Brewers' spent grain (BSG) is a protein-rich by-product of the brewing industry. The present study examined the in vitro bioactivity of a BSG protein enriched preparation and its associated enzymatic hydrolysates (assigned A-J). Cytotoxicity was measured using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay in U937 and Jurkat T cells. IC50 values were lower in the U937 cell line, ranging from 4.93 to 9.27% v/v versus a range of 4.11% v/v to undetectable in Jurkat T cells. The superoxide dismutase (SOD) and comet assays were performed on U937 cells pre-incubated with test samples and subsequently exposed to an oxidant. Hydrogen peroxide (H2O2) significantly reduced SOD activity by 37.7% and none of the test samples provided protection. None of the samples protected against DNA damage induced by tert-butylhydroperoxide (t-BOOH); hydrolysate H, prepared with Alcalase at 60 degrees C, protected against H2O2-induced DNA damage. The total phenolic content (TPC) was found to range from 0.021 to 0.055 mg GAE/mg dry powder. The effect of the BSG-derived test samples on cytokine production (IL-2, IL-4, IL-10, IFN-gamma) in Concanavalin A (conA) stimulated Jurkat T cells was measured using an enzyme linked immunosorbent assay (ELISA). The samples had no effect on IL-2 and IL-4 production. The unhydrolysed sample C significantly reduced IL-10, while the protein rich isolate, unhydrolysed control samples and hydrolysates D, E, F, and J significantly reduced IFN-gamma production. The BSG preparations possess little antioxidant potential and exhibit selective immunomodulatory effects that may be of benefit in the control of inflammatory diseases. (c) 2012 Elsevier Ltd. All rights reserved.