Non-covalent assembly of folic acid with lactoferrin: multi-spectroscopy, docking, and dynamics elucidate binding and...

Folic acid (FA) is important for human health, but it is sensitive to pH and can degrade during gastrointestinal digestion. Therefore, direct FA supplementation is limited. In this study, lactoferrin (LF) was used to prepare an LF-FA complex through a non-covalent interaction. LF significantly improves the pH stability (90.36 % at pH 2.1) and retention rate (59.19 %) of FA after in vitro digestion, compared with those of free FA (70.56 and 34.53 %). Furthermore, fluorescence and ultraviolet (UV) spectroscopy revealed that FA binding alters the secondary structure of LF, increasing the α-helix context and decreasing β-sheet structures. This interaction leads to complex formation through hydrogen bonding and hydrophobic interactions. Isothermal titration curves revealed that the LF-FA complex undergoes a spontaneous static quenching process, suggesting that the interaction is primarily driven by hydrogen bonds and van der Waals forces (ΔH = -97.37 kJ·mol^-1, ΔS = -224.42 J·mol^-1). Furthermore, molecular docking and molecular dynamics simulations indicated that FA stably binds to key residues of LF, such as Asp60 and Tyr192, through hydrogen bonds and hydrophobic interactions. This enhances the structural rigidity of the complex and stabilizes the free energy landscape. These results not only improve the shortcomings of directly adding FA, but also provide references for the development of other similar products and expand the application scenarios of FA.