Efficient purification and characterization of novel antioxidant peptides from protein hydrolysate of Malaysian fish sausage (Keropok Lekor) by-products

Biotransformation of fish by-products (FBPs) through microbial fermentation is an economical way to produce antioxidative fish protein hydrolysate. Conventional purification techniques involving column chromatography require several unit operations and are complicated. This study explored the use of an alternative bio-separation technique, an aqueous two-phase system (ATPS) in combination with ultrafiltration (UF) to purify the antioxidant peptides (APs) derived from fish sausage by-products hydrolysate. Optimised polymer–phosphate ATPS elevated peptide yield by 34.0% and the purification fold (PF) of antioxidant activities were 3.2 for ferrous chelating and 10.3 for ABTS radical scavenging. The most effective AP purification was achieved by the ATPS–UF (10 and 3 kDa) achieving 218- and 62.6-fold purification for the ferrous chelating and ABTS radical scavenging activities, respectively. Thirteen peptides have been identified and they were characterised using amino acid analysis and an antioxidant score. The antioxidant score indicated that 61.5% of the peptides identified were APs. Abundant polar and hydrophobic amino acids in the sequences and residues were the main elements that led to the antioxidant activity. This work demonstrated the potential of combining ATPS and UF to purify APs, which is simple and effective for industrial applications.