**3. Biological activities of milk proteins**

## **3.1 Antioxidant activities**

Overall, proteins have antioxidant activity through some amino acid residues such as cysteine, methionine and tryptophan. Indeed, these residues are involved in free radical-scavenging as they possess the highest antioxidant activity compared to the other amino acids [55]. Hence, the amino acid composition of proteins, their positioning and their accessibility are important in scavenging the free radicals [56].

Lactoferrin, representing between 1 and 2% of the total whey proteins, is characterized by its exceptional antioxidant capacity especially its ability to scavenge free radicals due to its sulfur-containing amino acids in its structure and the chelation of transition metals [57]. Native α-lactalbumin also exhibited significant antioxidant activities with respect to Ferric-reducing (FRAP), iron chelating and antiradical activities in both apo and holo forms with higher antioxidant activities for the apo form due to the greater exposure of antioxidant amino acids after calcium depletion [50]. Previous works indicated that caseins exhibited also important antioxidant activities. For instance, the β-casein samples showed significant iron chelating and antiradical activities depending on the protein concentration (0.1, 1 and 5 g/l) which could be explained by the higher content of antioxidant aminoacid residues in the β-casein protein [42].

Peptides generated from the enzymatic digestion of milk proteins are reported to have significant bioactivities such as antioxidant, antihypertensive, antidiabetic, immunomodulatory, antimicrobial, opioid properties. Indeed, peptides can be released through in vitro enzymatic hydrolysis, in vivo digestion approaches and fermentation, alone or in combination [58]. Antioxidant activities of native and hydrolyzed whey protein isolate were studied and compared to those of the major individual whey proteins (β-lactoglobulin, α-lactalbumin, serum albumin and lactoferrin) [59]. Antioxidant activities of whey proteins were significantly increased after enzymatic digestion compared with native proteins. The α-lactalbumin showed the highest FRAP (8.19 ± 1.19 μmol of Trolox equivalent/g) and ABTS free radical-scavenging activity (20.97 ± 1.44%) when compared of the other tested whey proteins with the release of the highest amount of the antioxidant peptides. These results lead to prefer the α-lactalbumin in food formulations to boost antioxidant defenses [59]. Investigations revealed that "Corolase PP", a commercial complex mixture of enzymes is the most appropriate enzyme in obtaining antioxidant hydrolysates from the pure α-lactalbumin [60]. The enzymatic hydrolysis of α-lactalbumin revealed a peptide having an IC50 inhibition value of 143 of superoxide radical-scavenging. This peptide was separated through a Sephadex G-200

column within a size-exclusion chromatography after peptic hydrolysis of whey filtrate [61, 62].
