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Brewer's spent grain (BSG) is the major waste generated by breweries, which contains 20-30% (w/w) proteins and 40-60% (w/w) of fibers. Technologies have been investigated to valorize BSG by extracting proteins from BSG; however, none of them has been implemented on an industrial scale due to the challenges including high drying cost of wet BSG, heavy chemical consumptions, and a large volume of secondary waste after protein extraction. Herein, a wet fractionation process was proposed to fully utilize BSG by simultaneously producing high protein product (HPP) and high fiber product (HFP) through the separation of proteins and fibers from BSG. The effect of different concentrations (1, 3, and 5%, w/w) of chemicals (sodium hydroxide and sodium bisulfite) and enzyme (Alcalase, 5, 20 and 35 µL/g dry BSG) treatments on product yield and composition was investigated to maximize the process separation efficiency and potentially reduce the chemical and enzyme consumptions. The sodium hydroxide and Alcalase treatments improved the protein and fiber separation compared to the sodium bisulfite treatment. Under the optimal condition (20 µL/g dry BSG) using Alcalase, the protein separation efficiency was 84%, and the protein concentration in HPP was 43% (w/w), almost double the protein concentration (23%, w/w) in original BSG. The fiber content of the HFP was more than 80% (w/w), of which hemicellulose is the dominant fiber component. [ABSTRACT FROM AUTHOR]