Daniela Barile

Sialylated Milk Oligosaccharides Promote Microbiota-Dependent Growth in Models of Infant Undernutrition

Identifying interventions that more effectively promote healthy growth of children with undernutrition is a pressing global health goal. Analysis of human milk oligosaccharides (HMOs) from 6-month-postpartum mothers in two Malawian birth cohorts revealed that sialylated HMOs are significantly less abundant in those with severely stunted infants. To explore this association, we colonized young germ-free mice with a consortium of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted Malawian infant and fed recipient animals a prototypic Malawian diet with or without purified sialylated bovine milk oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent augmentation of lean body mass gain, changed bone morphology, and altered liver, muscle, and brain metabolism in ways indicative of a greater ability to utilize nutrients for anabolism. These effects were also documented in gnotobiotic piglets using the same consortium and Malawian diet. These preclinical models indicate a causal, microbiota-dependent relationship between S-BMO and growth promotion.

Human milk and related oligosaccharides as prebiotics

Human milk oligosaccharides (HMO) are believed to have a range of biological activities beyond providing nutrition to the infant. Principal among these is that they may act as prebiotics. Prebiotics are dietary ingredients, usually oligosaccharides that provide a health benefit to the host mediated by the modulation of the human gut microbiota. While it is clear that such oligosaccharides may have potential applications in infants and adults alike, this potential is limited by the difficulties in manufacturing HMO. Consequently functional alternatives such as galacto-oligosaccharides (GOS) are under investigation. GOS are produced enzymatically from lactose for commercial use in food applications--including addition to infant formulae--as similar to breast milk oligosaccharides, they encourage a gut bacteria population that promotes health and reduces the incidence of intestinal infections. New methods for separation and concentration of complex, breast milk-like oligosaccharides from bovine milk industrial streams that contain only low amounts of these valuable oligosaccharides are providing the opportunity to investigate other viable sources of specific oligosaccharides for use as prebiotics in supplements or food products.

Bovine Milk as a Source of Functional Oligosaccharides for Improving Human Health

Human milk oligosaccharides are complex sugars that function as selective growth substrates for specific beneficial bacteria in the gastrointestinal system. Bovine milk is a potentially excellent source of commercially viable analogs of these unique molecules. However, bovine milk has a much lower concentration of these oligosaccharides than human milk, and the majority of the molecules are simpler in structure than those found in human milk. Specific structural characteristics of milk-derived oligosaccharides are crucial to their ability to selectively enrich beneficial bacteria while inhibiting or being less than ideal substrates for undesirable and pathogenic bacteria. Thus, if bovine milk products are to provide human milk-like benefits, it is important to identify specific dairy streams that can be processed commercially and cost-effectively and that can yield specific oligosaccharide compositions that will be beneficial as new food ingredients or supplements to improve human health. Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk. With further refinements to dairy stream processing techniques and functional testing to identify streams that are particularly suitable for enriching beneficial intestinal bacteria, the future of oligosaccharides isolated from dairy streams as a food category with substantiated health claims is promising.

A Molecular Basis for Bifidobacterial Enrichment in the Infant Gastrointestinal Tract

Bifidobacteria are commonly used as probiotics in dairy foods. Select bifidobacterial species are also early colonizers of the breast-fed infant colon; however, the mechanism for this enrichment is unclear. We previously showed that Bifidobacterium longum subsp. infantis is a prototypical bifidobacterial species that can readily utilize human milk oligosaccharides as the sole carbon source. MS-based glycoprofiling has revealed that numerous B. infantis strains preferentially consume small mass oligosaccharides, abundant in human milks. Genome sequencing revealed that B. infantis possesses a bias toward genes required to use mammalian-derived carbohydrates. Many of these genomic features encode enzymes that are active on milk oligosaccharides including a novel 40-kb region dedicated to oligosaccharide utilization. Biochemical and molecular characterization of the encoded glycosidases and transport proteins has further resolved the mechanism by which B. infantis selectively imports and catabolizes milk oligosaccharides. Expression studies indicate that many of these key functions are only induced during growth on milk oligosaccharides and not expressed during growth on other prebiotics. Analysis of numerous B. infantis isolates has confirmed that these genomic features are common among the B. infantis subspecies and likely constitute a competitive colonization strategy used by these unique bifidobacteria. By detailed characterization of the molecular mechanisms responsible, these studies provide a conceptual framework for bifidobacterial persistence and host interaction in the infant gastrointestinal tract mediated in part through consumption of human milk oligosaccharides.

Current peptidomics: applications, purification, identification, quantification, and functional analysis.

Peptidomics is an emerging field branching from proteomics that targets endogenously produced protein fragments. Endogenous peptides are often functional within the body—and can be both beneficial and detrimental. This review covers the use of peptidomics in understanding digestion, and identifying functional peptides and biomarkers. Various techniques for peptide and glycopeptide extraction, both at analytical and preparative scales, and available options for peptide detection with MS are discussed. Current algorithms for peptide sequence determination, and both analytical and computational techniques for quantification are compared. Techniques for statistical analysis, sequence mapping, enzyme prediction, and peptide function, and structure prediction are explored.

Annotation and structural elucidation of bovine milk oligosaccharides and determination of novel fucosylated structures

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies.

Extensive in vivo Human Milk Peptidomics Reveals Specific Proteolysis Yielding Protective Antimicrobial Peptides

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 predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mothers mammary gland and her nursing offspring from infection.

Neutral and acidic oligosaccharides in Holstein-Friesian colostrum during the first 3 days of lactation measured by high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry

Oligosaccharides (OS) from bovine milk are a class of bioactive molecules that are receiving increasing commercial attention for their potential health benefits. In the present work we measured, comprehensively and systematically, free milk OS in the colostrum of 7 Holstein-Friesian cows during the first 3 d of lactation in 12-h intervals by HPLC-chip/time-of-flight mass spectrometry to determine the biological variation of free milk OS in early lactation. The high sensitivity and resolution of the analytical technique made it possible to monitor all OS species, thus providing a comprehensive and quantitative analysis of OS variations during colostrum production. This study confirmed that although sialyllactose is the major OS in bovine colostrum, several neutral OS species are present in significant abundance even at the third day of lactation. Furthermore, variation in terms of OS species and relative abundances of OS between cows suggest individual animal variation. These variations are likely due to genetic factors because environmental factors such as nutrition, lactation number, and accommodation were the same for all cows. This investigation revealed that colostrum milk from Holstein-Friesian cows is a rich source of neutral and acidic OS for the food and pharmaceutical industries.

The Impact of the Milk Glycobiome on the Neonate Gut Microbiota

Human milk is a complete source of nourishment for the infant. Exclusive breastfeeding not only sustains the infants development but also guides the proliferation of a protective intestinal microbiota. Among the many components of milk that modulate the infant gut microbiota, the milk glycans, which comprise free oligosaccharides, glycoproteins, and glycolipids, are increasingly recognized as drivers of microbiota development and overall gut health. These glycans may display pleiotropic functions, conferring protection against infectious diseases and also acting as prebiotics, selecting for the growth of beneficial intestinal bacteria. The prebiotic effect of milk glycans has direct application to prevention of diseases such as necrotizing enterocolitis, a common and devastating disease of preterm infants. In this article, we review the impact of the human (and bovine) milk glycome on gut health through establishment of a milk-oriented microbiota in the neonate.

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 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).