Dual Decoration of Ferritin Nanocages by Caffeic Acid and Betanin with Covalent and Noncovalent Approaches: Structure and Stability Analyses

Ferritin is a cage-like protein with modifiable outer and inner surfaces. To functionalize ferritin with preferable carrier applications, caffeic acid was first covalently bound to the soybean ferritin outer surface to fabricate a caffeic acid-ferritin complex (CFRT) by alkali treatment (pH 9.0). A decreased content of free amino acid (0.34 mu mol/mg) and increased polyphenol binding equivalent (63.76 nmol/mg) indicated the formation of CFRT (ferritin/caffeic acid, 1:80). Fluorescence and infrared spectra verified the binding of caffeic acids to the ferritin structure. DSC indicated that the covalent modification enhanced the thermal stability of CFRT. Besides, CFRT maintained the typically spherical shape of ferritin (12 nm) and a hydration radius of 7.58 nm. Moreover, the bioactive colorant betanin was encapsulated in CFRT to form betanin-loaded CFRT (CFRTB), with an encapsulation rate of 15.5% (w/w). The betanin stabilities in CFRTB were significantly improved after heat, light, and Fe3+ treatments, and its red color retention was enhanced relative to the free betanin. This study delves into the modifiable ferritin application as nanocarriers of dual molecules and gives guidelines for betanin as a food colorant.