*3.3. Selection of Protease*

To find a targeted peptide that consists of active amino acid residues at the N- or C- terminus, the database ExPASy ENZYME integrating available information about proteolytic sites and enzymes was used to select appropriate protease. The use of ExPASy ENZYME allows us easily to determine the cleavage site between all pairs of amino acids in the N- or C-terminal [34]. According to the preferential cleavage sites searched from ExPASy ENZYME (https://enzyme.expasy.org/enzyme-search-ec.html, accessed on 13 August 2020) and from a review [35], the alkaline proteinase (EC number: 3.4.21.62), chymotrypsin (EC number: 3.4.21.1) and pepsin A (EC number: 3.4.23.1) are likely to hydrolyze proteins to the peptides with Tyr, Trp, or Met as C-terminus or N-terminus (Seen in Table 1). Among the three proteases, alkaline hydrolysates exhibited the highest inhibition effect on linoleic acid oxidation [36]. To further reveal the preference of the proteases for producing Tyr, Trp, and Met residues, alkaline and neutral proteinases were chosen to "really" hydrolyze hazelnut protein. It was seen from Table 1 that alkaline proteinase hydrolysates exhibited a high inhibition rate of 95.11 ± 0.17% after 1 mL of the hydrolysates were added to linoleic acid emulsions incubated at 40 ◦C for 48 h. However, the inhibition rate of linoleic acid by neutral hydrolysates was 81.44 ± 1.94%. In addition, protein hydrolysates hydrolyzed by alkaline + neutral proteinase showed an in-between inhibition rate (83.35 ± 1.02%). Similar to the result reported by Ngamsuk [37], that alkaline proteinase was found to give high activity hydrolysate compared to neutrase and mix.

**Table 1.** Preferential cleavage sites of several proteinases and inhibition rate of hazelnut protein hydrolysates against oxidation of linoleic acid.


Note: 1 means not analyzed.

Furthermore, the purity of hazelnut hydrolysates processed by alkaline proteinase was 73.66 ± 2.50%. It was clear from our data that the proteinase alkaline could be appropriately selected for processing the antioxidant peptides from hazelnut protein. The alkaline protease hydrolysates were freeze-dried for further study.
