Background:Brewers’ spent grain (BSG) typically contains 20％–29％crude protein (CP) with high concentrations of glutamine, proline and hydrophobic and non-polar amino acid, making it an ideal material for producing value-added products like bioactive peptides which have antioxidant properties. For this study, protein was extracted from BSG, hydrolyzed with 1％alcalase and flavourzyme, with the generated protein hydrolysates (AlcH and FlaH) showing antioxidant activities. This study evaluated the effects of AlcH and FlaH on gas production, ruminal fermentation characteristics, nutrient disappearance, microbial protein synthesis and microbial community using an artificial rumen system (RUSITEC) fed a high-grain diet. (Continued from previous page) Results:As compared to the control of grain only, supplementation of FlaH decreased (P<0.01) disappearances of dry matter (DM), organic matter (OM), CP and starch, without affecting fibre disappearances;while AlcH had no effect on nutrient disappearance. Neither AlcH nor FlaH affected gas production or VFA profiles, however they increased (P<0.01) NH3-N and decreased (P<0.01) H2 production. Supplementation of FlaH decreased (P<0.01) the percentage of CH4 in total gas and dissolved-CH4 (dCH4) in dissolved gas. Addition of monensin reduced (P <0.01) disappearance of nutrients, improved fermentation efficiency and reduced CH4 and H2 emissions. Total microbial nitrogen production was decreased (P <0.05) but the proportion of feed particle associated (FPA) bacteria was increased with FlaH and monensin supplementation. Numbers of OTUs and Shannon diversity indices of FPA microbial community were unaffected by AlcH and FlaH; whereas both indices were reduced (P <0.05) by monensin. Taxonomic analysis revealed no effect of AlcH and FlaH on the relative abundance (RA) of bacteria at phylum level, whereas monensin reduced (P <0.05) the RA of Firmicutes and Bacteroidetes and enhanced Proteobacteria. Supplementation of FlaH enhanced (P <0.05) the RA of genus Prevotella, reduced Selenomonas, Shuttleworthia, Bifidobacterium and Dialister as compared to control; monensin reduced (P <0.05) RA of genus Prevotella but enhaced Succinivibrio. Conclusions: The supplementation of FlaH in high-grain diets may potentially protect CP and starch from ruminal degradation, without adversely affecting fibre degradation and VFA profiles. It also showed promising effects on reducing CH4 production by suppressing H2 production. Protein enzymatic hydrolysates from BSG using flavourzyme showed potential application to high value-added bio-products.