Premature vs. term infant milk protein digestome project

Each year, over half a million infants are born prematurely in the United States. Although most of these infants now survive due to the immense advances in medicine, they are at high risk for a variety of conditions, including necrotizing enterocolitis, learning and behavioral problems, metabolic bone syndrome, chronic lung disease, and cerebral palsy. Preterm infants require more protein per kilogram of body weight than normal term infants to ensure proper growth and development. Traditionally, milk protein was considered a simple source of amino acids for protein synthesis in order to meet that need, but there are hundreds of unique proteins found in milk. Many of these milk proteins, despite much research, have not been ascribed functions.

We hypothesized that many milk proteins serve as a source for encrypted bioactive peptides that are only released during infant digestion. In order to explore the digestion of human milk within the infant, we first had to assess milk for any peptides. Drs. David Dallas, Andres Guerrero and Carlito Lebrilla collaborated to develop a state-of-the-art analytical mass spectrometry and data analysis platform to identify the released peptides. With this technology, we’ve now discovered thousands of naturally-occurring peptides in human milk. Our enzymatic analyses, performed in collaboration with Dr. Nora Khaldi and Evan Parker, are showing that milk proteins are partially pre-digested within the mammary gland by specific milk enzymes. Our bioinformatic comparisons are showing that many of these peptides could have antimicrobial or immunomodulatory functions. Working with Dr. Chuck Bevins, we discovered that the pool of human milk peptides inhibited the growth of Escherichia coli and Staphylococcus aureus.

Next, we worked to analyze what happens to milk proteins and peptides in the infant stomach. Dr. Mark Underwood, head of the Neonatal Intensive Care Unit in the UC Davis Medical School, provides samples from inside the stomach of human term and preterm infants. Dr. Mark Underwood realized that samples could be obtained from infants that were fed via oro- or naso gastric tubing, and provided the sample collection needed for the research. Previously, it was thought that little digestion happened in newborn babies’ stomachs because of the high post-feeding pH, which normally would inhibit the action of the main gastric protease, pepsin. We analyzed gastric extracts from infants who had been fed human milk. Hundreds of novel peptides are released in the infant stomach, and bioinformatic enzyme analysis has revealed that pepsin is active, despite the high pH. Perhaps more interestingly, this analysis revealed that the mother’s milk proteases are active in the infant stomach and are responsible for a majority of digestion there! This shows that the mother continues to control digestion even within her infant’s stomach.

Now, we are working to analyze the peptides produced in digestion in term and preterm infants. Using mass spectrometry (ESI-Orbitrap MS/MS), naturally occurring peptides from mothers’ milk will be compared to those present in the infant stomach, urine and stool to identify patterns of proteolysis. The identified peptides can then be annotated for function, and differences between the term and preterm groups will highlight deficiencies in the digestive ability of preterm infants. This data will then provide a target for future work aiming to compensate for the specific nutritional needs of preterm infants.