Functionality of the gastrointestinal tract is essential for growth and development of newborns. Preterm infants have an immature gastrointestinal tract, which is a major challenge in neonatal care. This study aims to improve the understanding of gastrointestinal functionality and maturation during the early life of preterm infants by means of gastrointestinal enzyme activity assays and metaproteomics. In this single-center, observational study, preterm infants born between 24 and 33 weeks (n = 40) and term infants born between 37 and 42 weeks (n = 3), who were admitted to Isala (Zwolle, the Netherlands), were studied. Enzyme activity analyses identified active proteases in gastric aspirates of preterm infants. Metaproteomics revealed human milk, digestive and immunological proteins in gastric aspirates of preterm infants and feces of preterm and term infants. The fecal proteome of preterm infants was deprived of gastrointestinal barrier-related proteins during the first six postnatal weeks compared to term infants. In preterm infants, bacterial oxidative stress proteins were increased compared to term infants and higher birth weight correlated to higher relative abundance of bifidobacterial proteins in postnatal week 3 to 6. Our findings indicate that gastrointestinal and beneficial microbial proteins involved in gastrointestinal maturity are associated with gestational and postnatal age.

Background & Aims The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. Methods Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. Results We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. Conclusions Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning.

Donor human milk is generally processed by holder pasteurization (HoP) at 62. 5°C for 30 min. This temperature-time combination is sufficient for eliminating pathogens in donor milk, but also negatively affects several bioactive milk components. Long heating up times may further affect the bioactive properties of pasteurized milk. High-Temperature-Short-Time (HTST), a treatment with shorter processing times (72°C for 15 sec), was investigated as a suitable alternative to HoP. In addition, pasteurization methods that follow the same temperature regime but with varying heating up times were compared. Human milk samples from four different donors were combined into one pool, which was then used to perform all analyses. The effects of these methods on the levels and functionality of immunoglobulin A, lactoferrin, lysozyme and bile salt-stimulated lipase, were evaluated with LC-MS/MS-based proteomics and activity assays, while the pasteurization efficacy was evaluated with an alkaline phosphatase test. HoP, a treatment with long processing times, times, caused the highest reduction in all proteins studied (reduced by 50–98%). Compounds such as lactoferrin and bile salt-stimulated lipase that are more sensitive to heat treatments were better retained with HTST, but their levels and functionality were still significantly lower than those of untreated donor milk (52 and 81% reduction of lactoferrin and bile salt-stimulated lipase activity, respectively). Our findings showed that a treatment with considerably shorter processing times, such as HTST, may reduce the thermal damage caused to the bioactive proteins compared to HoP, without affecting pasteurization efficacy. Since the vast majority of the donor human milk banks that are currently operating on a global level apply HoP to donor milk, our findings may provide relevant information for the optimization of donor milk processing.