Baby Unable to Process Protien Enzyme in Mothers Milk

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Peer-reviewed

Inquiry Article

Release of functional peptides from female parent'due south milk and fortifier proteins in the premature babe tummy

• Søren D. Nielsen,
• Robert L. Beverly,
• Mark A. Underwood,
• David C. Dallas

10

• Published: November 29, 2018
• https://doi.org/ten.1371/journal.pone.0208204

Figures

Abstract

Digestion of milk proteins in the premature baby tummy releases functional peptides; nevertheless, which peptides are present has not been reported. Premature infants are often fed a combination of human milk and bovine milk fortifiers, only the variety of functional peptides released from both homo 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 confronting 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 nigh agile protease in milk, while pepsin or cathepsin D were predicted to be most agile in the stomach. Alignment of the peptide distribution showed a different digestion pattern between human being and bovine proteins. The number of peptides with loftier 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 being and bovine milk proteins may bear on overall wellness outcomes in premature infants.

Commendation: Nielsen SD, Beverly RL, Underwood MA, Dallas DC (2018) Release of functional peptides from female parent'due south milk and fortifier proteins in the premature infant stomach. PLoS ONE xiii(eleven): e0208204. https://doi.org/10.1371/journal.pone.0208204

Editor: Joseph J. Barchi, National Cancer Plant at Frederick, United states

Received: July 21, 2018; Accustomed: November 13, 2018; Published: November 29, 2018

Copyright: © 2018 Nielsen et al. This is an open up access commodity distributed nether the terms of the Creative Eatables Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original writer and source are credited.

Data Availability: The data have been deposited to the jPOST repository (ID: PXD010502).

Funding: This study was supported by the K99/R00 Pathway to Independence Career Award, Eunice Kennedy Shriver Institute of Child Health & Evolution of the National Institutes of Wellness (R00HD079561) (DCD), the USDA National Institute of Food and Agriculture and The Gerber Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have alleged that no competing interests be.

Introduction

Homo 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. All the same, milk proteins provide more than than the ideal amino acids for infants. In vitro studies advise that peptides encrypted within parent milk proteins possess a variety of bioactive functions, including antimicrobial [2], angiotensin-converting enzyme (ACE) inhibition [3], immunomodulation [iv, 5], antioxidant [6], opioid [seven] and calcium delivery [eight]. Many of these bioactive peptides are released from milk proteins during digestion within the mammary gland by native milk proteases and past proteases inside the babe gut. Our previous work demonstrated that mother'due south 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 [xiv–16]. These encrypted peptides may be functional units with biological furnishings within the babe that evolved to be released based on the specificity of proteases in the normal term mother'south milk and the term baby's gut.

During early years of the evolutionary timescale, infants born prematurely (< 37 weeks gestational historic period) were unlikely to have exerted much selective pressure level on milk composition and structure, every bit 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 wellness outcome associations, including reduced risk of necrotizing enterocolitis (NEC) [19, 20] and sepsis [21], improved cognitive skills [22] and decreased fourth dimension to hospital discharge [23]. However, the energy and protein content of human milk alone do non ensure optimal growth in preterm infants. Therefore, homo milk fed to preterm infants is typically fortified to encounter their poly peptide needs, which range from 2.v to four g protein/kg body weight/twenty-four hours depending on gestational age at nascence and twenty-four hours of life [24]. Human milk fortifiers (HMFs) can derive from either human or bovine milk [25, 26]. While limited testify suggests that homo milk-based fortifiers may reduce risk of NEC [27], bovine fortifiers are commonly used due to their lower cost and college availability [28]. The different amino acid sequences of bovine proteins may pb to differential degradation in the infant. The processing—particularly the heat treatments used to ensure sterility—of milk proteins to prepare fortifiers tin alter protein structure, which tin can change 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 electrophoresis protein profiling [31, 32], however whether the specific peptides released differ betwixt these poly peptide sources has not been adamant, particularly in human infants.

To determine which functional peptides premature infants are exposed to during digestion of consumed milk, we used peptidomics to analyze the peptides released from human and bovine milk proteins based on homology with known functional peptides in our recently created Milk Bioactive Peptide Database [33]. We assessed the peptidomics data to determine which proteases were about probable responsible for their release. This initial work can lead to insights on how the food type can bear upon the bioactive potential of peptides inside protein fragments that bear on the preterm infant's overall wellness.

Materials and methods

Participants and sample collection

Man milk samples and their matching gastric milk samples were collected from five mother–premature babe pairs, every bit approved by the UC Davis Institutional Review Board. Written consents were obtained from all mothers. Milks from mothers of infants in the Neonatal Intensive Care Unit of measurement (NICU) of the UC Davis Medical Center were nerveless by pumping on-site or at home with sterile breast pumps and stored immediately at –20°C. Milks pumped at home were transported to the NICU on ice and stored at –40°C. Milks were transported from the NICU to the laboratory on dry ice and stored at –80°C. The infants of these same mothers were sampled for their stomach contents at 2 h later initiation of feeding the female parent's milk. Due to their need for more concentrated nourishment, these premature infants were fed mother'due south milk enriched with a bovine milk protein-based fortifier (Abbott Similac HMF powder (1 0.ix-g packet per 25 mL human milk). Samples were taken via suction from already in-place oro- or naso-gastric feeding tubes. Infants selected for this study had no known digestive functional problems. Infants ranged from 24 to 32 weeks gestational historic period and from 11 to 45 days of life. Subsequently drove, gastric samples were immediately stored at –twoscore°C. Samples were transferred to the laboratory on dry water ice and stored at –80°C.

Sample preparation

Aliquots of milk and gastric aspirates (between 8 and xx μL) from the v mother–infant pairs were thawed on ice for approximately 30 min. A sample of the HMF was dissolved in nanopure water at the same concentration as was added to the infant feeds. HMF was processed according to the steps described for the milk and gastric samples. To remove the milk fat, samples were centrifuged at 3,000 10 g for 10 min at 4°C and placed on water ice while the infranate were collected by pipette. Milk proteins were precipitated from the skimmed milk by addition of 240 g/L trichloroacetic acrid (1:1 sample:solution). After mixing, the samples were centrifuged (four,000 x g for x min at 4°C) and the peptide-containing supernates were nerveless.

Trichloroacetic acid, salts, oligosaccharides and lactose were removed from the peptide solution and peptides were extracted via C18 reverse-stage preparative chromatography 96-well plates (Glygen, Columbia, MD) as previously described [34]. The purified peptide solutions were frozen at –80°C and lyophilized using a freeze dry arrangement (Labconco FreeZone 4.5 Fifty, Kansas Urban center, MO). After drying, the samples were rehydrated in 0.1% formic acid in water to their original book.

Liquid chromatography nano-electrospray ionization mass spectrometry

The liquid chromatography separation was performed on a Waters nanoAcquity Ultra-Performance Liquid Chromatography (UPLC) (Waters Corporation, Milford, MA, Us) with a nanospray source. Ane microliter of each sample was loaded onto a 180 μm × twenty mm, v-μm bead 2G nanoAcquity UPLC trap cavalcade (opposite stage) for online desalting and enrichment, and and then onto a 100 μm × 100 mm, ane.7-μm dewdrop Acquity UPLC Peptide BEH C18 cavalcade (Waters) for analytical separation. The LC eluent was initially set to 97% solvent A (0.1% FA in h2o) and three% solvent B (99.9% ACN, 0.1% FA) with a flow rate of 500 nL/min. Then, the LC slope increased from 3–10% solvent B over iii min, and then 10–30% solvent B over 99 min, then xxx–90% solvent B over 3 min, and then 90% solvent B for 4 min, and so 90–iii% solvent B over ane min and finally kept at 3% solvent B for x min. Each sample run was followed past a 30-min column wash.

The peptides were profiled with a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer. Spectra were obtained in positive-ionization mode with an electrospray voltage set to 2,400 V. The MS browse range was 400–1500 m/z at a resolution of 120K. The fragmentation mode was set to collision-induced dissociation and the collision free energy was 35%. The MS bicycle time was 3 s with information-dependent analysis and automated precursor tiptop option. Forerunner ions were excluded after i fragmentation for 60 s and exclusion within a 10 ppm mass error window. Precursors were selected for fragmentation based on the following criteria: about intense peaks, ion-intensity threshold 5.0 × 103 and charge state two–7. Fragments were detected with the ion trap with an automatic scan range.

Spectra were analyzed by database searching in Thermo Proteome Discoverer (v2.1.0.81) using an in-business firm human and bovine milk protein sequence database as previously described in particular [35]. Potential modifications allowed included phosphorylation of serine and threonine and oxidation of methionine. Proteome Discoverer percolator was used to discriminate between correct and incorrect spectrum identifications using a decoy database search. Based on this, merely peptides identified with high confidence (P < 0.01) were included in the results (FDR < 0.01). Peptide sequences with multiple modifications were grouped into a single peptide for counts and ion intensity. "Counts" represents the number of unique peptide sequences identified in a sample and "ion intensity" represents the area under the bend of the eluted tiptop. The peptide ion intensity, is used as an approximation of the corporeality of peptides in the samples. The data have been deposited to the jPOST repository [36] (ID: PXD010502).

Data analysis

Peptides were mapped to the parent protein sequence of β-casein, α_s1-casein and osteopontin using an in-house tool (PepEx) [37], bachelor at http://mbpdb.nws.oregonstate.edu/pepex/. This mapping provides a visual overview of the position of peptides released within the parent poly peptide sequence by totaling the ion intensity of each amino acid from the peptidomic data. The sequences of bovine and homo milk proteins were aligned using the Protein BLAST alignment tool at blast.ncbi.nlm.nih.gov. This data was combined with the Pepex output to compare the digestion of proteins from either homo or bovine origin. The protein hydrophobicity distribution was determined using ProtScale (https://spider web.expasy.org/protscale/) which employs the method described by Tanford [38]. The hydrophobicity was calculated equally an average of seven amino acids and a linear weight variation with 25% relative weight of the amino acids at the window border.

Excel was used to create tables of amino acids present in the P1 and P1' positions at the N- and C-terminal cleavage sites of each peptide identified. The nomenclature for a protein cleavage site was formulated by Schecter and Berger [39]. P1 is the beginning amino acrid positioned before a cleavage site. P1' is the first amino acrid positioned after a cleavage site.

Proteasix, an online tool that predicts enzymes involved in peptide cleavage using the cleavage site-specificity matrices for proteases within the Merops protease cleavage site database [40], was used to predict the proteases involved in peptide cleavage inside the 5 milk and v gastric samples. The peptide data were searched against the following proteases: cathepsin D (CTSD; P07339), neutrophil elastase (ELANE; P08246), thrombin (F2; P00734), kallikrein half dozen (KLK half dozen; Q92876), kallikrein 11 (KLK 11; Q9UBX7), plasmin (PLG; P00747) and pepsin (PGA3; P0DJD8). Proteasix searches the database of known peptide cleavages against the peptidomic data, identifying instances where the exact cleavage site has previously been observed (experimental data) and attributed to a specific enzyme. The results from Proteasix are given equally experimentally known cleavages (observed) and predicted cleavages based on the cleavage site amino acids positioned at P4P3P2P1-P1'P2'P3'P4'. Equally predictions based on 8 amino acids might be too stringent, Proteasix allows for upwardly to 3 mismatches (from loftier to low conviction). However, in all cases cleavage site restrictions are obeyed. All predicted and observed cleavages were combined in the final results [39].

Identified peptides in milk, HMF and gastric samples were examined for homology with literature-identified bioactive peptides using the Milk Bioactive Peptide Database (MBPDB, http://mbpdb.nws.oregonstate.edu/) [33], which is a comprehensive collection of all milk bioactive peptides. The search was performed as a sequence search that searches for bioactive peptides matching the input peptide amino acid sequence. The similarity threshold was set to 80% and the amino acid scoring matrix was fix to identity. "Get extra output" was selected to obtain the specific percent similarity between the query sequence and the bioactive peptide sequence.

Protein concentration

The protein concentration in human being milk and gastric samples were measured with the bicinchoninic acrid assay poly peptide assay.

Statistical analysis

Analyses were carried out using the statistical program RStudio version 1.0.136. A linear mixed model with Tukey's HSD mail hoc test was used to adjust for multiple comparisons between bovine and human milk peptides. Pregnant deviation was divers as P < 0.05. Results are presented as least square ways ± standard error.

Results

Demographic details for the five female parent-baby pairs are presented in Table one. Each 25 mL of human milk was fortified with 0.25 chiliad of bovine milk proteins. Sampling of human being milk for this analysis was performed prior to HMF addition for baby feeding.

The poly peptide concentration of human milk was fifteen.9 ± ane.0 mg/mL and the gastric aspirates two hours subsequently feeding human milk fortified with bovine milk proteins (x mg/mL) contained 17.iii ± ane.8 mg/mL.

Peptide profile

Peptide profiling by mass spectrometry identified a total of 5,264 unique peptides deriving from human and bovine milk proteins (14,413 when counting modification variants) across all human milk, HMF, and gastric samples. From the total number of identified peptides, ane,722 and 3,399 originated from bovine and human being milk proteins, respectively. One hundred and thirty-viii peptides derived from protein regions that have identical sequences for human and bovine milk proteins and, thus, their origin could not exist distinguished. The peptides that were indistinguishable were mostly attributed to relatively low affluence milk proteins such equally actin, xanthine dehydrogenase, fatty acrid-binding protein, and lipoprotein lipase.

The five human milk samples contained an average of 719.6 ± 77.2 peptides with a total ion intensity 7.62 × ten^xi ± 1.xix× x^xi. Bovine milk-based HMF measured lonely (non mixed with human being milk) contained 538 peptides with a full ion intensity of 1.1 × x¹¹, whereas infant gastric samples two hours afterwards initiation of feeding contained an average of 1,720.eight ± 108.2 peptides accounting for a full ion intensity of 1.67 × 10¹² ± 2.85 × 10^xi. Of the boilerplate number of peptides identified in the gastric sample, 980.eight ± 101.0 derived from man milk proteins with nine.21 × 10^eleven ± 1.49 × 10¹¹ total ion intensity and 691.6 ± 7.2 peptides derived from bovine milk proteins with 7.39 × 10^xi ± 1.35 × 10^xi total ion intensity. The number of peptides identified from human milk proteins increased significantly from human milk before feeding to the gastric 2 hours after initiation of feeding.

One hundred lxxx-five peptides were plant in all five milk samples, making up 25.7% of the total number of peptides identified in human milk (Fig 1A). 3 hundred ten peptides were establish in all v infant gastric samples: 168 from bovine milk, 137 from human milk, and 5 that were indistinguishable (Fig 1A). Combined, these peptides accounted for 18% of the total number of peptides identified in the gastric samples. A greater number of peptide sequences were thus released from homo milk proteins (as represented past the lower pct of conserved sequences) than from bovine fortifier proteins from the standardized HMF post-obit preterm gastric digestion.

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

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

https://doi.org/10.1371/periodical.pone.0208204.g001

The majority of the peptide content (by ion intensity) in both milk and gastric samples was accounted for past peptide sequences nowadays in samples from all five of the infants (82.4% and 67.7% respectively) (Fig 1B). In the gastric samples, 63.viii% of the man milk protein-derived peptide ion intensity and 72.5% of the bovine milk protein-derived peptide ion intensity was comprised of peptide sequences nowadays in all samples. For both human and bovine proteins, most of the affluence derived from peptides present in all samples, but human milk peptides were slightly less conserved across infants.

Several of the human milk peptides identified in the human milk samples were also identified in the gastric samples from the corresponding infant. Peptides identified in both milk and gastric accounted for 21.vii ± v.2% of the total number of peptides deriving from human being milk proteins identified in the gastric, and accounted for 32.2 ± 6.1% of the peptide full ion intensity. The list of peptides tin can be found in S1 Table.

Next, nosotros examined the extent of digestion of individual proteins. To compare between sample and feed types, we represented each protein'southward peptides relative to the total homo or bovine peptide profile in that sample (Fig 2). In HMF, peptides deriving from β-casein contributed with the highest ion intensity (26.7% of total), whereas the highest number of peptides was identified from α_s1-casein (26.2% of total). In both the milk and gastric samples, virtually peptides where identified from β-casein. Human β-casein made upward 39.ix% of the total number of identified peptides and 43.8% of the full ion intensity of peptides in the homo milk samples, and 33% of total number of peptides and 71.2% of total ion intensity of homo milk protein-derived peptides in the gastric samples. Bovine β-casein made up 30.3% of the full number of peptides and 35.6% of the total ion intensity of bovine milk protein-derived peptides in the gastric samples. For human being milk protein-derived peptides in the gastric samples, the next ii highest contributors were α_s1-casein and osteopontin, and each accounted for less than one-third of β-casein's count and less than 1-tenth of its ion intensity. The distribution of bovine protein contribution to the peptide counts and ion intensity was more equivalent for the top three proteins (β-casein, α_s1-casein, and κ-casein) compared with man milk proteins. In addition to proteins present in both human and bovine milk, peptides from proteins unique to bovine milk were identified in the gastric samples, including β-lactoglobulin (count, ion intensity; 10.ii%, 3.9%) and α_s2-casein (7.7%, 3.3%).

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

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

https://doi.org/10.1371/periodical.pone.0208204.g002

Peptide distribution

The protein sequences of β-casein, α_s1-casein and osteopontin were aligned between the human being and bovine protein sequences using the online bioinformatics software Protein Boom. The peptides identified in the gastric samples were then distributed across the aligned poly peptide sequence according to their ion intensity using the PepEx software. Ion intensity of peptides also identified in human milk or HMF were subtracted from those present in the gastric samples to better correspond merely peptides released in the stomach. The hydrophobicity score was added above the peptide distribution every bit it impacts protein folding and potential accessibility for proteases (Fig 3). In full general, the observed cleavage sites were generally dissimilar between bovine and homo milk proteins. The largest cleavage site in bovine α_s1-casein by ion intensity was between Leu²⁰(P1)—Leu²¹(P1') (amino acid number is counted without the signal peptide). This site is not present in the human α_s1-casein sequence. Similarly, the largest cleavage site in homo α_s1-casein (Asn³⁵-Arg³⁶) is not nowadays in bovine α_s1-casein. Several of the virtually abundant sequences of cleavage sites were conserved between the ii species yet not cleaved to the aforementioned caste, eastward.g. the highly broken site Phe¹⁷⁹-Ser¹⁸⁰ in bovine α_s1-casein is nowadays as Phe¹⁴⁹-Ser¹⁵⁰ in the human protein, but it is not highly cleaved in the man protein despite their highly similar hydrophobicity patterns. Similar results were constitute for β-casein. The largest cleavage site of bovine β-casein was Leu¹⁶³-Ser¹⁶⁴, which was non nowadays in the human β-casein sequence; and the largest site of human β-casein was Leu¹⁸⁷-Leu¹⁹⁰, which was nowadays in the bovine β-casein sequence. Interestingly, the major cleavage sites of osteopontin were mostly conserved between the two species as was their hydrophobicity pattern (notation: despite the sequence similarities, none of the osteopontin peptide sequences were identical for bovine and human. The largest cleavage site in bovine osteopontin was Phe³⁸-Leu³⁹, which was present in human osteopontin as Leu³⁸-Leu³⁹; whereas the largest in homo osteopontin was Thr²⁶-Trp²⁷, which was exactly conserved in bovine osteopontin. An additional site was conserved at Ala²⁵-Thr²⁶ for both species.

Fig 3. Mapping of identified peptide on milk proteins.

Full ion intensity of peptides identified in baby gastric samples from bovine and human milk β-casein (A), α_s1-casein (B) and osteopontin (C). The ion intensity of each peptide too present in the human being 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 oestrus map with dark-green as the about hydrophobic, red equally the near hydrophilic.

https://doi.org/10.1371/journal.pone.0208204.g003

Enzyme cleavage

Each of the 692 ± vii and 981 ± 101 peptides identified from bovine and human being milk proteins in the gastric samples of infants, respectively, have both an North- and C-terminus, resulting in potentially double the number of cleavage sites equally peptides identified. Withal, of these peptides, 83 ± six bovine milk peptides and 82 ± 5 human milk peptides had a C- or N-terminus, which was the same equally the parent protein's C- or N-terminus, and therefore did not count as a cleavage site. The amino acids nowadays before and later a cleavage site were matched to the cleavage site specificity of proteases to determine which proteases nigh likely broken each peptide bond. For virtually proteases, the amino acids at P1 and P1' are of virtually importance for peptide cleavage specificity. The most common P1 position amino acids in human milk (past relative count and relative ion intensity, respectively) were Lys (17.7%, thirty.0%), Arg (xi.five%, 16.five%), Leu (8.ix%, 0.1%) and Ser (7.7%, 3.viii%). The high level of P1 Lys and Arg observed matches the sequence specificity for plasmin. The remaining cleavage site P1 positions were distributed among all other amino acids except for Cys, which was never present at the P1 position of a cleavage site (Fig 4 and Fig five).

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 5 human milk and baby gastric samples distributed by amino acrid.

https://doi.org/ten.1371/periodical.pone.0208204.g004

Fig five. 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 homo milk and infant gastric samples distributed past amino acrid.

https://doi.org/10.1371/periodical.pone.0208204.g005

In the baby gastric samples, for peptides deriving from homo milk proteins, the nigh mutual P1 site amino acids were Leu (21.2%, 38.seven%), Tyr (9.4%, 8.vii%)), Phe (eight.3%, iii.7%) and Ala (vii.2%, 9.6%). For the bovine peptides in the infant gastric samples, the most common P1 site amino acids were Leu (xix.1%, 31.ix%), Phe (11.1%, 18.viii%), Glu (seven.7%, 5.4%), Tyr (half-dozen.8%, five.6%) and Trp (half-dozen.viii%, 5.six%) (Fig 4 and Fig 5). Cysteine was never present at the P1 or P1' position of a cleavage site. Phe and Leu are preferred amino acids at P1 for pepsin and cathepsin D.

Proteasix is an online software that predicts the protease responsible for peptide cleavage based on the P4–P4' amino acids positioned effectually the cleavage site [40]. In the Proteasix analysis, virtually peptides identified in the milk and gastric samples were not assigned to a specific protease. Of those matched, Proteasix predicted most of the cleavage sites of human milk proteins to derive from the action of plasmin (49 ± iii cleavage sites) and elastase (23 ± 4 cleavage sites). Additional cleavage sites matched to thrombin (x ± 1 cleavage sites) and kallikrein 6 (3 ± 0 cleavage sites). The relative count of cleavage sites matched to each protease is shown in Tabular array two.

Proteasix predicted that most of the cleavage sites of milk proteins from gastric aspirates derived from the activity of cathepsin D (93 ± v in bovine milk proteins, 75 ± 12 in human milk proteins) and pepsin (64 ± 3, 67 ± 14). Many of the cleavage sites could be assigned to either cathepsin D or pepsin, every bit the cleavage site specificities of these two enzymes overlap and cannot e'er exist distinguished past this method. On average, 47 ± ii and 105 ± 25 of bovine and human milk protein cleavage sites, respectively, could be assigned to both cathepsin D and pepsin. Some gastric peptide cleavage sites matched to elastase (48 ± two in bovine proteins, lx ± two in homo), and a low number matched to plasmin (five ± 1, 27 ± 4), thrombin (3 ± 0, half-dozen ± 1) and kallikrein half-dozen (1 ± 0, 3 ± ane).

Bioactive peptides

Searching the identified peptides against the MBPDB revealed 58 peptides that were identical with known bioactive peptides: 5 bioactive peptides deriving from human milk proteins, 50 from bovine milk proteins, and 3 that could not be distinguished between the two species due to identical sequences (Tabular array 3). All five of the homo milk peptides were identified in the gastric samples, but just two were identified in the milk samples. No bioactive human milk poly peptide-derived peptides were present in either all v of the milk samples or all five of the gastric samples. Eleven of the 100% homologous bovine peptides were identified in all 5 gastric samples and are listed in bold in Table three.

In addition to the identical sequences, multiple peptides identified in the milk and gastric samples were highly homologous (≥ fourscore% sequence lucifer) to known bioactive peptides. Several of these peptides were identified with more than i part. The loftier degree of sequence similarity suggests the possibility for these peptides to have bioactive function as well. We identified 85 bovine milk peptides in the HMF with ≥ 80% homology to a known bioactive peptide. In the human milk, 31.2 ± 3.iii peptides with ≥ lxxx% homology to a known bioactive peptide were identified as deriving from human being milk proteins. Of those, xi peptides were identified in all v milk samples. In the gastric samples, a total of 206.4 ± 8.6 peptides with ≥ 80% homology to a known bioactive peptide were identified. From the total, 165.6 ± 5.9 were identified from bovine proteins, and 40.8 ± 3.8 were identified from human proteins. 50 bovine peptides and 11 homo peptides were present in all five gastric samples.

The counts and total ion intensity of human-derived bioactive peptides significantly increased from human milk to the gastric samples (P = 0.0438 and P = 0.00287, respectively). The count and total ion intensity of bovine-derived bioactive peptides increased from HMF to gastric samples, 212% and 2268%, respectively; however, equally only one sample of HMF was analyzed, no statistical ability could be calculated. Peptides highly similar to known antimicrobial peptides were the virtually often identified, including 31 from human milk, 48 from HMF, 36 from homo milk proteins in the gastric samples and 166 from bovine milk proteins in the gastric samples (Fig 6). Peptides with sequences closely related to known ACE-inhibitory peptides were often identified: 11, 36, 31 and 152 possible ACE-inhibitory peptides were identified in human milk, HMF, gastric (human) and gastric (bovine) samples, respectively. Additional peptides with potential antioxidant and cell-proliferation stimulatory effects were identified from human milk proteins. From bovine milk proteins, bioactive peptides with a greater diversity of functions were identified, including DPP-4-inhibitory, antithrombotic, cytomodulatory, immunomodulatory, opioid and anxiolytic peptides (S1 Tabular array).

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

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

https://doi.org/10.1371/journal.pone.0208204.g006

Give-and-take

This study profiled the peptides in human milk, a bovine milk-based fortifier and the gastric contents of preterm infants after fortified human milk feedings. Peptides from both human being milk proteins and bovine fortifier proteins increased from the undigested feed to the stomach. The total count of human milk protein-derived peptides increased from the milk to gastric samples, and the total count of bovine milk protein-derived peptides increased from the HMF to gastric samples. Total peptide ion intensity also increased from milk/HMF to the gastric samples for both protein types. A previous report in term infants likewise showed an increase in peptide counts and ion intensity from milk to gastric samples [fourteen]. Use of the Orbitrap Fusion Lumos mass spectrometer allowed u.s. to place far more than peptides than were previously discovered (5,264 compared to 418 [xiv]). The current study used database searching for the identification of peptides. This approach will not identify peptides shorter than seven amino acids, simply it is likely that gastric digestion results in several short peptides that were not detected.

Peptides that were identified from all five mother-infant pairs accounted for the majority of the peptide ion intensity for both milk and gastric samples. The finding that the bulk of the man milk peptide ion intensity derived from peptides plant in all five milks matches previous findings from foremilk and hindmilk [34]. In the gastric samples, the majority of both human and bovine poly peptide-derived peptide ion intensity was conserved in all five infants. All the same, the majority of peptide count from both species was composed of peptides that were found in fewer than all five of the samples, with human milk poly peptide-derived peptides being slightly less conserved. The differences in count probable resulted from pocket-sized variations in protease activity that differentially released peptides from the parent proteins. Though the abundance of these peptides was not large, there is the possibility that the sequence differences had varying bioactivity.

Caseins stand for about xx% of milk proteins in early lactation and 45% of milk proteins in belatedly lactation [41]. Of the human milk peptides, β-casein accounted for 71.ii% of the total human peptide ion intensity, with α_s1-casein and κ-casein combining for an boosted 11.7%. Also, HMF has a blend of milk protein and whey proteins designed to lucifer the casein:whey ratio of homo milk [42], nonetheless the HMF-derived bovine peptides from caseins accounted for 84.ix% of the gastric peptide ion intensity. Using peptide ion intensity as an indicator for relative gastric protein digestion, the casein proteins in both HMF and human milk, and β-casein in particular, were digested more efficiently than the whey proteins. These findings match, in office, with findings of the previous report on term babe gastric digestion [14], except that in that report, lactoferrin-derived peptides fabricated up a relatively larger proportion of the peptides released in the stomach. That divergence could signal that the term infant stomach has greater potential to dethrone whey proteins than the preterm infant breadbasket. A caveat is that using peptide ion intensity tin can but exist used as an estimate of true abundance, as peptide ionization efficiency varies between peptides.

The combination of human milk and bovine-based fortifier in the sampled preterm infants' diets allowed for comparison of gastric digestion betwixt human- and bovine-derived proteins. The protein profile closely followed that establish in previous human being and bovine milk studies, and in vitro gastric digestion studies [37, 43, 44]. β-casein from both species was the most digested protein based on peptides released, while α_s1-casein-derived peptides from bovine milk were relatively more abundant than the human analogue. Though osteopontin was partially digested in man milk and was the third nigh hydrolyzed poly peptide in the gastric samples, by count, few peptides from bovine osteopontin were identified in the gastric samples. The cleavage pattern between β-casein, α_s1-casein and osteopontin was highly dissimilar between man and bovine proteins, as shown past alignment of the peptide distribution.

Previously, we measured the concentration and activity of proteases in both human being milk and gastric aspirates of infants [9]. Our findings in this report contribute to determining the end-products of protease activity in the preterm babe stomach. From the P1–P1' amino acid annotations in human milk, we institute that Lys and Arg were mostly observed at the P1 position of a cleavage site. Plasmin, kallikrein and thrombin are enzymes known to be active in human milk [9, 16] that prefer to cleave after these amino acids. Hence, it is not easy to distinguish between these enzymes based on the P1 amino acid. The bioinformatics tool Proteasix uses a more complex algorithm to identify enzyme activity and takes into account amino acids from P4 to P4'. This analysis constitute that most cleavage sites in the milk samples matched to the specificity of plasmin (in a higher place that of kallikrein half dozen and thrombin). A recent peptidomics report of preterm and term milk samples likewise indicated that, based on cleavage sites, plasmin was highly active inside milk [16].

In the gastric samples, the P1–P1' amino acid annotations switched from generally Lys and Arg in human milk to Leu and Phe in the tum, which matches the P1 amino acid cleavage specificity of pepsin/cathepsin D. The Proteasix P4 to P4' analysis showed that both cathepsin D and pepsin are likely highly active in cleaving both bovine and human milk proteins in the preterm infant stomach. Every bit cathepsin D and pepsin both preferentially cleave subsequently a Leu/Phe amino acrid, a large portion of the predicted cleavages sites could exist assigned to either cathepsin D or pepsin. We previously observed that milk cathepsin D is inactive within human milk but becomes agile when exposed to the more acidic pH of the baby stomach [sixteen]. This activation is expected equally acidic weather lead to autoactivation of the inactive procathepsin D to the active pseudocathepsin D [45]. Pepsin is known to be produced in the infant stomach as early as 16 weeks of gestation [46]. These data demonstrate a articulate shift in the release of milk peptides from the activity of mostly plasmin in man milk to by and large pepsin and cathepsin D in the stomach. The addition of fortifier is unlikely to contribute to the protease action, equally these enzymes would be most probable inactivated by the heat-treatment during processing [47].

A large number of cleavage sites could non exist assigned to the proteases examined. These cleavage sites could potentially exist due to the activity of cytosol aminopeptidase and carboxypeptidase B2, which are exopeptidases that sequentially release amino acids from the N- and C-termini, respectively. These enzymes are known to exist present in milk [nine], and based on peptide profiling, are thought to exist major contributors to milk poly peptide digestion [48]. Human milk protein-derived peptides and bovine milk protein-derived peptides had similar numbers of cleavage sites that could be due to exopeptidase activity.

This study demonstrated that a large number of potentially bioactive peptides are released by milk proteases in the mammary gland and the preterm infant breadbasket. Potential bioactive peptides increased from the undigested milk/fortifier to the stomach for both human and bovine proteins. Whether these peptides exert their bioactive effects depends on whether they attain their site of action. Further proteolytic activity in the intestine due to the action of pancreatic proteases and castor-border peptidases is likely to break downwardly existing peptides and release new ones from the milk proteins [49]. Studies investigating the systemic effect of milk-derived bioactive peptides are limited, only several of the identified predicted bioactive peptides may be relevant for the gut, such equally those with antimicrobial and mucin-stimulatory properties.

A college number of potential bioactive peptides from bovine milk proteins were identified compared with those from human being milk proteins. The predominance of bovine milk-derived bioactive peptides about likely derives from the fact that bovine milk has been more than comprehensively studied for bioactivity than has human milk rather than whatsoever biological reality. The MBPDB contains 713 known bovine milk bioactive peptides and only 106 known human milk bioactive peptides [33]. Notwithstanding, though more than twice as many ACE-inhibitory peptides than antimicrobial peptides are described in the literature (and present in the database) [33], more peptides homologous to known antimicrobial peptides were identified in the samples in the present study, which demonstrates that determining homology does not reflect a simple probability distribution. Several literature-described bioactive peptides have a brusque sequence of fewer than vii amino acids, which is below the size threshold for the peptide analysis approach used in this study and therefore are not identified.

In conclusion, proteins from human milk and bovine milk proteins from HMF were similarly digested in the preterm baby breadbasket. Both human milk and HMF contain bioactive peptides, and the number, abundance and functional variety of both human and bovine-derived peptides increased with gastric digestion in preterm infants. Studies of the potential clinical impact of bioactive peptides released in the premature babe stomach will be of particular importance, including bear on on the many diseases of prematurity in which inflammation and/or oxidants play a role such as peri-ventricular leukomalacia, bronchopulmonary dysplasia, retinopathy of prematurity, and necrotizing enterocolitis. Also of interest will be the affect of released proteins on more typical nutritional outcomes such as growth, neurodevelopment, anemia of prematurity and metabolic os disease of prematurity. The addition of analysis of gastric aspirates for bioactive peptides to current accomplice studies focused on the diseases unique to premature infants has great potential value.

Supporting information

S1 Table. The consummate listing of peptides identified in the HMF, milk, and gastric samples.

Sequence lists the peptide sequence. Species lists the peptide's parent poly peptide species. Protein Name lists the parent protein. Start and Stop listing the amino acid position the peptide is derived from in the parent protein. HMF, Milk, and Gastric list the number of samples the peptide was identified in. Modifications lists all modifications the peptide was identified with (O = Oxidation, P = Phosphorylation, A = Acetylation). Highest Homology Match lists the bioactive peptide from the MBPDB that the peptide about closely matched by sequence. % Alignment lists the sequence alignment. Part lists the bioactive functions of homologous peptides. DOI lists the journal article of the Highest Homology Match.

https://doi.org/x.1371/journal.pone.0208204.s001

(XLSX)

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