Identification of Bitter Peptides in Lilium lancifolium Thunb.; Peptidomics, Computational Simulation and Cellular Functional Assays

<i>Lilium lancifolium</i> Thunb., as a predominant variety of medicinal and edible lilies, has long been renowned in traditional medicine for "moistening the lungs, relieving coughs, and calming the mind to soothe the heart." The bitter taste formation in <i>L. lancifolium</i> is predominantly attributed to secondary metabolites such as alkaloids, this study explores an alternative mechanism underlying taste divergence among <i>Lilium brownii</i> var. <i>viridulum</i>, and <i>Lilium pumilum</i> DC, proposing a foundational scientific question: Are peptides one of the important sources of bitterness in <i>Lilium lancifolium</i> Thunb.? Peptidomic analysis identified 8479 peptide sequences, with 46.27% upregulated in <i>L. lancifolium</i> flesh. Through high-throughput molecular docking with the bitter taste receptor TAS2R14, 214 candidate bitter peptides were identified, showing the strongest average binding affinity (-119.73 kcal/mol). Molecular dynamics simulations further demonstrated that four of these peptides formed stable interactions with key residues in TAS2R14. Cellular assays confirmed TAS2R14 activation by these peptides, as indicated by enhanced EGFP reporter fluorescence, upregulation of downstream signaling molecules (<i>GNAT1</i>, <i>PLCB2</i>, <i>TRPM5</i>), decreased cAMP levels, and increased IP₃ accumulation. Transcriptomic analysis further indicated that bitter peptides mediate taste transduction primarily through neuroactive receptor interaction pathways. These findings represent the first identification of bitter peptides as a key source of bitterness in <i>L. lancifolium</i> and elucidates their transduction mechanism combining peptidomics, computational simulation, and cellular validation. Our study provides a methodological framework for exploring flavor substances in other plant-derived foods.