- Premature birth interrupts the typical development of the human fetus, leaving the infant born with underdeveloped gastrointestinal and immune systems. Preterm infants have reduced stomach acidity, reduced digestive protease activity, more permeable intestinal membranes, impaired innate and adaptive immune response, and poor microbiome development. Due to these factors, preterm infants are at increased risk for developing a variety of infections and complications, such as necrotizing enterocolitis, sepsis, retinopathy of prematurity, and respiratory distress syndrome. Furthermore, preterm infants are born at an earlier stage of gestation at which they would still be reliant upon the maternal placenta for nutrient delivery and thus are less well equipped to handle the introduction of large quantities of nutrients directly into their gastrointestinal system. The preterm infant’s inability to efficiently digest the nutritious components of milk, such as protein, may contribute to their increased risk for developing infectious diseases, as they are less able to release bioactive factors that contribute to their gastrointestinal development. Human milk proteins are cleaved into thousands of peptides as they progress through the gastrointestinal system, many of which have been identified with bioactivities beneficial to the infant, such as antimicrobial, bifidogenic, immunomodulatory, and antioxidant activities. For these peptides to be active in the infant, they must first be released from their parent protein and then survive additional digestion before reaching their site of activity, often in the intestinal tract or bloodstream. However, if and where along the infant gastrointestinal tract milk bioactive peptides are released is currently unknown. Furthermore, it is unknown how preterm infants may differ from term infants in their ability to release these peptides. The central hypothesis of this research is that preterm infants, due to their impaired gastrointestinal function, will have reduced bioactive peptide release throughout the gastrointestinal system compared with term infants. To test this hypothesis, I performed a series of peptidomic experiments identifying and categorizing the human milk peptides at various stages of infant digestion.
The results of the first experiment identifying human milk and bovine milk fortifier peptides in the human milk and the preterm infant over a period of three hours are presented in Chapter 2. Milk peptide release increased significantly from milk to the stomach and in the stomach over time. The rate of bioactive and potentially bioactive peptide release increased in a similar manner as total peptides. These results contribute to the understanding of how milk proteins are digested and peptides are released in the infant stomach. They also establish the initial release of bioactive peptides as milk is fed to the infant.
The results of the second experiment identifying human milk peptides in preterm and term infant stool are presented in Chapter 3. Whereas the gastric contents represent the beginning of digestion and release of peptides, the stool represents the terminus and everything that has survived digestion. Over one hundred milk peptides were present in the stool, and thousands more that could derive from proteins known to be in human milk. One bioactive milk peptide was present, and thus may have had the opportunity to be active in the intestinal tract. The stool peptide profiles of preterm and term infants were distinct from each other, with several peptides significantly higher in abundance in one group over the other. This study was the first to determine the capacity for bioactive human milk peptides to survive the entirety of gastrointestinal digestion.
Human milk peptide release was compared from milk, to the stomach, to stool and between term and preterm infants at each site in Chapter 4. Though peptide release was equivalent between infant groups in the milk and stool, peptide abundance was significantly higher in the preterm infants than the term infants. However, term infants had higher abundance of specific milk peptides with antimicrobial activity in the stomach. There was a significant increase in peptide count abundance from milk to stomach, then a decrease from stomach to stool for all infants. These results are the first to compare peptide release after gastrointestinal digestion between preterm and term infants. They suggest that term infants are able to cleave off specific bioactive peptides from the C-terminus of β-casein at higher rates than preterm infants.
Finally, the release of peptides in the infant intestinal tract and the identification of novel antimicrobial peptides are presented in Chapter 5. The bulk peptide extracts of the duodenal/jejunal fluid of preterm and term infants were incubated with Staphylococcus aureus and Escherichia coli to identify antimicrobial peptides, and Bifidobacterium infantis to identify bifidogenic peptides. Several infants had bulk peptide extracts that were highly antimicrobial or bifidogenic. From comparing these extracts with those that had no activity, seven novel human milk antimicrobial peptides were identified. These results are the first to profile milk peptide release in the intestinal tract and confirm that bioactive peptides are present and potentially active therein.
The summation of these studies is the most detailed map of human milk peptide release across the infant gastrointestinal system to date. Until this point, peptides had only been found in milk and the infant stomach from preterm infants or after in vitro digestions of milk. The relevance of the bioactive peptides identified in the literature so far to infant health has not been entirely understood, in part because it was unknown whether the infant was able to release those specific peptides. Peptides in the milk and stomach likely undergo further proteolysis before they reach the intestinal tract where they can act locally or be absorbed in the bloodstream, and peptides from in vitro digests may not accurately represent those in in the infant gastrointestinal system, particularly for preterm infants whose systems are less developed and understood. The central hypothesis that preterm infants release fewer bioactive peptides throughout the gastrointestinal system than term infants could only be partly answered, as intestinal samples from only two term infants were able to be collected. In the stomach, it was shown that term infants release one known bioactive peptide at higher abundances than preterm infants and several more potentially bioactive peptides. In the stool, the only identified bioactive peptide was present at similar abundance for both preterm and term infants, though they differed in their total peptide profiles. These studies combine to show that a simple answer for whether term infants release more bioactive peptides than preterm infants may not exist, as peptide release is complicated by stage of digestion, day of life of the infant, starting milk protein/peptide profile, and potentially many other factors.