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The α_(s1)-and β-casein were purified by DEAE-cellulose chromatography and digested with alkaline protease from Bacillus subtilis. Bitter fractions from the hydrolyzates were isolated using n-butanol extraction, Sephadex G-25 gel chromatography, and high performance liquid chromatography. Peptide mixtures were separated by reverse-phase octadecyl silica column with linear gradient of 0-80% acetonitrile containing 0.1% trifluoroacetic acid. Major peaks were combined from replicate chromatographies and the bitterness of each peak was evaluated. The bitter-tasting peaks were rechromatograpied until isolated peaks were obtained. Three different bitter peptides(BP- I , BP-II, BP-III) were obtained from the α_(s1)-casein hydrolyzate. BP- I was eluted at 34% acetonitrile and BP-II, 35%, BP-III, 26%, respectively. Two bitter peptides(BP-IV, BP-V) were isolated from the β-casein hydrolyzate: BP-IV was eluted at 40% acetonitrile and BP-V, 42%. BP-V was the most hydrophobic peptide in the five bitter peptides. However, BP- I and BP-II tasted more bitter than BP-IV and BP- V.
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