Characterization of bitter peptides in casein hydrolysates using comprehensive two-dimensional liquid chromatography

• A comprehensive 2D-LC method for the analysis of casein peptides was developed. • Peptides were separated according to their size and hydrophobicity. • A region of interest was defined using six standard peptides of varying bitterness. • A correlation between bitterness and 2D-LC profile of casein hydrolysates is shown. Casein hydrolysates are important additives to foods for elderly and sports nutrition. However, due to the enzymatic generation of so-called bitter peptides, their application is limited. Therefore, the procedure needs to be optimized in order to restrict their occurrence. For this, extensive sensory evaluations are necessary. By combining two separation techniques using comprehensive two-dimensional liquid chromatography, we present a novel method for estimating the bitter taste of hydrolysate samples on the basis of their elution pattern. Using a size exclusion column in the first and a reversed phase column in the second dimension allows for a detailed sample evaluation regarding peptide size and relative hydrophobicity. The results obtained for different casein hydrolysates were correlated with the sensory evaluation. We found that hydrolysates with increasing bitterness contain a higher amount of peptides of high hydrophobicity and a molecular weight less than 6.5 kDa.