Discovery and Putative Perception Mechanisms of Novel Umami Peptides from Ruditapes philippinarum Cooking Liquid: In Silico Screening, Molecular Docking, Sensory Evaluation, and STC-1 Cell-Based Validation

Cooking liquid from Manila clam (Ruditapes philippinarum) is an underutilized byproduct rich in water-soluble taste compounds, representing a potential source of natural umami peptides. In this study, peptide fractions were separated from the cooking liquid. A total of 764 peptide sequences were identified from the most potent fraction, F3 (<3 kDa), by UPLC-ESI-Q-TOF-MS/MS. Machine learning prediction and molecular docking were further used for screening. Five candidate peptides were selected: TQDTVVALDA, KEY, YKD, RND, and GEAF. Sensory evaluation (on a 0–5 scale) and electronic tongue measurements independently confirmed that peptide YKD possessed the strongest taste profile, with an electronic tongue relative umami score of 8.81 ± 0.22. Furthermore, cell-based assays demonstrated that YKD effectively up-regulated the transcriptional expression of taste-related receptors, including GPRC6A, in STC-1 cells, revealing a multi-receptor synergetic mechanism for umami perception. In STC-1 cells, all peptides induced intracellular Ca2+ responses and showed no obvious cytotoxicity at 0.5–8.0 mmol/L. YKD produced the highest fluorescence response (0.59) at 1.0 mmol/L. Quantitative RT-PCR analysis suggested that YKD was associated with T1R1/T1R3-related expression, whereas TQDTVVALDA induced stronger CaSR expression. These findings elucidate the specific peptide sequence that engages multiple receptors to create complex tastes, providing a theoretical basis for converting seafood processing byproducts into natural flavor enhancers.