β-Lactoglobulin-naringenin complexes: Nano-vehicles for the delivery of a hydrophobic nutraceutical

Naringin, a glycosylated polyphenol of the flavanones class and its aglycone, naringenin, have been shown to possess many health benefits, therefore they show promise for supplementation of staple foods and beverages, as a mean of preventive medicine. Major obstacles to the successful implementation of these flavanones as nutraceuticals are their low water-solubility and the related low oral bioavailability. Naringin is more soluble but is also very bitter. To allow the enrichment of clear beverages with these important health promoters, we have used native and preheated β-Lactoglobulin (β-Lg) based nanovehicles. Using UV spectrophotometry and intrinsic fluorescence methods, we have found that naringenin forms complexes with preheated and non-preheated β-Lg, with K_a in the order of 10⁴M^-1. No binding was found between naringin and β-Lg, probably due to the lower hydrophobicity of naringin and the steric interference of its sugar. Naringenin: β-Lg binding stoichiometry was 2:1, suggesting naringenin was probably bound at the two main hydrophobic binding sites of the protein, the calyx and the cleft near the alpha-helix. The complex formation with both native and preheated protein solubilized naringenin up to 3 times its solubility limit and prevented crystal formation in the aqueous medium. Such crystallization is responsible for both reduced bioavailability and hampered visual attractiveness of the product. Dynamic light scattering revealed the existence of nanoparticles of ~10nm, marginally larger than those of the pure protein (~7nm), therefore the solution was clear, hence suitable for clear beverages. Reconstitution after freeze drying was good, but only after quench freezing.