosity versus shear stress for 10% (w/ w) oil-in-water emulsions with various β-lg fibril concentrations in the aqueous phase and various ionic strengths. The closed symbols refer to a 0.01 M ionic strength, while the open symbols indicate an ionic strength of 0.07 M. (2) 0% β-lg; (b) 0.14% β-lg; ([) 0.28% β-lg; (9) 0.6% β-lg; (4) 0% β-lg; (O) 0.14% β-lg; (]) 0.35% β-lg; (0) 0.67% β-lg.
Figure 3. Apparent viscosity versus shear stress for 0.6% β-lg fibrils at a 0.01 M ionic strength (O); solution of 0.6% β-lg fibrils at a 0.01 M ionic strength containing 10% emulsion droplets (b); 0.67% β-lg fibrils at a 0.07 M ionic strength (4); solution of 0.67% β-lg fibrils at a 0.07 M ionic strength containing 10% emulsion droplets (2).
Newtonian behavior and has a viscosity of about 10-3 Pa‚ s for both ionic strengths. The behavior changes from Newtonian to shear thinning when fibrils are added. The viscosity increases with increasing fibril concentration over the whole spectrum of shear stresses measured. The viscosity of emulsions at a similar fibril concentration is higher at a 0.07 M ionic strength than at a 0.01 M ionic strength. No substantial hysteresis was observed for both ionic strengths. In addition, we measured the viscosity of only the fibril solutions and compared these to the viscosity of the same fibril solutions that contained 10% emulsion droplets. Figure 3 shows that the fibril solutions show shearthinning behavior at about 0.6 wt %, similar to the emulsion-fibril mixtures. The viscosity of the emulsionfibril mixtures is substantially higher than the viscosity of the fibril solutions. This result indicates a contribution of the emulsion droplet network to the viscosity. 4.3. Theoretical Prediction of Depletion-Flocculation. The experimental results on the demixing were
Figure 4. Curves of the interaction potential between two emulsion droplets in the presence of 0.6 wt % of protein fibrils for (A) long fibrils (0.01 M ionic strength) and (B) short fibrils (0.07 M ionic strength).
compared with theoretically predicted fibril concentrations, cff, required to induce depletion-flocculation. To predict these values, we used the equations given in section 2. Table 1 shows the parameters of the protein fibril (Lc, Lp, and Mfibril), the calculated values for rg and ∆, and the resulting values for cff. 5. Discussion The sequence observed in the type of demixing as a function of fibril concentration (Figure 1) indicates that flocculation is caused by a depletion mechanism. In addition, the absence of hysteresis observed in steady shear measurements (Figure 2) shows that the network structure is reversible, also suggesting flocculation by depletion. Moreover, good agreement was found between theoretical predictions and experimentally obtained values of the minimum fibril concentration necessary for depletion-flocculation at a 0.07 M ionic strength (Table 1 and
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Figure 1). For a 0.01 M ionic strength, an exact comparison could not be made between theory and experiment, but the order of magnitude predicted by theory was not in disagreement with our experiments. When we compare cff for the short and the long fibrils (high and low ionic strength, respectively), the long fibrils give a cff that is two orders of magnitude lower, showing the importance of the length and flexibility of the fibrils. The emulsion containing the long fibrils (0.01 M ionic strength) shows a lower viscosity at equal shear rates/ stresses than the emulsion containing the short fibrils (0.07 M ionic strength). This observation is remarkable, because the minimum concentration for flocculation is much lower in the emulsion that contains the long fibrils. A possible explanation for this lower viscosity might be a difference in the interaction potential curve for the long and short fibrils (Figure 4). The well of the interaction potential appears much deeper for the long fibrils, but both curves have a similar shape. However, the range of the droplet-droplet interaction is much larger for the long fibrils than for the shorter ones. As a result of this larger range, droplets can rearrange more easily during flow compared to the short-ranged bonds in the short-fibril system, resulting in a lower viscosity. In addition, for the
Blijdenstein et al.
long fibrils a stronger alignment of the fibrils is expected than for the short fibrils at equal shear rates/stresses. A stronger alignment gives a lower effective volume of the fibrils, also resulting in a smaller contribution to the viscosity. Conclusion Addition of β-lg fibrils, above a minimum concentration, leads to flocculation of the oil droplets by a depletion mechanism. This was deduced from demixing experiments and viscosity measurements. Good agreement between experiment and theory was found for the minimum fibril concentration to induce depletion-flocculation. Comparing two different fibril lengths, the minimum fibril concentration for flocculation is two orders of magnitude higher for the short fibrils than for the long ones. The viscosity increase of emulsion-fibril mixtures compared to fibril solutions is higher for the short fibrils. The results of this work show that fibrillar structures form candidates to efficiently induce depletion-flocculation. By varying the length of the fibrils, the minimum concentration required for depletion-flocculation can be tuned. LA0497447
