Bread waste represents a significant fraction of organic waste in Europe, creating both environmental challenges and opportunities for valorization. Solid-state fermentation (SSF) with filamentous fungi is a proven approach for upcycling such residues into protein-rich biomass. This study aimed to utilize SSF with the edible fungus Neurospora intermedia to convert stale bread into a mycelium-bread product with enhanced nutritional qualities for potential applications in both human food and animal feed. A five-day solid-state fermentation was optimized by systematically varying key environmental parameters (temperature, substrate moisture, and relative humidity). The product generated under optimal conditions was then subjected to a comprehensive nutritional analysis, including its amino acid and fatty acid profiles and mineral content, to evaluate its potential for food and feed applications. Under optimal conditions (30 °C, 60% initial moisture and 50% relative humidity, the crude protein content of the mycelium-bread product increased from 12.2 g/100g dry matter (DM) in the initial bread to 34.8 g/100g DM. The nutritional profile of the optimized product was substantially improved, featuring a marked increase in the essential amino acid lysine and the bioconversion of starch (from 61.6 to 1.44%) into 15.2 g/100g DM of functional fungal fiber. Moreover, a substantial improvement was observed in the lipid quality, evidenced by the PUFA/SFA ratio increasing from 0.72 to 2.10 after the fermentation. This study highlights the potential of SSF using N. intermedia to upcycle organic residues such as stale bread into a sustainable protein source with multi-nutrient potential for food and feed applications.

Global food waste management necessitates circular bioeconomy solutions to transform organic residues into high-value nutrients to address nutritional demands. This study investigated the valorization of two abundant waste streams, stale bread and sunflower oil through solid state fermentation using food-grade filamentous fungi. Three strains, Neurospora intermedia, Aspergillus oryzae and Rhizopus oryzae were evaluated for the bioconversion of stale bread. Oil supplementation levels of 10, 20 and 30% (g/100 g dry matter) using both fresh and spent sunflower oil were tested to assess changes in proximate composition, characterizing fungal growth dynamics and mycelial development. Furthermore, modifications in fatty acid profiles and hydrolytic enzyme activities were analyzed to determine species responses to oil source and concentration. The results demonstrated that N. intermedia achieved peak protein levels of 36% (g/100 g) alongside efficient starch catabolism, while 10% fresh oil supplementation induced a significant protein increase (26%) in A. oryzae. Regarding lipid accumulation, 10% spent oil supported higher fat content in R. oryzae (19%) compared to fresh oil (17%). PUFA/SFA ratio reached its maximum in A. oryzae with the highest of 5.91 ± 0.56 under 10% fresh oil. Enzymatic analysis identified A. oryzae as the most efficient lipase producer, reaching a maximum activity of approximately 0.10 U/g at 10% spent oil supplementation. Conversely, R. oryzae lipase activity peaked at 20% supplementation (0.08 U/g), reflecting its high capacity for lipid accumulation. These findings establish a potent bioprocess for upcycling mixed food wastes into enhanced functional ingredients for sustainable food and feed systems.

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.