Study on internal structure of casein micelles in reconstituted skim milk powder

Abstract

In the current study, the internal structure of casein micelles (CMs), primary casein cluster, has been studied by size-exclusion chromatography (SEC) coupled with small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations. The casein cluster featured a hierarchical structure predominately consisting of αs-CN and β-CN molecules and a trace of κ-CN. ITC profile showed a typical enthalpogram of CMs with a critical micelle concentration (CMC) of ~0.85 μg/mL. The casein cluster exhibited apparent characteristics of intrinsically disordered proteins (IDPs) with a secondary structure content of 24 % in α-helix, 35.4 % in antiparallel-parallel, 20.2 % in β-turn and 20.4 % in random coil. SAXS results revealed a slightly elongated and tortuous worm-like conformation for the casein cluster in solution with an aspect ratio of 1.36 and an estimated molecular weight of 162.7 kDa. Further scattering data analysis proposed three coexisted species (αs1-β-αs2-CN, αs1-CN and αs1-β-αs2-CN dimer) with a volume fraction of 57.4 %, 30.1 %, and 12.5 % respectively in the casein cluster. TEM observation was consistent with the results of spectra, SAXS and MD that calcium sequestration resulted in a more extended and loose structure, instead, EDTA chelation induced a more compact conformation of the casein cluster.