PROJECT SUMMARY Human milk, whether maternal or pasteurized donor human milk, is considered the ideal nutrition for preterm infants. In addition to serving as a source of amino acids to support infant growth, human milk proteins encode cryptic bioactive peptides released during infant digestion. These peptides can be found in the infant intestine luminal fluid and have an array of biological activities, including antimicrobial, bifidogenic, immunomodulatory, and effects on intestinal physiology. Although peptides responsible peptides our initial work demonstrates that milk released and present in the infant intestine have beneficial activities, identifying the specific peptides for these gut-related actions has been limited. Thus, there is a critical need to determine the specific released within the infant's gastrointestinal tract that mediate these bioactivities. Our long-term goal is to leverage the biology of human milk to promote optimal development and growth of preterm infants ex utero. The overall objective of this proposal is to demonstrate the presence and identities of gut health-promoting human milk protein-derived peptides within the preterm infant intestine. Our central hypothesis is that bioactive peptides are released in the infant intestine to modulate gut physiology through the microbiome, intestinal epithelium, and immune system. Our specific aims are to determine the 1) antimicrobial and bifidogenic activity, 2) intestinal epithelial cell (IEC)-specific effects (barrier function, proliferation, differentiation) and 3) macrophage-immunomodulatory activity of peptides in the intestinal contents of preterm infants fed human milk. For each of these activities, we will identify and validate specific peptides. Our approach will be to collect intestinal samples from preterm neonates in the neonatal intensive care unit (NICU) after feeding human milk, extract the peptide component from these samples, test their antibacterial, bifidogenic, IEC-modulatory and macrophage-immunomodulatory activity in vitro, and identify candidate bioactive peptides via mass spectrometry, database searching, and statistical techniques to be synthesized and tested for function. As a sub-aim, we will perform this analysis for maternal and pasteurized donor human milk. This research is innovative because it identifies, for the first time, bioactivities of peptides released during in vivo infant digestion, provides a means for identifying candidate bioactive peptides and establishes a pipeline for testing candidate peptides in relevant model systems, including neonatal human enteroids. At the conclusion of this project, we expect to 1) identify the gut health-related bioactivity of human milk protein-derived peptide extracts from the preterm infant intestine and 2) identify specific peptides present in the infant intestine that can modulate the microbiome, IECs, and macrophages. The positive impact of this research is that it will provide the basis for leve...