The aim of this study was to investigate factors affecting total, true protein and lactoferrin (Lf) concentrations in human milk (HM) and to evaluate the changes in protein concentrations over the course of lactation (first to sixth month postpartum). HM samples were collected from exclusively breastfeeding mothers during six time periods (1-6 months postpartum); 198 breast milk samples were collected in total. The concentrations of total and true protein in HM were determined using the MIRIS human milk analyzer (HMA). The assessment of HM protein content was also performed in skim HM samples and quantified by bicinchoninic methods with the Bicinchoninic Acid Protein Assay Kit. In turn, Lf content in skim HM samples was determined by the enzyme-linked immunosorbent assay (ELISA) in accordance with a slightly modified procedure. In the first month of lactation total protein concentration was negatively correlated with maternal pre-pregnancy BMI (r = - 0.397; p = 0.022), whereas in the third month postpartum, positive correlation with maternal age was found (r = 0.399; p = 0.021). Considering Lf concentration, in the first month of lactation, it was positively correlated with baby's birth weight (r = 0.514; p = 0.002). In the next months (from second to sixth) no relationships between Lf concentration and maternal and infants' factors were observed. The concentration of protein and Lf in HM changes dynamically throughout lactation. Maternal and infant characteristics may impact the HM protein and Lf content, especially in the first month postpartum.
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