Abstract
Determination of key bitterness compounds of cigar tobacco leaves (CTLs) is of great importance for their quality control, flavor regulation, and agricultural production, but the key bitterness compounds of CTLs are not known clearly. In this study, three bitterness CTLs along with one reference CTL were chosen. The key potential bitterness compounds were screened out through sensory evaluation, analysis of mainstream smoke compounds, orthogonal partial least squares discriminant analysis (OPLS-DA), partial least squares regression (PLSR), and correlation heatmap analysis. The identified key bitterness components were further verified by sensory evaluation, molecular docking, and kinetic simulation. It was found that CTLs F447-1, F166-1, and B065-2 possessed strong bitterness taste. Totally 33 potential bitterness compounds were screened out from mainstream smoke components of 4 CTLs, and the contents of nicotinamide, 2,3’-bipyridine, 3-ethyl-pyridine, myosmine, and cotinine in the test bitterness CTLs were significantly higher than those in the reference CTL Chuxue 14. Seven potential compounds with VIP > 1 and P < 0.05 were screened out by OPLS-DA. PLSR and heatmap correlation analysis further suggested that nicotine, myosmine, 2,3’-bipyridine, cotinine, nicotinamide, and 3-ethyl-pyridine were positively correlated with bitterness taste. Four key bitterness compounds were further confirmed as nicotine, 2,3’-bipyridine, myosmine, and nicotinamide through sensory evaluation verification. Molecular docking indicated that the four bitterness compounds mainly interacted with bitter receptor TAS2R14 through hydrogen bond, π-alkyl, and alkyl interaction. The key mutual binding sites were revealed as SER-194, PHE-198, TYR-107, and TRP-89. Kinetic studies showed that bitter components could form a stable complex with TAS2R14. This is the first report about elucidation of the key bitterness compounds of CTLs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-39473-9.