In an effort to increase antioxidant properties of

In an effort to increase antioxidant properties of peptides from WP, Le Maux et al. (2016) altered the hydrolysis conditions (pH, enzyme type, reaction time, and temperature). Hydrolysis of WP (81% protein) test samples with papain (EC 3.4.22.2) at a constant pH of 7.0 gave significantly higher ORAC values (285.32 ± 36.71 μmol of TE/g of powder) than those obtained from hydrolysates generated under noncontrolled pH conditions (192.54 ± 42.61 μmol of TE/g of powder, P < 0.05). To characterize the functional fraction of whey, WPC was hydrolyzed by several enzymes and the resultant hydrolysates were fractioned by size using gel or membrane filtration (Peng et al. 2009; Önay-Ucar et al. 2014; Tarango-Hernández et al. 2015). The ORAC results showed that fractions containing peptides with smaller molecular weight (subtilisin hydrolysate 1-kDa permeate = 0.91 μmol of TE/mg of powder) exhibited more antioxidant activity than those fractions containing larger peptides (subtilisin hydrolysate 5-kDa permeate = 0.75 μmol of TE/mg of powder; O'Keeffe and FitzGerald, 2014). Mass spectrometry analysis revealed the AA sequences of peptides in antioxidant fractions (Table 3). Several peptides from β-LG were identified in antioxidant fractions produced by enzymatic hydrolysis of whey products. Many of these peptides occurred within 3 location hotspots (42–61, 77–110, and 123–135 AA). Interestingly 125 to 135 AA contains the iron-binding peptide TPEVDDEALEK (Cruz-Huerta et al., 2016). Bertucci et al. (2015) also discovered several α-LA peptides in fractions exhibiting antioxidant activity. In this case, peptides from location 15 to 23 AA were frequently identified. To date none of these peptides have been synthesized and tested in DPPH, ABTS, ORAC, or FRAP assays for antioxidant activity. The free AA present in these fractions, some of which may contribute to the antioxidant activity (e.g., Trp, Phe, Tyr, Cys, and His) have also not been described. In Gastrodin (Table 3), Hernández-Ledesma et al. (2005) identified several peptides and AA from a 3-kDa permeate of the β-LG hydrolyzed by Corolase PP (AB Enzymes, Darmstadt, Germany). Three peptides, MHIRL, YVEEL, and WYSLAMAASDI, were synthesized and exhibited antioxidant activity by ORAC (MHIRL = 0.306 µmol of TE/µmol of peptide; YVEEL = 0.799 µmol of TE/µmol peptide; and WYSLAMAASDI = 2.621 µmol of TE/µmol of peptide). In particular, the antioxidant activity of WYSLAMAASDI is comparable to the synthetic antioxidant butylated hydroxyanisole (2.43 µmol of TE/µmol pure compound), but 1.7 to 4 fold lower than the plant polyphenols catechin and quercetin (14.9 and 10.5 µmol of TE/µmol of compound, respectively; Dávalos et al., 2004). Additionally, Hernández-Ledesma et al. (2007) identified 3 synthetic peptides derived from β-LG (19–24 AA; WY, WYS, and WYSLAM) that exhibited ORAC values [4.45 (WYS) to 7.67 (WY) µmol of TE/µmol of peptide] higher than equimolar mixtures of their corresponding free AA. Nongonierma and Fitzgerald (2013) identified a synthetic dipeptide WC present in α-LA (60–61 AA) and lactoferrin (8–9 and 347–348 AA) that exhibited 50% DPPH scavenging capacity at 0.26 mM, equivalent to 17.2 nM Trolox. Moreover, purified peptides, LDQW and INYW, derived from thermolysin (EC 3.4.24.27) hydrolysis of α-LA were capable of a 100% ABTS radical inhibition at 2.5 μM (Sadat et al., 2011). In addition to processing, whey origin may also play a role in antioxidant activity. Salami et al. (2010) hydrolyzed camel WP with either chymotrypsin, trypsin, proteinase K (EC 3.4.21.64), or thermolysin. Camel liquid whey is rich in α-LA and lysozyme, but lacks β-LG. Camel WP exhibited 40% higher antioxidant activity by ABTS than bovine WP (Salami et al., 2010). In addition, sheep WP was found to be more active against the DPPH radical, requiring 3.1 ± 0.09 mg/mL to inhibit 50% of the radical compared with 8.2 ± 0.77 mg of bovine WP/mL (Kerasioti et al., 2014). It also exhibited greater iron-reducing power than bovine WP, although, in this case, ABTS data was similar (Kerasioti et al., 2014). Indeed, conflicting data between different radical scavenging methods (ORAC, DPPH, ABTS) is common to the majority of studies (Adjonu et al., 2013; Kerasioti et al., 2014), indicating the need to perform several antioxidant assays to be confident of results. Interestingly, other components in milk appear to synergistically enhance the antioxidant activity of WP. Zulueta et al. (2009) showed higher ORAC values for pasteurized milk (13,935 μM TE) than from whey obtained after casein precipitation of pasteurized milk (1,078 μM TE). In this regard, Conway et al. (2013) observed that hydrolysates from buttermilk protein (54.6% protein content) were more effective (P < 0.05) at scavenging free radicals than those from WPC (74.5% protein content). The ORAC values were 1,319.6 ± 46.7 µmol TE/g of protein for buttermilk compared with 782.5 ± 34.8 µmol TE/g of protein for WPC. Although buttermilk is unlikely to contain large quantities of WP, analysis revealed 4 β-LG peptides (VAGTWY, TKIPAVFK, IPAVF, and VLVLDTDYK) that were proposed to contribute to the antioxidant activity (Conway et al., 2013).