A multi-step strategy to study trub, a beer by-product, using LC-MS/MS and feature-based molecular networking, evaluating its antioxidant potential.

Trub, a brewing by-product, is a metabolite-rich material containing bioactive compounds that remain largely underexplored. Our multi-step strategy, which integrates exhaustive extraction, LC-HRMS/MS analyses of the extracts, feature-based molecular networking (FBMN) for rapid dereplication, unsupervised multivariate analysis, and bioactivity profiling, was applied to bioprospect this waste stream. We found that the ethanol extract contains ∼700 clustered metabolites, 18 putatively annotated α- and β-acid analogue candidates expanding the known chemical diversity of hop bitter acids, and displays antioxidant potential. Overlaying bioactivity trends onto the molecular network, together with PCA-based exploratory patterns that identify the directions in which the data varies the most, suggested metabolite clusters potentially associated with the observed antioxidant activity. Overall, this study provides a multi-step strategy that enables a broader and faster assessment of chemical diversity, making it particularly effective for evaluating reuse-derived materials, supporting broader applications in the nutraceutical, pharmaceutical, and sustainable waste-upcycling sectors.