Mechanistic Study on Ethanol-Induced Sweetness Enhancement: Combining Cellular and Computational Approaches to Uncover Its Dual Role as a Weak Agonist and Allosteric Stabilizer.

The growing consumer demand for low-sugar and low-alcohol beverages has spurred increased research into flavor-enhancement strategies. This study systematically investigated the sweet taste enhancement effect of low-concentration ethanol (4%) using a combined approach of T1R2/T1R3-expressing HEK293 cellular models and computational simulations. The fluorescence change ratio (ΔF/F0) was measured to quantify cellular response to sweeteners and assess perceived sweetness intensity. The observation of a classic dose-dependent response to sucrose confirmed the functionality and reliability of the cellular assay system. Furthermore, 4% ethanol was found to significantly enhance the relative sweetness intensity of glucose (Glc) and ethyl cyclopentenolone (ECP) by 28.29% and 46.63%, respectively, compared to aqueous solutions. Molecular modeling of T1R2/T1R3 receptor in complex with Glc or ECP, conducted in both aqueous and ethanol environments, revealed that low-concentration ethanol directly activated sweet taste receptors (STRs), stabilized receptor conformations, and enhanced ligand-binding affinity through optimized interaction networks-as evidenced by reduced root mean square deviation (RMSD) values, increased hydrogen bond formation, lower radius of gyration (Rg), and improved binding energy (ΔEbind). Furthermore, ethanol functioned as a potent sweet taste enhancer with a more pronounced effect on ECP, likely attributable to its unique structural interactions with the T1R2/T1R3 receptor. These findings provided molecular-level insights into ethanol's role as a sweetness modulator in low-alcohol beverages, offering a theoretical foundation and practical parameters for developing reduced-sugar alcoholic products.