A multi-modal workflow identifies non-γ-glutamyl kokumi peptides in plant proteins

Plant proteins are increasingly incorporated into sustainable foods, yet their sensory performance falls short of consumer expectations. Kokumi sensation is typically associated with γ-glutamyl peptides, which enhance mouthfulness and flavour continuity via activation of the calcium-sensing receptor (CaSR). This study develops a multi-modal workflow to discover plant-derived CaSR-active peptides beyond γ-glutamyl motifs. Hemp, quinoa, and black gram were profiled using untargeted proteomics and in silico cryptide prediction. Black gram protein concentrate demonstrated the highest kokumi-generating potential and was subjected to protease A hydrolysis, followed by γ-glutamination, or fermentation with Lactococcus lactis strains. Peptidomics identified >8900 peptides across treatments, their kokumi relevance was predicted through molecular docking, and validated using a CaSR assay. While γ-glutamination generated potent CaSR allosteric agonists, multi-modal screening revealed novel non-γ-glutamyl peptides, including Tyr-Phe-Tyr, capable of activating CaSR. These findings expand the diversity of plant-derived kokumi relevant peptides and support flavour optimisation of plant-based foods.