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Langmuir 2008, 24, 3794-3800
Diffusing Wave Spectroscopy Study of the Colloidal Interactions Occurring between Casein Micelles and Emulsion Droplets: Comparison to Hard-Sphere Behavior Zafir Gaygadzhiev, Milena Corredig, and Marcela Alexander* Department of Food Science, UniVersity of Guelph, Ontario, Canada N1G 2W1 ReceiVed October 19, 2007. In Final Form: January 16, 2008 Understanding the underlying processes that govern interparticle interactions in colloidal systems is fundamental to predicting changes in their bulk properties. In this paper we discuss the colloidal behavior of casein micelles and protein-stabilized fat globules individually and in a mixture. The colloidal interactions were observed by transmission diffusing wave spectroscopy. The turbidity parameter, l*, and the diffusion coefficients of the samples studied were measured experimentally and compared to the theoretically calculated parameters for a hard-sphere system. The light scattering properties of casein micelles (volume fraction φ ) 0.1-0.2) dispersed in milk permeate showed no deviation from the theoretically predicted model. Whey protein isolate (WPI)-stabilized emulsions (φ ) 0.025-0.1) prepared either in milk permeate or in 5 mM imidazole buffer at pH 6.8 showed a behavior identical to that of the hard-sphere model. Similarly to the WPI-stabilized fat globules, the sodium caseinate (NaCas)-stabilized emulsions (φ ) 0.0250.1) prepared in milk permeate also showed resemblance to the theory. In contrast, NaCas-stabilized emulsions prepared in 5 mM imidazole buffer exhibited some discrepancy from the theoretically calculated parameters. The deviation from theory is attributed to the enhanced steric stabilization properties of these droplets in a low ionic strength environment. When recombined milks made from concentrated milk and WPI- and NaCas-stabilized droplets prepared in permeate (φ ) 0.125-0.2) were studied, the experimental data showed a significant deviation from the theoretical behavior of a hard-sphere model due to mixing of two different species.
Introduction Milk can be described as a fluid composed of colloidal particles (mostly proteins and fat globules) dispersed in an aqueous solution of lactose, vitamins, and other low molecular weight compounds.1 Understanding the principal colloidal interactions occurring between the dispersed particles in milk, mostly casein micelles and milk fat globules, would contribute greatly to predicting changes in the bulk properties of milk. Casein micelles can be described as aggregates of individual casein molecules (Rs1-, Rs2-, β-, and κ-casein) and calcium phosphate.2,3 The calcium-sensitive Rs1-, Rs2-, and β-casein are mostly present in the interior of the micelle. This protein particle is stabilized by κ-casein located predominantly on the outer surface. Most studies agree that κ-casein provides steric and electrostatic stabilization of the casein micelles through the protruding part of its polypeptide chain.3-6 The negatively charged groups, situated along the C-terminal of κ-casein, impart a ζ-potential of about -20 mV (at the normal pH of milk) to the casein micelle and contribute to the electrostatic repulsion between these protein particles.3,4,7,8 Milk fat is also a significant colloidal fraction in milk. The native fat globules are coated with a very complex and fragile membrane and characterized by a wide particle size distribution (from 0.2 to 15 µm, with an average of about 4 µm).1,9,10 Because of the compositional complexity and the polydispersity of the * To whom correspondence should be addressed. Phone: (519) 8244120. Fax: (519) 824-6631. E-mail:
[email protected]. (1) Fox, P. F.; Guinee, T. P.; Cogan, T. M.; McSweeney, P. L. H. Fundamentals of Cheese Science; Aspen Publishers, Inc.: Gaithersburg, MD, 2000. (2) Holt, C. AdV. Protein Chem. 1992, 43, 63. (3) Horne, D. S. Curr. Opin. Colloid Interface Sci. 2006, 11, 148. (4) Dalgleish, D. G. In AdVanced Dairy Chemistry; Fox, P. F., Ed.; Elsevier Applied Science Publishers: London; 1992; Vol. 1, p 579. (5) Dalgleish, D. G. J. Dairy Sci. 1998, 81, 3013. (6) Holt, C.; Horne, D. S. Netherlands Milk Dairy J. 1996, 50, 85. (7) de Kruif, C. G. J. Dairy Sci. 1998, 81, 3019. (8) de Kruif, C. G.; Zhulina, E. B. Colloids Surf., A 1996, 117, 151.
native milk fat globules, it is quite difficult to study the interparticle interactions present in the very complex homogenized milk system. For this reason, studies on model emulsion systems are necessary at first to be able to draw conclusions on the colloidal interactions existing between caseins and model emulsion droplets in a milk environment. Presently, there are a number of products available to the consumer which incorporate different ingredients to a dairy system, such as fortified acidified drinks, for example. Consumers are also demanding more and more nutritious foods or foods with “added” nutritional value. The incorporation of healthbenefit oils or proteins into these drinks or modifications and additions to the simple homogenized milk would be welcomed by both consumer and industry alike. However, to achieve this, a more in-depth knowledge of the interactions occurring between components under realistic conditions in these highly complex systems is needed (which may include the concentrated state and the presence of salts in milk serum, to name a few). There is a wealth of knowledge about the interactions between casein micelles in their native state,11-13 and also some work has been done on nondiluted emulsion systems.14 However, there is no investigation (to our knowledge) on the combination of these two very common industrial ingredients. For this reason, this work involves a simplified milk-plus-emulsion system to try to begin to understand the complex interfacial properties between droplets under the influence of concentration and a highly ionized milk environment. (9) Fox, P. F.; McSweeney, P. L. H. Dairy Chemistry and Biochemistry; Blackie Academic & Professional: London, U.K., 1998; Chapter 3. (10) Singh, H. Curr. Opinion Colloid Interface Sci. 2006, 11, 154. (11) de Kruif, C. G. Int. Dairy J. 1999, 9, 183. (12) Tuinier, R.; de Kruif, C. G. J. Chem. Phys. 2002, 117, 1290. (13) Alexander, M.; Rojas-Ochoa, L. F.; Leser, M.; Schurtenberger, P. J. Colloid Interface Sci. 2002, 253, 35. (14) Ruis, H. G. M.; van Gruijthuijsen, K.; Venema, P. van der Linden, E. Langmuir 2007, 23, 1007.
10.1021/la703265c CCC: $40.75 © 2008 American Chemical Society Published on Web 03/07/2008
Casein Micelle and Emulsion Droplet Interaction
In the present study we investigate model sodium caseinate (NaCas)- or whey protein isolate (WPI)-stabilized emulsion systems individually and in recombined systems containing casein micelles. The static and dynamic behaviors of the colloidal particles (casein micelles and emulsion droplets) were observed using transmission diffusing wave spectroscopy (DWS). The experimentally obtained particle behavior (measured by DWS) was compared to a theoretically predicted behavior for a hard-sphere model. The system is considered a hard sphere when the only interactions occurring among the particles arise from concentration effects (crowding). This is a simple theoretical model that can be applied to assess colloidal systems assuming that a behavior deviating from the hard-sphere model may be an indication for the existence of additional interparticle forces besides the interactions determined solely by the particle crowding.15 In this paper we attempt to characterize the colloidal behavior of recombined milk (prepared to represent as close as possible the “real” conditions of native milk) containing casein micelles and model fat globules. The contribution of both scatterers to the scattering intensity and their role in determining the structural and dynamic properties of recombined milks in an industrially realistic concentrated state have never been reported. Materials and Methods Emulsion and Recombined Milk Preparation. Fresh raw milk was obtained from the Elora Dairy Research Station (Elora, ON, Canada) of the University of Guelph. Sodium azide (0.02% w/v) was added to prevent microbial growth. Milk was centrifuged at 4000g for 20 min at 4 °C to separate the milk fat portion (Beckman J2-21 centrifuge, Beckman Coulter, Mississauga, Canada). Skimmed milk was filtered four times through a Whatman fiberglass filter (Fisher Scientific, Whitby, Canada) to ensure practically complete removal of fat globules. Skimmed milk was then concentrated to a 2× volume fraction by ultrafiltration (PLGC 10k regenerated cellulose cartridge, Millipore Corp., Bedford, MA). The permeate (i.e., milk serum) was also collected. Samples containing various volume concentrations of casein micelles (φ ) 0.1-0.2) were prepared by recombining appropriate volumes of the concentrated skim milk and the milk permeate. Appropriate amounts of NaCas (Alanate 180, New Zealand Milk Products, Lemoyne, PA) or WPI (Land O’Lakes, St. Paul, MN) were dissolved in buffer or milk permeate (to ensure that, when recombined with the fresh milk, the addition of fat globules would not modify the native milk environment). The protein solutions were stirred for 2 h at room temperature and then stored overnight at 4 °C. Coarse emulsions (2% (w/w) protein, 25% (w/w) milk fat) were prepared by mixing the proper amounts of melted anhydrous milk fat (Parmalat Food Inc., London, Canada) and protein solution with a high-speed blender (PowerGen 125, Fisher Scientific, Mississauga, ON, Canada). The emulsification was achieved by passing these coarse emulsions four times through a high-pressure homogenizer (Emulsiflex C5, Avestin, Ottawa, Canada) at a pressure of 350 bar. The homogenization process was carried out at 45 °C by immersing the laboratory homogenizer in a temperature-controlled water bath (Versa-Bath, Fisher Scientific). The emulsions were diluted with milk permeate (pH ≈ 6.8) or buffer (5 mM imidazole buffer, pH 6.8) to obtain volume concentrations of emulsion droplets in the range of 0.025-0.1. Recombined milks (φ ) 0.125-0.2) containing a constant casein micelle concentration (φ ) 0.1) and various volume fractions of fat globules (φ ) 0.025-0.1) were prepared by adding the appropriate volume of emulsions to concentrated skim milk. Diffusing Wave Spectroscopy Experiments. DWS is a light scattering technique that permits the investigation of the interparticle interactions in situ, avoiding the necessity of extensive dilution, (15) Salgi, P.; Rajagopalan, R. AdV. Colloid Interface Sci. 1993, 43, 169.
Langmuir, Vol. 24, No. 8, 2008 3795 which may alter the “true” behavior of the colloidal suspensions.16-19 DWS relies on many scattering events happening when the light passes through a colloidal dispersion.16,20,21 The light propagation through the sample is assumed to occur in a diffusive fashion. Similarly to dynamic light scattering, DWS measures the intensity fluctuations of the transmitted scattered light caused by the Brownian motion of colloidal particles. In transmission DWS, detected intensity fluctuations can be mathematically represented by the following autocorrelation function holding true when t , τ and L/l* > 10:16 L 4 6t + ( l* 3)( τ ) (t) ≈ (1 + 3τ8t ) sinh[l*L (6tτ ) ] + 34[6tτ cosh[l*L (6tτ ) ]] 1/2
g(1)
1/2
1/2 1/2
(1)
where L is the sample thickness, τ the decay time, and l* the transport mean free path. This exponential correlation function, g(1)(t), is described with a characteristic decay time τ ) 1/ko2D, where ko is the wave vector (ko ) 2πn/λ, where n is the refractive index of the continuous phase and λ is the wavelength) and D is the particle diffusion coefficient. This holds true only for freely diffusive motion; therefore, a direct correlation between the decay time and the diffusion coefficient would only be valid before strong interparticle interactions can be detected. Once the particle dynamics is changed to a subdiffusive motion (e.g., the point after which a liquid-like colloidal suspension is converted to a more gel-like state), the quantity t/τ in eq 1 must be substituted by ko〈∆r2(t)〉/6.16,20 DWS detects the mobility of scatterers over a length scale much shorter than the wavelength of the laser used to illuminate the sample.20 As mentioned above, each of the numerous occurring scattering events contributes to the complete dephasing of the light. Thus, each particle has to move a relatively short distance to collectively achieve the complete randomization of incident light. The photon transport mean free path, l*, represents the length scale over which the direction of the light passing through a sample has been fully randomized. Generally, l* is determined by the scattering form factor F(q) and the structure factor S(q):16
∫
l* ∝ ( F(q) S(q) q3 dq)-1
(2)
In a highly dilute system in which particles are far from each other and interparticle interactions are negligible, S(q) is assumed to be equal to 1. In more concentrated dispersions, however, the interparticle forces and the spatial correlation between particles cannot be ignored. A change in the value of l* in such a system can be attributed to the alteration of particle-particle interactions, if all other physical properties of the scatterers such as particle size, concentration, and refractive index contrast remain constant. The turbidity parameter, 1/l*, then describes the positional organization of the particles within the system and the arising interparticle forces. In summary, DWS allows the investigation of the static and dynamic behavior of colloidal particles in fairly concentrated suspensions (φ ≈ 0.1). Static properties of the sample are reflected by the temporal interparticle spatial organization, as determined by the value of l*. Dynamic properties of the sample are represented by the average particle self-diffusion coefficients (D) obtained by probing the colloidal mobility over a very short length scale. Static and dynamic properties of colloidal particles were observed by diffusing wave spectroscopy. The sample (∼1.5 mL) was poured into an optical glass cuvette (Hellma Canada Ltd., Concord, Canada) specified with a 5 mm path length. The cuvette was placed in a (16) Weitz, D. A.; Pine, D. J. Dynamic Light Scattering: The Method and Some Applications; Oxford University Press: Oxford, U.K., 1993; p 652. (17) Rojas-Ochoa, L. F.; Romer, S.; Schefold, F.; Schurtenberger, P. Phys. ReV. 2002, 65, 051403. (18) Alexander, M.; Dalgleish, D. G. Langmuir 2005, 21, 11380. (19) Alexander, M.; Dalgleish, D. G. Food Biophys. 2006, 1, 2. (20) Weitz, D. A.; Zhu, J. X.; Durian, D. J.; Gang, H.; Pine, D. J. Phys. Scr. 1993, T49B, 610. (21) Romer, S.; Urban, C.; Bossig, H.; Strander, A.; Schefold, F.; Schurtenberger, P. Philos. Trans. R. Soc. London 2001, A359, 977.
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Table 1. Particle Size (z-Average) and ζ-Potential of Casein Micelles and NaCas- or WPI-Stabilized Fat Globules, Prepared in Milk Permeate (pH ≈ 6.8) or 5 mM Imidazole Buffer, pH 6.8
casein micelles NaCas-stabilized fat globules prepared in milk permeate WPI-stabilized fat globules prepared in milk permeate NaCas-stabilized fat globules prepared in buffer WPI-stabilized fat globules prepared in buffer NaCas-stabilized fat globules prepared in milk permeate and diluted in buffer WPI-stabilized fat globules prepared in milk permeate and diluted in buffer
thermostated water bath at a temperature of 30 °C. The sample was illuminated by a solid-state laser light with a wavelength of 532 nm and a power of 100 mW (Coherent, Santa Clara, CA). Scattered light intensity was collected in transmission mode. The signal was fed into a cross-correlator (FLEX2K-12x2, Bridgewater, NJ), and the data were processed by a computer. Measurements were acquired every 4 min during a 28 min experimental time period. Details about the experimental setup of DWS equipment used in this study can be found elsewhere.18,22 Acquired experimental data were analyzed using software developed specifically for our equipment (Mediavention Inc., ON, Canada). Particle Size and ζ-Potential Determination. The particle size of the emulsion droplets was measured, under very dilute conditions, by dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments, Malvern, U.K.). The same instrument was used to calculate the ζ-potential of the fat globules by measuring the electrophoretic mobility and applying Henry’s equation.23 The emulsion samples were diluted ∼2000 times in either milk permeate or imidazole buffer depending on the continuous phase of the corresponding emulsion. All chemicals used in this work were purchased from Fisher Scientific (Whitby, ON, Canada). All experiments were carried out in triplicate, and the average and standard deviations are reported.
Results The particle sizes of casein micelles and emulsion droplets were derived from correlation functions (using eq 1) from DWS experiments of the most dilute concentration of the skim milk (φ ) 0.1) or the emulsions (φ ) 0.025). It was assumed that at these concentrations the systems were in full, free-diffusive motion.13 From the fitted characteristic decay time, τ, of the acquired autocorrelation function, the diffusion coefficient of the particle was calculated as explained in the DWS section. This diffusion coefficient, D, was used to calculate the radius of the particles according to the Stokes-Einstein relation:
R)
kT 6πηD
DWS, Do (nm)
DLS, Dapp (nm)
ζ-potential (mV)
204 ( 5 272 ( 5 434 ( 25 306 ( 8 262 ( 8 n/a n/a
n/a 320 ( 14 1164 ( 36 256 ( 18 238 ( 23 288 ( 14 297 ( 16
n/a -15.1 ( 1.2 -14.2 ( 0.8 -58.5 ( 0.3 -55.4 ( 0.9 n/a n/a
where Do is the diffusion coefficient of a particle at infinite dilution and φ is the volume fraction. Do was then determined from the experimentally measured D at the lowest φ used in these experiments, and from Do the particle radius was calculated using eq 3. As a means of comparison, DLS was also used to measure the radii of the particles. Table 1 summarizes the results for the particle size obtained from the DWS and DLS experiments. The value of the ζ-potential for the various particles is also shown. In general, the two light scattering techniques yielded comparable sizes, except for the WPI-stabilized emulsion prepared and measured in milk permeate. The sizes obtained from the DLS measurements of this emulsion were quite variable and ill-defined. The disagreement between the values measured by DWS and the larger diameter obtained from DLS measurements suggests that the extensive dilution required in DLS alters the specific dynamic equilibrium existing between the droplets. Though DLS is an excellent tool for sizing particles in suspension, the drawback of this technique is the necessity for dilution in order to be in the single-scattering regime. For this reason, the behavior of some components, especially in the presence of salts, can be quite different under concentration effects. This shortcoming is avoided by the use of DWS. Static and Dynamic Properties of Casein Micelle Suspensions. The static behavior of casein micelles as a function of their concentration (φ ) 0.1-0.2) was observed by following the change of the values of 1/l* (Figure 1). The experimental data are compared to theoretical values (line). The theoretical calculations of turbidity were performed using Mie scattering theory for the determination of the structure factor F(q) and the Percus-Yevick closure relation in the calculation of the effective structure factor S(q). To calculate the theoretical values of 1/l*, several input parameters were required such as the size, concentration, wavelength of the incoming light, and finally index
(3)
where k is the Boltzmann constant, T the temperature, and η the viscosity of the medium. However, it is important to note that this calculated radius is not at zero dilution as eq 3 requires. Therefore, the Beenakker-Mazur formalism was used to correct the self-diffusion coefficient as a function of the volume fraction assuming, as required, a hard-sphere model. The concentration dependence of the normalized self-diffusion coefficient of a hardsphere system can be written, within approximation, as24
D(φ) ) (1 - 1.83φ + 0.88φ2)Do
(4)
(22) Alexander, M.; Corredig, M.; Dalgleish, D. G. Food Hydrocolloids 2006, 20, 325. (23) Malvern Instruments Ltd. Zetasizer Nano Series User Manual; Worcestershire, England, 2004. (24) Beenakker, C. W. J.; Mazur, P. Physica A 1984, 126, 349.
Figure 1. Changes of 1/l* as a function of the casein micelle volume fraction. The average and standard deviation of three independent measurements are presented. The solid line corresponds to the theoretical behavior fitted to the theory for a hard-sphere system with the following parameters: diameter of the casein micelle, 204 nm; refractive index of the casein micelle, 1.386; refractive index of the solvent (milk permeate), 1.34.
Casein Micelle and Emulsion Droplet Interaction
Langmuir, Vol. 24, No. 8, 2008 3797
Figure 2. Changes in the self-diffusion coefficient (Ds) as a function of the casein micelle volume fraction. The average and standard deviation of three independent measurements are presented. The solid line corresponds to the theoretical dynamic behavior fitted to the theory for a hard-sphere system.
of refraction of both the medium and the particle. All parameters except the latter were obtained experimentally in peripheral experiments. Therefore, when the theoretical calculations of 1/l* were performed, only the index of refraction of the particle, n, was not fixed. Increasing the concentration of casein micelles led to a corresponding gradual growth of 1/l*. The best fit between the experimental data and the theory was achieved when using n ) 1.386. A similar refractive index of casein micelles was reported for reconstituted skim milk powder.13 Experimentally obtained values of 1/l* showed a good agreement with the theoretically predicted behavior for hard-sphere systems. The average deviation between the experimental and theoretical 1/l* was 4.5%. The dynamic properties of casein micelle dispersions in relation to the particle concentration were studied by observing the changes of the self-diffusion coefficients, Ds (Figure 2). The dynamic behavior of a hard-sphere system, as calculated from eq 4, is also shown. The diffusivity of casein micelles, with an average diameter of 204 nm, was gradually slowed with an increase of the volume fraction from 0.1 to 0.2. The average discrepancy between theory and experiment was 2.6%. These results showed that the concentration dependence of the short-time self-diffusion coefficient of the casein micelles can be understood solely on the basis of hard-sphere behavior. These findings are also in agreement with the dynamics of the casein micelle suspensions prepared from skim milk powder.13 Static and Dynamic Properties of Emulsion Droplets. The structural behavior of NaCas- or WPI-stabilized fat globules dispersed in milk permeate or in buffer is shown in Figure 3. Increasing the concentration of the emulsion droplets (φ ) 0.0250.1) caused a respective change of the system’s turbidity, as indicated by the increase of 1/l*. It can be noticed that NaCasor WPI-stabilized emulsions demonstrated some dissimilarity in their light scattering properties. The turbidity parameter for NaCas-stabilized emulsions showed slightly higher values than those for WPI-stabilized emulsions at all concentrations. This is most likely caused by size differences between the scatterers as well as slight differences in the effective refractive index of the particles since the interfaces have a different composition. Figure 3a shows the experimental concentration dependence of 1/l* of emulsion droplets dispersed in milk permeate superimposed with the theoretically predicted behavior of 1/l*. The theoretical behavior was calculated using particle diameters of 272 and 434 nm for NaCas- and WPI-stabilized fat globules, respectively (see Table 1). The experimental concentration dependence of 1/l* for both emulsions followed appreciably the
Figure 3. Changes of 1/l* as a function of the volume fraction for NaCas-stabilized (b) or WPI-stabilized (2) emulsions prepared in milk permeate (A) or 5 mM imidazole buffer (B). The average and standard deviation of three independent measurements are presented. The solid and dashed lines correspond to the theoretical behavior of a hard-sphere system, fitted to the experimental data for NaCasand WPI-stabilized emulsions, respectively. Parameters: (A) 272 and 434 nm, diameter of NaCas and WPI-stabilized fat globules, respectively; 1.34, refractive index of milk permeate; (B) 306 and 262 nm, diameter of NaCas- and WPI-stabilized fat globules, respectively; 1.33, refractive index of imidazole buffer; (in both systems) 1.456 and 1.444, refractive index of the droplet for NaCasand WPI-stabilized emulsions, respectively.
theoretical pattern for hard-sphere systems. The refractive indices of the NaCas- and WPI-stabilized fat globules obtained from the theoretical data were 1.456 and 1.444, respectively. This slight discrepancy between the refractive indices may result from the difference in the interfacial composition between the two emulsions; however, the difference between the refractive indices is much smaller than the experimental error; this makes it impossible to determine, at this point, whether there is a true difference in refractive indices between the emulsion droplets. It is noteworthy that when the same values of the refractive indices were used for the emulsions prepared in buffer, there was also a good theoretical fit of the experimental data. Figure 3b shows the theoretical and experimental behavior of NaCas- or WPI-stabilized emulsions prepared in 5 mM imidazole buffer (pH 6.8). The experimental concentration dependence of 1/l* was fitted to the hard-sphere system theory using a particle diameter of 306 or 262 nm, respectively (see Table 1). WPIstabilized fat globules dispersed in low ionic strength buffer demonstrated, again, hard-sphere behavior in the range of concentrations probed in this work. However, in the case of NaCas-stabilized emulsions, the low ionic strength buffer caused a significant deviation at higher concentrations (φ ) 0.075 0.1) from the theoretically predicted values. Figure 4 illustrates the dynamic properties of NaCas- or WPIstabilized emulsions, prepared in milk permeate or in imidazole buffer and as a function of concentration. Once again, the experimental data are compared to theoretical predictions. All emulsion systems exhibited, as expected, a reduction of the self-
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Figure 5. Changes of 1/l* as a function of the volume fraction for recombined milk containing caseins (φ ) 0.1) and NaCas-stabilized (b) or WPI-stabilized (2) fat globules. The average and standard deviation of three independent measurements are present. The solid and dashed lines correspond to the theoretical prediction for a hardsphere system.
Figure 4. Changes of the self-diffusion coefficient (Ds) as a function of the volume fraction for NaCas-stabilized (b) or WPI-stabilized (2) emulsions dispersed in milk permeate (A) or imidazole buffer (B). The average and standard deviation of three independent measurements are present. The solid and dashed lines correspond to the theoretical dynamic behavior of a hard-sphere system.
diffusion coefficients with increasing concentration of the fat globules. The experimentally obtained particle diameters were in accordance with the corresponding self-diffusion coefficients: larger fat globules led to slower particle mobility and vice versa. Both WPI- and NaCas-stabilized emulsions prepared in milk permeate demonstrated a behavior closely resembling that predicted from a hard-sphere model. However, a slight deviation from theory could be noticed at φ ) 0.1 for NaCasstabilized fat globules (Figure 4a). In contrast to WPI-stabilized emulsion droplets, the dynamic behavior of NaCas-stabilized droplets in imidazole buffer largely deviated from the theoretical expectation of the hard-sphere model, especially at higher concentrations (Figure 4b). This behavior was in agreement with the discrepancy found between the theoretical and the experimental values of 1/l* shown in Figure 3b. Static and Dynamic Properties of Recombined Casein Micelles and Emulsion Droplets. Figure 5 illustrates the concentration dependence of 1/l* for recombined milks (φ ) 0.125-0.2) containing a fixed amount of casein micelles (φ ) 0.1) and a range of concentrations of emulsion droplets (φ ) 0.025-0.1) stabilized by NaCas or WPI. A steady increase of 1/l* was observed for both systems with an increase of the total volume concentration. Similarly to what is shown in Figure 3 for the individual emulsion systems, recombined milk containing NaCas-stabilized fat globules manifested higher values of 1/l* than the mixed system containing WPI-emulsified emulsion droplets. The theoretically calculated dependence of 1/l* for mixed hard-sphere systems is also shown in Figure 5. In the case of uncorrelated particles, the calculated total, effective transport mean-free path, l*eff, is given by16
1 l*eff
)
1
∑i l*
(5) i
Figure 6. Changes of the self-diffusion coefficient (Ds) as a function of the volume fraction for recombined milk containing NaCasstabilized (b) or WPI-stabilized (2) fat globules. The average and standard deviation of three independent measurements are present. The solid and dashed lines correspond to the theoretical calculation of the dynamic behavior for a hard-sphere system for recombined milk containing NaCas- and WPI-stabilized fat globules, respectively.
where l*i is the transport mean-free path of each individual species (i.e., casein micelles or fat globules). The theoretical static calculations were generated using the l* values obtained from the single-scatterer systems shown in Figures 1 and 3. The experimental results of the recombined system can be fairly well described by the “expected” results obtained when diffusing wave spectroscopy theory is applied (eq 5). The dynamic behavior was also tested for consistency by the generation of a theoretical curve. Again, according to DWS theory, the effective, average diffusion coefficient of noninteracting, multicomponent systems can be described by16
Deff l*eff
)
Di
∑i l*
(6) i
where Di represent the diffusion coefficients of the individual components. Figure 6 shows the calculated results (line) superimposed to the experimental results (filled symbol). A decrease in the particles’ diffusion coefficients was registered for both recombined milks when the total particle volume fraction was increased; however, there was a more pronounced difference between the experimental particles’ mobilities and the dynamic properties of hard spheres predicted from the theoretical computation. The dynamic behavior of systems containing
Casein Micelle and Emulsion Droplet Interaction
interacting species is not as well understood as in the static case. There are currently no calculations for the collective, q-dependent diffusion coefficient of the collective motion of particles that interact hydrodynamically or otherwise.16 This discrepancy between experiment and theory could be attributed to other than hard-sphere interactions between the particles in the recombined system.
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Static and Dynamic Properties of Casein Micelles. The observed increase of the 1/l* value in relation to the casein micelle concentration can be attributed to the change of the system’s turbidity (Figure 1). Since l* represents the length scale over which the scattered light has been fully randomized, the presence of more particles per unit space would result in complete dephasing of the incoming light over a shorter length scale. An identical concentration dependence behavior of 1/l* was also reported for skim milk powder suspensions in the volume fraction range used in the present study.13 Simultaneously, the mobility of casein micelles was slowed by hydrodynamic interactions with an increasing number of particles occupying a unit volume (Figure 2). The magnitude of electrostatic forces or the steric stabilization of the κ-casein layer in the concentration range of this study did not seem to have a considerable impact on the static or dynamic properties of the casein micelles. Otherwise, the casein micelles’ behavior would show a deviation from the hard-sphere model as no other interactionssother than hydrodynamicsare assumed to be present. The natural environment of casein micelles (i.e., milk serum) is characterized with an ionic strength of about 100 mM.25 The ions present in milk serum cause a significant screening effect which reduces the range of the electrostatic interactions pushing the casein micelles to react as hard spheres.26 In conclusion, the structural and the dynamic properties of casein micelles (φ ) 0.1-0.2) dispersed in their natural environment (milk serum) considerably resembled the typical behavior for hard-sphere systems. Static and Dynamic Properties of Emulsion Droplets Stabilized with NaCas or WPI. The WPI-stabilized emulsion droplets prepared and dispersed in milk serum showed a larger particle size compared to the WPI-stabilized emulsion droplets prepared and dispersed in 5 mM imidazole buffer (Table 1). In particular, DLS measurements in buffer contained large errors and were very ill-defined. The presence of Ca2+ in the milk permeate is most likely responsible for the manifested behavior of the WPI-stabilized fat globules. Positively charged calcium ions bind to the available oppositely charged groups of whey proteins adsorbed at the interface.27 This screening effect caused by the ions present in permeate leads to a considerable decrease in the electrostatic repulsion between the fat globules, as shown by differences in the ζ-potential (Table 1). This allows for a closer approach between two neighboring emulsion droplets, leading to flocculation.27-29 A certain degree of flocculation of WPI-stabilized fat globules in the high ionic strength environment of the milk serum was confirmed by the large difference observed in the particle size of these emulsions (detected by DLS and DWS measurements) compared to the size of WPI-stabilized fat globules prepared and measured in low ionic strength imidazole buffer (Table 1). Both these measurements were carried out at
the same pH value of 6.8. These results clearly demonstrate the impact of various ionic species contained in the milk permeate on the stability of the system. The significant decrease in magnitude of the net interfacial electrostatic charges contributes to the domination of intersurface attractive forces, leading to particle flocculation. To further confirm whether this attraction led to bridging flocculation or whether it was merely an electrostatic effect, DLS measurements were performed on the WPI-stabilized fat globules prepared in serum but this time diluted buffer (Table 1). It is clearly seen from this table that dilution in a low ionic strength medium leads to the disruption of these flocs, yielding a particle size comparable to that of the WPIstabilized fat globules prepared and measured in buffer (Table 1). This experiment confirms that the WPI-emulsified fat globules in low ionic strength buffer are stabilized by electrostatic repulsive forces. The corollary of this experiment is the demonstration of the advantages of DWS as a technique to study the colloidal systems at realistic (nondiluted) concentrations. No visible creaming was shown for WPI-stabilized emulsions prepared in milk serum for several days, and surprisingly, these aggregates can be regarded as hard spheres with an apparent size equal to what can be called the average floc size (Figure 3). In contrast, the environmental conditions (milk permeate or imidazole buffer) did not lead to an extreme difference in the droplet size of NaCasstabilized fat globules. The slight variation between DWS measurements and DLS measurements as well as the differences in size between the droplets made and measured in serum or made in serum and measured in buffer might be due to dilution effects (DWS measures the solution in a concentrated state, while DLS needs high dilution) or the slight differences in the quality of the medium. In any case, the sizes obtained for both methods are quite similar, implying that the ionic strength and the calcium ion concentration in milk permeate (10 mM25) were not sufficient to destabilize these emulsions, at least over the time of the observations. The variation between the behavior of the two emulsions prepared in milk permeate might be attributed to the difference in the surface properties of fat globules stabilized by caseins or whey proteins. The structure of the interfacial surface stabilized by NaCas has been described as a train-loop-tail structure, in which the more hydrophobic fractions of casein polypeptide chains are in direct contact with the interface or form an association with the hydrophobic regions of other casein molecules.30 The hydrophilic portions of the caseins extend into the aqueous surroundings, providing steric and electrostatic stabilization.30,31 Even when the surface charge decreased because of ion screening (as shown by the reduction in the ζ-potential magnitude for droplets measured in milk permeate, Table 1), the stability of such an emulsion was maintained by the steric hindrance provided by the flexible casein chains located on the interface. On the other hand, the WPI-stabilized interface could be visualized as a layer of closely packed, highly interacting globular proteins, giving more electrostatic repulsion than steric stability of the emulsion droplets.32 Though once at the interface WPI undergoes some extent of denaturation, the change in the secondary structure for globular proteins is limited.33 It has been shown that β-lactoglobulin retains some of its native structure in an emulsified state.34 This protein can be regarded as a densely
(25) Holt, C. Eur. Biophys J. 2004, 33, 421. (26) de Kruif, C. G. Int. Dairy J. 1999, 9, 183. (27) Agboola, S. O.; Dalgleish, D. G. J. Food Sci. 1995, 60, 399. (28) Kulmyrzaev, A.; Chanamai, R.; McClements, D. J. Food Res. Int. 2000, 33, 15. (29) Kulmyrzaev, A.; Sivestre, M. P. C.; McClements, D. J. Food Res. Int. 2000, 33, 21.
(30) Dickinson, E. J. Chem. Soc., Faraday Trans. 1992, 88, 2973. (31) Dagleish, D. G. Trends Food Sci. Technol. 1997, 8, 1. (32) Dickinson, E. J. Dairy Sci. 1997, 80, 2607. (33) Martin, A. H.; Meinders, M. B. J.; Bos, M. A.; Cohen, Stuart, M. A.; van Vliet, T. Langmuir 2003, 19, 2922. (34) Shimizu, M. In Food Macromolecules and Colloids; Dickinson, E., Lorient, D., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1983; p 34.
Discussion
3800 Langmuir, Vol. 24, No. 8, 2008
2-D packed layer.35 Therefore, the spatial organization of fat globules in the WPI-stabilized emulsion was affected to a greater extent by the ionic contribution, especially Ca2+ ions, since an interface composed of whey proteins provides minor (if any) steric stability, allowing fat globules to approach and form small flocs. Although the fat globules dispersed in low ionic strength buffer (whether NaCas- or WPI-stabilized) were significantly more charged compared to those suspended in milk serum, both emulsions exhibited rather different behaviors. While the WPIstabilized fat globules still behaved as hard spheres, NaCasstabilized fat globules showed a pattern different from that of the hard-sphere model. Therefore, it can be suggested that, in addition to electrostatic repulsion, steric stabilization plays a significant role in determining the behavior of NaCas-stabilized fat globules in buffer. Most likely the spatial conformation of the “hairs” on the interface is different depending on the ionic strength. This is probably due to different phenomena at play. Possibly, the low ionic strength environment allows the hydrophilic parts of the casein polypeptide chains to adopt a more extended conformation compared to that of a “salted brush” where more screening of the charges occurs. The presence of more extended highly charged hairs would lead to an increase of the steric repulsion between interfaces. At the same time, variability in the suspending medium can affect molecular interactions via changes in the second virial coefficient. This quantity reflects the nature of protein-protein interactions, and a negative value can be interpreted as a predominantly attractive interaction among protein molecules at an interface, leading to attractive forces between the droplets. It has been shown36 that the absence of salt in an aqueous solution can lead to protein charges moving toward their isoelectric point with the corresponding surface charge fluctuations. This attractive force may explain the increased interactions (downward trend of 1/l*) shown in Figure 3b. The lack of lactose in the buffer solution could lead to folding and association changes of the surface protein,37 although the lactose concentration in milk might be too low for this effect to take place. Nevertheless, more work will be carried out to look at this variable. The combination of two or more of these different phenomena may explain the deviation of NaCasstabilized fat globules from the theoretical model when dispersed in 5 mM imidazole buffer. Static and Dynamic Properties of Recombined Casein Micelles and Emulsion Droplets. Like in single-scatterer systems, the increase of the oil concentration led to a gradual increase of 1/l* of the mixed system (Figure 5), indicating an increase of turbidity. The higher values of 1/l* observed for the system containing NaCas-stabilized fat globules could be attributed to the inherent factors characterizing the NaCasstabilized emulsion. In general terms, the experimental results seem to follow the theoretical calculations relatively well within experimental error. The dynamic properties of mixed systems, however, significantly differed from the behavior calculated from the DWS theory (Figure 6). The presence of two scatterers (casein micelles and fat globules), whose dynamic behavior was identical to that of hard spheres when measured in isolation, did not lead to the formation of a mixed system with similar behavior. Depletion effects can be excluded since for this phenomenon to take place, the ratio of the particle sizes must be around 1:10.38 As seen in Table 1, we are clearly well outside this critical region. (35) Dickinson, E. Colloids Surf., B 2001, 20, 197. (36) Kirkwood, J. G.; Timasheff, S. N. Arch. Biochem. Biophys. 1956, 65, 50. (37) Timasheff, J. G. Biochemistry 2002, 41, 13473. (38) Radford, S. J.; Dickinson, E. Colloids Surf., A 2004, 238, 71.
GaygadzhieV et al.
It could be speculated that the interactions occurring among the scatterers (casein micelles and emulsion droplets) at the higher volume fractions might contribute to the observed deviation from the theoretically predicted pattern. Other types of interactions (besides those arising from the hydrodynamic effect) are present at these high concentrations, accounting for the deviation from the hard-sphere behavior. The deviation of the experimental results from theoretical computations also points out the need for theoretical models which incorporate a q-averaged diffusion coefficient with other than hydrodynamic interactions.
Conclusions DWS has been successfully utilized to follow the static and dynamic changes observed in concentration effects of skim milk, emulsion solutions, and mixed systems. We could detect some similarities as well as differences among these three. It was confirmed that the casein micelles behaved very similarly to a hard-sphere system, as previously suggested.13,26,39 Similarly, both NaCas- and WPI-coated emulsion droplets when dispersed in milk permeate also behaved as hard spheres up to a 10% volume fraction. Interestingly, the effective size of the WPI emulsion was larger than for the NaCas case. This indicates that the WPI-coated droplets exist, under concentration, in a flocculated state. This is the first reported evidence of the instability of concentrated WPI-stabilized emulsions in the presence of salts at realistic volume fractions and could have important implications in the design of novel food products. However, these flocs seem to behave as hard spheres with a size equivalent to that of the flocculated state. This is quite surprising if we take into consideration the obvious, nonideal shape of a floc. When dispersed in salt-free buffer, NaCas-stabilized emulsions deviated from hard-sphere behavior. Probably, the absence of salts allows for the straightening of the “hairy” layer, which enhances the steric stabilization properties of the emulsion droplet. It seems that the electrostatic interactions alone do not play a role at these concentrations. It is, however, noteworthy that on the basis of the hard-sphere analogy we were able to quantitatively calculate the optical properties of the individual scattering components. It is quite remarkable to us that such a complex system can still be mostly explained in terms of simplified, ideal models. However, when the emulsion droplets are mixed with casein micelles, both systems deviate dynamically from the theoretical prediction for hard-sphere behavior. In these systems, the total volume fraction is higher than that of the individual components, and it could be that, at these concentrations, the presence of two different charged spheres at high volume fractions will show a repulsive effect and a subsequent deviation from the hard-sphere model. Further studies are required in complex mixed systems. We are currently upgrading our facilities to incorporate highpower lasers to be able to probe higher sample turbidities. In this paper we have attempted to explain, for the first time, the nature of the interfacial interactions in a real, concentrated, and mixed food system. Further work is needed on these realistic and complex food systems to understand the local structure and dynamics that drive a system to (in)stability, which will eventually enable us to design new food products. Acknowledgment. This work has been partly funded by the Ontario Dairy Council and the Natural Sciences and Engineering Research Council of Canada. LA703265C (39) Dickinson, E. Int. Dairy J. 1999, 9, 305.
