Study on β-Casein Depleted Casein Micelles: Micellar Stability, Enzymatic Cross-Linking, and Suitability as Nanocarriers

beta-Casein is an amphiphilic protein and thus considered as multilaterally bound in casein micelles. Its polar molecule part, in particular the phosphoserine residues, can interact electrostatically with colloidal calcium phosphate (CCP) to form nanoclusters and its nonpolar molecule part enhances micellar stability by forming hydrophobic bonds to other caseins. Because cooling weakens hydrophobic interactions, a substantial portion of beta-casein can be irreversibly removed from the casein micelle by repeated depletion steps, including cooling and subsequent ultracentrifugation. Although this effect of cooling on the micellar beta-casein concentration has been well known for decades, the influence of depletion on the main characteristics of casein micelles has been less investigated yet. Therefor; we aimed to analyze the consequences of fi-casein depletion on the stability as well as the functionality of casein micelles to evaluate the suitability of depleted compared to native casein micelles as nanocarriers. Up to 43.2% of the total beta-casein was irreversibly sequestered from native casein micelles by repeated cooling and ultracentrifugation steps. Depletion showed no effect on size distribution as well as polydispersity and particle concentration of micelle suspensions as measured via dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), respectively. Furthermore, the stability of the micelles against ethanol or the chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was not influenced by beta-casein depletion. Notwithstanding, depleted micelles were less susceptible to enzymatic cross-linking by microbial transglutaminase (mTG), indicating narrowed water channels due to depletion. Additionally, loading experiments showed that depleted micelles could be loaded with linoleic acid (LA) as intensively as native micelles, whereupon LA displaces up to 81.3% of beta-casein from native micelles. Our results confirm that depletion does not enhance the ability of the casein micelle to act as a nanocarrier for hydrophobic substances but could support the understanding of the casein micelle structure. Based on the observed unchanged stability against EGTA, the hindered enzymatical cross-linking, and the efficient displacing of beta-casein by LA, we suggest that the major portion of micellar beta-casein is hydrophobically incorporated into the micelle structure without impact on the formation of calcium phosphate nanoclusters. The main role of beta-casein for the casein micelle structure, therefore, might be to facilitate the high hydration of the interior and thus the high permeability of casein micelles.