Achieving stable blue coloration of anthocyanins through pH-Modulated protein complexation

INTRODUCTION: Natural blue colorants are exceptionally scarce, and achieving stable blue hues from anthocyanins typically requires structural features such as extensive acylation or metal complexation, which limits their broader application. Developing alternative stabilization strategies therefore represents a critical challenge in anthocyanin-based blue pigment research. OBJECTIVES: This study introduces a novel strategy to induce vivid and stable blue coloration of anthocyanins through pH-modulated, non-covalent complexation with protein, and elucidates the underlying mechanisms. METHODS: Malvidin-3-O-glucoside (M3G) and bovine serum albumin (BSA) were employed as a model system. Complex formation was investigated across different pH values using UV-Vis spectroscopy, isothermal titration calorimetry, competitive binding assay, and computational simulations to characterize blue color development, absorption spectral shifts, proton transfer equilibria of M3G quinoidal bases, binding thermodynamics, and molecular interactions. Both blue color and anthocyanin stability were further evaluated during storage at 5 and 22 °C using UV-Vis spectroscopy and HPLC. RESULTS: The BSA-M3G complexes exhibited pronounced bathochromic and hyperchromic shifts in absorption spectra, yielding highly stable blue hues under alkaline conditions. Mechanistic investigations revealed that BSA preferentially binds the negatively charged quinoidal base of M3G, shifting the proton transfer equilibrium toward enhanced formation of bluish dianionic species. The binding was enthalpy-driven and dominated by electrostatic interactions and hydrogen bonding. This pH-dependent interaction conferred remarkable improvements in both color retention and chemical stability, with negligible color loss or anthocyanin degradation after 28 days at pH 9 and 5°C. CONCLUSION: These findings uncover a protein-mediated mechanism for stabilizing anthocyanin-derived blue hues and establish a broadly applicable framework for developing natural blue colorants suitable for food, cosmetic, and pharmaceutical applications.