The aim of this research is to propose simple and scalable processes to obtain bioactive peptides extensively hydrolyzed starting from a tuna mixed biomass. The upcycling of this powdered biomass is challenging since it comes from the unsorted industrial side streams of the tuna canning process (cooked residues from fillet trimming) after a patented mild dehydration useful for preventing its degradation until its exploitation. Two different protocols were proposed, with and without the inclusion of an exogenous enzyme (Enzymatic-Assisted Extraction, EAE), with no relevant differences in yields (24% vs. 22%) and a comparable amino acid composition. Nevertheless, the former protocol (with EAE) provided peptides with an average molecular weight of 1.3 kDa, and the second one (without EAE) provided peptides with an average molecular weight of 2.2 kDa. The two corresponding types of tuna protein hydrolysates (Enzymatic Hydrolysates (EH) and Non-Enzymatic Hydrolysates (NEH)) were characterized by proximate compositions, pH, color profile, amino acid analysis, FTIR spectra, and molecular weight distribution. In addition, several biological analyses were performed to assess their potential use as nutraceutical supplements: special attention has been paid to antioxidant activity using three different methods to quantify it. EH showed the most promising antioxidant activity which could be exploited also in other fields (e.g., biomaterials, cosmetics).

The fish processing industry produces substantial quantities of by-products with significant potential for valorization into bioactive compounds. This study investigates the complete valorization of fish by-products coming from tuna cooking processes through the utilization of their two main fractions: lipids and proteins. Supercritical CO2 was evaluated as a novel, sustainable and non-invasive technique to recover oil and, more specifically, the omega-3 fatty acids. Response surface methodology was used to evaluate the effect of pressure and temperature on the extraction yield, the oil quality, and the omega-3 content. The most suitable conditions were achieved at 550 bar and 50 °C, resulting in a 70% extraction efficiency and a 97% recovery of total omega-3 fatty acids, without compromising oil acidity or peroxide values. Moreover, enzymatic hydrolysis that has been also optimized by response surface methodology yielded protein hydrolysates with significant antioxidant and hepatoprotective activities. Optimal hydrolysis conditions were obtained at an enzyme concentration of 0.785% (w/w) and a liquid-to-solid ratio of 9.58 mL/g, achieving a hydrolysis yield of 54%. These conditions were validated at a larger scale, confirming good reproducibility, with an experimental hydrolysis yield of 47%. Overall, the integrated use of supercritical CO2 and enzymatic hydrolysis provides a dual, complementary and sustainable cascade-based valorization strategy for tuna by-products, enabling the sequential recovery of functional oils and peptides. This combined approach promotes the efficient exploitation of marine resources and supports the creation of new value chains through the production of high-value ingredients for food and nutraceutical applications.