Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.
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$\text{L} $ -Fucose is the most widely distributed $\text{L} $-hexose in marine and terrestrial environments and presents a variety of functional roles. $\text{L} $-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-$\text{L} $-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α-$\text{L} $-fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2'- and 3-fucosyllactose, while Fp284 degraded 2'-fucosyllactose and Fuc(α1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s-1 and Km = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.
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Human milk is the optimal nutrition source for infants, and oligosaccharides represent the third most abundant component in milk after lactose and fat. Human milk oligosaccharides (HMO) are favorable macromolecules which are, interestingly, indigestible by the infant but serve as substrates for bacteria. Hypothesizing that the maternal diet itself might influence HMO composition, we sought to directly determine the effect maternal diet on HMO and the milk bacteria. Employing a human cross-over study design, we demonstrate that distinct maternal dietary carbohydrate and energy sources preferentially alter milk concentrations of HMO, including fucosylated species. We find significant associations between the concentration of HMO-bound fucose and the abundance of fucosidase (a bacterial gene that digests fucose moieties) harbored by milk bacteria. These studies reveal a successive mechanism by which the maternal diet during lactation alters milk HMO composition, which in turn shapes the functional milk microbiome prior to infant ingestion.

 Background: The etiology and the mechanism behind atropine treatment of progressive myopia are still poorly understood. Our study addressed the role of scleral and choroidal fibroblasts in myopia development and atropine function.
Fibroblasts treated in vitro with atropine or 7-methylxanthine were tested for ECM production by Western blotting. Corneal epithelial cells were treated with atropine in the presence or absence of colostrum or fucosyl-lactose, and cell survival was evaluated by the MTT metabolic test.
Atropine and 7-methyl-xanthine stimulated collagen I and fibronectin production in scleral fibroblasts, while they inhibited their production in choroidal fibroblasts. Four days of treatment with atropine of corneal epithelial cells significantly decreased cell viability, which could be prevented by the presence of colostrum or fucosyl-lactose.
Our results show that atropine may function in different ways in different eye districts, strengthening the scleral ECM and increasing permeability in the choroid. The finding that colostrum or fucosyl-lactose attenuate the corneal epithelial toxicity after long-term atropine treatment suggests the possibility that both compounds can efficiently blunt its toxicity in children subjected to chronic atropine treatment.

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2'-fucosyllactose (2'FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose - the two precursors of 2'FL - by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2'FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2'FL from yeast, which is a key consideration for any biocatalyst for 2'FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2'FL efflux. Finally, we demonstrated production of 2'FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2'FL production.
Copyright © 2019 DowDuPont Specialty Products (DuPont) Division. Published by Elsevier Inc. All rights reserved.