Molecular mechanism of synergistic tyrosinase inhibition by blueberry and black chokeberry anthocyanidins: Optimal synergistic formulation for multi-berry beverage development

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. • Synergistic tyrosinase inhibition by blueberry and black chokeberry (1:1 ratio). • AUF-UPLC-MS identified 16 inhibitors, with D3G and C3G as key anthocyanins. • The 4:1 anthocyanin mix acts as a reversible, mixed-type non-covalent inhibitor. • The mixture shows stronger, stable inhibition under various processing conditions. • Provides a basis for using defined berry blends in food and skincare products.