Sustainable Cultivation of Cordyceps militaris Using Coffee and Pineapple By-Products: Implications for Cordycepin Production and Substrate Valorization

Agricultural by-products are increasingly recognized as sustainable resources for mushroom cultivation and biomass valorization. This study evaluated pineapple stubble and coffee pulp as functional substrate supplements for Cordyceps militaris cultivation. Fruiting bodies were successfully produced on brown rice substrates supplemented with 5–15% pineapple stubble or coffee pulp, either alone or in combination. Substrate composition markedly influenced fungal morphology, biological efficiency (BE), and cordycepin production. Moderate supplementation improved cultivation performance, whereas excessive supplementation reduced BE. Cordycepin accumulation was generally higher in the substrate than in the fruiting bodies. The highest cordycepin content was observed in the substrate supplemented with 15% coffee pulp, reaching 3.93%, approximately twofold higher than that of the brown rice control. Correlation analysis revealed that carbon content and the carbon-to-nitrogen (C/N) ratio were positively associated with BE, while nitrogen content was positively correlated with cordycepin accumulation in the substrate. In addition, coffee pulp extract exhibited notable adenosine deaminase inhibitory activity, suggesting that substrate-derived bioactive compounds may contribute to enhanced cordycepin accumulation during cultivation. After cultivation, the spent mushroom substrate showed reduced C/N ratios and increased nitrogen content, indicating active fungal metabolism-mediated substrate transformation. These findings demonstrate that coffee and pineapple by-products can serve as sustainable supplements to improve cordycepin production in C. militaris. Furthermore, the resulting spent substrate, enriched in nitrogen and bioactive compounds, may represent a valuable secondary resource for agricultural and industrial applications.