From by-product to resource: Matrix-guided processing and protein enrichment of brewer's spent grain

Brewer's spent grain (BSG) is an abundant brewing by-product with high nutritional potential; however, its dense lignocellulosic matrix restricts nutrient accessibility, extractability, and functional utilization. This study investigated the effects of physical (ultrasound), thermal (steam explosion), and biological (spontaneous and solid-state fermentation) pretreatments on the structural, compositional, molecular, and functional properties of BSG and evaluated protein enrichment through triboelectric dry fractionation. Compared with untreated BSG, ultrasound induced cavitation-driven surface disruption, increasing soluble dietary fiber to 10.10% and improving water-holding and oil-binding capacities. Steam explosion caused thermo-mechanical disruption, enhancing fiber solubilization and lipid extractability, resulting in a fat content of 9.38% and phenolic release (8.33 mg GAE/g). Spontaneous fermentation promoted moderate enzymatic hydrolysis, resulting in localized fiber loosening and increased soluble dietary fiber and bioactive compounds. Solid-state fermentation using Pleurotus ostreatus resulted in significant increases in protein content (28.14%), soluble dietary fiber (11.80%), total phenolics (12.51 mg GAE/g), mineral composition, and total amino acids with notable increases in glutamic acid, proline, leucine and lysine. Microstructural, FTIR, and TGA analyses confirmed pretreatment-induced disruption of the lignocellulosic structure. Multistage milling and sieving redistributed chemical constituents, concentrating protein and bioactive compounds in the finer fractions. Triboelectric separation enriched protein to 27.4–36.9%, with yields of 15.6–28.7%, and separation efficiencies of 22.0–33.8%. These findings demonstrate that pretreatment-induced matrix modification combined with multistage milling and triboelectric separation enables solvent-free production of protein-rich BSG fractions, providing a sustainable strategy for BSG valorization.