Human milk (HM) is the first dietary exposure in infancy and the best nutritional option for growth and healthy development of the newborn and infant. The concentrations of macronutrients, namely proteins, carbohydrates and lipids, change during lactation stages. HM has been studied extensively in the first 6 months of lactation, but there are limited data regarding milk composition beyond 1 or 2 years postpartum. The aim of our study was to describe longitudinal changes in HM macronutrient concentrations during the prolonged lactation of healthy mothers from the 1st to the 48th month. For the macronutrient content of milk of mothers breastfeeding for longer than 18 months, fat and protein increased and carbohydrates decreased significantly, compared with milk expressed by women breastfeeding up to 12 months. Moreover, the concentration of fat, protein and carbohydrates in HM over 2 years of lactation from the 24th to the 48th month remained at a stable level. However, analyzed macronutrients were positively (carbohydrate, r = 0.51) or negatively (fat, r = −0.36 and protein, r = −0.58) correlated with the amount of feeding. Our results create a greater potential for understanding the nutritional contribution of HM over 2 years of lactation and showed that the source of calories in breast milk for older children is mainly fat, while carbohydrates play a greater role in infant nutrition in the early stage. The observed changes of macronutrient concentrations from the 1st to the 48th month of lactation are probably related to the adaptation of milk composition to the increased energy demand of the intensively growing child.

Mother's milk immunoglobulins (Igs) delivered to infants during breastfeeding are crucial in shaping and modulating immature infants' immune system and provide efficient protection against pathogens. The aim of the study was to evaluate the immunoglobulin concentrations in milk of 116 lactating mothers over prolonged lactation from the 1st to the 48th month using the ELISA method. The concentration of proteins, SIgA and IgG, but not IgM, showed a positive correlation (r = 0.69, p < 0.005; r = 0.54, p < 0.05; and r = 0.27, p < 0.05, respectively) with lactation from the 1st to the 48th month. The lowest concentrations of SIgA and IgG were observed for the first year (2.12 ± 0.62 g/L and 14.71 ± 6.18 mg/L, respectively) and the highest after the 2nd year of lactation (7.55 ± 7.16 g/L and 18.95 ± 6.76 mg/L, respectively). The IgM concentration remained stable during 2 years (2.81 ± 2.74 mg/L), but after 24 months it was higher (3.82 ± 3.05 mg/L), although not significantly. Moreover, negative correlations of protein (r = −0.24, p < 0.05) and SIgA (r = −0.47, p < 0.05) concentrations with the number of feedings were found. Human milk after the 2nd year of lactation contains significantly higher concentrations of protein, SIgA, and IgG. High concentration of immunoglobulins and protein during prolonged lactation is an additional argument to support breastfeeding even after introducing solid foods and should be one of the overarching goals in the protection of children's health.

Among the immunologically important bioactive factors present in human milk, lactoferrin (Lf) has emerged as a key player with wide-ranging features that directly and indirectly protect the neonate against infection caused by a variety of pathogens. The concentration of Lf in human milk is lactation-stage related; colostrum contains more than 5 g/L, which then significantly decreases to 2–3 g/L in mature milk. The milk of mothers who are breastfeeding for more than one year is of a standard value, containing macronutrients in a composition similar to that of human milk at later stages. The aim of this study was to evaluate lactoferrin concentration in prolonged lactation from the first to the 48th month postpartum. Lactating women (n = 120) up to 48 months postpartum were recruited to the study. The mean value of lactoferrin concentration was the lowest in the group of 1–12 months of lactation (3.39 ± 1.43 g/L), significantly increasing in the 13–18 months group (5.55 ± 4.00 g/L; p < 0.006), and remaining at a comparable level in the groups of 19–24 month and over 24 months (5.02 ± 2.97 and 4.90 ± 3.18 g/L, respectively). The concentration of lactoferrin in mother’s milk also showed a positive correlation with protein concentration over lactation from the first to the 48th month (r = 0.3374; p = 0.0002). Our results demonstrate the high immunology potential of human milk during prolonged lactation and that Lf concentration is close to the Lf concentration in colostrum. Evidence of stable or rising immunoprotein levels during prolonged lactation provides an argument for foregoing weaning; however, breastfeeding must be combined with solid foods meet the new requirements of a rapidly growing six-month or older baby.