The Impact of Holder Pasteurisation on the Human Milk Proteome and Its Functionality Inferred by Pathway Analysis

Donated human milk (DHM) obtained from milk banks is the preferred feeding alternative when a mother’s milk is unavailable for premature or otherwise medically compromised babies. In order to ensure its microbiological safety, milk banks pasteurise DHM by Holder Pasteurisation (HoP) at 62.5 °C for 30 minutes. In this study, the effect of HoP-treatment on the proteome of HM was analysed by Fourier-Transform Infrared (FTIR) Spectroscopy, MALDI-TOF MS and nanoscale liquid chromatography with electrospray ionisation coupled to tandem mass spectrometry (nanoLC-ESI-MS/MS). FTIR analysis showed a reduction in amide-II and an increase in amide-III functional groups, suggesting protein conformational changes, and further revealed changes in protein-lipid wavebands. Proteome analysis via MALDI-TOF MS indicated changes in peptide profiles had occurred for HoP-treated samples. A more detailed analysis by nanoLC-ESI-MS/MS identified 2162 proteins, of which 2015 were shared between raw (untreated) and HoP-treated samples. Although Principal Component Analysis (PCA) and hierarchical clustering showed no clear distinctions, pathway enrichment analysis showed that platelet activation and energy metabolism pathways were enriched in HoP-treated HM, whereas non-pasteurised (raw) HM contained a higher proportion of proteins associated with protein synthesis, cellular signalling and immune responses, including production of IL-12. These findings suggest that HoP affects pathways associated with the immunomodulatory properties of HM; however, since these changes to HM functional properties were inferred from pathway analyses, further direct experimental studies are needed to verify these potential effects.