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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.
... To address some of these knowledge gaps, recently Dallas and coworkers investigated peptides released inside the gastrointestinal tract and/or present in the stool of infants fed milk (Beverly et al., 2020;Beverly, Huston, et al., 2021). By analyzing stool samples with an untargeted nanoLC-ESI-MS/MS approach, authors identified 8132 peptides, among which 118 exclusively deriving from milk CNs and α-La, including molecules with known or potential antimicrobial properties eventually active within the final tract of the intestine (Supplementary Table 5). ...
... When stool samples were compared with corresponding gastric counterparts according to peptide abundance and count values, it was evident that molecular levels decreased from stomach to stool. To test the hypotheses that the milk peptide content of stools is different between preterm and term infants at different days of life, authors also performed a comparative peptidomic analysis between the different groups based on corresponding peptide counts and abundances (Beverly et al., 2020). Although many individual milk peptides were significantly different between preterm infants at 8/9 and 21/22 days of life, and between preterm and term infants, total peptide abundance and count values were similar for all groups. ...
... In addition to blood, urine and milk, other biofluids and tissues have the potential to provide important physiological information related to food-derived peptides. In this context, peptidomic studies reporting analysis of human stool samples from individuals fed specific diets have been recently reported (Sillner et al., 2019;Beverly et al., 2020;Beverly, Huston, et al., 2021). Generally, stool samples for peptidomic analysis were prepared by carefully dissolving material in water and removing solid, cell debris and lipid contaminants by centrifugation. ...
Background
Various bioactive peptides are present in foods and food protein hydrolysates, or are generated in the stomach/intestine of organisms after digestion of dietary proteins. Those resisting gastrointestinal degradation can exert local effects in the gut or systemic effects in the organism body as result of their transport across the intestinal epithelium in the bloodstream, and subsequent adsorption in various organs. For most of these molecules, no concentration data regarding body fluids/tissues are available; this information is essential to rationalize their bioavailability and putative bioactivity.
Scope and approach
The main purpose of this study is to provide an exhaustive overview of the bioactive food-derived peptides identified in the gastrointestinal tract, blood, body tissues, urine, breastmilk and feces of animal models or humans fed specific diets, as well as a description of the adsorption mechanisms and metabolic processes eventually affecting their fate. Untargeted and targeted peptidomic methods used for their quali-quantitative description are also reported, together with recent technological advances that have partially solved various analytical challenges in this research field and have disclosed future promising scenarios in nutrition and physiology.
Key findings and conclusions
Available information emphasizes that organism tissues/body fluids are pervaded of food-derived species resulting from the digestion of dietary proteins, including some already proved having a specific biological activity. For some for which blood concentration was measured, ascertained data highlight levels in the nanomolar range, which are lower than those generally used for in vitro functional assays. Conversely, few peptides have shown concentration values compatible with a substantial molecular bioavailability and a putative bioactivity. Thus, it remains uncertain if the presence of bioactive food-derived peptides in the body fluids/tissues can be associated with a significant functional effect. Accordingly, the actual study of these exogenous peptides in the human body is more relevant than ever, with the ultimate aim of tangling the complex relationship between diet and health.
... Human LF is more resistant to gastric digestion than bovine LF, but both are rapidly proteolyzed in the intestine (Zhao et al., 2023). Approximately 2-6 % of the LF obtained from breastmilk is secreted in infants faeces, decreasing with maturation of the infant's gastrointestinal tract (Beverly et al., 2020;Davidson & Lonnerdal, 1987;Lönnerdal, 2017). However, at acidic pH, LF loses part of its three-dimensional structure, and this unfolding may expose specific regions of the protein, making them more susceptible to enzymatic digestion; as a result, its bacteriostatic activity may be reduced (Abdallah & el Hage Chahine, 2000;Goulding et al., 2021;Sreedhara et al., 2010;Wang et al., 2017). ...
... The formation of ~ 48-and ~ 41-kDa fragments during peptic and tryptic digestion of LF was consistent with the reports of Goulding (2021) and Lönnerdal et al. (2021), and warrants further investigation into potential biofunctional properties. Other studies conducted in vivo demonstrated that human LF partially resists infant digestion (Beverly et al., 2020;Davidson & Lonnerdal, 1987). In addition, human LF has been shown to be more resistant to gastric digestion than bovine LF (Lönnerdal et al., 2011;Zhao et al., 2023). ...
olloidal structure and infant in vitro gastrointestinal digestibility of casein complexes simulating caseins and minerals compositions of human casein micelles were studied. κ- and β-Caseins were fractionated from bovine micellar casein concentrate (MCC), and mixed with MCC to increase their ratios to 20% and 68%, followed by adding citrate (Cit), calcium (Ca), and inorganic phosphate (Pi) to obtain the casein complexes. The complexes enriched with κ- and β-caseins showed higher percentages of serum Ca, Pi and caseins, larger particle size, and looser internal structure. During gastric digestion, the complexes enriched with κ- and β-caseins formed smaller and looser coagula, which promoted degradation of intact caseins and formation of free amino groups and small peptides. During initial intestinal digestion, the complexes enriched with κ- and β-caseins formed more free amino groups and smaller peptides. These results offered a potential strategy to form human casein micelles analogues for use in infant formula.
... In agreement with our findings, other studies have shown lower proteolytic capacity of gastric enzymes for human milk proteins in preterm infants compared to full-term infants 288 . Milk peptides, including caseins, can survive gastrointestinal digestion, which we could confirm in our study in a quantitative and longitudinal manner 324 . As milk peptides are an important source of peptides and amino acids for rapidly growing infants, the impaired degradation and/or absorption of human milk proteins in preterm infants could have serious consequences on energy acquisition and subsequent growth in early life 325 . ...
... Our findings confirmed the survival of (maternal) immunoglobulins as indicated by the persistent presence of them in the metaproteomes of gastric aspirates and feces of (pre)term infants (chapter 3). Congruently, immunoglobulins-particularly the secretory component, IgM and IgGhave previously been reported to remain intact throughout the gastrointestinal tract of preterm infants 324,438 . The work within this thesis described the identification of fragments of IgA, IgM and IgG in gastric aspirates and feces, although their persistence was not assessed. ...
... The overall abundant phosphorylation of β-casein is thought to contribute to these functions. Moreover, there has also been antimicrobial functionality attributed to the C-terminal peptide derived from βcasein, ( 200 QELLLNPTHQYPVTQPLAPVHNPISV 226 ), in recent human milk peptidomics studies [14,[21][22][23][24][25][26]. Additionally, this peptide has been found to survive infant digestion, indicating that it could exert antimicrobial functionality in the distal part of the infant gut [24]. ...
... The overall abundant phosphorylation of β-casein is thought to contribute to these functions. Moreover, there has also been antimicrobial functionality attributed to the C-terminal peptide derived from β-casein, (200QELLLNPTHQYPVTQPLAPVHNPISV226), in recent human milk peptidomics studies [14,[21][22][23][24][25][26]. Additionally, this peptide has been found to survive infant digestion, indicating that it could exert antimicrobial functionality in the distal part of the infant gut [24]. ...
Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.
... Several peptidomic studies have revealed that hundreds of bioactive peptides are released in human milk and the stomach of breast milk-fed infants [27,33]. Furthermore, bioactive milk peptides have been identified to survive to infant stool [34], but little is known about the presence or activity of bioactive peptides inside the infant intestinal tract. Bioactive peptides present in the intestinal tract have the highest potential to positively impact infant health, either by absorption into the infant's circulation or by local activity on the intestinal cells and bacteria. ...
... Peptides from 20 µL of each intestinal sample were extracted as described above and dissolved in 20 µL of nanopure water after freeze drying for LC-MS analysis. LC-MS was performed as previously described [34] with the following change: as a number of the infants were fed milk with Neosure® bovine-based fortifier, peptides were identified using Proteome Discoverer 2.2.0.388 with a Sequest HT search against a database that contained both human and bovine milk proteins. Dynamic peptide modifications only included phosphorylation of serine and threonine and oxidation of methionine. ...
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.
... However, comparisons of preterm and term infants are sparse, even using in vitro and animal models. The only comparisons of total milk peptide release between preterm and term infants were performed on human milk and infant stool [16,17], and only milk immunoglobulin peptides have been compared in preterm and term gastric samples [18]. ...
... Peptidomics enables determination of specifically how proteins are digested within in vivo samples. Peptidomics has previously been applied to identify milk peptides in human milk [16,30,31], infant formula [32], preterm infant gastric samples [13][14][15], and infant stool samples [17]. However, to date, no peptidomics studies have been performed to compare milk protein digestion and the release of bioactive peptides within the stomachs of term and preterm infants. ...
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 breakdown 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.
... DD results from the "diaper environment", including irritant exposure (urine, feces), over-hydration, altered skin pH, stool composition, chronological and gestational age, barrier immaturity, antibiotic therapy, and systemic illnesses [7][8][9][10][11][12][13]. Fecal proteases and lipases can cause DD via damage to epidermal proteins and lipids [8-11, 14, 15]. Protease distributions in stool vary with GA and postnatal age [16], e.g., elastase is lower for infants <28 weeks at postnatal week 2 but comparable across gestations by week six [17]. Stool frequency and perianal erythema severity were positively associated with increasing GA and enteral feeds (type, volume) [6]. ...
Objective
Quantify the evolution and severity of neonatal skin injury, specifically diaper skin compromise, by embedding a validated skin integrity evaluation into the electronic health record (EHR).
Methods
Retrospective longitudinal cohort analysis of 747 patients stratified by gestation: 22–27, 28–31, 32–24, and 35–37 weeks, from birth to discharge. Primary outcomes were time to first perineal erythema, duration as percent days with erythema, and severity as maximum score. Data were analyzed using generalized linear models and multiple linear regression methods.
Results
Seventy percent had erythema and, of these, 34% had at least one high score with bleeding. Days with erythema ranged from 34–44% (p < 0.05). Days to first erythema were inversely correlated with gestational age. Risks for severe injury included short time to first erythema, 5 or more stools/day, infection, and Caucasian race/ethnicity.
Conclusions
The EHR-based scale can be readily implemented to mitigate diaper skin compromise in premature infants.
... From the above data, the chain length of peptides with molecular weight less than 1000 Da is less than nine (Table S1), and this characteristic of chain length has two positive effects on the α-glucosidase inhibitory activity of peptides. First, because the α-glucosidase inhibitor peptide exerts its action at the brush border of small intestinal epithelial cells [26], the transport of peptides with chain length longer than four on small intestinal epithelial cells is usually transcytosis and passive transcellular diffusion, with limited transport efficiency [27,28]. This is conducive to the accumulation of peptides at the brush edge of small intestinal epithelial cells, and a greater probability of contact with α-glucosidase plays an inhibitory function. ...
Hydrolysates of coix seed prolamins (CHPs) have an excellent hypoglycemic effect and can effectively inhibit α-glucosidase, which is the therapeutic target enzyme for type 2 diabetes mellitus. However, its hypoglycemic components and molecular mechanisms remain unclear, and its stability in food processing needs to be explored. In this study, four potential α-glucosidase inhibitory peptides (LFPSNPLA, FPCNPLV, HLPFNPQ, LLPFYPN) were identified and screened from CHPs using LC-MS/MS and virtual screening techniques. The results of molecular docking showed that the four peptides mainly inhibited α-glucosidase activity through hydrogen bonding and hydrophobic interactions, with Pro and Leu in the peptides playing important roles. In addition, CHPs can maintain good activity under high temperatures (40~100 °C) and weakly acidic or weakly alkaline conditions (pH 6.0~8.0). The addition of glucose (at 100 °C) and NaCl increased the inhibitory activity of α-glucosidase in CHPs. The addition of metal ions significantly decreased the inhibitory activity of α-glucosidase by CHPs, and their effects varied in magnitude with Cu2+ having the largest effect followed by Zn2+, Fe3+, K+, Mg2+, and Ca2+. These results further highlight the potential of CHPs as a foodborne hypoglycemic ingredient, providing a theoretical basis for the application of CHPs in the healthy food industry.
... The initially published MBPDB included 737 unique peptide sequence-function combinations from 606 unique peptide sequences originating from the milks of ten different species . Use of the MBPDB allowed identification of hundreds of peptides with high homology to known bioactive milk peptides in human milk and infant formula (Nielsen et al. 2018) and in infant gastric digesta, intestinal digesta and stool after consuming human milk (Nielsen et al. 2020;Beverly et al. 2021Beverly et al. , 2020. ...
Partial digestion of milk proteins leads to the formation of numerous bioactive peptides. Previously, our research team thoroughly examined the decades of existing literature on milk bioactive peptides across species to construct the milk bioactive peptide database (MBPDB). Herein, we provide a comprehensive update to the data within the MBPDB and a review of the current state of research for each functional category from in vitro to animal and clinical studies, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, antioxidant, dipeptidyl peptidase (DPP)-IV inhibitory, opioid, anti-inflammatory, immunomodulatory, calcium absorption and bone health and anticancer activity. This information will help drive future research on the bioactivities of milk peptides.
... Commensal microbiota colonize the postnatal infant gastrointestinal tract (GIT) to participate in gut tissue maturation, interconnected metabolic operations, immunological development, and other functions that contribute to host homeostasis. 1 In addition to direct nourishment of the infant, exogenous molecules dissolved in milk interact with autochthonous microbial populations along the GIT to modulate host physiology. Accordingly, infant-indigestible human milk glycans, 2 and potentially other solutes such as peptides, 3 dictate the form and function of the infant gut microbiome. In turn, compounds derived from microbial metabolism (e.g. ...
Human milk guides the structure and function of microbial commensal communities that colonize the nursing infant gut. Indigestible molecules dissolved in human milk establish a microbiome often dominated by bifidobacteria capable of utilizing these substrates. Interestingly, urea accounts for ~15% of total human milk nitrogen, representing a potential reservoir for microbiota that may be salvaged for critical metabolic operations during lactation and neonatal development. Accordingly, B. infantis strains are competent for urea nitrogen utilization, constituting a previously hypothetical phenotype in commensal bacteria hosted by humans. Urease gene expression, downstream nitrogen metabolic pathways, and enzymatic activity are induced during urea utilization to yield elevated ammonia concentrations. Moreover, biosynthetic networks relevant to infant nutrition and development are transcriptionally responsive to urea utilization including branched chain and other essential amino acids. Importantly, isotopically labeled urea nitrogen is broadly distributed throughout the expressed B. infantis proteome. This incisively demonstrates that the previously inaccessible urea nitrogen is incorporated into microbial products available for infant host utilization. In aggregate, B. infantis possesses the requisite phenotypic foundation to participate in human milk urea nitrogen recycling within its infant host and thus may be a key contributor to nitrogen homeostasis early in life.
... Moreover, the partial digestion of many milk proteins releases an array of peptides. Infant gastric and intestinal digestion releases a large array of peptides highly homologous with known bioactive peptides with antimicrobial, calcium-binding, antihypertensive, immunomodulatory, and opioid activity [94][95][96][97][98][99] . This work suggests that milk is providing more that simple AA nourishment. ...
... Many of these proteins are resistant to proteolysis, which, together with a developing neonatal digestive system, facilitates the survival of biologically active forms of milk protein in the infant gastrointestinal tract [17,18]. It has been described how IgA from serum prevents infection of the intestinal mucosa by binding microorganisms and preventing them from being transported through the mucosa [19,20], just as it modulates the function and integrity of the gastrointestinal tract [21][22][23]. ...
Breast milk is a complex and dynamic biological fluid and considered an essential source of nutrition in early life. In its composition, the proteins have a relevant biological activity and are related to the multiple benefits demonstrated when compared with artificial milks derived from cow’s milk. Understanding human milk composition provides an important tool for health care providers toward the management of infant feeding and the establishment of breastfeeding. In this work, a new technique was developed to increase the knowledge of human milk, because many of the components remain unknown. To isolate minor proteins present in breast milk by using WGA lectin, breast milk was centrifuged to remove cells and separate the fat phase from the serum phase. The serum obtained was separated into two groups: control (n = 3; whole serum sample from mature milk) and WGA lectin (n = 3; sample processed with WGA lectin to isolate glycosylated proteins). The samples were analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). A total of 84 different proteins were identified from all of the samples. In the WGA lectin group, 55 different proteins were isolated, 77% of which had biological functions related to the immune response. Of these proteins, there were eight WGA lectin group exclusives, and two had not previously been described in breast milk (polyubiquitin-B and POTE ankyrin domain family member F). Isolation by WGA lectin is a useful technique to detect minor proteins in breast milk and to identify proteins that could not be observed in whole serum.
... Our research group has identified thousands of human milk-protein-derived peptides in human infant gastric, 2 intestinal, 3 and stool samples. 4 Human milk-protein-derived peptides exhibit an array of bioactivities, including immunomodulatory activity, antimicrobial activity, and angiotensin-converting enzyme (ACE)inhibitory activity. 5 Hundreds of milk-protein-derived peptides have been identified in infant digestive samples by mass spectrometry, and many of these peptides are highly homologous with known bioactive peptides when their homology is assessed against a database of known functional peptidesthe Milk Bioactive Peptide Database. ...
Human milk-protein-derived peptides exhibit an array of bioactivities. Certain bioactivities cannot be exerted unless the peptides are absorbed across the gastrointestinal lumen into the bloodstream. The purpose of study was to determine which peptides derived from in vitro digestion of human milk could cross human intestinal Caco-2 cell monolayers. Our results showed that the numbers of peptides absorbed by the Caco-2 cell monolayer were different at different concentrations (44 peptides out of 169 peptides detected at 10 μg/mL, 124 peptides out of 204 peptides detected at 100 μg/mL, and 175 peptides out of 236 peptides detected at 1000 μg/mL). Four peptides (NLHLPLP (β-casein [138-144]), PLAPVHNPI (β-casein [216-224]), PLMQQVPQPIPQ (β-casein [148-159]), and FDPQIPK (β-casein [126-132])) crossed to the basolateral chamber of the Caco-2 monolayer incubated with peptides at all three concentrations. Among the peptides identified in the basolateral chambers, three peptides (NLHLPLP (β-casein [138-144]), LENLHLPLP (β-casein [136-144]), and QVVPYPQ (β-casein [182-188])) are known ACE-inhibitors; one peptide (LLNQELLLNPTHQIYPV (β-casein [197-213])) is antimicrobial, and another peptide (QVVPYPQ (β-casein [182-188])) has antioxidant activity. These findings indicate that specific milk peptides may be able to reach the bloodstream and exert bioactivity.
... The susceptibility of α-La to hydrolysis by digestive enzymes is still controversial and debated (Dupont & Tomé, 2020). Several α-La peptides have been identified in the stool of lactating infants, suggesting that they might be scarcely degraded and adsorbed (Beverly, Huston, Markell, McCulley, Martin, & Dallas, 2020). In general, the degradation of α-La and formation of corresponding peptides was reported being highly dependent on the digestive conditions as well as on the other concomitant nutrients, leading to inter-and intra-individual fluctuations. ...
A dedicated two-step purification procedure prior to nanoliquid chromatography-electrospray-tandem mass spectrometry analysis enabled the identification of bovine milk-derived peptides absorbed and circulating in the plasma of three healthy volunteers who received 250 mL of pasteurized milk after a 10-days washout. The appearance and clearance of milk peptides in plasma were monitored at various time points. Overall, 758, 273 and 212 unique peptides derived from 15, 15 and 18 bovine milk proteins, respectively, were identified in the plasma of these volunteers, evidencing a substantial inter-individual variability. Peptides encrypting possible bioactive and/or immunogenic molecules originating from caseins, β-lactoglobulin and minor milk proteins were detected. Peptide representation data revealed the combined action of endoproteases involved in primary hydrolysis during gastroduodenal digestion and exopeptidases that hydrolyse peptides in the small intestine. It remains to be established whether the half-life and concentration ranges of circulating milk-derived peptides may have any impacts on human health.
... Proteins in human milk are related to many biological processes, including assistance in nutrient uptake [17], immune function and playing extensive roles in cellular processes and defense mechanisms in infants [18]. Moreover, some bioactivities are only exerted when protein fragments are released as bioactive peptides following enzymatic digestion by a variety of proteases either present in the milk or secreted by the infant's gastrointestinal tract [19]. For instance, Singh et al. [20] identified the function of human milk-derived biologically active b-casomorphins peptides in insulin regulation and b-cell mass regeneration. ...
Purpose of review:
Preterm birth is one of the most pressing clinical problems in obstetrics and neonatology worldwide. One of the most sophisticated components of human milk is the proteome and a better understanding of it can lead to precision guides for feeding preterm infants. In this review, we will examine recent research focused on the human milk proteome and individualized protein fortification of human milk.
Recent findings:
In both preterm and term birth, the protein content in mother's own milk dropped rapidly in the early postnatal period. Preterm milk had a higher protein content and contained different protein and endogenous peptide compositions compared with term milk. The peptides in gastrointestinal fluids of preterm infants still need further investigation. Individualized fortification is more superior to standard fortification, but it only focuses on the total protein amount. There is no data concerning the composition and posttranslational modifications of proteins and endogenous peptides with fortification and their long-term effects.
Summary:
Comprehensive identification and characterization of the human milk proteome have led to the targeted breast milk fortification theory of preterm infant feeding and has also been enriched by clinical trials. However, to achieve fortification of key proteins and/or endogenous peptides, as standard clinical practice, requires additional studies. Future research should explore the long-term effect of protein fortification and pay more attention to quality rather than quantity in relation to infant body composition and growth outcomes.
... In agreement with our findings, other studies have shown lower proteolytic capacity of gastric enzymes for human milk proteins in preterm infants compared to term infants 6 . Milk peptides, including caseins, can survive gastrointestinal digestion 29 , which we could confirm in our study in a quantitative and longitudinal manner. As milk peptides are an important source of peptides and amino acids for rapidly growing infants 30 , the impaired degradation and/or absorption of human milk proteins in preterm infants could have serious consequences on energy acquisition and subsequent growth in early life. ...
Functionality of the gastrointestinal tract is essential for growth and development of newborns. Preterm infants have an immature gastrointestinal tract, which is a major challenge in neonatal care. This study aims to improve the understanding of gastrointestinal functionality and maturation during the early life of preterm infants by means of gastrointestinal enzyme activity assays and metaproteomics. In this single-center, observational study, preterm infants born between 24 and 33 weeks (n = 40) and term infants born between 37 and 42 weeks (n = 3), who were admitted to Isala (Zwolle, the Netherlands), were studied. Enzyme activity analyses identified active proteases in gastric aspirates of preterm infants. Metaproteomics revealed human milk, digestive and immunological proteins in gastric aspirates of preterm infants and feces of preterm and term infants. The fecal proteome of preterm infants was deprived of gastrointestinal barrier-related proteins during the first six postnatal weeks compared to term infants. In preterm infants, bacterial oxidative stress proteins were increased compared to term infants and higher birth weight correlated to higher relative abundance of bifidobacterial proteins in postnatal week 3 to 6. Our findings indicate that gastrointestinal and beneficial microbial proteins involved in gastrointestinal maturity are associated with gestational and postnatal age.
... Besides their critical importance in terms of nutrition and source of amino acids, proteins have potential effects on the developing microbiota of neonates. Although milk protein digestibility is very high, undigested dietary protein or fractions of these proteins, associated with endogenous proteins may be fermented by preterm gut microbiota (Boudry et al., 2016;Beverly et al., 2019). Indeed, for low birth weight piglets characterized with intestinal immaturity, the quantity of protein intake, specifically when fed formula with high protein level, induced sustained modifications of gut microbiota composition in the first and 5 months of age (Chatelais et al., 2011;Boudry et al., 2013). ...
Gut microbiota and the central nervous system have parallel developmental windows during pre and post-natal life. Increasing evidences suggest that intestinal dysbiosis in preterm infants predisposes the neonate to adverse neurological outcomes later in life. Understanding the link between gut microbiota colonization and brain development to tailor therapies aimed at optimizing initial colonization and microbiota development are promising strategies to warrant adequate brain development and enhance neurological outcomes in preterm infants. Breast-feeding has been associated with both adequate cognitive development and healthy microbiota in preterms. Infant formula are industrially produced substitutes for infant nutrition that do not completely recapitulate breast-feeding benefices and could be largely improved by the understanding of the role of breast milk components upon gut microbiota. In this review, we will first discuss the nutritional and bioactive component information on breast milk composition and its contribution to the assembly of the neonatal gut microbiota in preterms. We will then discuss the emerging pathways connecting the gut microbiota and brain development. Finally, we will discuss the promising microbiota modulation-based nutritional interventions (including probiotic and prebiotic supplementation of infant formula and maternal nutrition) for improving neurodevelopmental outcomes.
... A small percentage of proteins in food is not absorbed and is eliminated as such with feces (1%-5% depending on food/protein). To this purpose, milk specific peptides have been recently characterized in the stool of both preterm and term infants, sampled at 8/9 and 20/21 days of life, confirming that fragments of dietary proteins can escape complete degradation and cross the entire gastrointestinal tract, regardless of the maturation of the digestive apparatus (Beverly et al., 2019). ...
Digestomics can be defined as the systematic characterization of the biomolecular complement produced by food digestion. Protein digestomics, that essentially corresponds to tracking the fate of dietary proteins in human body, assumes special relevance because food-derived peptides are virtually capable of inducing positive (health-promoting) or negative (allergy, adverse reactions) effects. In general, food digestion generates extremely heterogeneous mixtures, which include free amino acids and variously sized (poly) peptides, in addition to products from other macro- and micronutrients.
Modern proteomics and peptidomics have enabled the comprehensive study of complex digestomes. On the other hand, several gaps still need to be bridged to define kinetics and dynamics of food-derived peptides, also as a result of food structure, in order to ultimately assess the effects of dietary proteins on human health. This chapter surveys the recent advances in the comprehension of mechanisms of food degradation, highlighting research trends and still unaddressed issues.
... The initially published MBPDB included 737 unique peptide sequence-function combinations from 606 unique peptide sequences originating from the milks of ten different species . Use of the MBPDB allowed identification of hundreds of peptides with high homology to known bioactive milk peptides in human milk and infant formula (Nielsen et al. 2018) and in infant gastric digesta, intestinal digesta and stool after consuming human milk (Nielsen et al. 2020;Beverly et al. 2021Beverly et al. , 2020. ...
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.
... Many of these proteins are resistant to proteolysis, which, together with a developing neonatal digestive system, facilitates the survival of biologically active forms of milk protein in the infant gastrointestinal tract [17,18]. It has been described how IgA from serum prevents infection of the intestinal mucosa by binding microorganisms and preventing them from being transported through the mucosa [19,20], just as it modulates the function and integrity of the gastrointestinal tract [21][22][23]. ...
Contents
The mammalian oviduct is an anatomical part of the female reproductive tract, which plays several important roles in the events related to fertilization and embryo development. This review examines and compares several studies related to the proteomic and transcriptomic profile of the oviduct in different domestic animals. This information could be important for clarifying the role of oviductal factors in different events regulating fertilization and early embryo development, as well as for improving synthetic media for in vitro maturation/ in vitro fertilization/embryo culture techniques (IVM/IVF/EC).
α-Lactalbumin (α-LA) is an active protein with multiple biological functions, which can provide the body with abundant essential amino acids, such as Trp and Lys. It is the main whey protein in human milk, accounting for about 22% of its total protein, and provides energy and nutrition for infants’ body and brain development. However, the α-LA in bovine milk is relatively low, so bovine α-LA is often added as a supplement to improve α-LA content and the balance of amino acids in infant formula. In addition, more studies have focused on the health benefits of α-LA in humans, proposing novel physiological roles or supplementing more substantial research evidence, including improving metabolism-related chronic diseases, promoting intestinal health, strengthening bone and muscle health, reducing oxidative stress, delaying aging, and enhancing sleep and cognitive performance. Nonetheless, a considerable proportion of the research remains confined to cell and animal experiments, and it remains uncertain whether these findings will be replicated in humans, and at what dosage. This review describes the basic properties and the structural characteristics focusing on α-LA and specifically discusses its digestion and absorption in infants, adults, and the elderly. Further, the review summarizes the beneficial effects and mechanisms of action of α-LA supplementation in both infants and adults, as well as the future applications of α-LA are also discussed.
Background: β-casein is the main casein constituent in human milk (HM) and a source of bioactive peptides for the developing gastrointestinal tract and immune system. Infant formulas contain less β-casein than HM, but whether different levels of β-casein affect tolerability and gut and immune maturation in newborns is unknown.
Objective: Using near-term piglets as a model for newborn infants, we investigate whether increasing the β-casein fraction in bovine-based formula is clinically safe and may improve gut and immune maturation.
Methods: Three groups of near-term pigs (96% gestation) were fed formula with bovine casein and whey protein (ratio 40:60): 1) Standard skim milk casein (BCN-standard, 35% β-casein of total casein, n=18), 2) β-casein enrichment to HM levels (BCN-medium, 65%, n=19), and 3) high β-casein enrichment (BCN-high, 91%, n=19). A reference group was fed 100% whey protein concentrate as protein (WPC, n=18). Intestinal and immune parameters were assessed before and after euthanasia on day 5.
Results: Clinical variables (mortality, activity, body growth, diarrhea) were similar among the groups, and no differences in intestinal or biochemical parameters were observed between BCN-standard and BCN-medium pigs. However, pigs receiving high amounts of β-casein (BCN-high) had lower small intestine weight, and tended to have more intestinal complications (highest gut pathology score, permeability, IL-8) than the other groups, particularly those receiving no casein (WPC pigs). Blood lymphocyte, thrombocyte and reticulocyte counts were increased with higher β-casein, while eosinophil counts were reduced. In vitro blood immune cell responses were similar among groups.
Conclusions: β-casein enrichment of bovine-based formula to HM levels is clinically safe, as judged from newborn, near-term pigs, while no additional benefits to gut maturation were observed. However, excessive β-casein supplementation, beyond levels in HM, may potentially induce gut inflammation together with increased blood cell populations relative to natural β-casein levels or pure whey-based formula.
The centrifugation presterilizing UHT (C-UHT) sterilization method removes 90% of the microorganism and somatic cells from raw milk using high-speed centrifugation following UHT treatment. This study aimed to study the changes in protein composition and plasmin in the UHT and C-UHT milk. The digestive characteristics, composition, and peptide spectrum of milk protein sterilized with the 2 technologies were studied using a dynamic digestive system of a simulated human stomach. The Pierce bicinchoninic acid assay, laser scanning confocal microscope, liquid chromatography-tandem mass spectrometry, and AA analysis were used to study the digestive fluid at different time points of gastric digestion in vitro. The results demonstrated that C-UHT milk had considerably higher protein degradation than UHT milk. Different processes resulted during the cleavage of milk proteins at different sites during digestion, resulting in different derived peptides. The results showed there was no significant effect of UHT and C-UHT on the peptide spectrum of milk proteins, but C-UHT could release relatively more bioactive peptides and free AA.
Human breast milk is the optimal nutrition for all infants and is comprised of many bioactive and immunomodulatory components. The components in human milk, such as probiotics, human milk oligosaccharides (HMOs), extracellular vesicles, peptides, immunoglobulins, growth factors, cytokines, and vitamins, play a critical role in guiding neonatal development beyond somatic growth. In this review, we will describe the bioactive factors in human milk and discuss how these factors shape neonatal immunity, the intestinal microbiome, intestinal development, and more from the inside out.
As a subset of the proteome, peptidomics is the best tool to analyze peptides in different food matrices. Milk peptides are produced by protease hydrolysis in the mammary gland, which plays an important role in the growth and development of infants. However, methods of probing endogenous peptides are limited because the concentration of peptides is low, the peptides are easily lost during extraction/enrichment, and database searches are complicated. Based on the peptidomics analysis process, this paper reviews the research progress and applications of endogenous peptides with a focus on extraction, fractionation, identification, and bioinformatics analysis. Additionally, in-depth analysis of endogenous peptides in milk and milk-related products using joint strategies (a combined analysis of proteome/peptidomics/metabolomic approaches) was also introduced. New methodologies are provided to study peptides through bioinformatics and tracking the release of bioactive peptides from various milk sources by in vitro digestion models, which can better track the utilization of endogenous peptides in vivo. Challenges and future perspectives are also discussed, with a goal of exploring more bioactive peptides in milk and dairy products as well as simulating human milk in formula milk powder in the future.
Background and aims
Plasmin in human milk partially hydrolyzes milk proteins within the mammary gland and may enhance the hydrolysis of milk proteins within the infant’s stomach. This study examined the effects of extremely preterm (EP)-, very preterm (VP)-, and term-delivery on plasmin activity and the concentrations of plasminogen activators [urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA)], plasminogen activator inhibitor type 1 (PAI-1) and the complexes of PAI-1/uPA and PAI-1/tPA in human milk.
Materials and methods
Human milk samples were collected from mothers who delivered extremely preterm infants [24–27 weeks gestational age (GA), n = 20], very preterm infants (28–32 weeks GA, n = 12), and term infants (38–39 weeks GA, n = 8) during 2–72 days postnatally. Plasmin activity was determined using fluorometric substrate assay, whereas concentrations of uPA, tPA, PAI-1, the PAI-1/uPA complex and the PAI-1/tPA complex were quantified by ELISA.
Results
Plasmin activity, uPA and tPA were detected in all human milk samples, PAI-1 and the PAI-1/uPA complex were present in 42.5 and 32.5% of milk samples, respectively, and the PAI-1/tPA complex was not detected. Plasmin activity was correlated negatively with postnatal age and postmenstrual age (PMA) in the VP group and positively with postnatal age in the term group. uPA and tPA concentrations decreased with increasing postnatal age in both EP and VP groups but did not correlate in the term group. uPA concentration was correlated positively with GA in the VP group and tended to be elevated with increasing GA in the combined three groups. In contrast, tPA concentrations were correlated negatively with GA and PMA in the combined three groups ( P < 0.008) and with PMA in the EP and VP groups. PAI-1 concentration tended to be correlated positively with postnatal age in the combined three groups. No correlation was detected with the PAI-1/uPA complex.
Conclusion
Premature delivery impacted the plasmin activity and the concentrations of uPA, tPA, and PAI-1 in human milk. Whether these changes in milk plasminogen activators and inhibitors have a role in balancing the proteolytic digestion of premature infants remains to be investigated.
Potential endogenous hypoglycemic peptides derived from breast milk were screened by in silico approaches against intestinal glucose absorption- and metabolism-related membrane proteins (i.e., SGLT1, ATPase, and GPR40), and their inhibitory effects on glucose uptake were compared using the human intestinal epithelial Caco-2 cell model. A total of 762 endogenous peptides were obtained from breast milk, and 5 peptides (YPFVEPIPYGFL, LLNQELLLNPTHQIYPV, SPTIPFFDPQIPK, QHWSYGLRPG, and YPVTQPLAPVHNPIS) were shortlisted based on PeptideRanker and HPEPDOCK scores. Further flow cytometer analysis of 2-NBDG uptake showed the remarkable ability of these five peptides to inhibit glucose uptake, in particular YPVTQPLAPVHNPIS. More importantly, the in silico and in vitro gastrointestinal digestion of YPVTQPLAPVHNPIS combined with LC-QTOF-MS/MS demonstrated that the resulting hexapeptide PVTQPL had strong inhibitory activity on glucose uptake and transport (57% and 13% inhibition, respectively). Molecular docking indicated that PVTQPL bound to SGLT1 by forming two hydrogen bonds with Trp257 through the NH2 group and Ile253 through the carbonyl group, ATPase with Phe139 via one arene-H interaction, and GPR40 with Thr39, Ser41, Arg104, Arg2218 and Arg2221 residues through eight hydrogen interactions of its carbonyl groups and hydroxyl groups. The findings of this work open up the possibility of employing endogenous peptides from human milk as the hypoglycemic compounds for the prevention and treatment of diabetes.
This study aimed to explore nutritional compositions and proteomics of soft-shelled turtle (SST) egg, as well as identify potential antidiabetic oligopeptides with α-glucosidase inhibitory property. Results revealed that SST egg is a promising source of highly nutritious proteins and minerals (54.64% and 5.81% of dry matter, respectively). Further proteomic analysis showed SST egg proteins contained at least 9 protein families, such as transferrin/iron binding protein and immunoregulation-related protein. Hydrolysis by different enzymes, especially papain, remarkably increased α-glucosidase inhibitory activity and scavenging activity for ABTS, DPPH, hydroxyl and oxygen radicals of SST egg proteins. Peptides from papain hydrolysate were fractionated using ultrafiltration followed by reverse phase chromatography, and 16 peptides were identified in the most active fraction by LC-QTOF-MS/MS. Molecular docking revealed that 14 of these peptides could easily dock into the substrate-binding pocket and/or inhibitor binding sites of α-glucosidase with the docking score below -150 kcal/mol, indicating their potential α-glucosidase inhibitory properties. The five most abundant oligopeptides with potent interaction with α-glucosidase were further synthesized, and oligopeptides HNKPEVEVR, ARDASVLK and SGTLLHK strongly inhibited the activity of α-glucosidase (IC50 of 56, 195 and 289 µmol/L, respectively). Therefore, oligopeptides from enzymatic hydrolysate of SST egg protein exhibit potential antidiabetic activity, making it a promising functional food ingredient.
The gastrointestinal tract represents a specialized interface between the organism and the external environment. Because of its direct contact with lumen substances, the modulation of digestive functions by dietary substances is supported by a growing body of evidence. Food-derived bioactive peptides have demonstrated a plethora of activities in the organism with increasing interest towards their impact over the digestive system and related physiological effects. This review updates the biological effects of food proteins, specifically milk and soybean proteins, associated to gastrointestinal health and highlights the study of digestion products and released peptides, the identification of the active form/s, and the evaluation of the mechanisms of action underlying their relationship with the digestive cells and receptors. The approach towards the modifications that food proteins and peptides undergo during gastrointestinal digestion and their bioavailability is a crucial step for current investigations on the field. The recent literature on the regulation of digestive functions by peptides has been mostly considered in terms of their influence on gastrointestinal motility and signaling, oxidative damage and inflammation, and malignant cellular proliferation. A final section regarding the actual challenges and future perspectives in this scientific topic is critically discussed. This article is protected by copyright. All rights reserved.
Background and 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 β-casein that is conserved from milk through stool and from which are released known and potential antimicrobial peptides.
BACKGROUND
Milk proteins are widely used in food production and are often glycated by reducing sugar. Although many studies have reported the digestibility of glycated milk protein, most have focused on measuring degree of hydrolysis (DH), showing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) image of digests. Detailed information on the changes in peptide composition of digests has seldom been revealed. Therefore, in addition to measuring the DH and showing the SGS‐PAGE images of digests, we also analyzed the peptidomics in digests using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) and Mascot database in this work to further reveal the influence of glycation on protein nutrition.
RESULTS
Compared with β‐lactoglobulin and bovine serum albumin (BSA), DH of β‐casein was suppressed to a lesser extent by glycation in both gastric and intestinal stages. Aggregates of glycated BSA were less sensitive to the action of digestive enzymes throughout gastrointestinal digestion according to SDS‐PAGE images. Changes in the peptide composition of digests induced by glycation were distinctly displayed, showing both absence of peptides and occurrence of new peptides, based on the results obtained from LC‐ESI‐MS/MS.
CONCLUSIONS
Glycation can greatly change the peptide composition in digests of milk protein. The nutritional impact of the change in the peptide composition requires further investigation, and the impact of MRPs in unabsorbed digests on the gut flora should be an interesting field for further studies. © 2018 Society of Chemical Industry
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.
The gastrointestinal tract houses millions of microbes, and thus has evolved several host defense mechanisms to keep them at bay, and prevent their entry into the host. One such mucosal surface defense is the secretion of secretory immunoglobulins (SIg). Secretion of SIg depends on the polymeric immunoglobulin receptor (pIgR), which transports polymeric Ig (IgA or IgM) from the basolateral surface of the epithelium to the apical side. Upon reaching the luminal side, a portion of pIgR, called secretory component (SC) is cleaved off to release Ig, forming SIg. Through antigen-specific and non-specific binding, SIg can modulate microbial communities and pathogenic microbes via several mechanisms: agglutination and exclusion from the epithelial surface, neutralization, or via host immunity and complement activation. Given the crucial role of SIg as a microbial scavenger, some pathogens also evolved ways to modulate and utilize pIgR and SIg to facilitate infection. This review will cover the regulation of the pIgR/SIg cycle, mechanisms of SIg-mediated mucosal protection as well as pathogen utilization of SIg.
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.
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.
The total IgA, IgG, IgM and lactoferrin concentrations in human milk from 89 donors were studied at three lactational stages: early transitional (3 to 8 d postpartum), transitional (10 to 14 d postpartum) and mature (30 to 47 d postpartum). The effects of processing and storage on these components in composite samples of mature human milk were determined. There were no significant diurnal variations in any of the four protective factors at either the transitional or mature stages. Concentrations of total IgA, IgM and lactoferrin decreased significantly as time postpartum increased, whereas the IgG content showed no significant changes. The total IgA, IgM and lactoferrin levels were significantly decreased by all heat treatments (62.5°C for 30 min, 72°C for 15 s, 88°C for 5 s, and 100°C for 5 min). Heating at 62.5°C for 30 min did not affect the IgG content; however, the other heat treatments significantly reduced IgG concentration. At the times and temperatures selected for this study, the two lower tempe...
Bioactive milk peptides are reported to illicit a range of physiological benefits and have been proposed as potential functional food ingredients. The objective of this study was to characterize the anti-inflammatory properties of sodium caseinate (NaCAS), its enzyme hydrolysate (EH) and peptide-enriched fractions (5 kDa retentate [R], 1 kDaR and 1 kDa permeate [P]), both in vitro using a Caco-2 cell line, and also ex vivo using a porcine colonic tissue explant system. Caco-2 cells were stimulated with tumour necrosis factor alpha (TNFα) and co-treated with casein hydrolysates for 24 h. Following this, interleukin (IL)-8 concentrations in the supernatant were measured using enzyme-linked immunosorbent assay. Porcine colonic tissue was stimulated with lipopolysaccharide and co-treated with casein hydrolysates for 3 h. The expression of a panel of inflammatory cytokines was measured using qPCR. While dexamethasone reduced the IL-8 concentration by 41.6%, the 1 kDaR and 1 kDaP fractions reduced IL-8 by 68.7% and 66.1%, respectively, relative to TNFα-stimulated Caco-2 cells (P < 0.05). In the ex vivo system, only the 1 kDaR fraction elicited a decrease in IL1-α, IL1-β, IL-8, TGF-β and IL-10 expression (P < 0.05). This study provides evidence that the bioactive peptides present in the 1 kDaR fraction of the NaCAS hydrolysate possess anti-inflammatory properties in vitro and ex vivo. Further in vivo analysis of the anti-inflammatory properties of the 1 kDaR is proposed.
Lactoferrin (Lf) is an iron-binding glycoprotein of the transferrin family, which is expressed in most biological fluids with particularly high levels in mammalian milk. Its multiple activities lie in its capacity to bind iron and to interact with the molecular and cellular components of hosts and pathogens. Lf can bind and sequester lipopolysaccharides, thus preventing pro-inflammatory pathway activation, sepsis and tissue damages. Lf is also considered a cell-secreted mediator that bridges the innate and adaptive immune responses. In the recent years much has been learned about the mechanisms by which Lf exerts its activities. This review summarizes the recent advances in understanding the mechanisms underlying the multifunctional roles of Lf, and provides a future perspective on its potential prophylactic and therapeutic applications.
In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray liquid chromatography separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from β-casein. The numbers of peptides from β-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile salt-associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688).
Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus. The pre-digestion of milk proteins and the consequent release antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection.
Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin.
Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals for which various biological functions have
been reported, such as antimicrobial activity and bifidogenic activity. In this study, we compared the bifidogenic activity
of bovine lactoferrin (bLF) and pepsin hydrolysate of bLF (bLFH), isolated bifidogenic peptide from bLFH, and investigated
the bifidogenic spectra of bLF, bLFH, and its active peptide against 42 bifidobacterial strains comprising nine species. Against
Bifidobacterium breve ATCC 15700T, minimal effective concentrations of bLF and bLFH were 300 and 10 μg/ml. Against Bifidobacterium longum subsp. infantis ATCC 15697T, the minimal effective concentration of bLFH was 30 μg/ml, and bLF did not show bifidogenic activity within 300 μg/ml. As
an active peptide, a heterodimer of A1-W16 and L43-A48 linked by a disulfide bond was isolated. Previously, this peptide was identified as having antibacterial activity. An amino
acid mixture with the same composition as this peptide showed no bifidogenic activity. The strains of each species whose growth
was highly promoted (>150%) by this peptide at 3.75 μM were as follows: B. breve (7 out of 7 strains [7/7]), B. longum subsp. infantis (5/5), Bifidobacterium bifidum (2/5), B. longum subsp. longum (1/3), Bifidobacterium adolescentis (3/6), Bifidobacterium catenulatum (1/4), Bifidobacterium pseudocatenulatum (0/4), Bifidobacterium dentium (0/5), and Bifidobacterium angulatum (0/3). Growth of none of the strains was highly promoted by bLF at 3.75 μM. We demonstrated that bLFH showed stronger bifidogenic
activity than natural bLF, especially against infant-representative species, B. breve and B. longum subsp. infantis; furthermore, we isolated its active peptide. This is the first report about a bifidogenic peptide derived from bLF.
The aim of this study was to compare the digestion of milk proteins from different species using an in vitro gastrointestinal model. Raw and heated milks from bovine, caprine, human and equine species were digested by human digestive enzymes. Digestion was performed in two 30-min sequential steps by digestive juices from the stomach (pH 2.5/37 °C) and from the duodenum (pH 8.0/37 °C). The degradation patterns of the milk proteins were visualized by SDS-PAGE and quantified using the ImageQuant program. Caseins in the equine milk were rapidly digested by the gastric juice in contrast to the caseins from the other species. During the subsequent digestion by the duodenal juice most of the caseins from all species were degraded within 5 min, and within 30 min only traces of caseins were detected. The mean casein micellar size varied between species in the range of 146.0–311.5 nm (equine > caprine > bovine > human). The α-lactalbumin from all species appeared to be very resistant to both gastric and duodenal digestions. A similar trend was shown for β-lactoglobulin from bovine and caprine milks, of which ∼ 60% intact protein remained, while only 25% remained intact in equine milk after total digestion. Equine milk contained a high amount of lysozyme, of which 60% remained intact in the present study. In heated milks from all species, only α-lactalbumin degradation increased approximately 12–20% in comparison to the raw milk. This study shows that equine milk with fast digestible proteins could be considered as a replacement for bovine milk in the diet of people with special needs, such as infants and the elderly.
There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial alpha-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial alpha-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.
Plasmin is the major endogenous protease present in milk. The level of plasmin activity is controlled by the availability of the precursor plasminogen and by the levels of plasminogen activators and inhibitors. Recently, a differential distribution of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) has been demonstrated in bovine milk. To assess whether this distribution pattern is a general feature, the occurrence of components of the plasminogen activation system in different fractions of human milk was investigated.
Milk samples were separated into the following fractions; milk fat, skim milk, and milk cells by centrifugation. The different fractions were detected for the presence of plasminogen and plasminogen activators by immunoblotting and zymography. The distribution of t-PA and u-PA was investigated by ligand binding analysis. t-PA-catalyzed plasminogen activation was examined by a coupled chromogenic assay.
A differential distribution of plasminogen, t-PA, and u-PA was found. Casein micelles were found to exhibit t-PA and plasminogen binding activity, whereas the u-PA receptor was identified as the u-PA binding component in the cell fraction. Furthermore, human casein enhanced t-PA-catalyzed plasminogen activation, comparable to the enhancing effect obtained with fibrinogen fragments.
The finding of a differential distribution of u-PA and t-PA in milk suggests that the two activators may have different physiological functions, which involve protection against invading microorganisms and maintenance of patency and fluidity in the ducts of mammary gland, respectively.
In light of the need for new antifungal agents, the candidacidal activities of human lactoferrin (hLF) and synthetic peptides representing the first, hLF(1-11), and second, hLF(21-31), cationic domains of its N terminus were compared. The results revealed that hLF(1-11) was more effective in killing fluconazole-resistant Candida albicans than hLF(21-31) and much more effective than lactoferrin, as determined microbiologically and by propidium iodide (PI) staining. By using hLF(1-11) and various derivatives, it was found that the second and third residues of the N terminus of hLF(1-11) were critical for its candidacidal activity. Detailed investigation to elucidate the mechanism of action of hLF(1-11) revealed a dose-dependent release of ATP by Candida upon exposure to hLF(1-11). Our observations that sodium azide reduced the PI uptake and candidacidal activity of hLF(1-11) and that, upon exposure to hLF(1-11), the fluorescent dye rhodamine 123 first accumulated inside the mitochondria and later was released into the cytoplasm indicate that the peptide triggers the energized mitochondrion. Furthermore, oxidized ATP, which interferes with the interaction of ATP with its extracellular receptors, blocked the candidacidal action of hLF(1-11), as measured microbiologically and by PI staining. Addition of ATP (or analogues) was not a sufficient stimulus to kill C. albicans or to act synergistically with suboptimal concentrations of the peptide. The main conclusions are that the first two arginines at the N terminus of hLF are critical in the candidacidal activity of hLF(1-11) and that extracellular ATP is essential but not sufficient for the peptide to exert its candidacidal activity.
The synthetic peptides Lfpep and kaliocin-1 include the sequences from positions 18 to 40 and 153 to 183 of human lactoferrin,
respectively. Lfpep is a cationic peptide with bactericidal and giardicidal effects, whereas kaliocin-1 is a novel bactericidal
peptide that corresponds to a highly homologous sequence present in the transferrin family of proteins. Both peptides presented
fungicidal activity against Candida spp., including fluconazole- and amphotericin B-resistant clinical isolates. Lfpep exhibited higher antifungal activity (8-
to 30-fold) and salt resistance than kaliocin-1. The killing activity of Lfpep was mediated by its permeabilizing activity
on Candida albicans cells, whereas kaliocin-1 was unable to disrupt the cytoplasmic membrane, as indicated by its inability to allow permeation
of propidium iodide and the small amount of K+ released. The amino acid sequence of kaliocin-1 includes the “multidimensional antimicrobial signature” conserved in disulfide-containing
antimicrobial peptides and a striking similarity to brevinin-1Sa, an antimicrobial peptide from frog skin secretions, exhibiting
a “Rana box”-like sequence. These features may be of interest in the design of new antifungals.
Beyond being a source of key nutrients, bovine milk influences physiological functions by synthesising bioactive peptides during the process of digestion. Some of the claimed negative health outcomes associated with milk consumption, such as cardiovascular diseases and type 1 diabetes may be attributed to an opioid peptide, beta-casomorphin-7 (BCM-7), derived from A1 beta-casein. BCM-7 exerts its function by binding to the μ-opioid receptors in the body. It is hypothesised that activation of the μ-opioid receptors in the gut can alter gut microbial composition, impair gut barrier integrity and bile acid metabolism, in addition to increasing gastrointestinal transit time and gut inflammation. Further, it is hypothesised that BCM-7 may influence fractures and obesity via μ-opioid receptor pathways. In conclusion, it appears that BCM-7 might have multiple functions pertinent to human health; however, the evidence is limited and warrants further pre-clinical and clinical studies for hypothesis confirmation.
Variations in endogenous peptide profiles, functionality, and the enzymes responsible for the formation of these peptides in human milk are understudied. Additionally, there is a lack of knowledge regarding peptides in donor human milk, which is used to feed preterm infants when mother’s own milk is not (sufficiently) available. To assess this, 29 human milk samples from the Dutch Human Milk Bank were analyzed as three groups, preterm late lactation stage (LS) (n=12), term early (n=8) and term late LS (n=9). Gestational age (GA) groups were defined as preterm (24-36 weeks) and term (>37 weeks). LS was determined as days postpartum as early (16-36 days) or late (55-88 days). Peptides, analyzed by LC-MS/MS, and parent proteins (proteins from matched peptide sequences) were identified and quantified, after which peptide functionality and the enzymes responsible for protein cleavage were determined. A total of 16 different parent proteins were identified from human milk, with no differences by GA or LS. We identified 1104 endogenous peptides, of which, the majority were from the parent proteins β-casein, polymeric immunoglobulin receptor, αs1-casein, osteopontin, and κ-casein. The absolute number of peptide differed by GA and LS with 30 and 41 differing sequences respectively (p<0.05) Odds likelihood tests determined that 32 peptides had a predicted bioactive functionality, with no significant differences between groups. Enzyme prediction analysis showed that plasmin/trypsin enzymes most likely cleaved the identified human milk peptides. These results explain some of the variation in endogenous peptides in human milk, leading to future targets that may be studied for functionality.
Objectives:
Our previous studies suggested that human milk proteases begin to hydrolyze proteins in the mammary gland and continue within the term infant stomach. No research has measured milk protease and pepsin activity in the gastric aspirates of preterm infants after human milk feeding. This study investigated how the concentrations of human milk proteases and protease inhibitors changed in the premature infant stomach.
Methods:
Human milk and infant gastric samples were collected from 18 preterm-delivering mother-infant pairs (24-32 wk gestational age). Paired human milk and gastric samples were collected across postnatal age (2-47 days). Protease concentrations were determined by spectrophotometric or fluorometric assays, and the concentrations of protease inhibitors and bioactive proteins were determined by ELISA. Paired t-tests were applied to compare enzymes, antiproteases and bioactive proteins between human milk and gastric samples.
Results:
Our study reveals that although human milk proteases, including carboxypeptidase B2, kallikrein, plasmin, cathepsin D, elastase, thrombin and cytosol aminopeptidase are present in the preterm infant stomach, only plasmin and cathepsin D can actively hydrolyze proteins at gastric pH. ELISA and peptidomic evidence suggest that all milk antiproteases as well as lactoferrin and immunoglobulin A are partially digested in the preterm stomach.
Conclusions:
Most human milk proteases are active in milk but not at preterm infant gastric pH. Only cathepsin D and plasmin have potential to continue degrading milk proteins within the preterm infant stomach.
Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm- delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown.
Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age.
Methods: Milk samples were collected from 18 mothers aged 26–40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24–26 wk GA)-delivering mothers and 9 from late GA (LGA) (27–32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng mL21 d21 and 0.454 mg mL21 d21 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples.
Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not vary substantially across GA and postnatal age.
Microbial colonization of the human gastrointestinal tract plays an important role in establishing health and homeostasis. However, the time-dependent functional signatures of microbial and human proteins during early colonization of the gut have yet to be determined. To this end, we employed shotgun proteomics to simultaneously monitor microbial and human proteins in fecal samples from a preterm infant during the first month of life. Microbial community complexity increased over time, with compositional changes that were consistent with previous metagenomic and rRNA gene data. More specifically, the function of the microbial community initially involved biomass growth, protein production, and lipid metabolism, and then switched to more complex metabolic functions, such as carbohydrate metabolism, once the community stabilized and matured. Human proteins detected included those responsible for epithelial barrier function and antimicrobial activity. Some neutrophil-derived proteins increased in abundance early in the study period, suggesting activation of the innate immune system. Likewise, abundances of cytoskeletal and mucin proteins increased later in the time course, suggestive of subsequent adjustment to the increased microbial load. This study provides the first snapshot of coordinated human and microbial protein expression in a preterm infant's gut during early development. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
DNA-synthesis stimulatory activity was found in some tryptic fragments of human β-casein by using BALB/c3T3 cells, and two of them were identified as the β-casein fragments of Arg[l] to Lys[18] (β-CN(f1-18)) and of Gln[105] to Lys[117] (β-CN(f105-117)).
It has often been stated that premature infants as compared with full-term infants are handicapped in utilization of their food due to relative enzyme deficiency. This has been indicated above all to apply to the proteins, and therefore it has been recommended to supply their ordinary food with casein hydrolysates. In histological and chemical investigations of pancreas from premature infants 0-5 days old, Werner (1948) found that pancreas in infants with a birth weight below 2,000 gm. showed an extremely immature picture with little or no tryptic activity. She therefore concluded that the premature infant, as compared with the full-term infant is very poorly equipped for the task of protein digestion. Gschwind (1950) has investigated the presence of trypsin, lipase, and diastase in pancreas of premature and full-term infants of varying age. Especially the activity of lipase and diastase was reduced, but even the proteolytic activity was decreased. This author
Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile.
This study examines differences in peptides present between milk from women delivering at term and women delivering prematurely.
Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d).
Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides. Conclusion: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.
© 2015 American Society for Nutrition.
Human milk pepsin and pancreatin digests were separated using molecular membrane and reverse phase chromatography. Chemical screening of the resulting fractions using the ORAC antioxidant assay yielded a peptide fraction (PF-23) with high antioxidant activity (5207μM Trolox Equivalents (TE)/g). Tandem mass spectrometry allowed the identification of twenty peptides. Eight small molecular weight peptides from 4 to 6 amino acids were synthesised and screened for antioxidant properties using ORAC and linoleic acid emulsion. On ORAC, the peptides YGYTGA (5169μM TE/mmol) and ISELGW (4479μM TE/mmol) were the most active. At 250μM peptide ISELGW and its derivatives significantly reduced hydroperoxides formed during autoxidation of linoleic acid for 4days at 50°C. Further testing of these peptides may allow their inclusion in infant formulas to reduce the incidence of oxidative stress-mediated diseases in newborns.
1.1. Human lactoferrin in the iron-saturated form was treated with pepsin at pH 3.0, and a fragment with a single iron-binding site was isolated in a 90% pure state.2.2. Its mol. wt was near 33,000.3.3. Its N-terminal residue was alanine, and its iron-binding properties were identical to those of lactoferrin.4.4. The curve showing iron incorporation from lactoferrin and its fragment into reticulocytes showed a biphasic character.5.5. It is concluded that lactoferrin consists of two largely independent parts, which may have arisen by a gene duplication process.
In this study, the hypothesis that food-derived opioid peptides besides β-casomorphin 7 might modulate the production of mucin via a direct action on epithelial goblet cells was investigated in HT29-MTX cells used as a model of human colonic epithelium. Seven milk whey or casein peptides, a human milk peptide, and a wheat gluten-derived peptide with proved or probable ability to bind μ- or δ-opioid receptors were tested on the cell culture. Significantly increased secretion of mucins was found after exposure to six of the assayed peptides, besides the previously described β-casomorphin 7, as measured by an enzyme-linked lectin assay (ELLA). Human β-casomorphin 5 and α-lactorphin were selected to study the expression of mucin 5AC gene (MUC5AC), the HT29-MTX major secreted mucin gene. α-Lactorphin showed increased expression of MUC5AC from 4 to 24 h (up to 1.6-fold over basal level expression), although differences were statistically different only after 24 h of exposure. However, this increased expression of MUC5AC did not reach significance after cell treatment with human β-casomorphin 5. In conclusion, six food-derived peptides have been identifed with described or probable opioid activity that induce mucin secretion in HT29-MTX cells. Concretely, α-lactorphin is able to up-regulate the expression of the major secreted mucin gene encoded by these cells.
The pH of gastric juice, obtained 3 to 4 minutes after birth in 158 unselected neonates, varied between 7.5 and 8.5 in 8 meconium-containing specimens and ranged from 1.4 to 7.8 in 150 meconium-free samples. In mature infants of the latter group, pH was (1) significantly lower after vaginal delivery than after cesarean section; (2) tended to be lower after section preceded by labor than after elective section; and (3) was lowest after precipitate delivery. In premature infants, pH was above 7 regardless of mode of delivery. Three was no correlation between neonatal gastric pH and pH of simultaneously obtained maternal gastric juice, pH of amniotic fluid, pH of cord blood duration of rupture of membranes, birth weight, or Apgar score. It was concluded that the mature human fetus produces gastric acidity in response to stresses associated with labor and vaginal delivery. The possibility of a low gastric pH and the resultant pulmonary damage if aspirated must be considered in the initial care of thew newborn with poor muscle tone or reflex activity as well as in the anesthetic management of neonates undergoing emergency surgery.
The molecular forms of lactoferrin (LF) were examined in stools and urine collected at 2.5 or 5 wk of age from very low birth wt infants fed either a cow's milk formula or a fortified human milk preparation. LF was not found by Western blotting in excreta from infants fed cow's milk. In contrast, intact and fragmented forms of LF were detected in stools and concentrated urine of each infant who received human milk. Only intact LF was detected in the fortified human milk preparation, whereas many types of LF fragments were present in the stools and urine. The approximate molecular wt of the most prominent fragments were 44, 38, 34, and 32 kD. However, the stools also displayed lower molecular wt fragments that were not found in urines of those infants. The LF fragments in those excreta were similar in size to those produced in vitro by limited digestion of apo-LF with trypsin. Furthermore, fragments produced by in vitro proteolysis were immunoreactive in an ELISA for LF. Thus, the fragments of LF in stools of very low birth wt infants fed human milk appeared to be produced by in vivo proteolysis, and the close resemblance between the LF fragments in the stools and urine suggests that the urinary LF fragments originated in the gastrointestinal tract. It remains unclear, however, whether the whole LF molecules that were fragmented were derived solely from ingested LF in human milk or in part from LF produced by the infant in response to human milk feedings.
Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.
Several proteins in human milk are postulated to have physiological functions in the breast-fed infant. Therefore, survival of human milk proteins after passage through the gastrointestinal tract of the breast-fed infant was investigated. Fecal samples were collected from exclusively breast-fed term infants and milk samples from their mothers. Soluble proteins in the feces were extracted and analyzed for total protein, nitrogen, lactoferrin, secretory IgA, serum albumin and lysozyme. Significant amounts of lactoferrin and secretory IgA were excreted by the infants and this excretion decreased throughout the study period in a trend similar to the decreasing milk concentrations of these proteins. Gel filtration demonstrated excreted lactoferrin and secretory IgA to be intact. No serum albumin or lysozyme was detected in the fecal extracts. Crossed immunoelectrophoresis showed three human milk proteins to be present in the feces--the third was identified as alpha 1-antitrypsin. Excretion of these proteins indicates the total protein content of human milk is an over-estimation of the protein nutritionally available to the infant.
. Haneberg, B. and Finne, P. (Department of Pediatrics and the Broegelmann Research Laboratory for Microbiology, The University of Bergen, School of Medicine, Bergen, Norway). Lysozymes in feces from infants and children. Acta Paediatr Scand, 63: 588, 1974.—An agar diffusion technique seemed useful for the assay of lysozyme activity in serum, milk, and extracts of freeze-dried feces. Normal levels of this activity were established before the method was applied to extracts of feces from premature infants, receiving either human milk, with lysoyme activity of its own, or cow's milk with or without added albumen lysozyme. Results of these investigations, in addition to immunologic studies, using antisera to human and albumen lysozymes, indicated that human milk lysozyme and albumen lysozyme in cow's milk formula may pass through the infants' intestinal tract. Albumen lysozyme, however, will most often be inactivated before being excreted with feces. Low fecal pH was found in those receiving albumen lysozyme as well as in those receiving human lysozyme. This may reflect the intestinal activity of the lysozymes. Low intestinal pH, brought about by lactulose in the cow's milk feed, did not in itself lead to increased lysozyme activity. At present, no advantage of adding albumen lysozyme to cow's milk formula has been substantiated.
The susceptibility of lactoferrin in bovine colostrum and human milk to digestion by trypsin and chymotrypsin has been investigated. Neither enzyme had much effect on the lactoferrin-mediated antimicrobial activity of human milk, and the iron binding capacity of lactoferrin in the milk was only slightly reduced. Likewise both enzymes had only a slight effect on the iron-binding capacity of purified lactoferrin. Although iron-free (apo)lactoferrin was slightly more susceptible to digestion, especially by chymotrypsin, than the iron-saturated form, the difference was much less than has been found in earlier studies with other proteins of the transferrin class. In contrast, trypsin destroyed the antimicrobial activity of bovine colostrum, and, in line with earlier studies, appreciably reduced the iron-binding capacity of both colostrum and purified bovine apolactoferrin. Bovine iron-saturated lactoferrin was more resistant to digestion. The unusual resistance of human apolactoferrin to proteolysis may reflect an evolutionary development designed to permit its survival in the gut of the infant.
A hexapeptide obtained from human casein by enzymatic digestion has been purified, sequenced and synthesized; its structure is: Val-Glu-Pro-Ile-Pro-Tyr. In vitro this hexapeptide stimulates the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages. Administered intravenously to adult mice, it enhances the resistance to infection with Klebsiella pneumoniae.
In breast-fed infants, digestion of milk triglycerides, the major source of energy and long-chain polyunsaturated fatty acids, is catalyzed by a concerted action of gastric lipase, colipase-dependent pancreatic lipase, and bile salt-stimulated lipase (BSSL). The major part of BSSL is present in the milk and the lesser part originates in the infant's exocrine pancreas. Gastric lipase is important in initiating digestion of milk fat globule triglycerides in the stomach. BSSL shifts the final products of triglyceride digestion from monoglyceride and free fatty acid (the products of colipase-dependent pancreatic lipase) to glycerol and free fatty acid, which may promote efficient absorption. Moreover, BSSL is likely to promote efficient use of milk cholesteryl- and fat-soluble vitaminesters and long-chain polyunsaturated fatty acids (> C18). The cDNA sequence has shown that BSSL has a unique primary structure. The N-terminal half is highly conserved between species and shows striking homology to typical esterases, for example, acetylcholine esterase. In contrast, the C-terminal half, containing 16 proline-rich repeats of 11 amino acid residues, is unique to BSSL. Using several recombinant variants of BSSL, we have found that these unique repeats and the glycosylation are completely dispensable for activity. Thus all typical properties of BSSL reside in the N-terminal half of the molecule.
IgA antibodies reacting to enteropathogenic Escherichia coli (EPEC) antigens in human colostrum and their role in the inhibition of EPEC adherence to HEp-2 cells were studied. Colostrum IgA was isolated with a Sepharose anti-IgA column. IgA-depleted colostrum lost its inhibitory effect on EPEC adhesion, while the IgA-enriched eluate was a potent adherence inhibitor. The same eluate showed a significant loss of inhibitory activity after absorption with an EPEC strain showing localised adherence (LA+), but no alteration after absorption with an LA- strain. No bands were observed in Western blot analysis with LA+ absorbed eluate and with a crude extract of the EPEC strain, but the eluate absorbed with LA- showed a strong recognition of a 94-kDa band, a molecular weight equivalent to that of intimin. Colostrum antibodies reacting to non-protein antigens were not detected by Western blot analysis. The persistence of anti-EPEC IgA in the gastrointestinal tract was shown by the strong reactivity to the 94-kDa band in Western blot analysis of one mother's colostrum and her infant's faeces. These data confirm the role of colostrum antibodies in protecting the neonate against infections due to EPEC.
The large intestine of breast-fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. In this study we report for the first time the identification of human milk peptides that selectively stimulate the growth of bifidobacteria. Several bifidogenic peptides were purified chromatographically from pepsin-treated human milk and identified as proteolytically generated fragments from the secretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial effects. Hydrolysis of the identified peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss of bifidogenic activity, indicating their potential function in vivo. Sequential comparison revealed a similar structural motif within the identified peptides. A correspondingly designed small peptide (prebiotic lactoferrin-derived peptide-I, PRELP-I) was found to stimulate the growth of bifidobacteria as effectively as the native peptides. The combination of antimicrobial and bifidobacterial growth stimulatory activity in human milk proteins leads to highly specific compounds capable of regulating the microbial composition of infants' large intestine.
This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.
Food-derived peptides stimulate mucin Milk peptides are excreted in infant stool 9
- D Martínez-Maqueda
- B Miralles
- De Pascual-Teresa
- S Reverón
- I Muñoz
- R Recio
Martínez-Maqueda D, Miralles B, De Pascual-Teresa S, Reverón
I, Muñoz R, Recio I. Food-derived peptides stimulate mucin
Milk peptides are excreted in infant stool 9
secretion and gene expression in intestinal cells
secretion and gene expression in intestinal cells. J Agric Food Chem
2012;60(35):8600-5.