Abstract
Microbial protein represents a sustainable alternative to conventional animal protein, yet optimizing substrates for fungal cultivation remains critical. This study demonstrates the successful upcycling of chitin waste and aged rice into fungal protein through fermentation with Cordyceps militaris. Substrate formulations (0-20% chitin waste mixed with aged rice) were evaluated for their effects on fungal growth, yield, and metabolite profiles. Results revealed that aged rice alone supported fruiting body yields comparable to fresh rice (9.8 g vs. 9.8 g), with no significant differences in the morphology or growth rate. The addition of 5% chitin waste led to a 17% improvement in yield compared to the control, increasing the average fresh weight of fruiting bodies from 9.8 g to 11.5 g per bottle, while higher chitin levels (20%, T4) suppressed mycelial growth entirely. Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed chitin's structural complexity and nitrogen-rich composition, which slowed the substrate utilization but enriched secondary metabolites. Liquid chromatography-mass spectrometry (LC-MS) identified 1025 metabolites, including up-regulated bioactive compounds (e.g., cordycepin and piplartine) in chitin-amended substrates, linked to amino acid and lipid metabolism pathways. Safety assessments confirmed the absence of toxins, validating the substrates' suitability for food applications. These findings highlight chitin waste (≤5%) as a viable nitrogen supplement to aged rice, improving the fungal protein yield and bioactive compound synthesis. This approach advances sustainable biomass valorization, offering a scalable strategy to reduce agricultural waste while producing nutrient-dense fungal protein.