Biomacromolecules 2005, 6, 3107-3111
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Jamming and Gelation of Dense β-Casein Micelle Suspensions Maud Panouille´ , Dominique Durand,* and Taco Nicolai* Polyme` res, Colloı¨des, Interfaces, Unite mixte´ de recherche, Centre National de la Recherche Scientifique, Universite´ du Maine, 72085 le Mans Cedex 9, France Received June 23, 2005; Revised Manuscript Received September 15, 2005
The rheology of dense suspensions of β-casein micelles is investigated at pH 6. For a given temperature, the viscosity increases dramatically at a critical concentration (Cc) of about 100 g/L due to jamming of the micelles. For a given concentration close to and above Cc, the viscosity of dense suspensions decreases strongly with increasing temperature because Cc increases. The suspensions show weak shear thickening followed by strong shear thinning. At lower pH, that is, closer to the isoelectric point, spontaneous gelation is observed, which is favored by lowering the temperature and addition of sodium polyphosphate. The gelation process is studied at pH 5.5 by rheology and light scattering. β-Casein represents 40% of native casein, which constitutes the main milk protein. The other three major casein molecules that make up native casein are R1- (40%), R2(10%), and κ-casein (10%).1 β-casein is a rheomorphic and flexible molecule, with a molecular weight of 24 000 g‚mol-1. It contains phosphoseryl residues in the central region of the molecule and is therefore sensitive to the addition of calcium ions, which can lead to precipitation.2-4 Precipitation does not occur at 4 °C, suggesting that hydrophobic attraction is implicated in the aggregation process. β-Casein has a negatively charged hydrophilic N-terminal region and a strongly hydrophobic C-terminal region.5 It is thus an amphiphilic protein, which associates into micelles similarly to surfactant molecules. β-Casein association is driven by hydrophobic interactions and is limited by electrostatic repulsion due to negative charges on the hydrophilic extremity. Its association is thus favored by increasing temperature, which increases hydrophobic attraction, and by increasing ionic strength, which decreases electrostatic repulsion.6 The self-association mechanism of β-casein has been described as a monomer-micelle equilibrium:5,7-10 nβ T βn However, recently de Kruif et al.11-14 proposed a consecutive micellization, using the Kegeles shell model, to explain high sensitivity calorimetry results and the polydispersity of β-casein micelles: β + βi T βi+1 All reaction constants have the same value except the first one, which is smaller and constitutes the limiting step. The aggregation number and size of the micelles depend on the concentration, temperature, pH, and ionic strength.8,9,11,15 * Corresponding authors: e-mail
[email protected] (D.D.) or
[email protected] (T.N.).
They are usually described as spherical8,9,16,17 or ellipsoidal15 particles consisting of a dense hydrophobic core surrounded by a loose hydrophilic shell.14,15,17 β-Casein micelles have been studied extensively in dilute conditions but, as far as we are aware, not at high concentrations. In the present work we study the rheological properties of concentrated β-casein suspensions as a function of temperature and concentration. A comparison will be made with earlier results obtained on mixed micelles formed by all four caseins after removal of the colloidal calcium phosphate (CCP) that stabilizes the large native casein complex. These mixed micelles are similar in size to β-casein micelles and are called submicelles for historical reasons. Recently, we reported a study of the rheological properties of submicelle suspensions obtained by dissolving native casein in 20 g‚L-1 sodium polyphosphate at pH 6. We showed that the viscosity increases dramatically above a critical concentration close to 100 g‚L-1 caused by jamming of the submicelles. 18 However, the submicelles are not stable, and with time they aggregate and gel.19 Gelation is very slow at room temperature, but the aggregation process is accelerated by increasing temperature or casein concentration. At pH 6 no aggregation and gelation was observed for β-casein micelles. However, if the pH is reduced to 5.5, the systems gel even at concentrations as low as 8 g‚L-1. A preliminary study of the gelation process by light scattering and rheology is presented. Materials and Methods We used for this study β-casein supplied by Lactalis (Laval, France). The protein content is 92% of the dry material. The powder was dissolved in Millipore water (18 MΩ), with 200 ppm sodium azide added as a bacteriostatic agent, with stirring for about 1 h. The pH of the solutions was adjusted with 0.5 M HCl. Unless otherwise stated, 20 g‚L-1 sodium polyphosphate (Joha, BK Giulini Chemie) was added to facilitate comparison with earlier results obtained for casein submicelles.
10.1021/bm050438x CCC: $30.25 © 2005 American Chemical Society Published on Web 10/18/2005
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Figure 1. Concentration dependence of zero-shear viscosity at 20 °C for suspensions of β-casein micelles or casein submicelles in 20 g‚L-1 sodium polyphosphate. The solid line represents eq 1 with Cc ) 103 g‚L-1.
Static and dynamic light scattering measurements were made on an ALV-5000 multibit multi-τ correlator in combination with a Malvern goniometer and a Spectra Physics laser operating with vertically polarized light with wavelength λ ) 532 nm. Data were collected at scattering angles θ between 10° and 140°, which correspond to scattering wave vectors 2.7 × 10-3 < q < 3.0 × 10-2 nm-1 with q ) (4πn/λ) sin (θ/2), n being the refractive index. The temperature was controlled by a thermostat bath and was set at 20 °C. The apparent hydrodynamic radius was determined by dynamic light scattering as described elsewhere.18 The viscosity of suspensions with η < 10 Pa‚s was measured with a Contraves Low-Shear 40, using Couette geometry with an inner diameter of 12 mm and an outer diameter of 12.5 mm. Steady shear rate and oscillation measurements were made with a stress-imposed rheometer (TA Instruments Rheolyst AR1000) with cone (40 mm, 0.58°)-plate geometry. The temperature was controlled by a Peltier system, and paraffin oil was added to prevent water evaporation. Gelation was studied in situ at different temperatures by use of a strain-imposed rheometer (Rheometrics, ARES) with cone (40 mm, 0.58°)-plate geometry at 0.1 Hz in the linear regime. The temperature was controlled by a Peltier system, and mineral oil was added to prevent evaporation. Results and Discussion 1. Jamming of Dense β-Casein Suspensions. The rheological properties of β-casein suspensions were studied at pH 6 as a function of the casein concentration and the temperature. At this pH the hydrodynamic radius of the micelles determined at C ) 15 g‚L-1 is 14 nm. The concentration dependence of the viscosity extrapolated to zero shear rate (η0) at 20 °C is shown in Figure 1. It shows a dramatic increase when C approaches 100 g‚L-1. The increase is caused by jamming of the close-packed micelles and is similar to that observed for casein submicelles,18 also shown in Figure 1 for comparison. The initial increase of
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Figure 2. Shear-rate dependence of the viscosity of casein suspensions at different temperatures indicated in the figure for a casein concentration of 90 g‚L-1.
the viscosity can be described by the following semiempirical equation:20
(
η0 ) ηs 1 -
C Cc
)
-2
(1)
where ηs is the solvent viscosity. Above the critical concentration (Cc), η0 does not diverge, as is observed for hard spheres, but nevertheless increases very steeply. This behavior has already been observed and described for soft spheres21 or multiarm star polymers.22,23 The dependence of the dynamic viscosity as a function of the shear rate (γ˘ ) is shown in Figure 2 for a casein concentration of 90 g.L-1 and at different temperatures between 10 and 30 °C. The low shear viscosity increases strongly with decreasing temperature, similar to what has been reported for casein submicelles.18 For submicelles we found that increasing the temperature leads to an increase of Cc, due to a decrease in the effective volume of submicelles. A small increase of Cc has a strong effect on the viscosity of dense suspensions, because η0 increases very steeply with C/Cc close to and above Cc. In the same way a small temperature dependence of the thermodynamic volume of β-casein micelles can explain the strong temperature dependence of the viscosity for dense suspensions. With increasing shear rate we observed first shearthickening followed by strong shear-thinning. The same behavior is observed if the measurements are repeated. In the case of casein submicelles, only shear thinning was observed starting at critical shear rate (γ˘ c) that was inversely proportional to η0. The value of γ˘ c is determined by the time needed for a micelle to diffuse its own radius. At present we can only speculate that the origin of the shear thickening observed for pure β-casein suspensions could be caused by attraction between the close-packed micelles. The flow experiments at a fixed shear rate showed a constant welldefined viscosity except at shear rates much larger than γ˘ c, where the flow became irregular and large stress fluctuations are observed at a fixed shear rate. This behavior is similar to that of dense casein submicelles shown in Figure 5 of ref 18.
Jamming and Gelation of Dense β-Casein Micelles
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Figure 4. Time dependence of the scattered light intensity and the storage (solid symbols) and loss (open symbols) moduli for a casein solution (8 g‚L-1) in 20 g‚L-1 polyphosphate (pH 5.5) heated at 40 °C.
Figure 3. (a, top panel) Frequency dependence of the storage (solid symbols) and loss (open symbols) moduli at different temperatures indicated in the figure for a casein concentration of 90 g‚L-1. (b, bottom panel) Master curve of the data shown in panel a, obtained by normalizing the frequency f by a critical frequency fc. The lines indicate the limiting liquidlike behavior at low reduced frequencies.
Oscillatory shear measurements were done in the linear regime for C ) 90 g‚L-1 at different temperatures. The frequency dependence of the storage (G′) and loss (G′′) moduli is shown in Figure 3 (top). We observed a transition between a viscous liquidlike behavior at low frequencies and elastic gellike behavior at high frequencies. The transition shifts to lower frequencies with decreasing temperature. This means that the terminal relaxation time of the jammed suspensions decreases with increasing temperature. The curves obtained at different temperatures can be superimposed by temperature-frequency superposition (see Figure 3, bottom). A vertical shift was necessary only for the data obtained at 10 °C. The master curves are similar to those obtained for casein submicelles. However, the high-frequency elastic modulus is larger for dense suspensions of submicelles than for β-casein micelles, while the relaxation is faster at the same temperature and concentration. This means that the similar viscosity of the two systems is the result of a fortuitous compensation of effects. Nevertheless, although different in detail, both β-casein micelles and casein submicelles behave like jammed soft spheres at elevated concentrations. 2. Gelation. At pH 6, β-casein did not evolve in time, but if the pH of β-casein suspensions is reduced to pH 5.5,
gels are formed even at relatively low concentrations (8 g‚L-1) in the presence of 20 g‚L-1 sodium polyphosphate. When the pH is decreased, the charge density of β-casein decreases, because it has an isoelectric point at pH 5.2. The rate of gelation increases with increasing temperature and protein concentration. At 20 and 40 °C, the minimum concentration to observe gelation within a few days is between 4 and 8 g‚L-1 in the presence of 20 g‚L-1 sodium polyphosphate and about 45 g‚L-1 in pure water. It appears that polyphosphate plays a specific role in the gelation, because results obtained in 0.2 M NaCl are close to those obtained in pure water. Polyphosphate is a polyanion and could perhaps bridge between local positive sites of β-casein molecules of different micelles. At 4 °C, gelation was observed only above 45 g‚L-1 in sodium polyphosphate and above 60 g‚L-1 in pure water, suggesting that hydrophobic interactions play a role in the gel formation. At temperatures above 60 °C, the suspensions destabilize and precipitate. Gels could be formed at 40 °C up to pH 5.6 in pure water and up to pH 5.7 in 20 g‚L-1 sodium polyphosphate, but only at relatively high concentrations (C > 60 g‚L-1). The gelation process has been investigated by light scattering and rheology. Figure 4shows the evolution of the light scattering intensity (I) and the shear moduli for β-casein suspension with C ) 8 g‚L-1 at 40 °C. The scattered intensity increases because the β-casein micelles aggregate. At a critical time the intensity starts to show large fluctuations in time. These large fluctuations are caused by critical slowing down of the concentration fluctuations at the length scale of observation determined by the scattering wave vector (q ) 8 × 10-3 nm-1). Close to this time G′ and G′′ increase steeply, indicating the formation of a gel. At later times the shear moduli continue to increase more slowly. Dynamic light scattering studies were done to determine the apparent hydrodynamic radius (Rha) as a function of time. The hydrodynamic radius of the micelles at pH 5.5 is about 18 nm, that is, slightly larger than at pH 6. The measurements were done at 35 °C instead of 40 °C so that the aggregation is somewhat slower, which facilitates the experiment. Figure 5 shows an increase of Rha caused by the growth of the
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Figure 5. Time dependence of the apparent hydrodynamic radius of β-casein micelles heated at 35 °C (8 g‚L-1 casein, 20 g‚L-1 polyphosphate, pH 5.5).
Figure 6. Frequency dependence of the storage (solid symbols) and loss (open symbols) moduli for β-casein gels (8 g‚L-1 casein, 20 g‚L-1 polyphosphate, pH 5.5) formed at 40 °C before and after cooling at 20 °C.
β-casein micelle aggregates. Rha is close to the true z-average hydrodynamic radius as long as the aggregates are small, but the effect of interactions on the value of Rha increases with increasing aggregate size. Figure 6 shows the frequency dependence of the gel at 40 °C and after cooling to 20 °C. At both temperatures G′ is larger than G′′ and weakly dependent on the frequency. Cooling leads to an increase of shear moduli at a given frequency, which is clearly seen in Figure 7, where we have plotted the temperature dependence of G′ and G′′ at a fixed frequency (0.1 Hz). The temperature dependence is the same during cooling and heating. A reversible strengthening of the gels indicates that hydrogen bonding is involved in the junctions. The strain dependence of the shear moduli is shown in Figure 8. Strain hardening is observed above about 30%, but the gel breaks up irreversibly at a strain of about 100%. 3. Conclusion. Dense suspensions of β-casein micelles jam, which leads to a dramatic increase of the zero shear rate viscosity. The critical concentration where the micelles jam decreases with increasing temperature, which explains the strong decrease of η0 with increasing temperature. Dense suspensions show an initial shear thickening followed by
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Figure 7. Temperature dependence of the storage and loss moduli and tan δ for β-casein gels (8 g‚L-1 casein, 20 g‚L-1 polyphosphate, pH 5.5). The gel was formed at 40 °C, cooled to 10 °C, and subsequently reheated to 50 °C. The cooling (open symbols) and heating (solid symbols) ramps were done at a rate of 2 °C/ minute.
Figure 8. Strain dependence of the storage and loss moduli for β-casein gels at 20 °C (8 g‚L-1 casein, 20 g‚L-1 polyphosphate, pH 5.5).
strong shear thinning above a critical shear rate that is inversely proportional to η0. β-Casein micelles aggregate and gel if the pH is reduced to below 5.7. Gelation is favored by increasing the temperature and by the presence of sodium polyphosphate. The gel modulus increases with decreasing temperature, indicating the presence of hydrogen bonding. References and Notes (1) Schmidt, D. G., Association of caseins and casein micelle structure. In DeVelopments in Dairy Chemistry; Fox, P. F., Ed.; Applied Science Publishers: London, 1982; Vol. I, p 61. (2) Baumy, J. J.; Brule´, G., Effect of pH and ionic strength on the binding of bivalent cations to β-casein. Lait 1988, 68 (4), 409. (3) Parker, T. G.; Dalgleish, D. G. J. Dairy Res. 1981, 48, 71. (4) Guo, C.; Campbell, B. E.; Chen, K.; Lenhoff, A. M.; Velev, O. D. Colloids Surf. B: Biointerfaces 2003, 29 (4), 297. (5) Rollema, H. S. AdV. Dairy Chem. 1992, 1, 111. (6) Schmidt, D. G. Neth. Milk Dairy J. 1980, 34, 42. (7) Payens, T. A. J.; van Marwijk, B. W Biochim. Biophys. Acta 1963, 71, 517. (8) Andrews, A. L.; Atkinson, D.; Evans, M. T. A.; Finer, E. G.; Green, J. P.; Phillips, M. C.; Roberstson, R. N. Biopolymers 1979, 18, 1105. (9) Thurn, A.; Burchard, W.; Niki, R. Colloid Polym. Sci. 1987, 256, 653.
Jamming and Gelation of Dense β-Casein Micelles (10) Morris, G. A. Biotechnol. Genet. Eng. ReV. 2002, 19, 357. (11) de Kruif, C. G. Prog. Biotechnol. 2003, 23, 219. (12) Mikheeva, L. M.; Grinberg, N. V.; Grinberg, V. Y.; Khokhlov, A. R.; de Kruif, C. G. Langmuir 2003, 19, 2913. (13) de Kruif, C. G.; Grinberg, V. Y. Colloids Surf. A: Physicochem. Eng. Aspects 2002, 210, 183. (14) O’Connell, J. E.; Grinberg, V. Y.; de Kruif, C. G. J. Colloid Interface Sci. 2003, 258, 33. (15) Kajiwara, K.; Niki, R.; Urakawa, H.; Hiragi, Y.; Donkai, N.; Nagura, M. Biochim. Biophys. Acta 1988, 955, 128. (16) Buchheim, W.; Schmidt, D. G. J. Dairy Res. 1979, 46, 277. (17) Leclerc, E.; Calmettes, P. Physica B 1998, 241-243, 1141.
Biomacromolecules, Vol. 6, No. 6, 2005 3111 (18) Panouille´, M.; Benyahia, L.; Durand, D.; Nicolai, T. J. Colloid Interface Sci. 2005, 287, 468-475. (19) Panouille´, M.; Durand, D.; Nicolai, T.; Boisset, N.; Larquet, E. J. Colloid Interface Sci. 2005, 287, 85-93. (20) Quemada, D. J. Theor. Appl. Mech. 1985, 267 (special issue).. (21) Jones, D. A. R.; Leary, B.; Boger, D. V. J. Colloid Interface Sci. 1991, 150, (1), 84. (22) Roovers, J. Macromol. Symp. 1997, 121, 89. (23) Vlassopoulos, D.; Fytas, G.; Pispas, S.; Hadjichristidis, N. Physica B 2001, 296, 184.
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