Utilization of Skipjack tuna (Katsuwonus pelamis) canning by-products for antioxidative peptide production using neutrase and enzymatic membrane reactor

The hydrolysis of fish proteins has been extensively studied at the laboratory scale using conventional batch processes. However, the inherent limitations of batch systems have highlighted the need for alternative approaches such as continuous enzymatic membrane reactor (EMR) systems. EMRs offer a more efficient and convenient method for hydrolyzing proteins and producing bioactive peptides. Skipjack canning generates body juice during the pre-cooking stage. The body juice with a high COD value in water pollution is rich in water-soluble protein. These proteins have attracted considerable interest for their potential recovery and utilization as nutraceutical compounds, such as antioxidative peptides. This study aimed to produce antioxidative peptides from skipjack body juice using a continuous EMR system and evaluate their antioxidative activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2’-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) assays. The study was conducted in two stages: (1) determination of the optimal substrate-to-enzyme ratios (1% and 2% substrate with 15% enzyme and 2% substrate with 10%, 15%, and 20% enzyme) in a batch reactor, based on antioxidative peptide activities; and (2) production of antioxidative peptides from skipjack tuna cooking juice using a continuous EMR, with antioxidative activity measured via DPPH, FRAP, and ABTS assays. The results indicated that a substrate concentration of 2% with a 15% enzyme concentration yielded the highest antioxidative activities, with DPPH, FRAP, and ABTS values of 349.479 μM TE/mL, 655.400 μM TE/mL, and 90.93%, respectively. Hydrolysis of skipjack tuna cooking juice was subsequently performed in the continuous EMR system using a 2% substrate and 15% Neutrase enzyme for 7 h at 50℃. The system utilized a 10 kDa membrane oper-ated at a speed of 300 rpm with a constant flux. The IC₅₀ values for the substrate, batch process, and fractions of 10, 5, 4, and 2 kDa were 3.02 ± 0.12, 1.54 ± 0.08, 1.48 ± 0.02, 1.25 ± 0.04, 1.03 ± 0.02, and 1.36 ± 0.47 mg/mL, respectively. In summary, this study clearly demonstrates that the use of neutrase in a continuous EMR system is an effective approach for producing antioxidative peptides from skipjack tuna cooking juice, offering significant potential for the valorization of fish canning by-products in nutraceutical applications.