Processing methods modulate bioactive components in highland barley, including β-glucan, polyphenols, and γ-aminobutyric acid (GABA), influencing regulation of glucose and lipid metabolism. This study employed a germination-fermentation strategy, where highland barley was germinated (25 °C, 24 h), freeze-dried, ground, and fermented with a mixed lactic acid bacteria starter culture at 40 °C for 5 h, with dynamic changes in bioactive components and in vitro antioxidant, hypoglycemic, and hypolipidemic activities tracked during 28-day storage at 4 °C. Total phenols, total flavonoids, and GABA reached peak concentrations of 0.250 ± 0.01, 0.220 ± 0.03, and 0.720 ± 0.08 mg/mL during storage, respectively. Optimal antioxidant efficacy occurred at storage days 14–21 (1.00–1.11 times vitamin C equivalent), with a DPPH scavenging capacity of 98.5 ± 0.02% and a ferric reducing antioxidant power of 1.13 ± 0.07 mmol/L. The hypoglycemic and hypolipidemic efficacy peaked at day 14, achieving an α-glucosidase inhibitory capacity of 93.85 ± 0.93% and an α-amylase inhibition rate of 87.28 ± 1.92% (1.10–1.11 times acarbose equivalent), whereas hypolipidemic efficacy (1.05–1.13 times cholestyramine equivalent) manifested as a cholesterol esterase inhibitory capacity of 89.11 ± 1.99%, a pancreatic lipase inhibition rate of 87.77 ± 2.4%, and bile acid binding capacities of 96.5 ± 0.49% (sodium taurocholate), 88.46 ± 1.24% (sodium glycocholate) and 89.28 ± 1.49% (sodium cholate). Untargeted plant metabolomics identified germination-induced increases in amino acids and their derivatives (e.g., L-phenylalaninol), as well as alkaloids (e.g., N-cinnamoylserotonin), whereas flavonoids (e.g., phellatin) critically influenced physiological activities during storage. Thus, germination-fermentation demonstrates functional food development potential.
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