Amorphous Calcium Phosphate Maintains Casein Microarchitecture while Modulating Bacterial Growth Kinetics

Abstract Casein, the primary source of protein in ruminant milk, represents a family of proteins that self-assemble into globules composed of finer spherical subunits and stabilized by calcium phosphate (CP) nanoparticles. These globules act as holdase-type chaperones for unfolded or partially folded proteins, preventing their aggregation. Simultaneously, by integrating CP nanoparticles, they enable the bioavailability of calcium and phosphate beyond their solubility limits, thereby enhancing the nutritional value of milk. Using a range of physicochemical and biological characterization techniques, including scanning and transmission electron microscopy, organic and inorganic elemental analyses, SDS-PAGE, and vibrational, UV/Vis and NMR spectroscopies, it is demonstrated that native casein globules critically depend on CP nanoparticles for structural cohesion. Upon removal of the CP phase, the casein globules shrink, compromise their sphericity, and begin to disintegrate at both supramolecular and molecular levels. Concordantly, spectroscopic analyses revealed heightened stereochemical disorder in the microenvironments around spectrally active atoms or molecular groups following CP depletion, attesting to the gradual collapse of the casein structure. This structural collapse was also evident from the increase in the packing density of β-sheets induced by the removal of CP nanoparticles. CP nanoparticles within natural casein globules were furthermore confirmed to be structurally amorphous, lacking any long-range crystalline order. Additionally, demineralized casein delayed the growth of Escherichia coli, suggesting that casein-derived fragments generated during disintegration modulate bacterial physiology and growth behavior. The findings of the study open avenues for further research into casein-derived antimicrobial peptides and biomimetic designs that build upon protein-inorganic nanoparticle synergies for applications in drug delivery and beyond.