Antioxidant activity of in vitro digested bovine milk

The objective of the present study was to analyze the antioxidant properties of different types of bovine milk subjected to simulated gastro-pancreatic digestion. Whole, semi-skimmed, and skimmed milk were digested in sequence with pepsin (300 U/ml; pH 2.5; 2h) then with pancreatin (0.8 g/l; pH 7.5; 2h) and bile salts (5 mg/ml). At the end of digestion, samples were subjected to ultrafiltration (3 KDa) and the high (HMW) and low (LMW) molecular weight fractions analyzed with ABTS assay. All milk types contain antioxidants with whole milk having the highest amount (622.3 ± 44.5 mg vitamin C/L). About 90% of antioxidant activity was in the HMW fraction. The degree of protein hydrolysis (DH) after digestion was different considering the different milk types (20.8 ± 0.4, 24.3 ± 0.3 and 30.7 ± 0.8 in whole, semi-skimmed and skimmed milk, respectively). Pancreatic digestion caused an increase in the antioxidant activity for all the analyzed milk (post-pancreatic ABTS value of 3374.3 ± 104.6, 2657.1 ± 39.6 and 2751.2 ± 46.9, in whole, semi-skimmed and skimmed milk, respectively). The distribution of the antioxidant activity between the LMW and HMW fractions was different considering the diverse milk types. The percentage of antioxidant activity in the LMW fraction increased to 38%, 79% and 90% in whole, semi-skimmed and skimmed milk, respectively, according to the increase in the DH. HPLC analysis of LMW fractions revealed that all the digested milk had the same peptides pattern but the peptides concentrations in skimmed milk was higher than in semi-skimmed and whole milk. On-line HPLC-ABTS analysis of LMW fractions showed that two peptides are the major contributor to the antioxidant activity of this fraction. The two peptides were identified by MS/MS. Our data suggest that digestion of milk increased the antioxidant activity, potentially reducing the oxidative stress in the gastro-intestinal tract.