Raman spectra of amino acids showed complexity in the C-H stretching region (2800-3100 cm(-)(1)) attributed to diversity of CH, CH(2), and CH(3) groups in the side chains, ionization state, and microenvironment. The involvement of specific amino acids in the C-H stretching region of selected proteins, namely, lysozyme, alpha-lactalbumin, beta-lactoglobulin, and their binary mixtures, was investigated by deconvolution using maximum likelihood techniques. The main protein band near 2940 cm(-)(1) was attributed not only to aromatic and aliphatic amino acids but also to many other amino acids. A band near 3065 cm(-)(1) was assigned to aromatic residues, whereas bands near 2880 and 2900 cm(-)(1) corresponded primarily to aliphatic amino acids. Heating at 90 degrees C increased relative intensity near 2940 cm(-)(1) and decreased relative intensity at 2895-2902 cm(-)(1) for lysozyme and its mixtures with alpha-lactalbumin or beta-lactoglobulin. Additional bands at 2812 or 2838 and 3003 cm(-)(1) were observed after heating or in 8 M deuterated urea, reflecting changes upon denaturation.

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 liposomeoxidising system (50 µM FeCl 3 /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 lowermolecular-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.

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