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David A Mills - One of the best experts on this subject based on the ideXlab platform.
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digestion of human Milk Oligosaccharides by bifidobacterium breve in the premature infant
Journal of Pediatric Gastroenterology and Nutrition, 2017Co-Authors: Mark A. Underwood, Carlito B Lebrilla, David A Mills, Jasmine C C Davis, Karen M Kalanetra, Sanjay Gehlot, Sanjay Patole, Daniel J Tancredi, Karen SimmerAbstract:Objective The aim of this study was to measure consumption and absorption of human Milk Oligosaccharides (HMOs) in a cohort of premature infants treated with probiotic Bifidobacterium breve. Methods Twenty-nine premature infants (median gestational age 28 weeks, range 23-32 weeks) cared for in the neonatal intensive care unit of the King Edward and Princess Margaret Hospital in Perth, Australia, were treated with B breve at a dose of 1.66 billion organisms per day. Samples of feces, urine, and Milk were obtained at initiation of the probiotic and again 3 weeks later. 16S ribosomal RNA from the feces was analyzed by next-generation sequencing. Quantitation of HMO content of the Milk, urine, and feces was performed using nano-high-performance liquid chromatography-chip/time-of-flight mass spectrometry. Results There was heterogeneity in colonization with bifidobacteria. "Responders" received Milk with higher percentages of fucosylated HMOs and had higher percentages of bifidobacteria and lower percentages of Enterobacteriaceae in their feces than "nonresponders." Several individual HMOs in the Milk were associated with changes in fecal bifidobacteria over time. Changes over time in Milk, fecal, and urine HMOs suggested heterogeneity among HMO structures in consumption by microbes in the gut lumen and absorption from the intestine. Conclusions Colonization of the premature infant intestinal tract with probiotic B breve is influenced by prebiotic HMOs. B breve is a selective consumer of HMOs in the premature infant.
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human Milk Oligosaccharides in premature infants absorption excretion and influence on the intestinal microbiota
Pediatric Research, 2015Co-Authors: Mark A. Underwood, Bruce J German, David A Mills, Karen M Kalanetra, Stephanie C Gaerlan, Maria Lorna A De Leoz, Lauren M Dimapasoc, Danielle G Lemay, Carlito B LebrillaAbstract:Human Milk Oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota
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human Milk Oligosaccharides in premature infants absorption excretion and influence on the intestinal microbiota
Pediatric Research, 2015Co-Authors: Mark A. Underwood, Bruce J German, David A Mills, Karen M Kalanetra, Stephanie C Gaerlan, Maria Lorna A De Leoz, Lauren M Dimapasoc, Danielle G Lemay, Carlito B LebrillaAbstract:Human Milk Oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis (NEC) and sepsis, and the influence of HMOs on the microbiota is unclear.Milk, urine, and stool specimens from 14 mother-premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing to complement a previous analysis.Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces, and Milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes were noted in premature infants of nonsecretor mothers. Specific HMO structures in the Milk, urine, and feces were associated with alterations in fecal Proteobacteria and Firmicutes.HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and NEC, while other HMOs may increase dysbiosis in this population.
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bifidobacteria isolated from infants and cultured on human Milk Oligosaccharides affect intestinal epithelial function
Journal of Pediatric Gastroenterology and Nutrition, 2012Co-Authors: Maciej Chichlowski, Bruce J German, Guillaume De Lartigue, Helen E Raybould, David A MillsAbstract:Objectives Human Milk Oligosaccharides (HMO) are the third most abundant component of breast Milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in select bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of select bifidobacteria on HMO stimulates the intestinal epithelium.
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release and utilization of n acetyl d glucosamine from human Milk Oligosaccharides by bifidobacterium longum subsp infantis
Anaerobe, 2012Co-Authors: Daniel Garrido, Santiago Ruizmoyano, David A MillsAbstract:Human Milk contains high amounts of complex Oligosaccharides, which can be utilized especially by Bifidobacterium species in the infant gut as a carbon and energy source. N-acetyl-d-glucosamine is a building block of these Oligosaccharides, and molecular details on the release and utilization of this monosaccharide are not fully understood. In this work we have studied some of the enzymatic properties of three N-acetyl-β-D-hexosaminidases encoded by the genome of the intestinal isolate Bifidobacterium longum subsp. infantis ATCC 15697 and the gene expression of the corresponding genes during bacterial growth on human Milk Oligosaccharides. These enzymes belong to the glycosyl hydrolase family 20, with several homologs in bifidobacteria. Their optimum pH was 5.0 and optimum temperature was 37 °C. The three enzymes were active on the GlcNAcβ1-3 linkage found in lacto-N-tetraose, the most abundant human Milk oligosaccharide. Blon_0459 and Blon_0732, but not Blon_2355, cleaved branched GlcNAcβ1-6 linkages found in lacto-N-hexaose, another oligosaccharide abundant in breast Milk. Bifidobacterium infantis N-acetyl-β-D-hexosaminidases were induced during early growth in vitro on human Milk Oligosaccharides, and also during growth on lacto-N-tetraose or lacto-N-neotetraose. The up-regulation of enzymes that convert this monosaccharide into UDP-N-acetylglucosamine by human Milk Oligosaccharides suggested that this activated sugar is used in peptidoglycan biosynthesis. These results emphasize the complexity of human Milk oligosaccharide consumption by this infant intestinal isolate, and provide new clues into this process.
Lars Bode - One of the best experts on this subject based on the ideXlab platform.
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immunological effects of human Milk Oligosaccharides
Frontiers in Pediatrics, 2018Co-Authors: Vassilis Triantis, Lars Bode, R Joost J Van NeervenAbstract:Human Milk Oligosaccharides (HMOs) comprise a group of structurally complex, unconjugated glycans that are highly abundant in human Milk. HMOs are minimally digested in the gastrointestinal tract and reach the colon intact, where they shape the microbiota. A small fraction of HMOs is absorbed, reaches the systemic circulation, and is excreted in urine. HMOs can bind to cell surface receptors expressed on epithelial cells and cells of the immune system and thus modulate neonatal immunity in the infant gut, and possibly also sites throughout the body. In addition, they have been shown to act as soluble decoy receptors to block the attachment of various microbial pathogens to cells. This review summarizes the current knowledge of the effects HMOs can have on infections, allergies, auto-immune diseases and inflammation, and will focus on the role of HMOs in altering immune responses through binding to immune-related receptors.
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overcoming the limited availability of human Milk Oligosaccharides challenges and opportunities for research and application
Nutrition Reviews, 2016Co-Authors: Lars Bode, Anthony R Prudden, Geert-jan Boons, Daniela Barile, Nikhat Contractor, Nicola L B Pohl, Yong Su Jin, Stefan JenneweinAbstract:Human Milk Oligosaccharides (HMOs) are complex sugars highly abundant in human Milk but currently not present in infant formula. Rapidly accumulating evidence from in vitro and in vivo studies, combined with epidemiological associations and correlations, suggests that HMOs benefit infants through multiple mechanisms and in a variety of clinical contexts. Until recently, however, research on HMOs has been limited by an insufficient availability of HMOs. Most HMOs are found uniquely in human Milk, and thus far it has been prohibitively tedious and expensive to isolate and synthesize them. This article reviews new strategies to overcome this lack of availability by generating HMOs through chemoenzymatic synthesis, microbial metabolic engineering, and isolation from human donor Milk or dairy streams. Each approach has its advantages and comes with its own challenges, but combining the different methods and acknowledging their limitations creates new opportunities for research and application with the goal of improving maternal and infant health.
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The functional biology of human Milk Oligosaccharides
Early Human Development, 2015Co-Authors: Lars BodeAbstract:Human Milk Oligosaccharides (HMOs) are a group of complex sugars that are highly abundant in human Milk, but currently not present in infant formula. More than a hundred different HMOs have been identified so far. The amount and composition of HMOs are highly variable between women, and each structurally defined HMO might have a distinct functionality. HMOs are not digested by the infant and serve as metabolic substrates for select microbes, contributing to shape the infant gut microbiome. HMOs act as soluble decoy receptors that block the attachment of viral, bacterial or protozoan parasite pathogens to epithelial cell surface sugars, which may help prevent infectious diseases in the gut and also the respiratory and urinary tracts. HMOs are also antimicrobials that act as bacteriostatic or bacteriocidal agents. In addition, HMOs alter host epithelial and immune cell responses with potential benefits for the neonate. The article reviews current knowledge as well as future challenges and opportunities related to the functional biology of HMOs.
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human Milk Oligosaccharides protect against enteropathogenic escherichia coli attachment in vitro and epec colonization in suckling mice
Journal of Pediatric Gastroenterology and Nutrition, 2014Co-Authors: Carolin F Manthey, Chloe Autran, Lars Eckmann, Lars BodeAbstract:Breast-feeding reduces the risk of enteric bacterial infections in newborns in part because of human Milk Oligosaccharides (HMOs), complex glycans that are present in human Milk, but not in infant formula. Enteropathogenic Escherichia coli (EPEC) are attaching/effacing pathogens that cause serious diarrheal illness with potentially high mortality in infants. We isolated HMOs from pooled human Milk and found that they significantly reduce EPEC attachment to cultured epithelial cells. In suckling mice, administration of HMOs significantly reduced colonization with EPEC compared with untreated controls. These data suggest an essential role for HMOs in the prevention of EPEC infections in human infants.
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human Milk Oligosaccharides protect bladder epithelial cells against uropathogenic escherichia coli invasion and cytotoxicity
The Journal of Infectious Diseases, 2014Co-Authors: Ann Lin, Chloe A. Autran, Lars Bode, Sophia D Espanola, Victor NizetAbstract:The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human Milk Oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants.
Carlito B Lebrilla - One of the best experts on this subject based on the ideXlab platform.
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digestion of human Milk Oligosaccharides by bifidobacterium breve in the premature infant
Journal of Pediatric Gastroenterology and Nutrition, 2017Co-Authors: Mark A. Underwood, Carlito B Lebrilla, David A Mills, Jasmine C C Davis, Karen M Kalanetra, Sanjay Gehlot, Sanjay Patole, Daniel J Tancredi, Karen SimmerAbstract:Objective The aim of this study was to measure consumption and absorption of human Milk Oligosaccharides (HMOs) in a cohort of premature infants treated with probiotic Bifidobacterium breve. Methods Twenty-nine premature infants (median gestational age 28 weeks, range 23-32 weeks) cared for in the neonatal intensive care unit of the King Edward and Princess Margaret Hospital in Perth, Australia, were treated with B breve at a dose of 1.66 billion organisms per day. Samples of feces, urine, and Milk were obtained at initiation of the probiotic and again 3 weeks later. 16S ribosomal RNA from the feces was analyzed by next-generation sequencing. Quantitation of HMO content of the Milk, urine, and feces was performed using nano-high-performance liquid chromatography-chip/time-of-flight mass spectrometry. Results There was heterogeneity in colonization with bifidobacteria. "Responders" received Milk with higher percentages of fucosylated HMOs and had higher percentages of bifidobacteria and lower percentages of Enterobacteriaceae in their feces than "nonresponders." Several individual HMOs in the Milk were associated with changes in fecal bifidobacteria over time. Changes over time in Milk, fecal, and urine HMOs suggested heterogeneity among HMO structures in consumption by microbes in the gut lumen and absorption from the intestine. Conclusions Colonization of the premature infant intestinal tract with probiotic B breve is influenced by prebiotic HMOs. B breve is a selective consumer of HMOs in the premature infant.
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human Milk Oligosaccharides in premature infants absorption excretion and influence on the intestinal microbiota
Pediatric Research, 2015Co-Authors: Mark A. Underwood, Bruce J German, David A Mills, Karen M Kalanetra, Stephanie C Gaerlan, Maria Lorna A De Leoz, Lauren M Dimapasoc, Danielle G Lemay, Carlito B LebrillaAbstract:Human Milk Oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota
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Detection of Milk Oligosaccharides in plasma of infants
Analytical and Bioanalytical Chemistry, 2014Co-Authors: L. Renee Ruhaak, Carol Stroble, Mark A. Underwood, Carlito B LebrillaAbstract:Human Milk Oligosaccharides (HMO) are one of the major components of human Milk. HMO are non-digestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of Milk Oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3′SL, 6′SL, 3′SLN, and 6′SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine Milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast Milk. Our results indicate that free MO similar in structure to those found in human Milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.
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novel high molecular weight fucosylated Milk Oligosaccharides identified in dairy streams
PLOS ONE, 2014Co-Authors: Raj Mehra, Daniela Barile, Carlito B Lebrilla, Mariarosaria Marotta, Caroline S Chu, Bruce J GermanAbstract:Oligosaccharides are the third largest component in human Milk. This abundance is remarkable because Oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, Oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant Milk formula, the technologies to reproduce the variety and complexity of human Milk Oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable Oligosaccharides. Recent research has demonstrated that bovine Milk and whey permeate also contain Oligosaccharides. Thus, a thorough characterization of Oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine Milk Oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called "mother liquor", and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant Oligosaccharides present in human Milk. Notably 6 Oligosaccharides contained fucose, a sugar monomer that is highly abundant in human Milk, but is rarely observed in bovine Milk. This work shows that dairy streams represent a potential source of complex Milk Oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human Milk.
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annotation and structural elucidation of bovine Milk Oligosaccharides and determination of novel fucosylated structures
Glycobiology, 2013Co-Authors: Danielle Aldredge, Carlito B Lebrilla, Maria R Geronimo, Serenus Hua, Charles C Nwosu, Daniela BarileAbstract: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 structurerelated 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 isomerspecific 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.
Bruce J German - One of the best experts on this subject based on the ideXlab platform.
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human Milk Oligosaccharides in premature infants absorption excretion and influence on the intestinal microbiota
Pediatric Research, 2015Co-Authors: Mark A. Underwood, Bruce J German, David A Mills, Karen M Kalanetra, Stephanie C Gaerlan, Maria Lorna A De Leoz, Lauren M Dimapasoc, Danielle G Lemay, Carlito B LebrillaAbstract:Human Milk Oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota
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human Milk Oligosaccharides in premature infants absorption excretion and influence on the intestinal microbiota
Pediatric Research, 2015Co-Authors: Mark A. Underwood, Bruce J German, David A Mills, Karen M Kalanetra, Stephanie C Gaerlan, Maria Lorna A De Leoz, Lauren M Dimapasoc, Danielle G Lemay, Carlito B LebrillaAbstract:Human Milk Oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis (NEC) and sepsis, and the influence of HMOs on the microbiota is unclear.Milk, urine, and stool specimens from 14 mother-premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing to complement a previous analysis.Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces, and Milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes were noted in premature infants of nonsecretor mothers. Specific HMO structures in the Milk, urine, and feces were associated with alterations in fecal Proteobacteria and Firmicutes.HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and NEC, while other HMOs may increase dysbiosis in this population.
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novel high molecular weight fucosylated Milk Oligosaccharides identified in dairy streams
PLOS ONE, 2014Co-Authors: Raj Mehra, Daniela Barile, Carlito B Lebrilla, Mariarosaria Marotta, Caroline S Chu, Bruce J GermanAbstract:Oligosaccharides are the third largest component in human Milk. This abundance is remarkable because Oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, Oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant Milk formula, the technologies to reproduce the variety and complexity of human Milk Oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable Oligosaccharides. Recent research has demonstrated that bovine Milk and whey permeate also contain Oligosaccharides. Thus, a thorough characterization of Oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine Milk Oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called "mother liquor", and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant Oligosaccharides present in human Milk. Notably 6 Oligosaccharides contained fucose, a sugar monomer that is highly abundant in human Milk, but is rarely observed in bovine Milk. This work shows that dairy streams represent a potential source of complex Milk Oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human Milk.
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bifidobacteria isolated from infants and cultured on human Milk Oligosaccharides affect intestinal epithelial function
Journal of Pediatric Gastroenterology and Nutrition, 2012Co-Authors: Maciej Chichlowski, Bruce J German, Guillaume De Lartigue, Helen E Raybould, David A MillsAbstract:Objectives Human Milk Oligosaccharides (HMO) are the third most abundant component of breast Milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in select bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of select bifidobacteria on HMO stimulates the intestinal epithelium.
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bacteroides in the infant gut consume Milk Oligosaccharides via mucus utilization pathways
Cell Host & Microbe, 2011Co-Authors: Angela Marcobal, Mariana Barboza, David A Mills, Carlito B Lebrilla, Erica D Sonnenburg, Nicholas A Pudlo, Eric C Martens, Prerak T Desai, Bart C Weimer, Bruce J GermanAbstract:Summary Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human Milk Oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis , induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto- N -neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis , which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both Milk and future solid food digestion.
David S Newburg - One of the best experts on this subject based on the ideXlab platform.
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utilization of major fucosylated and sialylated human Milk Oligosaccharides by isolated human gut microbes
Glycobiology, 2013Co-Authors: Ceng Chen, David S NewburgAbstract:Human Milk Oligosaccharides (HMOS) are not digested in the proximal intestine. In distal intestine, HMOS collectively modify the microbiota, but the response of individual bacteria to individual components of the HMOS is not well defined. Here, each of 25 major isolates of the human intestinal microbiota was fed individual major fucosylated and sialylated HMOS in anaerobic culture. This allowed for an assessment of the influence of specific HMOS on the growth and metabolic products of individual microbiota bacteria. Most Bifidobacteria spp. and Bacteroides spp. grew, induced α-l-fucosidase activity, and produced abundant lactate or short-chain fatty acids (SCFAs) when fed 2′-fucosyllactose (2′-FL), 3-FL, and lactodifucotetraose (LDFT). Lactobacillus delbrueckii ATCC7830, Enterococcus faecalis ATCC19433, and Streptococcus thermophilus ATCC19258 exhibited slight growth, pH reduction, and lactate production when supplemented with 2′-FL or 3-FL, but not LDFT. Supplementation with 3′-sialyllactose (3′-SL) and 6′-SL promoted moderate growth of Bifidobacterium longum JCM7007, 7009, 7010, 7011, 1272, 11347, ATCC15708, Bacteroides vulgatus ATCC8482, and B. thetaiotaomicron ATCC29148; accordingly, these bacteria exhibited greater neuraminidase activity and produced copious lactate, SCFA, or both. Lactobacillus delbrueckii ATCC7830 also consumed 6′-SL. In contrast, Clostridium spp., L. rhamnosus ATCC53103, E. faecalis ATCC29200, Staphylococcus spp., Enterobacter spp., and Escherichia coli K12 did not consume Milk Oligosaccharides nor produce appreciable acidic fermentation products. Specific Bifidobacteria and Bacteroides differentially digest specific individual HMOS, with the major fucosylated Milk Oligosaccharides most strongly stimulating key species of mutualist symbionts. This suggests strategies for treating dysbiosis of the microbiota and associated inflammatory disorders.
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quantification of neutral human Milk Oligosaccharides by graphitic carbon high performance liquid chromatography with tandem mass spectrometry
Analytical Biochemistry, 2013Co-Authors: Yuanwu Bao, Ceng Chen, David S NewburgAbstract:Defining the biological roles of human Milk Oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of Milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral Oligosaccharides of human Milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon high-performance liquid chromatography (HPLC) with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights, allowing 11 peaks to be fully resolved and quantified by monitoring mass-to-charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 Oligosaccharides is linear from 0.078 or 0.156 to 20 μg/ml (R(2)>0.998). Precision (coefficient of variation) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, and accurate quantification for each of the 11 Milk Oligosaccharides and allows measurement of differences in Milk oligosaccharide patterns between individuals and at different stages of lactation.
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human Milk Oligosaccharides are associated with protection against diarrhea in breast fed infants
The Journal of Pediatrics, 2004Co-Authors: Ardythe L Morrow, Prasoon Chaturvedi, Mekibib Altaye, Guillermo M Ruizpalacios, Larry K Pickering, Xi Jiang, Lourdes M Guerrero, Jareen Meinzenderr, Tibor Farkas, David S NewburgAbstract:Abstract Objective To determine the association between maternal Milk levels of 2-linked fucosylated oligosaccharide and prevention of diarrhea as a result of Campylobacter, caliciviruses, and diarrhea of all causes in breast-fed infants. Study design Data and banked samples were analyzed from 93 breast-feeding mother-infant pairs who were prospectively studied during 1988-1991 from birth to 2 years with infant feeding and diarrhea data collected weekly; diarrhea was diagnosed by a study physician. Milk samples obtained 1 to 5 weeks postpartum were analyzed for oligosaccharide content. Data were analyzed by Poisson regression. Results Total 2-linked fucosyloligosaccharide in maternal Milk ranged from 0.8 to 20.8 mmol/L (50%-92% of Milk oligosaccharide). Moderate-to-severe diarrhea of all causes (n = 77 cases) occurred less often ( P = .001) in infants whose Milk contained high levels of total 2-linked fucosyloligosaccharide as a percent of Milk oligosaccharide. Campylobacter diarrhea (n = 31 cases) occurred less often ( P = .004) in infants whose mother's Milk contained high levels of 2′-FL, a specific 2-linked fucosyloligosaccharide, and calicivirus diarrhea (n = 16 cases) occurred less often ( P = .012) in infants whose mother's Milk contained high levels of lacto- N -difucohexaose (LDFH-I), another 2-linked fucosyloligosaccharide. Conclusion This study provides novel evidence suggesting that human Milk Oligosaccharides are clinically relevant to protection against infant diarrhea.
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fucosylated human Milk Oligosaccharides vary between individuals and over the course of lactation
Glycobiology, 2001Co-Authors: Prasoon Chaturvedi, Christopher D Warren, Mekibib Altaye, Ardythe L Morrow, Guillermo M Ruizpalacios, Larry K Pickering, David S NewburgAbstract:Specific human Milk Oligosaccharides, especially fucosylated neutral Oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral Oligosaccharides during lactation, a total of 84 Milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral Oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of Oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced Milk fucosylOligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In Milk samples from the remaining 11 donors, fucosylOligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosylOligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to Oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual Oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of Milk Oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human Milk could also vary among individuals and during lactation.