Folic acid is the synthetic form of vitamin B9. This water soluble vitamin is essential in numerous human metabolic pathways, such as DNA and RNA synthesis, and the correct development of neural tube. By this reason, the introduction of vitamin B9 in food or supplement formulations constitutes a strategy to prevent its deficiency. However, vitamin B9 is sensitive to UV light, hence its incorporation requires the application of techniques that prevent/reduce its deterioration. Encapsulation is a strategy for protection sensitive biomolecules, in which the proteins are one of the biopolymers most used like encapsulating matrix. Among them, the main protein fractions of whey: α-lactalbumin (α-lac), β-lactoglobulin (β-lg) and bovine serum albumin (BSA), constitute an interesting option to achieve this goal. Then, understanding how bioactive substances interacts with carrier agent, constitutes a key aspect into the design of innovative vehiculization and protection systems. 
Thus, the objective of this work was to study the interaction of folic acid with the main whey proteins of Bubalus bubalis through fluorescence and molecular docking. With this purpose, folic acid (FA) solutions were prepared (0, 5, 10, 20 and 50 μM) in saline phosphate buffer (pH 7.4), containing 20 μM of buffalo whey proteins (BWPC) with 63% proteins. Then, the fluorescence spectra were obtained in the range 305 - 400 nm with λex of 280 and 295 nm. Moreover, a FA conformer was docked against the crystal structures of α-lac (1F6S), β-lg (3NPO) and BSA (4F5S) using Autodock Vina. For both λex, the protein solution (in absence of FA) showed the highest fluorescence emission and this was observed at 334 nm. The intrinsic fluorescence of proteins decreased conform vitamin concentration increased (quenching), and this was attributed to the formation of complexes. The decrease occurred without changes in λmax, which indicated that the microenvironment of Trp and Tyr residues was not modified. Quenching was fitted with the Stern-Volmer equation (R2=0.99), and this allowed obtaining the constants 2.32x1012 M-1s-1 and 1.92x1012 M-1s-1 for the λex of 280 nm and 295 nm, respectively. These results enabled classify the quenching as a static type, that is, the complexes formed had a permanent character. The molecular docking showed that the FA binds in the cleft region of the α-lac, at the open end of β-barrel of the β-lg, and in site I of the subdomain IIA of the BSA. The binding energies were -6.4, -7.2 and -8.6 kcal/mol, respectively. These results contribute to characterize the interactions of the FA with the main whey proteins of Bubalus bubalis, which will allow to optimize the encapsulation of this vitamin using BWPC as matrix.

Folic acid complexation with Bubalus bubalis whey proteins

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