Abstract
Tyrosinase is the rate-limiting enzyme in melanogenesis and food browning, making its inhibitors crucial for health and food preservation. However, the specific mechanisms by which multi-berry formulations synergistically inhibit tyrosinase remain uncharacterized, particularly within the context of overcoming bioactivity limitations caused by standardized food ingredients. Our study revealed that blueberry-black chokeberry extracts at 1:1 ratio with superior synergistic inhibition in tyrosinase. Using affinity-ultrafiltration coupled with ultra-performance liquid chromatography-mass spectrometry (AUF-UPLC-MS), sixteen inhibitors were identified, notably delphinidin-3-O-galactoside (IC(50) = 45.06 ± 1.32 μM) and cyanidin-3-O-arabinoside (IC(50) = 55.54 ± 0.83 μM) displayed the highest binding affinities with synergistic inhibition at 4:1-ratio. This anthocyanin mixture exhibited reversible mixed-type inhibition, with Lineweaver-Burk analysis and multi-spectroscopic studies confirming stable complex formation. Molecular docking and dynamics simulations revealed the mixture sequence-specific inhibition mediated by enhanced hydrogen bonding that prevented drastic structural changes. The anthocyanin mixture also exhibited enhanced processing stability. These findings provided mechanistic foundations for developing multi-berry-derived anti-tyrosinase ingredients, enabling bioactivity maximization in industrial food processing.