Peptides Derived from In Vitro and In Vivo Digestion of Human Milk Are Immunomodulatory in THP-1 Human Macrophages.

BACKGROUND: Milk proteins contain many encrypted bioactive peptides. Whether these bioactive peptides are released in the infant intestine and exert immunomodulatory activity remains unknown. OBJECTIVE: This study examined in vitro immunomodulatory activities of peptides from in vitro- and in vivo-digested human milk. METHODS: Peptides were extracted from in vitro-digested human milk and pooled intestinal samples from 8 infants fed human milk. Peptides extracted from in vitro-digested samples were fractionated. The in vitro effects of these peptides and fractions on the secretion of TNF-α and IL-8 in LPS-treated human immune THP-1 macrophages were evaluated. The significance of differences between in vitro peptide fraction treatment and control on cytokine production was analyzed by t test. LC-MS/MS-based peptidomics was conducted to identify the peptides. The peptides were screened for potential bioactivity using a sequence homology search using the Milk Bioactive Peptide Database (MBPDB). RESULTS: Six fractions of the peptide mixture extracted from the in vitro-digested human milk significantly inhibited TNF-α production by LPS-challenged THP-1 macrophages. Fractions F4, F8, F11, F14, and F17 attenuated IL-8 secretion, and F6/7 and F18 increased IL-8 secretion. Peptides extracted from the pooled in vivo intestinal samples attenuated both TNF-α and IL-8 secretion. There were 266 and 418 peptides identified in the in vitro and in vivo samples, respectively. Among the peptides, 34 and 50 in the in vitro and in vivo samples, respectively, had >80% sequence similarity to bioactive peptides in the MBPDB. CONCLUSIONS: Peptides released by in vitro and in vivo infant digestion of human milk were immunomodulatory in human immune cells; fractions F4, F8, and F11 were anti-inflammatory; and F6/7 and F18 were proinflammatory. Thirteen peptides were present in all fractions with anti-inflammatory activity, and 38 peptides were present in all fractions with proinflammatory activity. These peptides potentially contributed to the observed immunomodulatory activity of the peptide mixtures.

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity.

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.

Differences in human milk peptide release along the gastrointestinal tract between preterm and term infants.

BACKGROUND & AIMS: Preterm infants are born with a gastrointestinal tract insufficiently developed to digesting large quantities of human milk proteins. Peptides released from the digestion of human milk proteins have been identified with bioactivities that may be beneficial to the developing infant. However, it is unknown how prematurity affects total and bioactive peptide release along the gastrointestinal tract. The aim of this study was to compare milk peptide release from milk to stomach to stool between preterm and term infants. METHODS: Milk, gastric, and stool samples were collected from preterm infants as early collection (days 8 and 9 of life) and late collection (days 21 and 22 of life), and from term infants as early collection. Milk peptides were extracted from the samples and identified using Orbitrap mass spectrometry. Peptide abundance and count were compared across digestion and between the three infant groups at each stage of digestion. RESULTS: Total milk peptide count and abundance increased from milk to stomach then decreased in stool. Total peptide release was similar among the three infant groups for milk and stool samples. In the stomach, preterm early collection had significantly higher peptide abundance and count than the other two groups. Patterns for peptide release from individual milk proteins were distinct from total peptide release both across digestion and among the infant groups. When analyzing single peptides, term early collection gastric samples had significantly higher peptide abundance than preterm early collection for a known antimicrobial peptide, QELLLNPTHQIYPVTQPLAPVHNPISV. CONCLUSIONS: Preterm and term infants digest milk proteins differently along their gastrointestinal tracts. While preterm infants released more total peptides in the stomach, term infants released specific bioactive peptides at higher abundance. We identified a region at the C-terminus of ß-casein that is conserved from milk through stool and from which are released known and potential antimicrobial peptides.

Differences and Similarities in the Peptide Profile of Preterm and Term Mother's Milk, and Preterm and Term Infant Gastric Samples.

Our previous studies revealed that milk proteases begin to hydrolyze proteins in the mammary gland and that proteolytic digestion continues within the infant stomach. No research has measured how the release of milk peptides differs between the gastric aspirates of term and premature infants. This study examined the presence of milk peptides in milk and gastric samples from term and preterm infants using an Orbitrap Fusion Lumos mass spectrometer. Samples were collected from nine preterm-delivering and four term-delivering mother-infant pairs. Our study reveals an increased count and ion abundance of peptides and decreased peptide length from mother's milk to the infant stomach, confirming that additional break-down of the milk proteins occurred in both preterm and term infants' stomachs. Protein digestion occurred at a higher level in the gastric contents of term infants than in gastric contents of preterm infants. An amino acid cleavage site-based enzyme analysis suggested that the observed higher proteolysis in the term infants was due to higher pepsin/cathepsin D activity in the stomach. Additionally, there was a higher quantity of antimicrobial peptides in term infant gastric contents than in those of preterm infants, which could indicate that preterm infants benefit less from bioactive peptides in the gut.

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

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.

Peptidomics Analysis of Milk Protein-Derived Peptides Released over Time in the Preterm Infant Stomach.

Over the course of milk digestion, native milk proteases and infant digestive proteases fragment intact proteins into peptides with potential bioactivity. This study investigated the release of peptides over 3 h of gastric digestion in 14 preterm infant sample sets. The peptide content was extracted and analyzed from milk and gastric samples via Orbitrap tandem mass spectrometry. The relative ion intensity (abundance) and count of peptides in each sample were compared over time and between infants fed milk fortified with bovine milk fortifier and infants fed unfortified milk. Bioactivity of the identified peptides was predicted by sequence homology to known bioactive milk peptides. Both total and bioactive peptide abundance and count continuously increased over 3 h of gastric digestion. After accounting for infant weight, length, and postconceptual age, fortification of milk limited the release of peptides from human milk proteins. Peptides that survived further gastric digestion after their initial release were structurally more similar to bioactive peptides than nonsurviving peptides. This work is the first to provide a comprehensive profile of milk peptides released during gastric digestion over time, which is an essential step in determining which peptides are most likely to be biologically relevant in the infant. Data are available via ProteomeXchange with identifier PXD012192.

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

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.

Survival of Immunoglobulins from Human Milk to Preterm Infant Gastric Samples at 1, 2, and 3 h Postprandial.

BACKGROUND: Human milk immunoglobulins (Ig) are an important support for the naïve infant immune system; yet the extent to which these proteins survive within the infant digestive tract, particularly for preterm infants, is poorly studied. OBJECTIVES: Our objective was to evaluate the survival of human milk Igs in the preterm stomach across postprandial time. METHODS: Human milk and infant gastric samples were collected from 11 preterm (23-32 weeks gestational age) mother-infant pairs within 7-98 days postnatal age. Preterm gastric samples were collected 1, 2, and 3 h after the beginning of the feeding. Samples were analyzed for concentration of total IgA (secretory IgA [SIgA]/IgA), total secretory component (SC/SIgA/SIgM), total IgM (SIgM/IgM), and IgG via enzyme-linked immunosorbent assay. Ig-chain fragment peptides were determined using peptidomic analysis. One-way analysis of variance with repeated measures followed by Tukey's multiple comparison tests was applied. RESULTS: Concentrations of total IgA were lower in the gastric contents at 3 h postprandial compared with human milk and gastric contents at 1 and 2 h. Human milk SC/SIgA/SIgM, IgG, and total IgM concentrations remained stable in the preterm stomach across postprandial time. Peptide counts from the Ig alpha-chain and the Ig gamma-chain increased in gastric contents from 1 to 2 h postprandial. Peptide counts from the human milk Ig-chain, Ig-chain, and SC were stable across postprandial time. These peptides from Ig-chains were not present in human milk but were released in the stomach due to their partial degradation. CONCLUSIONS: Human milk total SC (SIgA/SC/SIgM), total IgM, and IgG survived mostly intact through the preterm infant stomach, while total IgA was -partially digested.

Comparison of Human Milk Immunoglobulin Survival during Gastric Digestion between Preterm and Term Infants.

Human milk provides immunoglobulins (Igs) that supplement the passive immune system of neonates; however, the extent of survival of these Igs during gastric digestion and whether this differs between preterm and term infants remains unknown. Human milk, and infant gastric samples at 2 h post-ingestion were collected from 15 preterm (23⁻32 week gestational age (GA)) mother-infant pairs and from 8 term (38⁻40 week of GA) mother-infant pairs within 7⁻98 days postnatal age. Samples were analyzed via ELISA for concentration of total IgA (secretory IgA (SIgA)/IgA), total secretory component (SC/SIgA/SIgM), total IgM (SIgM/IgM), and IgG as well as peptidomics. Total IgA concentration decreased by 60% from human milk to the preterm infant stomach and decreased by 48% in the term infant stomach. Total IgM and IgG concentrations decreased by 33% and 77%, respectively, from human milk to the term infant stomach but were stable in the preterm infant stomach. Release of peptides from all Ig isotypes in the term infant stomach was higher than in the preterm stomach. Overall, the stability of human milk Igs during gastric digestion is higher in preterm infant than in term infants, which could be beneficial for assisting the preterm infants' immature immune system.

Peptides Released from Foremilk and Hindmilk Proteins by Breast Milk Proteases Are Highly Similar.

Human milk contains active proteases that initiate hydrolysis of milk proteins within the mammary gland. Milk expressed at the beginning of feeding is known as foremilk and that at the end of feeding is known as hindmilk. As hindmilk contains higher fat, vitamins A and E, and higher calories than foremilk, feeding only hindmilk initially and reserving foremilk for later are practiced in some neonatal intensive care units. This study investigated the difference in peptide profiles, predicted milk protease activities, and bioactive peptides between foremilk and hindmilk. Bioactive peptides are short fragments of proteins that influence biological processes. Four mothers pumped 10 mL of their foremilk and 10 mL of their hindmilk into iced containers prepared with antiproteases and the samples were immediately frozen. The peptide profile of each sample was analyzed by liquid chromatography nano-electrospray ionization Orbitrap Fusion tandem mass spectrometry. Peptide abundance (sum of ion intensities) and count (number of unique peptide sequences) in each milk sample were determined from this analysis. The specific enzymes that participated in peptide release were predicted based on the amino acids positioned at each cleavage site. Peptide bioactivity was predicted based on homology to a known functional peptide database and two bioactivity prediction algorithms. Hindmilk contained a higher count of peptides than foremilk. The higher number of unique peptide sequences in hindmilk was related to hydrolysis of ß-casein, osteopontin, αs1-casein and mucin-1 via plasmin and elastase cleavage, and possible aminopeptidase and carboxypeptidase activities. Though hindmilk contained a greater number of peptides than foremilk, the overall peptide abundance did not differ and most of the total peptide abundance derived from peptide sequences that were present in both milk types. The presence of higher numbers of predicted bioactive peptides in the hindmilk could indicate that the practice of providing hindmilk rather than foremilk to premature infants could positively impact health outcomes; however, as there are few differences in overall peptide abundance, the overall effect is likely limited.

Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization.

During processing and digestion, milk proteins are disassembled into peptides with an array of biological functions, including antimicrobial, angiotensin-converting enzyme inhibition, antioxidant, opioid, and immunomodulation. These functions are summarized in numerous reviews, yet information on which peptides have which functions remains scattered across hundreds of research articles. We systematically searched the literature for all instances of bioactive peptides derived from milk proteins from any mammalian source. The data were compiled into a comprehensive database, which can be used to search for specific functions, peptides, or proteins (http://mbpdb.nws.oregonstate.edu). To review this large dataset, the bioactive peptides reported in the literature were visually mapped on the parent protein sequences, providing information on sites with highest abundance of bioactive peptides.

Milk Proteins Are Predigested Within the Human Mammary Gland.

Previous work demonstrates that proteases present in human milk release hundreds of peptides derived from milk proteins. However, the question of whether human milk protein digestion begins within the mammary gland remains incompletely answered. The primary objective of this study was to determine whether proteolytic degradation of human milk proteins into peptides begins within the mammary gland. The secondary objectives were to determine which milk proteases participate in the proteolysis and to predict which released peptides have bioactivity. Lactating mothers (n = 4) expressed their milk directly into a mixture of antiproteases on ice followed by immediate freezing of the milk to limit post-expression protease activity. Samples were analyzed for their peptide profiles via mass spectrometry and database searching. Peptidomics-based protease prediction and bioactivity prediction were each performed with several different approaches. The findings demonstrate that human milk contains more than 1,100 unique peptides derived from milk protein hydrolysis within the mammary gland. These peptides derived from 42 milk proteins and included 306 potential bioactive peptides. Based on the peptidomics data, plasmin was predicted to be the milk protease most active in the hydrolysis of human milk proteins within the mammary gland. Milk proteases actively cleave milk proteins within the mammary gland, initiating the release of functional peptides. Thus, the directly breastfed infant receives partially pre-digested proteins and numerous bioactive peptides.

Domain analysis of a modular alpha-L-Arabinofuranosidase with a unique carbohydrate binding strategy from the fiber-degrading bacterium Fibrobacter succinogenes S85.

Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique ß-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k(cat) for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the K(m) for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.