The unique composition of human milk and its positive short- and long-term effects on health have been subject of scientific interest and extensive research over decades. In order to expand knowledge about proteins in human breast milk, a rapid and reliable RP-HPLC method was developed for separation and quantitation of individual caseins and whey proteins (i.e., β-casein, κ-casein, α-lactalbumin, lactoferrin, lysozyme and human serum albumin) in human milk samples. Furthermore, the same analytical system could be applied to separate bovine milk proteins (including their most common genetic variants). This HPLC method was validated for the analysis of target analytes regarding linearity, instrument precision, limits of detection and quantification, repeatability precision, and accuracy of results. Besides HPLC analysis, also the Kjeldahl method was applied for all 52 human breast milk samples. Furthermore, protein patterns of human milk samples obtained by SDS-PAGE were compared to reference samples from other animal species (i.e., cow, sheep, goat, mare). This study clearly showed that especially the results of the relative proportion of whey proteins in human milk, analyzed using either by Kjeldahl or HPLC, demonstrated the obvious methodological advantage of the HPLC method. In heat-treated milk samples, the Kjeldahl method falsely indicated elevated casein values and falsely decreased whey protein levels, whereas HPLC analysis did not show any difference between raw and pasteurized human milk samples (since the reducing sample buffer solution completely dissolved caseins and also denatured whey proteins). SDS-PAGE proved to be a rapid/reliable protein fingerprinting tool due to its straightforward sample preparation. • Same analytical HPLC-system for separation of human milk and bovine milk proteins • Accurate quantitation of individual caseins and whey proteins in human milk by HPLC • No difference between HPLC results of raw and heated milk (in contrast to Kjeldahl ) • Falsely elevated casein (decreased whey protein) in pasteurized milk using Kjeldahl • Wide range of protein concentrations in individual human breast milk samples (n=52)
Oever et al. (Sun,) studied this question.