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Herein, the antioxidant peptides from a Thai traditional semi-dried fermented farmed hybrid catfish (Clarias macrocephalus × Clarias gariepinus) catfish, Pla Duk Ra, were characterized. After extraction and deproteinization, Pla Duk Ra crude peptide extract (CPE) was fractioned using 2 connected Hitrap Sephadex-G25 columns, yielding two significant fractions, F1 with higher browning intensity (A420) and F2. CPE, F1, and F2 had different amino acid profiles, contents, and sequences evaluated by LC-MS/MS, which could be responsible for their antioxidant properties. F2 contained the highest numbers of hydrophobic amino acid (HBA) (47.45%) and aromatic amino acid (27.31%), followed by F1, and CPE. The peptides with 8–24 amino acid residues were detected in CPE and its fractions. In CPE, F1, and F2, there were 69, 68, and 85 peptides with varied HBA content, respectively. ARHSYGMLYCSCPPND (50% HBA), ALRKMGRK (37.5% HBA), and ANWMIPLM (87.5% HBA) were the most prevalent peptides found in CPE, F1, and F2. Overall, F2 was the most effective at inhibiting free radicals (DPPH● and ABTS●+) and reactive oxygen species (hydroxyl radical, singlet oxygen, and hydrogen peroxide), followed by F1 and CPE. The metal chelation of F1 was, however, superior to that of F2 and CPE. For the stability test, the effects of pH, heating temperature, and in vitro digestion on the DPPH● scavenging activity of F2 were investigated. The activity was boosted by lowering the pH and raising the heating temperature. In the gastrointestinal tract model system, however, roughly 50% of DPPH● scavenging activity reduced after digesting.

Characterization of Antioxidant Peptides from Thai Traditional Semi-Dried Fermented Catfish