Unlocking Bioactive, Peptide-Rich Extracts from Tomato Seeds Using Enzymatic-Assisted Extraction

Tomato processing generates large amounts of by-products, with seeds representing an underutilized yet protein-rich fraction. This study investigated direct enzyme-assisted protein extraction from non-defatted tomato seeds. Various enzymes, enzyme/substrate ratios, pre-treatments, and incubation temperatures were evaluated and optimized. An enzyme/substrate ratio of 5% (w/w) was found to be optimal, with proteases alone outperforming cell wall-degrading enzymes and two-step extraction strategies. Bromelain, Protamex, and Trypsin, for the first time applied directly to non-defatted tomato seeds, achieved the highest protein recoveries (average 110.56 mg BSA eq/g DW). Among them, Trypsin also produced the highest reducing sugar content (25.07 mg GLU eq/g DW), indicating effective cell wall disruption. Digestates obtained from defatted and non-defatted tomato seeds showed comparable protein contents, demonstrating that defatting was unnecessary. Avoiding the defatting step improved process sustainability by reducing solvent use and energy consumption without significantly affecting protein extraction efficiency. Incubation at 37 °C was preferred over 60 °C, as similar yields were achieved under milder conditions while also reducing energy consumption by approximately three-fold (54,340 kJ vs 150,480 kJ for a 1000 L water-based scale-up simulation). These digestates showed significantly higher antioxidant and, for the first time in tomato seed extracts, anti-tyrosinase activities compared with controls. Protamex-derived samples exhibited the highest bioactivities (7.40 mg AA eq/g DW; 101.36 μg KA eq/g DW). Compared to conventional alkaline–acid extraction followed by enzymatic digestion, the direct enzymatic approach provided higher protein recovery. Overall, this method represents a sustainable strategy for producing bioactive peptide-rich extracts for food and non-food applications.