The overall aim of this Ph.D. project was to investigate the potential of new colloidal casein particles
with targeted composition. It was of great focus to relate the colloidal and serum composition of the
micellar system to the structural changes occurring to the casein micelle particles. To understand the
structure of the native and modified casein micelles, a series of casein micelle suspensions, depleted
of whey proteins, were subject to mild to severe environmental changes, such as cooling, acidification
and dephosphorylation. The consequential compositional changes were related to the structural
changes probed by Light Scattering (LS) and Small-Angle X-ray Scattering (SAXS). To interpret the
scattering data in sufficient detail, a SAXS model was developed describing the structural levels within
the supramolecular structure of the casein micelle. The model was placed on an absolute scale using
colloidal casein and mineral concentrations as constraints, thereby establishing the exact cause for the
scattering observed at q = 0.08-0.1 Å-1, namely that it is due to the scattering from a combination of
both colloidal CaPO4 (CCP) and caseins. It was found that cool storage of casein micelles resulted in
the depletion of β-casein, and if suspensions were acidified to pH 6.0, it caused the release of CCP,
however, the release of one was not directly associated to the dissociation of the other. Interestingly, it
was found that cooling and acidification of skim milk, containing whey proteins, caused dissociation
of a significant amount of κ-, αS1- and αS2-caseins. By SAXS analyses of Ca- and β-casein depleted
casein micelles, it was found that the intensity in the intermediate scattering range (q =0.009-0.02 Å-1)
and the high-q shoulder (q = 0.08-0.1 Å-1) decreased, indicating that both Ca- and β-casein release
caused inner micellar rearrangements, especially by decreasing the heterogeneity at the intermediate
length scale. To understand the effect of decreasing ionic strength, casein micelles separated from skim
milk by ultracentrifugation were resuspended in whey protein-depleted serum with or without EDTA,
a Ca-chelating agent. It was found that resuspension of casein micelles in its native, although whey
protein depleted, serum phase, resulted in subtle internal changes which could only be quantified by
analyzing SAXS intensity data. By total replacement of the native serum phase with water, the pH
increased to 7.2, which resulted in an increased heterogeneity at the intermediate length scale. The
presence of EDTA in the serum phase caused substantial casein and mineral dissociation, a reduction
in the polydispersity of casein micelles and the formation of looser micellar structure compared to the
native, although with the characteristic micellar structural motifs still conserved. Dephosphorylation,
was also evaluated, by addition of a calf alkaline phosphatase extract. The enzymatic reaction resulted
in only about 13% dephosphorylation in the native micellar suspensions and had a larger impact in
more dissociated casein suspensions. These results confirmed that the micellar structure hinders the
enzyme’s accessibility to the inner micellar regions. SAXS analysis of the micellar suspensions
demonstrated that even low enzymatic treatment affected the internal structure of the micelles.
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Interestingly, the recombination of native casein micelles with additional β-casein, instead, did not
seem to alter the micellar structure.
These results are a clear demonstration of the novel toolbox created by combining structural
information with compositional analyses, to reveal new insights of the casein micelle structure.