Release of functional peptides from mother's milk and fortifier proteins in the premature infant stomach

RESEARCH ARTICLE Release of functional peptides from mother’s milk and fortifier proteins in the premature infant stomach Søren D. Nielsen ID1, Robert L. Beverly1, Mark A. Underwood2, David C. Dallas ID1* 1 Nutrition Program, School of Biological and Population Health Sciences, College of Public Health and Human Sciences, Oregon State University, Corvallis, OR, United States of America, 2 Department of Pediatrics, University of California, Davis, Sacramento, CA, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Nielsen SD, Beverly RL, Underwood MA, Dallas DC (2018) Release of functional peptides from mother’s milk and fortifier proteins in the premature infant stomach. PLoS ONE 13(11): e0208204. https://doi.org/10.1371/journal. pone.0208204 Editor: Joseph J. Barchi, National Cancer Institute at Frederick, UNITED STATES Received: July 21, 2018 Accepted: November 13, 2018 Published: November 29, 2018 Copyright: © 2018 Nielsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * Abstract Digestion of milk proteins in the premature infant stomach releases functional peptides; however, which peptides are present has not been reported. Premature infants are often fed a combination of human milk and bovine milk fortifiers, but the variety of functional peptides released from both human and bovine milk proteins remains uncharacterized. This study applied peptidomics to investigate the peptides released in gastric digestion of mother’s milk proteins and supplemental bovine milk proteins in premature infants. Peptides were assessed for homology against a database of known functional peptides—Milk Bioactive Peptide Database. The peptidomic data were analyzed to interpret which proteases most likely released them from the parent protein. We identified 5,264 unique peptides from bovine and human milk proteins within human milk, fortifier or infant gastric samples. Plasmin was predicted to be the most active protease in milk, while pepsin or cathepsin D were predicted to be most active in the stomach. Alignment of the peptide distribution showed a different digestion pattern between human and bovine proteins. The number of peptides with high homology to known functional peptides (antimicrobial, angiotensin-converting enzyme-inhibitory, antioxidant, immunomodulatory, etc.) increased from milk to the premature infant stomach and was greater from bovine milk proteins than human milk proteins. The differential release of bioactive peptides from human and bovine milk proteins may impact overall health outcomes in premature infants. Data Availability Statement: The data have been deposited to the jPOST repository (ID: PXD010502). Introduction Funding: This study was supported by the K99/ R00 Pathway to Independence Career Award, Eunice Kennedy Shriver Institute of Child Health & Development of the National Institutes of Health (R00HD079561) (DCD), the USDA National Institute of Food and Agriculture and The Gerber Foundation. The funders had no role in study Human milk composition is evolutionarily optimized to provide essential nourishment for the term infant [1]. Human milk proteins provide a balanced source of amino acids that are essential for the infant’s rapid growth. However, milk proteins provide more than the ideal amino acids for infants. In vitro studies suggest that peptides encrypted within parent milk proteins possess a variety of bioactive functions, including antimicrobial [2], angiotensin-converting enzyme (ACE) inhibition [3], immunomodulation [4, 5], antioxidant [6], opioid [7] and PLOS ONE | https://doi.org/10.1371/journal.pone.0208204 November 29, 2018 1 / 18 Digestion of mother’s milk and fortifier proteins in the infant stomach design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. calcium delivery [8]. Many of these bioactive peptides are released from milk proteins during digestion within the mammary gland by native milk proteases and by proteases within the infant gut. Our previous work demonstrated that mother’s milk contains a coordinated array of proteases and antiproteases that together release specific peptides from milk proteins within the mammary gland [9–13], and that in term infants, both milk proteases and infant digestive proteases release functional peptides within the stomach [14–16]. These encrypted peptides may be functional units with biological effects within the infant that evolved to be released based on the specificity of proteases in the normal term mother’s milk and the term infant’s gut. During early years of the evolutionary timescale, infants born prematurely (< 37 weeks gestational age) were unlikely to have exerted much selective pressure on milk composition and structure, as they rarely survived. Premature infants have a lower protein digestion capacity compared with term infants due to their lower gastric pepsin and intestinal protease activity [17, 18]. Therefore, the peptides released from milk proteins during premature infant digestion may be different from those released in term infants, which could impact the functional contribution of the peptides that affect infant health and development. Premature infants are typically not provided a single source of protein. Human milk is preferred over bovine milk-based formulas due to its positive health outcome associations, including reduced risk of necrotizing enterocolitis (NEC) [19, 20] and sepsis [21], improved cognitive skills [22] and decreased time to hospital discharge [23]. However, the energy and protein content of human milk alone do not ensure optimal growth in preterm infants. Therefore, human milk fed to preterm infants is typically fortified to meet their protein needs, which range from 2.5 to 4 g protein/kg body weight/day depending on gestational age at birth and day of life [24]. Human milk fortifiers (HMFs) can derive from either human or bovine milk [25, 26]. While limited evidence suggests that human milk-based fortifiers may reduce risk of NEC [27], bovine fortifiers are commonly used due to their lower cost and higher availability [28]. The different amino acid sequences of bovine proteins may lead to differential degradation in the infant. The processing—particularly the heat treatments used to ensure sterility—of milk proteins to prepare fortifiers can change protein structure, which can alter the susceptibility of the protein to proteolysis [29, 30] and, hence, release of bioactive peptides. A few studies that investigated the digestion of human milk proteins and bovine fortifier proteins using in vitro and rhesus macaque models found similar rates of digestion based on gel electrophor (...truncated)