Transferring caseins from the nutraceutical to the pharmaceutical world: the case of β-casein self-assembling

Caseins are the most abundant protein fraction in milk, representing approximately 50-80% of its total content. Specifically, they are a family of proteins constituted by α-caseins (αs-1 and αs-2 caseins), β-caseins and κ-caseins, which differ each other for the amino acid sequence and the type and extent of the post-translational modifications (mainly phosphorylation and glycosylation for -casein) [1]. They are present in milk as large colloidal aggregates, resulting from the supramolecular assembling of the different casein components, which are known as casein micelles. Their biological function is to carry large amounts of the insoluble salt calcium phosphate in an aqueous environment (as in milk), providing nutritional support to the infants [2]. Caseins have also a nutraceutical impact and health implications in all ages of human life. In fact, technologically-produced casein-derived hydrolysates and casein-derived bioactive peptides, formed during the gastrointestinal digestion, have been demonstrated to exert several biological effects and benefits both in physiological and pathological conditions [3]. Nevertheless, some concerns related to the immunogenicity and the role of caseins and their by-products on the acquisition of milk hypersensitivity still remain. This aspect has been mostly elucidated for bovine caseins but it represents an interesting field of research as regards casein proteins obtained from milk of different animal species. The possible use of dairy proteins from different animal sources has been reinforced by the discovery of the less allergenicity of caseins from donkey milk [4]. On the base of these assumptions, we have decided to isolate and purify the β-casein fraction of caseins from bovine, sheep, donkey, goat and human milk. Among all caseins, β-casein was chosen since it is the only casein showing a temperature- and concentration-dependent self-assembling behaviour, denoting how caseins also configure as versatile proteins, which can be exploited for different technological applications. The investigation of the self-assembling properties and the eventual less immunogenicity of β-caseins from milk of animal species different from bovine will broaden the use of these proteins from the nutraceutical to the pharmaceutical world, since β-caseins can also act as a nanocarrier for drug delivery applications. The casein fraction was firstly isoelectrically precipitated and, then, purified by cation ion exchange chromatography. The isolated β-caseins from milk of different animal origin, were resuspended in the isotonic 50 mM Tris/HCl+110 mM NaCl buffer. The self-assembling behaviour was investigated in term of micellar size and critical micelle temperature (CMT) by dynamic light scattering (DLS) measurements and in term of critical micelle concentration (CMC) by spectrofluorometric analyses using pyrene as a fluorescent probe. A similar self-assembling profile were found among all β-caseins in comparison to the previously characterized behaviour of bovine β-casein. The obtained results is auspicious for the second part of the work in which the immunogenicity of β-caseins will be evaluated through different in vitro and in vivo models.