Human milk (HM) is the ideal food for newborns. In some cases, when a mother's milk isn't available, donated HM (DHM) is the next best option, especially for at-risk babies (such as severely premature babies or those with enterocolitis in hospitals). A major contributor to the health benefits of HM is the protein fraction, which is divided into whey, casein, peptides, and other conjugates, including those found in the milk fat globule membrane (MFGM). To date, around 1800 proteins in HM have been described, and the peptidome consists of the product of protein's breakdown (mostly ĸ- and β-casein) by proteases present within the mammary gland (Dekker et al., 2022; Nielsen et al., 2017; Sari et al., 2021)
DHM is treated by Holder pasteurisation (HoP) by milk banks to ensure microbiological safety, but this may lead to the loss of essential nutrients and health-promoting properties, especially highly thermolabile components, such as proteins and peptides. As a result, babies fed DHM generally suffer poorer health outcomes than those provided with their mother's HM. Although large efforts have been made to describe the impact of pasteurization on the most abundant proteins in HM, only recently have more studies described the so-called “proteome” composition (made of the pool of molecules present in the protein fraction of HM) and the effect of HoP on its physiological properties (Escuder-Vieco et al., 2021; Zhang et al., 2023).
In this study, the protein and peptide complement of DHM (both raw and HoP-treated) was compared. MALDI-TOF mass spectrometry and analysis of proteome and peptidome spectra with ClinProTools (Bruker Daltonics) allowed comparisons of protein and peptide profiles. Overall protein and peptide concentration as measured with BCA assay, appeared to decrease following pasteurisation. Principal component analysis (PCA) and unsupervised clustering analysis (UCA) showed that HoP impacted the proteome profile (MW 5-20 kDa) of samples less than individual variations between mothers. In contrast, the peptidome profile (MW 0.7-2.0 kDa) was impacted to a greater extent, with changes in some peaks further indicated by virtual 2D-gels built from the spectra. These results suggest that HoP affects the DHM proteome and peptidome composition. To the best of our knowledge, this is the first report on the impact of HoP on the HM peptidome. Future studies will use multiple analytical platforms, such as liquid chromatography, to broaden the coverage and improve quantitative analysis of the various HM protein and peptide fractions.