Process-driven flavour modulation in soybean-based alternative proteins via Neurospora crassa coculture fermentation

Soybean-derived ingredients are widely utilised in plant-based foods and protein formulations but are often limited by lipid-derived off-flavours, particularly undesirable "beany" and "green" notes. Microbial fermentation offers a clean-label strategy to improve flavour while maintaining processing sustainability. In this study, a factorial experimental design was applied to optimise solid-state fermentation of soybeans using two distinct coculture systems: a fungal-fungal system (Neurospora crassa-Rhizopus oryzae), and a fungal-bacterial system (Neurospora crassa-Lactiplantibacillus plantarum). Fermentation temperature (25-30 °C), inoculum ratio (1,1,2,1,1,2), and duration (3-5 days) were evaluated to determine their effects on microbial dynamics, metabolite formation, aroma profiles, and sensory perception. Fermentation significantly reduced beany odorants, with hexanal levels decreasing by up to 95% compared with the uninoculated control. The two coculture systems exhibited distinct flavour regulation pathways. The fungal-bacterial system (N. crassa-L. plantarum) followed a carbohydrate-driven pathway characterised by controlled acidification and selective proteolysis, promoting enrichment of umami-related metabolites, including glutamic acid (up to 1.55 mg/g), umami-active peptides and nucleotides (guanosine monophosphate and inosine monophosphate), and moderated lactic acid production (<6.5 g/L). In contrast, the fungal-fungal system (N. crassa-R. oryzae) followed a lipid-driven pathway characterised by enhanced lipolysis and mobilisation of polyunsaturated fatty acids, leading to increased formation of lipid-derived volatiles such as 2,4-decadienal associated with fatty and savoury aromas. These contrasting metabolic pathways were supported by multivariate analysis, confirming distinct taste- and aroma-driven optimisation profiles, demonstrating that flavour development is strongly parameter-dependent. These findings establish a framework for fermentation-enabled flavour engineering in soybean-based alternative proteins.