Study of the interactions between whey proteins and pyranoanthocyanins using fluorescence spectroscopy and in silico analyses

Due to consumer demand and regulatory constraints, artificial colorants are increasingly being replaced by natural alternatives such as anthocyanins, water-soluble flavonoids known for their vibrant colors but limited stability in most food systems. Pyranoanthocyanins (PACN), formed through cycloaddition reactions involving anthocyanins and reactive cofactors, exhibit improved stability compared to their precursors. Previous studies indicated that whey proteins (WP) interact with anthocyanins, influencing color properties and enhancing tinctorial strength, with the extent of interaction depending on colorant structure. We hypothesized that WP, primarily composed of β-lactoglobulin, may similarly interact with PACN. This study aimed to characterize the interaction between WP and 10-catechyl-PACN at pH 3.0, relevant for beverages, using fluorescence spectroscopy and molecular modeling. Fluorescence quenching experiments revealed a concentration-dependent interaction between WP and PACN, occurring spontaneously (ΔG < 0) and dominated by hydrophobic forces (ΔH > 0, ΔS > 0), with a high quenching constant (Kq > 1010 M−1 × s−1). Docking simulations revealed that PACN bind near the hydrophobic region of β-lactoglobulin. However, WP addition did not significantly alter visible absorption spectra or overall color (ΔE*ab < 5). These findings confirmed WP – PACN interactions without notable color changes, suggesting potential for stabilizing natural colorants in acidic beverage systems.