*4.3. BIOPEP Database Analysis: Rules of Molecular Weight, GRAVY Value and Active Amino Acid Residue's Location*

Few reports have focused on how a molecular weight, GRAVY value, and position of amino acid residues affect the capacity of a peptide against oil oxidation yet. In our study, a peptide in the BIOEPE database falling within a molecular weight of 200 to 800, GRAVY value of −2 to 1, and active amino acid residues at N- or C- terminus produced a strong inhibition towards lipid oxidation, as shown in Table S1 and Figure S1.

Regarding molecular weight or numbers of amino acid residues, numerous studies have indicated that peptides containing amino acid residues between 2 and 11 [46] or weighing less than 1000 Da [47] will exhibit good antioxidant ability. Peptides with more molecular weights (>2000 Da) easily decrease in their antioxidant activity due to the hiding of the active site [47]. It is well-known that the interfacial phase, the contact region between the oil phase and the aqueous phase, is the critical region in the system with regard to the development of lipid peroxidation [48]. Thus, in a given emulsion system, GRAVY value could not be ignored for antioxidant estimation, because a higher GRAVY means higher hydrophobicity. Various researches have proposed that peptides with higher hydrophobicity can protect linoleic acid from oxidation by donating protons to hydrophobic peroxy-radicals [49,50]. In our study, it is seen that peptides from BIOPEP database which able to stop lipid oxidation have GRAVY values ranging from −2 to 1 (see Table S1 and Figure S1b). Thus, a GRAVY value of −2 to 1 was proposed to be an ideal criterion for looking for antioxidant peptides from protein hydrolysate. A recent study has overviewed the roles of amino acid composition and sequence in conferring the antioxidant activities of peptides. The phenolic hydroxyl of Tyr, the indolyl of Trp, the thiol group of Cys, and the thioether of Met are regarded to act as hydrogen donors for free radicals [2,39,51]. Our study has found that the most of antioxidative peptides which are searched from the database BIOPEPE have the residues Tyr, Trp, Cys, or Met. Similarly, the antioxidant activities of peptides LGFEY and LGFYY were attributed to the presence of Tyr residues [52]. Concerning the inhibition of lipid oxidation, most peptides searched from the BIOPEPE database are observed to have active amino acid residues located at C- terminus. In our case, a linoleic acid oxidation system was designed to confirm the strong antioxidant activity of the peptide FSEY having Tyr residue located at the C-terminal (see Figure 4a). HOMO analysis from the synthesized peptides FYSE and YFSE also presents evidence that the reaction sites are all located at Tyr (see Figure S2). Similarly, studies done by Guo et al. [53] and Torkova et al. [54] indicated that peptides having Tyr residue located at the C-terminus strongly scavenged hydroxyl-radical, hydrogen-peroxide, and peroxyl radicals. While

these peptides exhibited a better inhibition against ABTS cation radical when Tyr residue is located at the N-terminus [54]. Clearly, the types and positions of amino acid residues should be considered in searching for an antioxidant peptide from protein.

In conclusion, a wanted antioxidant peptide could be quickly screened by determining the types and location of amino acid residues as well as molecular weight and GRAVY, especially Tyr, Trp, Cys, and Met which act as H donors. Among the four amino acids, Tyr-containing peptides show a prominent antioxidant activity, especially when it is located at the C-terminus.
