Nanostructured Polymer Films

Biopolymeric nanostructures

With experimental techniques well known from synthetic polymers thin biopolymer films can be prepared on solid supports as well. We focus on different types of nanostructured biopolymer films, such as films made of milk proteins or cellulose.

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. Casein protein also has alternative non-food applications. 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 precipitation of casein which is known as lactic acid casein. Casein micelle in milk forms a unique biocolloid from calcium, phosphate, and proteins. Four main types of proteins are involved: alpha(s1)-casein (38 %), alpha(s2)-casein (10%), beta-casein (36 %) and kappa-casein (13%), which form hydrated casein micelles about 100-300 nm in size.

A consensus of opinion exists that an outer hairy-layer of kappa-casein ensures the stability of the casein micelle through a steric stabilization mechanism. The non-adsorbing part of kappa-casein can be regarded as a salted polyelectrolyte brush. In the extended state the brush is about 7 nm long and provides the steric stabilization, while in the collapsed state the stabilization is absent and flocculation and gel formation become possible. Calcium is essential for the micelle formation at all. The casein proteins divide themselves into two groups, the calcium-sensitive and the non-calcium sensitive, which also in mixtures prevent or inhibit the precipitation of the calcium-sensitive group by calcium. Kappa-casein is insensitive to calcium and alpha(s1)- and alpha(s2)-caseins and beta-casein are calcium sensitive.
Whereas most investigations address solutions at different calcium concentrations, in the most prominent applications besides milk, films containing caseins are used.

We focus on the effect of calcium concentration on the structure of casein micelles in thin films (prepared by spin-coating). CaCl2 was added, at room temperature, to casein micelles extracted from commercial-grade skim milk in a concentration range from 0 to 100 mM. With GISAXS and AFM thin casein films prepared with spin-coating are investigated. The addition of Ca changes the equilibrium between the steep repulsive interaction of two micelles and a short-range van der Waals attraction. With increasing amount of Ca added, the particle-stabilizing properties of the hairy-layer of kappa-casein become weaker and attraction between neighboring micelles becomes noticeable. Micelles can associate and build aggregates with larger hydrodynamical radii as observed in solution. In the thin films these aggregates are not dominant in number, but individual micelles are the dominant species. These micelles are close packed, but not merged into new very big micelles. It appears clear that the observed decrease in casein micelle correlation length is caused by calcium mediated transition from an expanded to a collapsed kappa-casein salted brush. As a consequence casein micelle size and distances between neighbored micelles decreases.
In addition to these structures created by the commonly observed casein micelles, a further type with significantly smaller diameter is probed. These mini-micelles are observed in solution as well as in the thin films. AFM allows to visualize them on the casein film surface directly and GISAXS proves the statistical relevance of this type of micelle. Both micelle types, the mini-micelles (diameter 20 nm without added Ca) and the commonly observed micelles (diameter 260 nm without added Ca) exist in coexistence. In solution this might be a dynamical equilibrium, with an exchange of caseins between individual micelles. In contrast, in thin films a snap shot of this equilibrium is frozen-in and thus aggregates, micelles and mini-micelles are detected all.

With the pH value of the casein solution a key parameter effecting the mean diameter of the casein micelles is addressed. In good agreement with the experimental observation using DLS, with increasing pH value the size also increases. A reduction in the electrostatic repulsion yields higher aggregation numbers of caseins and an enlargement in the micelle size.

Selected Publications:

  • 1. S.V.Roth, M.Rankl, G.R.J.Artus, S.Seeger, M.Burghammer, C.Riekel, P.Müller-Buschbaum
    Domain Nano-structure of thin cellulose layers investigated by microbeam grazing incidence small-angle x-ray scattering;
    Physica B 357, 190 (2005)
  • 2. P.Müller-Buschbaum, R.Gebhardt, E.Maurer, E.Bauer, R.Gehrke, W.Doster
    3. Thin casein films as prepared by spin-coating: Influence of film thickness and of pH;
    Biomacromolecules 7, 1773 (2006)
  • 4. P.Müller-Buschbaum, R.Gebhardt, S.V.Roth, E.Metwalli, W.Doster
    Effect of calcium concentration on the structure of casein micelles in thin films;
    Biophys. J. 93, 960 (2007)
  • 5. R.Gebhardt, M.Burghammer, C.Riekel, S.V.Roth, P.Müller-Buschbaum
    Structural changes of casein micelles in a calcium gradient film;
    Macromol. Biosci. 8, 347-354 (2008) link
  • 6. F.F.Rossetti, P.Panagiotou, F.Rehfeld, E.Schneck, M.Dommach, S.S.Funari, A.Timmann, P.Müller-Buschbaum, M.Tanaka
    Structures of regenerated cellulose films revealed by grazing incidence small-angle x-ray scattering;
    Biointerphases 3, 117-127 (2008) link
  • 7. E.Metwalli, J.-F.Moulin, R.Gebhardt, R.Cubitt, A.Tolkach, U.Kulozik, P.Müller-Buschbaum
    Hydration behavior of casein micelles in thin film geometry: A GISANS study;
    Langmuir 25, 4124-4134 (2009) link
  • 8. R.Gebhardt, S.V.Roth, M.Burghammer, C.Riekel, A.Tolkach, U.Kulozik, P.Müller-Buschbaum
    Structural changes of casein micelles in a rennien gradient film with simultaneous consideration of the film morphology;
    Internat. Dairy J. 20, 203-210 (2010) link