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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.

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

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

Immunomodulatory proteins from human milk may enhance the protection and development of the infant’s gut. This study compared the immunomodulatory effects of treatment with milk from preterm-(PM) and term-delivering (TM) mothers and pasteurized donor milk (DM) on cytokine gene expression in human macrophage-like cells derived from the monocytic cell line THP-1. The gene expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-12 (p40), IL-10 and GAPDH in macrophages treated with PM, TM and DM at steady and activated (inflammatory) states were measured using real-time reverse transcription-polymerase chain reaction. TNF-α and IL-6 in macrophages (both states) with DM were higher than PM or TM. IL-10 in steady state macrophages with DM was higher than PM whereas DM increased IL-10 in activated macrophages compared with TM. TM increased IL-6 and IL-12 (p40) in steady state macrophages compared with PM. IL-12 (p40) in activated macrophages with TM was higher than PM. IL-10 in steady state macrophages with TM was higher than PM. These results suggest that DM induces higher gene expression of pro-inflammatory and anti-inflammatory cytokines in macrophages compared with PM or TM. PM reduced gene expression of pro-inflammatory cytokines compared with TM, which may decrease the development of necrotizing enterocolitis and systematic inflammation.

Whey protein isolate (WPI) consists of an array of proteins and peptides obtained as a byproduct of the cheesemaking process. Research suggests that WPI, along with its peptides such as glycomacropeptide (GMP), possesses immunomodulatory properties. These properties hold potential for alleviating the adverse effects of inflammatory conditions such as inflammatory bowel disease. Although promising, the immunoregulatory properties of the digested forms of WPI and GMP—those most likely to interact with the gut immune system—remain under-investigated. To address this knowledge gap, the current study examined the effects of in vitro-digested WPI and GMP, in vivo-digested WPI, and undigested WPI and GMP on the secretion of pro-inflammatory cytokines (TNF-α and IL-1β) in lipopolysaccharide-stimulated macrophage-like cells. Our results indicate that digested WPI and GMP reduced the expression of TNF-α and IL-1β, two pro-inflammatory cytokines. Whole WPI had no effect on TNF-α but reduced IL-1β levels. In contrast, in vivo-digested WPI reduced TNF-α but increased IL-1β. Undigested GMP, on the other hand, increased the secretion of both cytokines. These results demonstrate that digestion greatly modifies the effects of WPI and GMP on macrophages and suggest that digested WPI and GMP could help mitigate gastrointestinal inflammation. Further clinical studies are necessary to determine the biological relevance of WPI and GMP digestion products within the gut and their capacity to influence gut inflammation.

Kappa-casein-derived caseinomacropeptide (CMP)—a 64-amino-acid peptide—is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. It also has two major amino acid sequences with different modifications, including glycosylation, phosphorylation, and oxidation. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract, where they can exert these functions. In this study, three solid-phase extraction (SPE) methods—porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), and C18 chromatography—were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples prior to LC-MS/MS acquisition. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples.

Caseinomacropeptide (CMP) is released from bovine kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate. CMP has in vitro anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications, including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of intact CMP. Full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In this study, we developed a top-down glycopeptidomics-based analytical method to profile CMP and CMP-derived peptides using Orbitrap mass spectrometry combined with nano-liquid chromatography with electron-transfer/higher-energy collision dissociation. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) spectra of CMPs were annotated to confirm peptide sequence, glycan composition and other post-translational modifications using automatic data processing. Fifty-one intact CMPs and 159 CMP-derived peptides were identified in four samples (one CMP standard, two commercial CMP products and one whey protein isolate). Overall, this novel approach provides comprehensive characterization of CMP and CMP-derived peptides and glycopeptides, and it can be applied in future studies of product quality, digestive survival and bioactivity.

Lactose intolerance, which affects about 65–75% of the world’s population, is caused by a genetic post-weaning deficiency of lactase, the enzyme required to digest the milk sugar lactose, called lactase non-persistence. Symptoms of lactose intolerance include abdominal pain, bloating and diarrhea. Genetic variations, namely lactase persistence, allow some individuals to metabolize lactose effectively post-weaning, a trait thought to be an evolutionary adaptation to dairy consumption. Although lactase non-persistence cannot be altered by diet, prebiotic strategies, including the consumption of galactooligosaccharides (GOSs) and possibly low levels of lactose itself, may shift the microbiome and mitigate symptoms of lactose consumption. This review discusses the etiology of lactose intolerance and the efficacy of prebiotic approaches like GOSs and low-dose lactose in symptom management.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder marked by chronic abdominal pain, bloating, and irregular bowel habits. Effective treatments are still actively sought. Kappa-casein glycomacropeptide (GMP), a milk-derived peptide, holds promise because it can modulate the gut microbiome, immune responses, gut motility, and barrier functions, as well as binding toxins. These properties align with the recognized pathophysiological aspects of IBS, including gut microbiota imbalances, immune system dysregulation, and altered gut barrier functions. This review delves into GMP’s role in regulating the gut microbiome, accentuating its influence on bacterial populations and its potential to promote beneficial bacteria while inhibiting pathogenic varieties. It further investigates the gut microbial shifts observed in IBS patients and contemplates GMP’s potential for restoring microbial equilibrium and overall gut health. The anti-inflammatory attributes of GMP, especially its impact on vital inflammatory markers and capacity to temper the low-grade inflammation present in IBS are also discussed. In addition, this review delves into current research on GMP’s effects on gut motility and barrier integrity and examines the changes in gut motility and barrier function observed in IBS sufferers. The overarching goal is to assess the potential clinical utility of GMP in IBS management.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that affects 10–15% of the global population and presents symptoms such as abdominal discomfort, bloating and altered bowel habits. IBS is believed to be influenced by gut microbiota alterations and low-grade inflammation. Bovine kappa-casein glycomacropeptide (GMP), a bioactive dairy-derived peptide, possesses anti-adhesive, prebiotic and immunomodulatory properties that could potentially benefit IBS patients. This pilot study investigated the effects of daily supplementation with 30 g of GMP for three weeks on gut health in five people with IBS. We assessed alterations in gut microbiota composition, fecal and blood inflammatory makers, and gut-related symptoms before, during and after the GMP feeding period. The results revealed no changes in fecal microbiota, subtle effects on systemic and intestinal immune makers, and no changes in gut-related symptoms during and after the GMP supplementation. Further research is needed to assess the potential benefits of GMP in IBS patients, including the examination of dosage and form of GMP supplementation.