*3.4. Screening of Antioxidant Peptides*

Peptides with molecular weights less than 3 kD, which produced from hazelnut protein were sequenced for the screening of potential antioxidants. Furthermore, the peptides which have Tyr, Trp, and Met residues, whose molecule weight is less than 800 and with GRAVY value of −2 to 1 are desirable for us, as sorted out in Table 2. It was seen that seven peptides from hazelnut protein, designed from No.1 to No.7, were screened as potential antioxidants due to up to the desired requirements. Next, they were artificially prepared as follows regarding the determined amino acid sequences. The five peptides FSEY, QIESW, SEGFEW, IDLGTTY, and GEGFFEM were artificially made based on the active amino acids

of their C-terminal. The peptide AHSVVYAIR (designed as No.6) was synthesized in terms of Tyr-containing residue in its middle position. The peptide NLNQCQRYM (named as No.7) was synthesized because of the existence of Tyr, Cys, and Met residues. Besides this, two reported peptides HLHSAT and ADGF from hazelnut protein were artificially synthesized according to their ability to scavenge ABTS and DPPH radical [22,23]. Thus, the four peptides NLNQCQRYM, AHSVVYAIR, HLHSAT, and ADGF were artificially prepared as the control, and they were designed to clarify the feasibility of amino acid residues in screening antioxidant peptides.


**Table 2.** Sequences of synthetic peptides containing Tyr, Trp, Cys, and Met residues.

Notes: A = Alanine, R = Arginine, N = Asparagine, D = Aspartic Acid, C = Cysteine, E = Glutamic Acid, Q = Glutamine, G = Glycine, H = Histidine, I = Isoleucine, L = Leucine, K = Lysine, M = Methionine, F = Phenylalanine, P = Proline, S = Serine, T = Threonine, W = Tryptophan, Y = Tyrosine,V=Valine. HLHSAT and ADGF are antioxidant peptides obtained from hazelnut in other studies [22,23].

As predicated by us, six synthesized peptides showed a significant impact on retarding the oxidation of linoleic acid (Figure 1d). Peptides FSEY and NLNQCQRYM showed the best antioxidant activity, followed by QIESW, SEGFEW, IDLGTTY, and GEGFFEM. However, the inhibition of these synthesized peptides against linoleic acid oxidation functioned in a dose-dependent manner. Peptide NLNQCQRYM with a concentration higher than 900 μg/mL performed excellent activity in stopping linoleic acid from oxidation, while inhibition rates of the peptides QIESW and SEGFEW which contain Trp residue were less than 80% when their concentrations exceeded 200 μg/mL. It was noted that peptide AHSVVYAIR showed a poor capacity, and its IR% was only 50 even when its concentration was elevated to 5000 μg/mL (Figure 1 not shown). Our work indicated that peptides HLHSAT and ADGF, which have been reported to have ABTS and DPPH radical scavenging capacity [22,23] did not show any inhibition against the oxidation of linoleic acid. Moreover, the peptides that contain Cys residue showed perfect O2 •− scavenging activity compared to others, as these dipeptides did (see Figure 1c). Clearly, data from the artificially synthesized peptides support our assumption that the occurrence of active amino acid residues plays a crucial role in promoting the antioxidant capacity of a peptide. Featuring the properties of the amino acid residues should be a simple and feasible tool for the quick selection of desirable antioxidant peptides from hazelnut protein. Besides this, despite the excellent impact on retarding linoleic acid oxidation of FSEY and NLNQCQRYM, FSEY was selected for further study as the peptide falls within a molecular weight of 200 to 800 and GRAVY value of −2 to 1, as well as containing Tyr residue at the C-terminal.
