RESUMO
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.
RESUMO
Gold nanostructures were synthesized by etching away gold from heat-treated mesoporous silica-coated gold nanorods (AuNR@mSiO2), providing an example of top-down modification of nanostructures made using bottom-up methodology. Twelve different types of nanostructures were made using this bottom-up-then-top-down synthesis (BUTTONS), of which the etching of the same starting nanomaterial of AuNR@mSiO2 was found to be controlled by how AuNR@mSiO2 were heat treated, the etchant concentration, and etching time. When the heat treatment occurred in smooth moving solutions in round-bottomed flasks, red-shifted longitudinal surface plasmon resonance (LSPR) was observed, on the order of 10-30 min, indicating increased aspect ratios of the gold nanostructures inside the mesoporous silica shells. When the heat treatment occurred in turbulent solutions in scintillation vials, a blue shift of the LSPR was obtained within a few minutes or less, resulting from reduced aspect ratios of the rods in the shells. The influence of the shape of the glassware, which may impact the flow patterns of the solution, on the heat treatment was investigated. One possible explanation is that the flow patterns affect the location of opened pores in the mesoporous shells, with the smooth flow of solution mainly removing CTAB surfactants from the pores along the cylindrical body of mSiO2, therefore increasing the aspect ratios after etching, and the turbulent solutions removing more surfactants from the pores of the two ends or tips of the silica shells, hence decreasing the aspect ratios after etching. These new stable gold nanostructures in silica shells, bare and without surfactant protection, may possess unique chemical properties and capabilities. Catalysis using heat-treated nanomaterials was studied as an example of potential applications of these nanostructures.