Novel Antioxidant Peptides from Grateloupia livida Hydrolysates: Purification and Identification

Grateloupia livida protein was hydrolyzed with various proteases (alkaline protease, Protamex and neutral protease) to obtain anti-oxidative peptides. Antioxidant activity of the enzymatic hydrolysates was evaluated by the DPPH radical scavenging, ABTS radical scavenging and reducing power assays. The results suggested that hydrolysates obtained by neutral protease 1 h hydrolysis displayed the highest antioxidant activity (DPPH IC₅₀ value of 3.96 mg/mL ± 0.41 mg/mL, ABTS IC₅₀ value of 0.88 ± 0.13 mg/mL and reducing power of 0.531 ± 0.012 at 8 mg/mL), and had low molecular weight distribution (almost 99% below 3 kDa). Three fractions (F1-F3) were then isolated from the hydrolysates by using semi-preparative RP-HPLC, and the fraction F3 showed the highest antioxidant ability. Four antioxidant peptides were identified as LYEEMKESKVINADK, LEADNVGVVLMGDGR, LIDDSFGTDAPVPERL, and GLDELSEEDRLT from the F3 by LC-MS/MS. Online prediction showed that the four peptides possessed good water solubility, non-toxic and non-allergenic characteristics. Moreover, the LYEEMKESKVINADK exhibited the highest antioxidant ability. Molecular docking revealed that these peptides could all well bind with Kelch-like ECH-associated protein 1 (Keap1), among which LYEEMKESKVINADK had the lowest docking energy (-216.878 kcal/mol). These results demonstrated that the antioxidant peptides from Grateloupia livida could potentially be used as natural antioxidant.