Re-assembled casein micelles and casein nanoparticles as nano-vehicles for ω-3 polyunsaturated fatty acids

Food enrichment with nutraceuticals is an important goal, but its effectiveness in preventing diseases depends on preserving the functionality and bioavailability of the bioactive nutraceuticals. Omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are important nutraceutical lipids, providing protection against cardiovascular and other diseases. Caseins are the major milk proteins whose biological function is to transport calcium, protein and phosphate from mother to the neonate. Our goal was to harness the natural self-assembly properties of caseins for protecting and delivering this important, but sensitive nutraceutical, DHA. Using spectrofluorescence we have shown, apparently for the first time, that casein can bind DHA with a relatively high affinity (K_b=(8.38±3.12)×10⁶M^-1), and the binding ratio was 3-4 DHA molecules per protein molecule on average. Moreover, DLS particle characterization experiments have shown the formation of nanoparticles upon addition of DHA (predissolved in ethanol) to a casein solution. When calcium and phosphate were added (at 4°C), DHA-loaded re-formed casein micelles (r-CM) with a size of 50-60nm were obtained and there was no significant effect of the thermal treatment (74°C, 20s) on particle size. When casein nanoparticles (CNP) were prepared (at room temperature and without adding calcium and phosphate), DHA-loaded CNP with a diameter of 288.9±9.6nm were formed. Both the DHA-loaded r-CM and the DHA-loaded CNP systems showed a remarkable protective effect against DHA oxidation, demonstrating good colloidal stability and bioactive conservation throughout shelf life at 4°C. These nanotechnologies may enable the enrichment of foods and beverages for promoting health of wide populations.