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Hydration Behavior of Casein Micelles in Thin Film Geometry: A GISANS Study† E. Metwalli,‡ J.-F. Moulin,§ R. Gebhardt,§ R. Cubitt,| A. Tolkach,⊥ U. Kulozik,⊥ and P. Mu¨ller-Buschbaum*,‡ Technische UniVersita¨t Mu¨nchen, Physik Department E13, James-Franck-Str.1, 85747 Garching, Germany, European Synchrotron Radiation Facility, bp 220, 38043 Grenoble Cedex, France, Institute Laue-LangeVin, 6 rue Jules Horowitz, bp 156, 38042 Grenoble, France, and Technische UniVersita¨t Mu¨nchen, Chair for Food Process Engineering and Dairy Technology, 85354 Freising-Weihnstephan, Germany ReceiVed August 10, 2008. ReVised Manuscript ReceiVed NoVember 20, 2008 The water content of casein micelle films in water vapor atmosphere is investigated using time-resolved grazing incidence small-angle neutron scattering (GISANS). Initial dry casein films are prepared with a spin-coating method. At 30 °C, the formation of a water-equilibrated casein protein film is reached after 11 min with a total content of 0.36 g of water/g of protein. With increasing water vapor temperature up to 70 °C, an increase in the water content is found. With GISANS, lateral structures on the nanometer scale are resolved during the swelling experiment at different temperatures and modeled using two types of spheres: micelles and mini-micelles. Upon water uptake, molecular assemblies in the size range of 15 nm (mini-micelles) are attributed to the formation of a high-contrast D2O outer shell on the small objects that already exist in the protein film. For large objects (>100 nm), the mean size increases at high D2O vapor temperature because of possible aggregation between hydrated micelles. These results are discussed and compared with various proposed models for casein micelle structures.
1. Introduction Milk and milk components are used to make related food products. One component of milk is casein protein, which plays an important biological role in stabilizing the colloidal form of calcium phosphate in milk and thereby inhibits crystal growth in the secretary cells of the glands.1 Casein protein also has alternative nonfood applications.2,3 Casein proteins exist predominately in the form of micelles and are recovered from skim milk by using a mineral acid at low pH (4.6, isoelectric point) or Lactobacillus that converts milk sugar to lactic acid and promotes the precipitation of casein, which is known as “lactic acid casein.” Casein-based glue was first used in ancient Egypt. Recently, aqueous mixtures of casein and alkaline materials such as lime were used to glue furniture, size canvas, and bind paint pigment. The formulation of modern casein4 glue uses a controlled proportion of lime that reacts with the casein protein to form calcium caseinate that is water-resistant. Additionally, active alkali metal salts such as sodium carbonate or sodium fluoride decompose to form alkali hydroxide and accomplish complete protein dispersion. Together, both alkaline and alkali salts convert casein granules to a slick, viscous consistency that is an excellent adhesive material.4 † Part of the Neutron Reflectivity special issue. * Corresponding author. Tel: +49 8928912451. Fax: +49 8928912473. E- mail:
[email protected]. ‡ Physik Department E13, Technische Universita¨t Mu¨nchen. § European Synchrotron Radiation Facility. | Institute Laue-Langevin. ⊥ Chair for Food Process Engineering and Dairy Technology, Technische Universita¨t Mu¨nchen.
(1) Holt, C. In DeVelopments in Dairy Chemistry-3; Fox, P. F., Ed.; Elsevier Applied Science Publishers: London, 1985. (2) Audic, J. L.; Chaufer, B.; Daufin, G. Lait 2003, 83, 417. (3) Southward, C. R.; Walker, N. J. N. Z. J. Dairy Sci. 1980, 15, 201. (4) Lambuth, A. In Coatings Technology Handbook, 3rd ed.; Tracton, A. A., Ed.; CRC Press: Boca Raton, FL, 2005; pp 64.
Casein micelles in milk form a unique biocolloid from calcium, phosphate, and proteins. Four main types of proteins are involved: Rs1-casein (38%), Rs2-casein (10%), β-casein (36%) and κ-casein (13%), which form hydrated casein micelles about 100-300 nm in size.5 Various models6 have been proposed to describe the casein micelle structure, including coat core, submicelle, and internal structure. The coat-core model describes the micelle as an aggregate of casein proteins with an inner layer that has a different composition than the outer layer. The submicelle model imagines the micelle to be made up of roughly spherical uniform submicelles that are linked together by calcium phosphate ion clusters. The internal structure model specifies that the mode of aggregation is through different caseins. Casein-associated water molecules are divided into different classes on the basis of the method used to study the system. In general, for dairy protein systems, they are classified7,8 as (1) structural water (i.e., water molecules directly involved in the stabilization of the protein structure); (2) hydration water, which refers to a monolayer of water molecules that is dynamically oriented and exhibits restricted motion because of a significant decrease in the translational and rotational modes of motion caused by macromolecular water interactions; and (3) hydrodynamic hydration water, which is transported with the protein during diffusion in an aqueous solution. The extent of the ordering of the monolayer hydration water molecules is quite different from those characterizing the fast, random motion of free or bulk water.8 Various techniques have been applied to investigate the structure, mobility, extent, and modes of binding water molecules in various systems. Among those are NMR spectroscopy,9-16 (5) Kinsella, J. E. CRC Crit. ReV. Food Sci. Nutr. 1984, 21, 197. (6) Phadungath, C. Songklanakarin J. Sci. Technol. 2005, 27, 201. (7) Kinsella, J. E.; Fox, P. F. CRC Crit. ReV. Food Sci. Nutr. 1986, 24, 91. (8) Anagnostopouloukonsta, A.; Pissis, P. J. Phys. D: Appl. Phys. 1987, 20, 1168. (9) Griffin, M. C. A.; Roberts, G. C. K. Biochem. J. 1985, 228, 273. (10) Moragutierrez, A.; Farrell, H. M.; Kumosinski, T. F. FASEB J. 1992, 6, A1284.
10.1021/la802602g CCC: $40.75 2009 American Chemical Society Published on Web 12/29/2008
Hydration BehaVior of Casein Micelles
thermally stimulated depolarization current,8 differential scanning calorimetry,17 and small-angle X-ray scattering.18 Generally, both the structural and hydration water are considered to be bound water because of their irreversible binding to the protein molecules. The “free” water is usually referred to as hydrodynamic hydration water, which can also exhibit fast exchange with the monolayer surface-bound water. The amount of water bound to casein micelles is still in question. For instance, small-angle X-ray scattering19 (SAXS) indicates a very high water interaction with casein protein (water/protein ratio >3 g/g); however, a nuclear magnetic resonance (NMR) relaxation experiment20 reveals a much lower hydration value (water/protein ratio 0.2) near the experimental resolution limit for the small micelles. The structure present in the solution used for spin coating, as determined by dynamic light scattering (data not shown), is consistent with that observed for the as-coated dry protein films as determined by the GISANS method. The casein micelle reaches an equilibrium mean size of about 100 nm. In solution and with X-rays, because of the low contrast between the solvent and the casein micelles, the detection of mini-micelles is less likely. Moreover, in solution because of the dynamic equilibrium between micelles and min-micelles, the unambiguous detection of minimicelles might be difficult. In the thin film geometry, the structure is frozen in snapshot-like, and the swelling with water vapor does not promote sufficient mobility. However, in previous work a possible indication of the coexistence of micelles and minimicelles can be found. Hansen et al.61 used the SANS technique to study casein micelles in solution by employing contrast variation and proved the presence of a large amount of polydispersity (40-50%) of the subunits forming a single 100-120 nm micelle. Though the authors described the subunits in the framework of the submicelle model, the study correlates with what we have described as mini-micelles with a wide size distribution rather than the unique size of the submicelle. For the large objects, an increase in the mean radius (up to 50% at 70 °C, Figure 8b) as a function of D2O vapor temperature is due not only to the formation of larger swollen micelles but also to the possible aggregation of mini-micelles of different sizes. Such aggregation is enhanced by local van der Waals attractions between neighboring hydrated micelles (Figure 9). The large aggregate grows progressively by increasing the number of hydrated micelles involved in such aggregates. Compared to polymer blends, our protein film is a phase-separated blend. The casein micelles are embedded in a lactose matrix. The good contrast between casein micelles and the lactose matrix has been demonstrated by developing the smaller objects (15 nm minimicelles) by increasing the water content of the protein film. (60) Gebhardt, R.; Burghammer, M.; Riekel, C.; Roth, S. V.; Mu¨llerBuschbaurn, P. Macromol. Biosci. 2008, 8, 347. (61) Hansen, S.; Bauer, R.; Lomholt, S. B.; Quist, K. B.; Pedersen, J. S.; Mortensen, K. Eur. Biophys. J. Biophys. 1996, 24, 143.
Hydration BehaVior of Casein Micelles
5. Conclusions In the present investigation, lactose casein films, called casein protein films, are explored with respect to swelling in water vapor. Using the GISANS technique and introducing contrast with the use of deuterated water, we succeed in probing the kinetics of water uptake at 30 °C and detailing the structure inside the D2O-vapor-equilibrated protein films. Modeling of GISANS data is performed in the framework of an approach using two objects of spherical shape but different sizes. The objects are identified as mini-micelles and micelles, and the chosen model is selected from previous work on thin spin-coated casein films in which the coexistence of both types of objects was shown. Thus, the large amount of lactose in the casein films used in the present investigation does not affect the basic structural motive of coexisting mini-micelles and micelles. Moreover, structural
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changes upon swelling with water are identified and attributed to the hydration layer around the micelles. Mini-micelles form aggregates by water bridges between neighboring ones. At 30 °C, a total water content of about 0.36 g of water/g of protein is found. This value of the water incorporated into the casein protein film is an intermediate value between previously reported values using solution-based experimental methods such as SAXS and NMR. The increased amount of water uptake as a function of water vapor temperature indicates a continuous structural reorganization of the film due to the correspondingly new equilibrium conditions. Acknowledgment. We obtained financial support from the DFG (MU 1487/8-1) within SPP1259. LA802602G
