Recently dairy proteins obtained from whey have received
Recently, dairy proteins obtained from whey have received considerable attention for their antioxidant bioactivity (Bayram et al., 2008; Haraguchi et al., 2011; Zhang et al., 2012). Bovine whey proteins (WP) are widely used in various foods for their nutritional, health-promoting, and functional values (Ramos et al., 2017). Bovine liquid whey is produced by enzymatic treatment of milk (sweet whey) or addition of organic acids or minerals (acid whey) with the precipitation and removal of casein (Yadav et al., 2015). Bovine WP account for 11 to 14.5% of dry whey; the other components of bovine whey powder are lactose (63–75%), fat (1–1.5%), minerals (8.2–8.8%), and vitamins (A, C, E, and B groups; Miller et al., 2006; Yadav et al., 2015). The protein component of whey provides a complete protein source and is rich in sulfur-containing AA (1.7%; Fox et al., 2015) and in branched-chain AA (26%; Ha and Zemel, 2003; Paul, 2009). It is composed of β-LG (50–60%), α-LA (15–25%), BSA (6%), lactoferrin (<3%), and 5-Methyl-CTP (<10%; de Wit, 1998; Madureira et al., 2007; Le Maux et al., 2014). β-Lactoglobulin is a small globular protein, composed of 162 AA with a molecular weight () of approximately 18,300 g/mol (Rade-Kukic et al., 2011). It contains all 20 EAA and is a rich source of sulfur. From a GSH precursor perspective, it has 5 Cys residues, 4 of them involved in disulfide bonds with the remaining 1 having a free reactive thiol group (Le Maux et al., 2014). α-Lactalbumin is a small protein with a MW of 14,200 g/mol consisting of 123 AA arranged in a single peptide chain (Konrad and Kleinschmidt, 2008). It has 8 Cys as 4 disulfide bonds and, therefore unlike β-LG, has no free thiol group (Konrad and Kleinschmidt, 2008; Pepe et al., 2013). Bovine serum albumin is composed of 583 AA with a MW of 66,430 g/mol (Hirayama et al., 1990). It contains 35 Cys groups making 17 disulfide bonds in addition to a free Cys that can facilitate intramolecular disulfide interactions (Madureira et al., 2007). Lactoferrin (MW 80,000 g/mol) is an iron-binding monomeric globular glycoprotein (Wakabayashi et al., 2006) that contains 708 AA, of which 34 are Cys and all of which participate in disulfide bonds (Marshall, 2004). In addition, each lactoferrin monomer can bind 2 Fe3+ ions, with a binding affinity of 10 to 20 M (Baker and Baker, 2004); this iron-binding capacity is likely to contribute to its antioxidant potential (Baker and Baker, 2004; Kim et al., 2013). Bovine WP also contains dilute concentrations of immunoglobulins [IgA, IgM, and IgG (IgG1 and IgG2)]. These are quaternary structure molecules, either monomers or polymers with 4 chains, consisting of 2 light polypeptide chains (MW 25,000 g/mol) and 2 heavy chains (MW between 50,000–70,000 g/mol) linked by disulfide bonds (Madureira et al., 2007). Several bovine whey products are produced commercially (Table 1) and differ primarily in protein content and lactose concentration.
DO WHEY PRODUCTS SHOW ANTIOXIDANT ACTIVITY IN VITRO? The antioxidant potential of WP has been assessed by different in vitro methodologies: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric-reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC). Table 2 details the most recent studies of the antioxidant activity of whey products after processing, enzymatic hydrolysis, or both. Other noteworthy studies have been reviewed previously (Power et al., 2013; Brandelli et al., 2015). The WP antioxidant activity was shown to be dose-dependent (20–100 mg/mL) by the DPPH assay, which measures the ability of a compound to scavenge the DPPH radical (Gad et al., 2011). Hydrolysis of preheated whey protein isolate (WPI) with the enzyme subtilisin (EC 184.108.40.206), a nonspecific endopeptidase purified from Bacillus licheniformis and commercially available as Alcalase (Novozymes A/S, Bagsvaerd, Denmark), with specific activities ranging from 0.6 to 2.5 U/g, increased its DPPH scavenging activity from 11.4 to 62.9% (Peng et al., 2010). Hydrolysates of whey protein concentrate (WPC) produced by subtilisin also showed significantly greater inhibition than WP hydrolysates (WPH) produced by other microbial enzymes (Dryáková et al., 2010; Lin et al., 2012; O'Keeffe and FitzGerald, 2014). Dryáková et al. (2010) demonstrated 35.5% greater inhibition of the ABTS radical with WPC hydrolyzed by subtilisin rather than by the enzymes as bacillolysin [EC 220.127.116.11, commercial source Neutrase (Novozymes A/S)] or Protamex (EC 18.104.22.168, Novozymes A/S). The WPC hydrolyzed by subtilisin also showed more ferric-reducing power (0.55 mM FeSO4 equivalents) than trypsin (EC 22.214.171.124), pepsin (EC 126.96.36.199), or leucyl aminopeptidase (EC 188.8.131.52, commercial source Flavourzyme, Novozymes A/S) hydrolysates (0.35 mM FeSO4 equivalents, P < 0.05). This activity was further increased by heat treatment (95°C, 5–10 min) of WPC before hydrolysis (Lin et al., 2012). However, Adjonu et al. (2013) observed that heat pretreatment (80°C, 15 min) did not improve antioxidant activity of WPI hydrolysates from pepsin [nonheated WPH = 0.32 ± 0.03 μmol of Trolox equivalents (TE)/mg of protein; heated WPH = 0.30 ± 0.03 μmol of TE/mg of protein] or chymotrypsin (EC 184.108.40.206; nonheated WPH = 0.27 ± 0.04 μmol of TE/mg of protein; heated WPH = 0.31 ± 0.02 μmol of TE/mg of protein). In Adjonu et al. (2013), ORAC methodology was employed, which scavenges peroxyl radicals and compares levels to the vitamin E analog Trolox.