Substrate dependent growth conditions and moisture dynamics drive aroma development in solid-state Aspergillus oryzae (koji) fermentations of cereal and oilseed processing side streams

Introduction Solid-state fermentation (SSF) with the filamentous fungus Aspergillus oryzae is widely used in traditional fermentations, yet the mechanisms governing aroma formation on agro-industrial side-streams remain poorly understood. This study investigated how substrate composition and dynamic moisture conditions influence physicochemical changes, metabolic activity, and volatile organic compound (VOC) formation during SSF. Methods Wheat bran, rye bran, rapeseed press cake, and pumpkin seed press cake were fermented with Aspergillus oryzae under varying moisture conditions. Fermentation progression was monitored through measurements of fungal growth, pH, water activity, reducing sugars, and primary amines. VOC profiles were analyzed over time to evaluate substrate-specific and moisture-dependent aroma development trajectories. Results Fermentation behavior differed markedly between substrates and moisture conditions. Wheat and rye bran followed similar trajectories characterized by decreasing pH, moderate moisture loss, and increased reducing sugars and primary amines. Rapeseed press cake showed alkalinization and strong amine accumulation, whereas pumpkin seed press cake displayed limited pH change but pronounced decreases in water activity and proteolytic signatures. VOC analysis revealed a directional transition from substrate-derived oxidative compounds toward fermentation-derived metabolites. Linear aldehydes associated with lipid oxidation declined rapidly in cereal substrates but persisted longer in oilseed matrices. Mid-fermentation in wheat and rye was associated with transient enrichment of alcohols, esters, and phenolic compounds, corresponding to increased aroma complexity before shifting toward amino acid-derived metabolites at later stages. Oilseed substrates retained branched and lipid-derived aldehydes longer, maintaining sharper and more pungent aroma characteristics. Rapeseed fermentation additionally generated nitriles and isothiocyanates from glucosinolate degradation. High-moisture conditions promoted formation of redox-related overflow metabolites, including acetic acid and vicinal diketones. Discussion The results demonstrate that aroma development during A. oryzae SSF is governed by a substrate- and moisture-dependent metabolic continuum, where evolving water activity and enzyme-mediated substrate conversion dynamically shape metabolic routing and volatilome composition. These findings highlight the importance of fermentation stage, substrate chemistry, and moisture dynamics in controlling aroma formation during koji fermentation of diverse agro-industrial side-streams.