In vitro digestibility evaluation of mycelium–bread meals as fish feed ingredient

The development of alternative feed ingredients is essential for the future sustainability of the aquaculture industry. This study aimed to evaluate the in vitro protein bioaccessibility and functional properties of mycelium‑based ingredients derived from two filamentous fungi, Aspergillus oryzae (AO) and Neurospora intermedia (NI). These ingredients were produced via submerged fermentation and solid‑state fermentation, with the latter utilizing stale bread as a novel, low‑value substrate. Protein bioaccessibility was assessed using two‑stage in vitro digestion models for rainbow trout (Oncorhynchus mykiss) and Nile tilapia (Oreochromis niloticus). The results showed that pure fungal biomass achieved significantly higher (p < 0.05) protein bioaccessibility than both fishmeal and soybean meal. In the trout model, amino acid release reached 83 and 72 mg AA/100 mg protein for pure mycelia (AO and NI, respectively) and 55 and 54 mg AA/100 mg protein for the mycelium–bread composites. Detailed fractional analysis indicated that high amino acid release was driven by both pre‑existing free amino acids and the fungal protease activity. Furthermore, antioxidant levels increased after digestion in mycelium–bread composites. Based on the in vitro findings, mycelium‑based ingredients represent highly bioaccessible protein sources with functional properties for aquafeed applications.