Human milk is the most complete and ideal form of nutrition for the developing infant. The composition of human milk consistently changes throughout lactation to meet the changing functional needs of the infant. The human milk proteome is an essential milk component consisting of proteins, including enzymes/proteases, glycoproteins, and endogenous peptides. These compounds may contribute to the healthy development in a synergistic way by affecting growth, maturation of the immune system, from innate to adaptive immunity, and the gut. A comprehensive overview of the human milk proteome, covering all of its components, is lacking, even though numerous analyses of human milk proteins have been reported. Such data could substantially aid in our understanding of the functionality of each constituent of the proteome. This review will highlight each of the aforementioned components of human milk and emphasize the functionality of the proteome throughout lactation, including nutrient delivery and enhanced bioavailability of nutrients for growth, cognitive development, immune defense, and gut maturation.

Background: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. Methods: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). Results: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. Conclusions: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Variations in endogenous peptide profiles, functionality, and the enzymes responsible for the formation of these peptides in human milk are understudied. Additionally, there is a lack of knowledge regarding peptides in donor human milk, which is used to feed preterm infants when mother's own milk is not (sufficiently) available. To assess this, 29 human milk samples from the Dutch Human Milk Bank were analyzed as three groups, preterm late lactation stage (LS) (n = 12), term early (n = 8) and term late LS (n = 9). Gestational age (GA) groups were defined as preterm (24–36 weeks) and term (≥37 weeks). LS was determined as days postpartum as early (16–36 days) or late (55–88 days). Peptides, analyzed by LC-MS/MS, and parent proteins (proteins from matched peptide sequences) were identified and quantified, after which peptide functionality and the enzymes responsible for protein cleavage were determined. A total of 16 different parent proteins were identified from human milk, with no differences by GA or LS. We identified 1104 endogenous peptides, of which, the majority were from the parent proteins β-casein, polymeric immunoglobulin receptor, α_s1-casein, osteopontin, and κ-casein. The absolute number of peptides differed by GA and LS with 30 and 41 differing sequences respectively (p < 0.05) Odds likelihood tests determined that 32 peptides had a predicted bioactive functionality, with no significant differences between groups. Enzyme prediction analysis showed that plasmin/trypsin enzymes most likely cleaved the identified human milk peptides. These results explain some of the variation in endogenous peptides in human milk, leading to future targets that may be studied for functionality.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.

Maternal supplementation with 1000 mg/day docosahexaenoic acid (DHA) provides third trimester DHA accretion levels in breast milk for the preterm infant. We hypothesized that DHA supplementation to mothers providing breastmilk for extremely preterm infants would result in decreased inflammatory markers, in the infant. Mother/infant dyads (n = 27) were enrolled at birth and mothers were assigned to receive 200 or 1000 mg/day of DHA. Milk and plasma samples were analyzed for fatty acids and inflammatory markers. Decreases in inflammation were observed in both maternal and infant plasma and correlated with red blood cell (RBC) DHA levels. The fact that maternal DHA supplementation decreases infant markers of inflammation implies that DHA, delivered through breastmilk, has the potential to decrease inflammation in the infant.

Background: Since the outbreak of COVID-19, many put their hopes in the rapid development of effective immunizations. For now patient isolation, physical distancing and good hygiene are the sole measures for prevention. Processed breast milk with antibodies against SaRS-CoV-2 may serve as additional protection. We aimed to determine the presence and neutralization capacity of antibodies against SaRS-CoV-2 in breastmilk of mothers who have recovered from COVID-19. Methods: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Serum and breastmilk was collected. To assess the presence of antibodies in breastmilk and serum, we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein, SARS-CoV-2 receptor binding domain (RBD) and with the SARS-CoV-2 nucleocapsid (N) protein for IgG and bridging ELISA with the SARS-CoV-2 RBD and N protein for total Ig. To assess the effect of pasteurization breastmilk was exposed to Holder Pasteurization and High Pressure Pasteurization. Results: Breastmilk contained antibodies against SARS-CoV-2 using any of the assays in 24 out of 29 (83%) proven cases, in six out of nine (67%) suspected cases and in none of the 13 controls. In vitro neutralization of SARS-CoV-2 clinical isolate virus strain was successful in a subset of serum (13%) and milk samples (26%). Although after pasteurization of the milk SARS-CoV-2 antibodies were detected with both methods of pasteurization, virus neutralizing capacity of those antibodies was only retained with the HPP approach. Conclusion: Breastmilk of mothers who recovered from COVID-19 contains significant amounts of IgA against SARS-CoV-2, both before and after pasteurization. Key Points: Question: Does breastmilk of mothers who have recovered from coronavirus disease 2019 (COVID-19) contain antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)? Findings: We provide multiple lines of evidence on the presence of a variety of antibodies against SARS-CoV-2, with no such antibodies present in the controls. These antibodies are capable of neutralizing a clinical isolate of SARS-CoV-2 in vitro . We furthermore show that high pressure pasteurization hardly affects antibody levels and efficacy. Meaning: Breastmilk, obtained from mothers who have recovered from COVID-19, may serve as a safe and widely applicable preventive strategy for vulnerable high risk populations