Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides

Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.     

Release and utilization of N-acetyl-D-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp. infantis

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.     

Oligosaccharide binding proteins from Bifidobacterium longum subsp. infantis reveal a preference for host glycans

Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process.     

Endo-β-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release Complex N-glycans from Human Milk Glycoproteins

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates

Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different α-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-α-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-α-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 °C. Notably, 80% activity remained after 30 min incubation at 80 °C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the α2,3-linkage rather than α2,6- and α2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-α-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.     

In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

Proteomic Analysis of Bifidobacterium longum subsp. infantis Reveals the Metabolic Insight on Consumption of Prebiotics and Host Glycans

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.     

Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways

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.     

Investigations of the in vitro transport of human milk oligosaccharides by a Caco-2 monolayer using a novel high performance liquid chromatography-mass spectrometry technique.

Complex lactose-derived oligosaccharides belong to the main components of human milk and are believed to exert multiple functions in the breast-fed infant. Therefore, we investigated the transepithelial transport of human milk oligosaccharides over Caco-2 monolayers. Main human milk oligosaccharides (HMOs) in the apical, basolateral, or intracellular compartment were separated by high performance liquid chromatography using a Hypercarb(TM) column and analyzed on line by mass spectrometry. This method allowed the identification and quantification of these components in intra- and extracellular fractions without prior purification. Using this technique we were able to show that acidic and neutral HMOs cross the epithelial barrier. The transepithelial flux of neutral, but not acidic, oligosaccharides was temperature-sensitive and partly inhibited by brefeldin A and bafilomycin A. Furthermore, net flux from the apical to the basolateral compartment was only observed for the neutral components. Similarly, apical cellular uptake was only found for neutral components but not for acidic oligosaccharides. Intracellular concentrations of neutral HMOs were significantly increased by inhibitors of transcytosis such as brefeldin A, N-ethylmaleimide, or bafilomycin A. The cellular uptake was saturable, and an apparent K(m) for lacto-N-fucopentaose I of 1.7 +/- 0.1 mmol/liter and for lacto-N-tetraose of 1.8 +/- 0.4 mmol/liter was determined. Furthermore, the uptake of lacto-N-fucopentaose I could be inhibited by the addition of the stereoisomer lacto-N-fucopentaose II but not by lacto-N-tetraose. These findings suggest that neutral HMOs are transported across the intestinal epithelium by receptor-mediated transcytosis as well as via paracellular pathways, whereas translocation of acidic HMOs solely represents paracellular flux.     

Lacto-N-tetraose, fucosylation, and secretor status are highly variable in human milk oligosaccharides from women delivering preterm

Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from 15 women delivering preterm and 7 women delivering at term using nanohigh performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene's test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2'-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments.     

Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.     

Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.     

Incorporation of orally applied (13)C-galactose into milk lactose and oligosaccharides

Biosynthesis and functions of human milk oligosaccharides (HMO) are not well known. A typical housekeeping enzyme, beta1,4-galactosyltransferase, links galactose to glucose to form lactose which is then used as backbone for the assembly of HMO. We investigated whether milk lactose and HMO may be labeled in vivo by an orally given (13)C-galactose bolus. Eleven exclusively breastfeeding mothers were given a (13)C-galactose bolus at the end of their breakfast. Milk and urine samples from each nursing up to 36 h were analyzed for carbohydrate composition by high-performance thin-layer chromatography, high-pH anion-exchange chromatography, and fast atom bombardment mass spectrometry. (13)C enrichment of milk fractions, urinary carbohydrates, lactose, and oligosaccharides as well as of breath CO(2) was determined by isotope ratio mass spectrometry. Up to 10% of the orally given galactose bolus was directly transported to the mammary gland and incorporated into milk components. Characteristic for most milk samples was the appearance of two (13)C-peaks, the first immediately after the (13)C-bolus was taken and the second on the next morning. The highest (13)C enrichment was found in lactose followed by neutral and acidic oligosaccharides. In breath samples, the (13)C-excretion followed the same pattern as in milk. (13)C nuclear magnetic resonance of isolated lactose revealed (13)C only at C(1)-atom of galactose and C(1)-atom of glucose. This label was without any exception at the same position as the (13)C-label of the orally applied galactose. Neutral and acidic HMO can easily be (13)C-labeled in vivo which facilitates investigations of their metabolic fate in infants.     

Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.     

Structural characterization of neutral and acidic oligosaccharides in the milks of strepsirrhine primates: greater galago, aye-aye, Coquerel’s sifaka and mongoose lemur

The structures of milk oligosaccharides were characterized for four strepsirrhine primates to examine the extent to which they resemble milk oligosaccharides in other primates. Neutral and acidic oligosaccharides were isolated from milk of the greater galago (Galagidae: Otolemur crassicaudatus), aye-aye (Daubentoniidae: Daubentonia madagascariensis), Coquerel's sifaka (Indriidae: Propithecus coquereli) and mongoose lemur (Lemuridae: Eulemur mongoz), and their chemical structures were characterized by (1)H-NMR spectroscopy. The oligosaccharide patterns observed among strepsirrhines did not appear to correlate to phylogeny, sociality or pattern of infant care. Both type I and type II neutral oligosaccharides were found in the milk of the aye-aye, but type II predominate over type I. Only type II oligosaccharides were identified in other strepsirrhine milks. α3'-GL (isoglobotriose, Gal(α1-3)Gal(β1-4)Glc) was found in the milks of Coquerel's sifaka and mongoose lemur, which is the first report of this oligosaccharide in the milk of any primate species. 2'-FL (Fuc(α1-2)Gal(β1-4)Glc) was found in the milk of an aye-aye with an ill infant. Oligosaccharides containing the Lewis x epitope were found in aye-aye and mongoose lemur milk. Among acidic oligosaccharides, 3'-N-acetylneuraminyllactose (3'-SL-NAc, Neu5Ac(α2-3)Gal(β1-4)Glc) was found in all studied species, whereas 6'-N-acetylneuraminyllactose (6'-SL-NAc, Neu5Ac(α2-6)Gal(β1-4)Glc) was found in all species except greater galago. Greater galago milk also contained 3'-N-glycolylneuraminyllactose (3'-SL-NGc, Neu5Gc(α2-3)Gal(β1-4)Glc). The finding of a variety of neutral and acidic oligosaccharides in the milks of strepsirrhines, as previously reported for haplorhines, suggests that such constituents are ancient rather than derived features, and are as characteristic of primate lactation is the classic disaccharide, lactose.     

Fucosylated but Not Sialylated Milk Oligosaccharides Diminish Colon Motor Contractions

Human milk oligosaccharides (HMO) are being studied by different groups exploring a broad range of potential beneficial effects to the breastfed infant. Many of these effects have been attributed to a growth promotion effect on certain gut organisms such as bifidobacteria. Additionally, evidence indicates that HMO are able to directly promote positive changes in gut epithelium and immune responses under certain conditions. This study utilizes a standardized ex vivo murine colon preparation to examine the effects of sialylated, fucosylated and other HMO on gut motor contractions. Only the fucosylated molecules, 2’FL and 3’FL, decreased contractility in a concentration dependent fashion. On the basis of IC₅₀ determinations 3’FL was greater than 2 times more effective than 2’FL. The HMO 3’SL and 6’SL, lacto-N-neotetraose (LNnT), and galactooligosaccharides (GOS) elicited no effects. Lactose was used as a negative control. Fucosylation seems to underlie this functional regulation of gut contractility by oligosaccharides, and L-fucose, while it was also capable of reducing contractility, was substantially less effective than 3’FL and 2’FL. These results suggest that specific HMO are unlikely to be having these effects via bifidogenesis, but though direct action on neuronally dependent gut migrating motor complexes is likely and fucosylation is important in providing this function, we cannot conclusively shown that this is not indirectly mediated. Furthermore they support the possibility that fucosylated sugars and fucose might be useful as therapeutic or preventative adjuncts in disorders of gut motility, and possibly also have beneficial central nervous system effects.