In vitro meat cultivation, a cutting-edge innovation in food science, may represent a more sustainable and ethical source of animal proteins compared with conventionally grown meat. An important challenge for meat cultivation lies in eliminating the use of fetal bovine serum (FBS) in cell culture media due to ethical concerns. Milk whey is a nutrient-rich liquid portion of the milk, derived as a byproduct of dairy industry. Similar to FBS, whey contains proteins that are crucial for nutrition, cell adhesion, and biomolecular transport. In this study, we investigated whether whey proteins (WP) can replace FBS in supporting muscle cell cultivation, using the C2C12 myoblast model. Accordingly, under serum-free conditions, cells were treated with 2 WP mixtures, grouping high (β-LG 1.25%, α-LA 1.25%, BSA 1.25%) and low (β-LG 0.07%, α-LA 0.15%, BSA 0.15%) selected concentrations of individual proteins that positively affected cell growth in a preceding dose-response study. Cells cultured in only basal Dulbecco's Modified Eagle Medium were included as a negative control, and cells cultured in 10% FBS as a positive control. Cells were maintained in the treatment media for 48 h (d 1 and 2) to support myoblast proliferation. Subsequently, all the treatments were replaced with a standard low mitogenic 2% horse serum (HS) medium until full differentiation (d 6). The treatment effects on morphology, viability, and lactate dehydrogenase release were assessed after d 1, 2, and 6, respectively. The results showed that WP stimulated cell proliferation under serum-free culture conditions, similar to the FBS control, and subsequently facilitated myotube formation when the WP or FBS treatments were switched to HS medium. After differentiation, these cells also exhibited increased expression of cell differentiation markers such as creatine kinase and citrate synthase and underwent morphological changes from spindle-shaped cells to fused elongated myotubes, in contrast to the negative control. This study demonstrates that WP are a promising and sustainable alternative for considerably replacing FBS-based growth supplements for use in cultivated animal products.

The Atlantic blue crab (Callinectes sapidus) is an opportunistic invasive species in the Mediterranean that is negatively affecting biodiversity, fisheries, and tourism. In Italy, it is appreciated for its good meat quality, but the processing yield is low (21.87 ± 2.38%), generating a significant amount of by-products (72.45 ± 4.08%), which are underutilized. Valorizing this biomass is in line with circular economy principles and can improve both environmental and economic sustainability. This study aimed to valorize Atlantic blue crab by-products (BCBP), producing protein hydrolysates and assessing their in vitro bioactivities, in order to plan applications in animal food and related sectors. BCBP hydrolysates were obtained by enzymatic hydrolysis using Alcalase and Protamex enzymes. The treatment with Alcalase resulted in a higher degree of hydrolysis (DH = 23% in 205 min) compared to Protamex (DH = 14% in 175 min). Antioxidant activity of the hydrolisates was evaluated through DPPH, ABTS, reducing power and FRAP assays, as well as in vitro test in fibroblasts (HS-68). At 10 mg/mL, hydrolysates from both enzymes exhibited the maximum radical scavenging activity in DPPH and ABTS assays. In HS-68 cells, 0.5 mg/mL hydrolysates protected against H₂O₂-induced oxidative stress, showing a cell viability comparable to cells treated with 0.5 mM N-acetyl cysteine (NAC), as an antioxidant. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls (SNK) or Games–Howell post hoc tests, with significance set at p < 0.05. Overall, both enzymes efficiently hydrolyzed BCBP proteins, generating hydrolysates with significant antioxidant activity and cytoprotective effects. These results demonstrate the potential to produce high-quality bioactive compounds from BCBPs, suitable for food, nutraceutical, and health applications. Scaling up this valorization process represents a viable strategy to improve sustainability and add economic value to the management of this invasive species, turning a problem in a resource.

The increasing global demand for sustainable protein sources calls for innovative biotechnological strategies to reduce reliance on animal-derived ingredients. In this context, the present study explored Pleurotus ostreatus as a promising fungal matrix for food and cellular agriculture applications. The objective was to assess its nutritional composition, antioxidant profile, and potential use as a functional component in cell culture media. Fungal biomass (FB) obtained through liquid submerged fermentation was characterised for its nutritional profile and functional activity after in vitro digestion (Infogest 2.0) in comparison with whey (WH) and yeast (YT); alternatives already used in food sector. Furthermore, a 100% aqueous extract of P. ostreatus (PL) submerged mycelium was tested on murine skeletal muscle cells (C2C12), as alternative to animal-origin ingredient in cell culture media, to evaluate its effects on viability, morphology, and gene expression. Results showed that fungal mycelium exhibited moderate protein content (21.33 ± 0.21 g/100 g), high fiber levels (19.05 ± 0.42 g/100 g), and notable antioxidant activity, with significantly higher phenolic content than YT. Despite lower digestibility than WH, FB demonstrated strong antioxidant potential after digestion, confirming the presence of bioactive compounds. In cell-based assays, PL enhanced C2C12 viability and proliferation at 48 h, particularly when combined with insulin–transferrin–selenium (ITS) and bovine serum albumin (BSA), resulting in improved morphology and upregulation of genes related to proliferation and differentiation (Cyclin D1, CDK4, MRF4). These findings indicate that FB can act as a sustainable, functional ingredient for both food systems and serum-reduced media in cultivated meat production, supporting a transition toward more ethical and circular protein production models. Graphical Abstract