Integrative Analysis of Widely Targeted Metabolomics and Flavoromics Reveals Dynamic Changes in Characteristic Metabolites at Different Processing Stages of Black Highland Barley Beer

Black highland barley, rich in phenolic compounds and β-glucans, is increasingly used in craft beer brewing, but the dynamic metabolic changes across its processing chain remain unclear. This study integrated GC×GC-TOF MS based flavoromics with UHPLC-QTRAP-MS based widely targeted metabolomics to track volatile and non-volatile metabolites from raw grain (K1) through germinated malt (K2) and roasted malt (K3) to finished beer (K4). A total of 256 volatile compounds and 615 non-volatile metabolites were identified. Alcohols and hydrocarbons dominated K1. Germination (K2) enriched aldehydes and reduced esters by 66%. Roasting (K3) generated Maillard reaction products, including 2,3,5-trimethylpyrazine (198.4-fold increase) and furfural (20.2-fold increase). Fermentation (K4) shifted the profile to ester dominance (40.95%), with isoamyl alcohol (ROAV = 71.59) and 2-undecanone (ROAV = 65.37) as the principal aroma contributors. Notably, 94.1% of phenolic and flavonoid compounds (168 of 184) exhibited higher abundance in finished beer than in raw grain. Their levels declined after roasting and then accumulated during fermentation. Cross-omics correlation indicated a strong association between furfural and its pentose phosphate precursor (ρ = 1.00, p < 0.001). These findings demonstrate that fermentation acts as the decisive metabolic turning point that simultaneously shapes the flavor profile and nutritional quality of black highland barley beer. The finished beer contained 250.45 mg GAE/L total phenolics and 97.91 mg RE/L total flavonoids, with ABTS and DPPH radical scavenging rates of 87.41% and 92.68%, respectively.