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

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.

Fig 1. Total number of peptides identified in human milk and infant gastric.

Total count (A) and abundance (B) of peptides identified in unfortified human milk and preterm infant gastric digests of fortified human milk from either bovine milk proteins, human milk proteins or either of the two. The graph is divided into sections of different shades of grey (from black to light grey) for the number of infants the peptides were identified in. Black represents peptides only identified in one infant and the lightest shade of grey representing peptide found in all 5 infants. Results are shown as mean ± standard error.

Fig 2. Relative number of peptides identified from proteins in human milk and infant gastric.

Relative count (A) and relative ion intensity (B) of peptides identified in human milk and preterm infants gastric from either bovine milk proteins (bovine), human milk proteins (human), sorted according to the total peptide release among all samples. Results are shown as mean ± standard error. CASB, β-casein; OSTP, osteopontin; CASA1, αs1-casein; PIGR, polymeric immunoglobulin receptor; CEL, bile salt-activated lipase; BT1A1, butyrophilin subfamily 1 member A1; TRFL, lactoferrin; CASK, κ-casein; XDH, xanthine dehydrogenase/oxidase; LALBA, α-lactalbumin.

Fig 3. Mapping of identified peptide on milk proteins.

Total ion intensity of peptides identified in infant gastric samples from bovine and human milk β-casein (A), α_s1-casein (B) and osteopontin (C). The ion intensity of each peptide also present in the human milk samples or HMF were subtracted from the gastric values prior to mapping to the sequence of the proteins. Results are shown as means, n = 5. The hydrophobicity score is shown as a heat map with green as the most hydrophobic, red as the most hydrophilic.

Fig 4. Distribution of amino acids at P1 and P1’ position by count.

Mean relative count of (A) P1 and (B) P1’ positions of human and bovine milk protein-derived peptides identified in five human milk and infant gastric samples distributed by amino acid.

Fig 5. Distribution of amino acids at P1 and P1’ position by ion intensity.

Mean relative ion intensity of (A) P1 and (B) P1’ positions of human and bovine milk protein-derived peptides identified in human milk and infant gastric samples distributed by amino acid.

Fig 6. Bioactive peptides identified in human milk or infant gastric.

Bioactive peptides identified in the (A) milk, (B) bovine-based HMF, (C) gastric samples (human-derived) and (D) gastric samples (bovine-derived) by searching the Milk Bioactive Peptide Database (MBPDB), with a threshold value of ≥ 80% homology.