Optimization and Identification of Antioxidant Peptide from Underutilized<i> Dunaliella salina Protein</i>: Extraction,<i> In Vitro</i> Gastrointestinal Digestion, and Fractionation

Abstract

DPPH• scavenging peptides (<3kDa) from underutilized <i>Dunaliella salina</i> protein were obtained by the following successive treatment, i.e., ultrasound extraction, simulated <i>in vitro</i> gastrointestinal digestion hydrolyzation, and membrane ultrafiltration classification. The optimal condition for ultrasound-assisted extraction was an ultrasound wave with 800 W of power treating a mixture of 60 mL of 1.0 mol L^-1 NaOH and 2 g algae powder for 15 min. A high correlation (r=0.8146) between DPPH• scavenging activity and yield of the intact peptides showed their antioxidant capacity. Simulated <i>in vitro</i> digestion assay resulted in excellent DPPH• scavenging activity of the total peptide, amounting to (86.5 ± 10.1)%, comparing with the nondigestion samples at (46.8 ± 6.5)%. After fractionation, the 500-1000 Da fraction exhibited the highest DPPH• scavenging activity (81.2 ± 4.0)%, increasing 1.5 times due to digestion. Then, the 500-1000 Da fraction was analyzed by RPLC-Q Exactive HF mass spectrometer, and 4 novel peptides, i.e., Ile-Leu-Thr-Lys-Ala-Ala-Ile-Glu-Gly-Lys, Ile-Ile-Tyr-Phe-Gln-Gly-Lys, Asn-Asp-Pro-Ser-Thr-Val-Lys, and Thr-Val-Arg-Pro-Pro-Gln-Arg, were identified. From these amino acid sequences, hydrophobic residues accounted for 56%, which indicated their high antioxidant property. The results indicated that underutilized protein of <i>Dunaliella salina</i> could be a potential source of antioxidative peptides through simulated <i>in vitro</i> gastrointestinal digestion.