Abstract

Brewer's spent grain (BSG) and brewer's spent yeast (BSY) are two major brewing by-products, which are low-cost raw materials with high yields and nutritional value. In this study, the antioxidant peptides from brewing by-products were separated, purified and identified by UF, GFC or HIC, semi-preparative RP-HPLC, and nano-HPLC-MS/MS. Thereafter, in silico analysis was applied to predict the bioactivity, toxicity, and bioavailability of the identified peptides, and a total of 23 potential novel peptides with antioxidant activity and bioavailability were excavated. Furthermore, molecular docking results revealed the potential antioxidant peptides had stable binding ability to Keap1, and Tyr334, Arg380, Arg415, Arg483, and Tyr525 residues contributed enormously to docking, all of which were located in the binding sites of Keap1-Nrf2 interaction. Peptides with the lowest binding energy (i.e. AGRLPW, DRLW, KPW, PLWWRHPR, RVWNPFR, and ANLPWLGK) were further synthesized and validated in vitro and at the cellular level to be bioactive. Western blot results showed that KPW, RVWNPFR, and ANLPWLGK could protect HaCaT cells from H2O2-induced oxidative damage by activating Keap1-Nrf2 pathway. These findings provided insights into the high value-added utilization of brewing by-products.

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