Fractionation and characterisation for antioxidant activity of hydrolysed whey protein

Whey protein isolate (WPI) was hydrolysed for 1 h using Alcalase, Protamex and Flavourzyme. Native WPI, hydrolysed WPI and two commercial WPI hydrolysates were subjected to fractionation by size exclusion chromatography. Antioxidant activity of WPI fractions was measured with a liposome-oxidising system (50 μM FeCl₃/0.1 μM ascorbate, pH 7.0). Lipid oxidation was measured as thiobarbituric acid-reactive substances (TBARS). Gel electrophoresis and amino acid analysis were run to identify the peptide composition. The influence of amino acid composition on antioxidant activity was evaluated using multivariate analysis methods (correlation analysis, principal component analysis, multiple linear regression and discriminant analysis). TBARS assays indicated the presence of antioxidant activity in all protein fractions, including non-hydrolysed WPI. For native and hydrolysed WPI samples the first fraction (>45 kDa) showed a higher TBARS inhibition effect (24-27%) when compared with lower-molecular-weight fractions and hydrolysate mixtures. In contrast, for commercial WPI hydrolysates a higher inhibitory effect was found in most of the lower-molecular-weight fractions (30-55%). The ability of WPI fractions to delay lipid oxidation was found to be related to the prevalence of histidine and hydrophobic amino acids.