A multi-glycomic platform for the analysis of food carbohydrates.

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.

Dietary fiber monosaccharide content alters gut microbiome composition and fermentation.

Members of the mammalian gut microbiota metabolize diverse complex carbohydrates that are not digested by the host, which are collectively labeled "dietary fiber." While the enzymes and transporters that each strain uses to establish a nutrient niche in the gut are often exquisitely specific, the relationship between carbohydrate structure and microbial ecology is imperfectly understood. The present study takes advantage of recent advances in complex carbohydrate structure determination to test the effects of fiber monosaccharide composition on microbial fermentation. Fifty-five fibers with varied monosaccharide composition were fermented by a pooled feline fecal inoculum in a modified MiniBioReactor array system over a period of 72 hours. The content of the monosaccharides glucose and xylose was significantly associated with the reduction of pH during fermentation, which was also predictable from the concentrations of the short-chain fatty acids lactic acid, propionic acid, and the signaling molecule indole-3-acetic acid. Microbiome diversity and composition were also predictable from monosaccharide content and SCFA concentration. In particular, the concentrations of lactic acid and propionic acid correlated with final alpha diversity and were significantly associated with the relative abundance of several of the genera, including Lactobacillus and Dubosiella. Our results suggest that monosaccharide composition offers a generalizable method to compare any dietary fiber of interest and uncover links between diet, gut microbiota, and metabolite production. IMPORTANCE: The survival of a microbial species in the gut depends on the availability of the nutrients necessary for that species to survive. Carbohydrates in the form of non-host digestible fiber are of particular importance, and the set of genes possessed by each species for carbohydrate consumption can vary considerably. Here, differences in the monosaccharides that are the building blocks of fiber are considered for their impact on both the survival of different species of microbes and on the levels of microbial fermentation products produced. This work demonstrates that foods with similar monosaccharide content will have consistent effects on the survival of microbial species and on the production of microbial fermentation products.

Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

Garlic essential oil ameliorates depression-like behaviors in unpredictable chronic mild stress by modulating the brain NLRP3 inflammasome pathway and influencing the gut barrier and microbiota.

Depression is a severe mental disorder, with approximately 300 million people suffering from it. Recent studies have demonstrated that chronic neuroinflammation is significantly associated with intestinal flora and barrier function in depression. As a therapeutic herb, garlic (Allium sativum L.) has detoxification, antibacterial activity, and antiinflammatory functions; however, its antidepressant effect through gut microbiota and barrier function has not been reported yet. The present study investigated the effect of garlic essential oil (GEO) and its active constituent diallyl disulfide (DADS) on depressive behavior by attenuating the NLRP3 inflammasome, alternating intestinal barrier function and gut microbiota in an unpredictable chronic mild stress (US) model in rats. This study found that dopamine and serotonin turnover rates were reduced significantly with a low dose of GEO (25 mg per kg bw). The GEO groups effectively reversed sucrose preference and increased the total distance traveled in the behavioral test. Moreover, 25 mg per kg bw GEO inhibited the UCMS-induced activated inflammatory response, reflected by reduced expression in the frontal cortex of NLRP3, ASC, caspase-1, and its downstream IL-1ß proteins, as well as the concentration of IL-1ß and TNF-α in the serum. Supplementation with GEO increased the expression of occludin and ZO-1 and the concentration of short-chain fatty acids to influence the impact of intestinal permeability in depressive conditions. The results revealed that GEO administration caused significant changes in the α and ß diversity and abundance of certain bacteria. At the genus level, GEO administration significantly increased the relative abundance, particularly beneficial SCFA-producing bacteria, and may improve depression-like behavior. In conclusion, these results indicated the antidepressant effects of GEO involved in the inflammatory pathway, short-chain fatty acids, intestinal integrity, and intestinal composition.

Rapid Quantification of Gut Microbial Short-Chain Fatty Acids by pDART-MS.

Short-chain fatty acids (SCFAs) are small molecules ubiquitous in nature. In mammalian guts, SCFAs are mostly produced by anaerobic intestinal microbiota through the fermentation of dietary fiber. Levels of microbe-derived SCFAs are closely relevant to human health status and indicative to gut microbiota dysbiosis. However, the quantification of SCFA using conventional chromatographic approaches is often time consuming, thus limiting high-throughput screening tests. Herein, we established a novel method to quantify SCFAs by coupling amidation derivatization of SCFAs with paper-loaded direct analysis in real time mass spectrometry (pDART-MS). Remarkably, SCFAs of a biological sample were quantitatively determined within a minute using the pDART-MS platform, which showed a limit of detection at the µM level. This platform was applied to quantify SCFAs in various biological samples, including feces from stressed rats, sera of patients with kidney disease, and fermentation products of metabolically engineered cyanobacteria. Significant differences in SCFA levels between different groups of biological practices were promptly revealed and evaluated. As there is a burgeoning demand for the analysis of SCFAs due to an increasing academic interest of gut microbiota and its metabolism, this newly developed platform will be of great potential in biological and clinical sciences as well as in industrial quality control.