Antioxidant peptides are able to scavenge radical oxygen species. They may be generated from milk proteins during gastrointestinal digestion or during milk fermentation (Tsai et al., 2008; $anlidere Aloglu and Oner 2011; Abubakr et al., 2012). Many fermented dairy products (e.g., kefir) show significant antioxidant activity (Liu et al., 2005), yet the ability of releasing antioxidant peptides from milk proteins is strain-dependent. It would seem that no direct relationship exists between antioxidant activity and degree of proteolysis (Virtanen et al., 2007). For instance, among 25 lactic acid bacteria, L. helveticus ATCC CNRZ32 caused the highest proteolysis during milk whey fermentation, but the resulting product showed intermediate radical scavenging activity (Virtanen et al., 2007).
Since dairy products are complex matrices, the in vitro antioxidant activity should be measured using at least two different methods. Even the same antioxidant peptide can show different activity using different methods (Virtanen et al., 2007). Radical (e.g., superoxide, hydroxyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylb- enzothiazoline-6-sulphonic acid (ABTS)) scavenging, inhibition of low-density lipoprotein oxidation, reduction of metal ions (e.g., from ferric to ferrous ion) are among the most common methods for measuring antioxidant activity in vitro (Carocho and Ferreira, 2013). However, due to their limited similarity to physiological conditions, in vitro assays are very restrictive. Thus, the reported effect needs to be ascertained using animal models and/or in vivo assays on humans (Hsieh et al., 2015).
Few in vivo studies have been carried out to test antioxidant activity of dairy products. Anti-peroxidative activity was reported in rats fed on a vitamin E-deficient diet but consuming fermented milk whey preparation (Zommara et al., 1998). Whey protein concentrate could prevent chemically induced damage of liver in rats (Gad et al., 2011). Administration (150 g/die, 21 days) of goat milk fermented with Lactobacillus fermen- tum ME-3 to healthy humans lowered the levels of oxidized low-density lipoprotein, isoprostanes, and the glutathione redox ratio, resulting in enhanced anti-atherogenicity (Kullisaar et al., 2003). Whey protein isolate (WPI) administered (110 g/die, 40 days) to healthy overweight young men participating in a resistance training program caused increased plasma total antioxidant capacity and glutathione (GSH) level (Sheikholeslami and Ahmadi Kani Golzar, 2012). On the contrary, no effect on plasma antioxidant status was reported following administration (33 g/die, 50 days) of whey protein (WP) to healthy subjects participating in a resistance-training program (Brown et al., 2004). Patients suffering from nonalcoholic steatohepatitis consuming (80 days) 20 g/die of WPI rich in cysteine showed an increase of plasma total antioxidant activity and plasma GSH (Chitapanarux et al., 2009). Although in most in vivo studies no specific compound responsible for the observed effects was identified, in this study the antioxidant activity could be attributed to cysteine, a well-known antioxidant amino acid (Chitapanarux et al., 2009). However, peptides released from milk proteins during either fermentation or gastrointestinal transit could play a key role in the antioxidant effect reported in the few in vivo studies performed so far. VLPVPQK, derived from P-casein and showing antioxidant activity in vitro, was identified in the human jeuje- num following ingestion of milk proteins (Boutrou et al., 2013). The antioxidant peptide VLPVPQ was isolated in the <3 kDa fraction of bovine casein fermented for 24 hours by Bifidobacterium longum KACC91563 (Chang et al., 2013).