Premature Infants have Lower Gastric Digestion Capacity for Human Milk Proteins than Term Infants.

OBJECTIVES: Whether premature infants have lower gastric protein digestive capacity than term infants and the extent to which human milk proteases contribute to overall gastric digestion are unknown and were investigated in this study. METHODS: Human milk and infant gastric samples were collected from 16 preterm (24-32 wk gestational age) and 6 term (38-40 wk gestational age) mother-infant pairs within a range of 5 to 42 days postnatal age. For each pair, an aliquot of human milk was adjusted to pH 4.5 and incubated for 2 hours at 37 °C to simulate the gastric conditions without pepsin (milkinc). Their gastric protein digestion capacity was measured as proteolysis (free N-terminals) and protease activities. Two-way analysis of variance followed by Tukey post hoc test was applied to compare measurements between preterm and term infants as well as among human milk, milkinc, and gastric samples. RESULTS: Measurements of gastric protein digestion were significantly lower in preterm infants than term infants. Overall milk protease activity did not differ between human milk samples from term- and preterm-delivering mothers. As protease activity did not increase with simulated gastric incubation, milk proteases likely contributed minimally to gastric digestion. CONCLUSIONS: Preterm infants have lower gastric protein digestion capacity than term infants, which could impair nutrient acquisition. Human milk proteases contribute minimally to overall gastric digestion. The limited activity of milk proteases suggests that these enzymes cannot compensate for the premature infant's overall lower gastric protein digestion.

Changes in Proteases, Antiproteases, and Bioactive Proteins From Mother's Breast Milk to the Premature Infant Stomach.

OBJECTIVE: 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 week 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 enzyme-linked immunosorbent assay. 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. Enzyme-linked immunosorbent assay 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.

Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

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 · mL-1 · d-1 and 0.454 µg · mL-1 · d-1 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.

Probiotic Administration in Infants With Gastroschisis: A Pilot Randomized Placebo-Controlled Trial.

OBJECTIVES: Infants with gastroschisis often require long periods of gastric suctioning and hospitalization. The impact of these interventions on the intestinal microbiota and attempts to alter the microbial community have not been studied. We sought to determine how the intestinal microbiota is influenced by the current treatment of gastroschisis and whether alteration of the intestinal microbiota with a probiotic microbe will influence length of hospitalization. METHODS: We performed a randomized, placebo-controlled pilot study of administration of probiotic Bifidobacterium longum subsp. infantis in 24 infants with gastroschisis. The primary outcome was changes in the fecal microbiota, and the secondary outcome was length of hospital stay. RESULTS: Administration of the probiotic or placebo was well tolerated, even during the period of gastric suctioning. The overall microbial communities were not significantly different between groups, although analysis of the final specimens by family demonstrated higher Bifidobacteriaceae, lower Clostridiaceae, and trends toward lower Enterobacteriaceae, Enterococcaceae, Staphylococcaceae, and Streptococcaceae in the probiotic group. Clinical outcomes, including length of hospital stay, did not differ between groups. CONCLUSIONS: In this pilot study, there was significant in infants with gastroschisis that was partially attenuated by the administration of B longum subsp. infantis.

Following the digestion of milk proteins from mother to baby.

Little is known about the digestive process in infants. In particular, the chronological activity of enzymes across the course of digestion in the infant remains largely unknown. To create a temporal picture of how milk proteins are digested, enzyme activity was compared between intact human milk samples from three mothers and the gastric samples from each of their 4-12 day postpartum infants, 2 h after breast milk ingestion. The activities of 7 distinct enzymes are predicted in the infant stomach based on their observed cleavage pattern in peptidomics data. We found that the same patterns of cleavage were evident in both intact human milk and gastric milk samples, demonstrating that the enzyme activities that begin in milk persist in the infant stomach. However, the extent of enzyme activity is found to vary greatly between the intact milk and gastric samples. Overall, we observe that milk-specific proteins are cleaved at higher levels in the stomach compared to human milk. Notably, the enzymes we predict here only explain 78% of the cleavages uniquely observed in the gastric samples, highlighting that further investigation of the specific enzyme activities associated with digestion in infants is warranted.

A peptidomic analysis of human milk digestion in the infant stomach reveals protein-specific degradation patterns.

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 LC 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 ß-casein. The numbers of peptides from ß-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).