Milk Peptides Survive In Vivo Gastrointestinal Digestion and Are Excreted in the Stool of Infants

Abstract

Background: Human milk peptides released by gastrointestinal proteases have been identified with bioactivities that can benefit the infant but must first reach their respective sites of activity. Peptides in the stool either survived to or were released inside the intestinal tract, and thus had the opportunity to exert bioactivity there. However, it is unknown whether any milk peptides, bioactive or not, can survive in the stool of infants.

Objective: The aim of this study was primarily to identify milk peptides in infant stool samples and secondarily test the hypotheses that the milk peptide profiles of stools are different between preterm infants at different days of life and between preterm and term infants.

Methods: Infant stool samples were collected from 16 preterm infants (<34 weeks gestational age) at 8 or 9 and 21 or 22 days of life (DOL), and from 10 term infants (>34 weeks gestational age) at 8 or 9 DOL. Milk peptides were isolated from the stool samples and identified using tandem MS. The peptide counts and abundances were compared between infant groups.

Results: In total, 118 exclusively milk-derived peptides from the caseins and α-lactalbumin were present in the stool samples, including some peptides with known or potential bioactivity. The remaining 8014 identified peptides could be derived either from milk or endogenous proteins. Although many individual milk peptides were significantly different between preterm infants at 8/9 and 21/22 DOL and between preterm and term infants, total peptide abundance and count were similar for all 3 groups.

Conclusions: This is the first study to confirm the survival of milk peptides in the stool of infants. Some of the peptides had potential bioactivities that could influence infant gut development. These results are important to understand the physiological relevance of human milk peptides to the infant.

Keywords: bioactive; human milk; infant; peptides; stool.

FIGURE 1

Pie chart of the breakdown of milk peptides identified in the infant stool samples. Numbers underneath the pie section labels represent the number of unique peptides identified from all infant stool samples, n = 33. BSSL, bile salt-stimulated lipase; PIgR, polymeric Ig receptor; XDH, xanthine dehydrogenase.

FIGURE 2

Boxplots of the abundance (A) and count (B) of total potential milk peptides from the stool of preterm infants at 8/9 DOL and 21/22 DOL and term infants at 8/9 DOL. Preterm 8/9 n = 12, preterm 21/22 n = 11, and term n = 10. DOL, days of life.

FIGURE 3

Stacked column charts of the abundance (A) and count (B) of peptides from individual milk proteins and protein groups. Stacking is based on the percentage of infants in each group that the peptides were identified in. Data are represented as means, n = 33. BSSL, bile salt-stimulated lipase; PIgR, polymeric Ig receptor; XDH, xanthine dehydrogenase.

FIGURE 4

Volcano plots of total peptide abundance for preterm 8/9 and preterm 21/22 (A) and preterm 8/9 and term 8/9 (B), lactoferrin peptide abundance for preterm 8/9 and preterm 21/22 (C), and preterm 8/9 and term 8/9 (D), and combined α-lactalbumin and casein peptide abundance for preterm 8/9 and preterm 21/22 (E), and preterm 8/9 and term 8/9 (F). Filled dots represent peptides that were both significantly different (P < 0.05) and present in 1 of the groups at 5-fold increased abundance. For lactoferrin and α-lactalbumin/casein, white dots with black borders represent peptides with ≥80% sequence homology to a known bioactive peptide from the MBPDB. Light gray arrows represent the number of peptides increased in the numerator, and dark gray arrows represent the number of peptides increased in the denominator. Preterm 8/9 n = 12, preterm 21/22 n = 11, and term n = 10. MBPDB, milk bioactive peptide database.

FIGURE 5

Variation in the peptide profiles of each infant. (A) Heatmap of the fold-change in peptide abundance from a specific protein or group of proteins for each infant compared with the mean. (B) Percent contribution of each protein or group of proteins to the total peptide profile of each infant. BSSL, bile salt-stimulated lipase; PIgR, polymeric Ig receptor; XDH, xanthine dehydrogenase.