Recently whey products have been added to nutritional

Recently, whey products have been added to nutritional beverages to boost their antioxidant capacity. Supplementation of a lemon drink with 1% WP hydrolyzed by subtilisin increased the antioxidant activity of the beverage from 0.75 to 7.79 mmol of TE/L (Athira et al., 2014). In addition, a flavored milk beverage fortified with 1 or 2% WPH from different enzymes [leucyl aminopeptidase, subtilisin, or Corolase PP (AB Enzymes)] increased the ABTS radical inhibition of the beverage by 21 and 33%, respectively. Interestingly, adding intact WPC (1–2%) to the beverage did not alter ABTS values (Mann et al., 2015). A polyphenol rich beverage [chlorogenic wee1 (0.01%) or catechin (0.01%)], thermally treated (121°C, 10 min) at pH 3.7, exhibited ABTS values of 0.45 to 1.22 mM TE/L respectively. However, the addition of WP (0.2%; ABTS = 0.45 mM TE/L) to this model beverage did not result in additive antioxidant activity, although ABTS results were higher than the beverage with polyphenol values alone (0.90–1.77 mM TE/L; He et al., 2015). Indeed, addition of WP (0.5, 2.0, 4.0, or 6.0%) did not significantly change (P > 0.05) the antioxidant activity of another beverage with 0.0032% lutein, a carotenoid antioxidant (Rocha et al., 2017). Interestingly, the combination of WPC and the algae Spirulina platensis, rich in carotenoids, tocopherol, and phycocyanin, showed less antioxidant power (125 TE mg/L of sample) than Spirulina platensis (100 mg/100 mL) alone (170 TE mg/L of sample), which indicates that whey products can exert an antagonistic effect on the antioxidant activity of other compounds (Gad et al., 2011). How WP compare in terms of their antioxidant activity to other proteins and known antioxidant compounds has been investigated (Dávalos et al., 2004; Hernández-Ledesma et al., 2007; Castro and Sato, 2014). Intact WP showed significantly lower DPPH radical inhibition (17.13 ± 2.33%) than soy protein isolate (27.18 ± 0.15%) or egg white protein (33.39 ± 0.26%; Castro and Sato, 2014), although the purity of each protein was not described. Interestingly, no significant differences in DPPH inhibition were found between WP (29.81 ± 0.48%) and egg (31.50 ± 0.24%) hydrolysates using leucyl aminopeptidase (Castro and Sato, 2014). In contrast, ORAC values for WPH were lower (160.72 ± 26.26 µmol of TE/g) than results obtained for their counterparts from egg (546.45 ± 55.75 µmol of TE/g) or soy (1,157.18 ± 134.66 µmol TE/g), which again underlines the inconsistencies across antioxidant assays (Castro and Sato, 2014). It is noteworthy that 100 g of WPC (79.0% protein) results in ORAC values of 13,662 ± 1,018 µmol of TE (Power-Grant et al., 2015), whereas 100 g of concentrated green tea extract results in 758,000 µmol of TE (de la Luz Cádiz-Gurrea et al., 2014). However, as a protein, WP can be added to foods at concentration of 22.2% (Chavan R. S. et al., 2015), whereas green tea extract is usually added to foods at concentrations less than 0.04% (Maruyama et al., 2017).
CAN WHEY PRODUCTS BOOST INTRACELLULAR ANTIOXIDANT DEFENSES IN VITRO? According to the Swedish Agency for Health Technology Assessment and Assessment of Social Services and cited by the World Health Organization, boosting antioxidants capabilities (GSH, CAT, and SOD) in cells by the diet will achieve long life and well-being (SBU, 1997). At cellular levels, GSH (1) directly scavenges free radicals (for example hydroxyl radicals); (2) is a substrate for the antioxidant enzymes glutathione peroxidase and glutathione transferase; (3) facilitates transport of AA, specifically Cys, across the plasma membrane; (4) regenerates antioxidants (e.g., vitamins C and E) to their functional form; and (5) forms conjugates with toxic electrophilic compounds, catalyzed by glutathione transferase, which are excreted from cells (Pastore et al., 2003; Masella et al., 2005; Valko et al., 2007). Tseng et al. (2006) reported that the rat renal cell line, PC12, pretreated with WPC at 10 mg/L for 24 h before an ethanol stress, produced 59.4 mM GSH/mg of protein compared with 29.9 mM GSH/mg of protein (P < 0.05) for cells with no WPC pretreatment. This indicates whey products may offer a protective benefit to cells when stressed. In agreement, stressing myoblast cells C2C12 with 0.3 mM tert-butyl hydroperoxide (t-BHP) for 30 min decreased GSH levels by 31.5% compared with control, as measured by flow cytometry. These t-BHP stressed C2C12 when pretreated for 24 h with sheep WP at 1.56, 3.12, and 6.24 mg increased GSH levels 112.9, 118.0, and 138.0%, respectively, compared with levels of t-BHP–stressed cells (Kerasioti et al., 2014). In a recent study (Table 4), t-BHP was also used to stress human hepatocytes (HepG2) for 2 h after 24 h of WPC treatment (100 μg/mL; Pyo et al., 2016). The WPC treatment increased GSH levels (130%) from basal conditions and also recovered GSH levels from stressed cells (80%). In an attempt to identify which whey fraction is responsible for increasing GSH, O'Keeffe and FitzGerald (2014) used enzymatically hydrolyzed WPC to treat human umbilical vein endothelial cells (HUVEC) and GSH levels were monitored. The WPC was hydrolyzed by subtilisin, bacillolysin, Corolase PP (AB Enzymes), and leucyl aminopeptidase and the resulting peptide fractions were separated according to size using 0.2 μm, 10 kDa, 5 kDa, and 1 kDa cut-off membranes. Whey hydrolysate fractions by subtilisin, bacillolysin, Corolase PP, and leucyl aminopeptidase at 1 mg/mL significantly increased intracellular GSH in HUVEC cells (P < 0.05) after 48 h of incubation compared with HUVEC cells cultured in media alone. The 1-kDa permeate of hydrolysate from subtilisin treatment increased GSH levels by 153% in HUVEC cells compared with media alone (P < 0.001). Kent et al. (2003) incubated prostate epithelial cells (RWPE-1) for 48 h with (1) 0.5 mg/mL of hydrolyzed WPI, (2) 0.5 mg/mL of hydrolyzed casein, (3) 500 μM buthionine sulfoximine (GSH synthesis inhibitor), or (4) 500 μM N-acetylcysteine (GSH stimulant). N-Acetylcysteine increased GSH by 88% in RWPE-1 cells. Hydrolysates of WP with trypsin, chymotrypsin, and peptidase increased GSH by 64% compared with hydrolyzed casein-treated and control cells (P < 0.05). Interestingly, the 50% reduction of GSH levels in RWPE-1 cells by buthionine sulfoximine could not be reversed by co-treatment with WPH, but could be reversed with N-acetylcysteine (Kent et al., 2003). Vilela et al. (2006) evaluated a combination of high hydrostatic pressure processing and low-MW whey peptide fractions, but did not observe a boost in GSH levels in human tracheal epithelial cells (9HTEo cell line).