Two-step enzymatic hydrolysis reshapes metabolite profiles and enhances aroma characteristics of Lentinula edodes stipe powder

Shiitake (Lentinula edodes ) stipes, despite accounting for nearly one-third of the fruiting body, are routinely discarded due to their rigid cell wall that restricts nutrient release. Understanding how enzymatic hydrolysis reshapes their metabolite profile and sensory properties is important for valorizing this biomass resource in food applications. Many studies have examined enzymatic treatments of mushroom tissues, but the molecular basis linking metabolite changes to nutrition/aroma enhancement has remained unclear. Here we show that a two-step enzymatic hydrolysis strategy markedly increased amino acids and lipid-related metabolites of L. edodes stipes. Multi-omics profiling revealed a broad enhancement of nutritional quality, with 599 small-molecule metabolites increased. Computational analyses identified 22 human physiological receptors with potential interactions with the increased metabolites. These receptors were significantly enriched in the renin–angiotensin system, suggesting possible influences on blood pressure homeostasis. Volatilomics further showed that the enzymatic hydrolysis reinforced mushroom-like, fruity and woody notes while diminishing undesirable waxy odors. These increased VOCs exhibit strong predicted interactions with human olfactory receptors, particularly OR2W1 and OR8D1. Together, these findings demonstrate that enzymatic hydrolysis transforms the underutilized stipes into a nutrient-dense and flavor-enhanced ingredient, contributing to sustainable food-resource utilization. • Enzymatic hydrolysis enhanced nutrient release and reshaped metabolite profiles. • Cellulase and Flavourzyme boosted polysaccharides, proteins, and amino acids. • Metabolomics identified 599 increased metabolites and enriched amino-acid and nitrogen metabolism. • Eight key aroma-active VOCs increased, enhancing mushroom/fruity/green notes. • The increased metabolites showed potential interactions with human physiological and olfactory receptors, including enrichment in the renin–angiotensin system.