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β-glucans found in cereal grains have been previously demonstrated to improve blood glucose control; however, current understanding points to their high viscosity as the primary mechanism of action. In this work, we present a novel, highly soluble, low-viscosity β-glucan fiber (HS-BG fiber) and a preclinical dataset that demonstrates its impact on two mechanisms related to the prevention of hyperglycemia. Our results show that HS-BG inhibits the activity of two key proteins involved in glucose metabolism, the α-glucosidase enzyme and the SGLT1 transporter, thereby having the potential to slow starch digestion and subsequent glucose uptake. Furthermore, we demonstrate in a multi-donor fecal fermentation model that HS-BG is metabolized by several different members of the gut microbiome, producing high amounts of short-chain fatty acids (SCFAs), known agonists of GPR43 receptors in the gut related to GLP-1 secretion. The production of SCFAs was verified in the translational gut model, SHIME ® . Moreover, HS-BG fiber fermentation produces compounds that restored permeability in disrupted epithelial cells, decreased inflammatory chemokines (CXCL10, MCP-1, and IL-8), and increased anti-inflammatory marker (IL-10), which could improve insulin resistance. Together, these data suggest that the novel HS-BG fiber is a promising new functional ingredient that can be used to modulate postprandial glycemic responses while the high solubility and low viscosity enable easy formulation in both beverage and solid food matrices.

Carbohydrates play essential roles in a variety of biological processes that are dictated by their structures. However, characterization of carbohydrate structures remains extremely difficult and generally unsolved. In this work, a de novo mass spectrometry-based workflow was developed to isolate and structurally elucidate oligosaccharides to provide sequence, monosaccharide compositions, and glycosidic linkage positions. The approach employs liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods in a 3-dimensional concept: one high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) analysis for oligosaccharide sequencing and two ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ MS) analyses on fractionated oligosaccharides to determine their monosaccharides and linkages compositions. The workflow was validated by applying the procedure to maltooligosaccharide standards. The approach was then used to determine the structures of oligosaccharides derived from polysaccharide standards and whole food products. The integrated LC-MS workflow will reveal the in-depth structures of oligosaccharides.

The identification of polysaccharide structures in complex samples remains a unique challenge complicated by the lack of specific tools for polymeric mixtures. In this work, we present a method that depolymerizes polysaccharides to generate diagnostic oligosaccharide markers that are then analyzed by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS). Rapid identification of food polysaccharides was performed by aligning the identified oligosaccharides with a library of oligosaccharide markers generated from standard polysaccharides. Measurements of standard and food polysaccharides were performed to obtain the contributions of the identified polysaccharides using percent peak coverage and angle cosine methods. The method was validated using a synthetic mixture of standard polysaccharides while the reproducibility was confirmed with experimental triplicates of butternut squash samples, where standard deviation was less than 3% for the relative abundance of oligosaccharides. The method was further employed to examine diverse set of food samples., (Published by Elsevier Ltd.)

Polysaccharides are the most abundant biomolecules in nature, but are the least understood in terms of their chemical structures and biological functions. Polysaccharides cannot be simply sequenced because they are often highly branched and lack a uniform structure. Furthermore, large polymeric structures cannot be directly analyzed by mass spectrometry techniques, a problem that has been solved for polynucleotides and proteins. While restriction enzymes have advanced genomic analysis, and trypsin has advanced proteomic analysis, there has been no equivalent enzyme for universal polysaccharide digestion. We describe the development and application of a chemical method for producing oligosaccharides from polysaccharides. The released oligosaccharides are characterized by advanced liquid chromatography-mass spectrometry (LC-MS) methods with high sensitivity, accuracy and throughput. The technique is first used to identify polysaccharides by oligosaccharide fingerprinting. Next, the polysaccharide compositions of food and feces are determined, further illustrating the utility of technique in food and clinical studies.

Background: The stable isotope deuterium dose-to-mother (DTM) technique to estimate nonbreast milk water intake demonstrates that maternal self-report methods of infant feeding overestimate the true prevalence of exclusively breastfeeding practices., Objective: We aimed to determine potential monosaccharide and oligosaccharide markers that distinguish between exclusively breastfed (EBF) versus nonexclusively breastfed (non-EBF) infants utilizing LC-MS-based methods., Methods: Data for the analysis were collected as part of a larger, longitudinal study of 192 breastfed Indonesian infants aged 2 mo and followed up at 5 mo. Feces samples were collected from infants aged 2 mo (n = 188) and 5 mo (n = 184). EBF and non-EBF strata at each time point were determined via the DTM technique. Feces samples were analyzed to determine monosaccharide content using ultra-high-performance LC-triple quadrupole MS (UHPLC-QqQ MS). Relative abundances of fecal oligosaccharides were determined using nano-LC-Chip-quadrupole time-of-flight MS (nano-LC-Chip-Q-ToF MS)., Results: At age 2 mo, monosaccharide analysis showed the abundance of fructose and mannose were significantly higher (+377% and +388%, respectively) in non-EBF compared with EBF infants (P <0.0001). Fructose and mannose also showed good discrimination with areas under the curve (AUC) of 0.86 and 0.82, respectively. Oligosaccharide analysis showed that a 6-hexose (Hex6) isomer had good discrimination (AUC = 0.80) between EBF and non-EBF groups at 5 mo., Conclusion: Carbohydrate products, particularly fecal mono- and oligosaccharides, differed between EBF and non-EBF infants aged under 6 mo and can be used as potential biomarkers to distinguish EBF versus non-EBF feeding practices., (Copyright © The Author(s) 2020.)

The extensive characterization of glycosidic linkages in carbohydrates remains a challenge because of the lack of known standards and limitations in current analytical techniques. This study encompasses the construction of an extensive glycosidic linkage library built from synthesized standards. It includes an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of glycosidic linkages derived from disaccharides, oligosaccharides, and polysaccharides present in complicated matrices. We present a method capable of the simultaneous identification of over 90 unique glycosidic linkages using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ MS) operated in dynamic multiple reaction monitoring (dMRM) mode. To build the library, known monosaccharides commonly found in plants were subjected to partial methylation to yield partially derivatized species representing trisecting, bisecting, linear, and terminal structures. The library includes glycosidic linkage information for three hexoses (glucose, galactose, and mannose), three pentoses (xylose, arabinose, and ribose), two deoxyhexoses (fucose and rhamnose), and two hexuronic acids (glucuronic acid and galacturonic acid). The resulting partially methylated monosaccharides were then labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by separation and analysis by UHPLC/dMRM MS. Validation of the synthesized standards was performed using disaccharide, oligosaccharide, and polysaccharide standards. Accuracy, reproducibility, and robustness of the method was demonstrated by analysis of xyloglucan (tamarind) and whole carrot root. The synthesized standards represent the most comprehensive group of carbohydrate linkages to date.

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer , which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx )., Competing Interests: JW, ED, XB, CS, AW, MA, AK No competing interests declared, (© 2019, Wexler et al.)

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

Carbohydrates make up the largest component of plant-based foods and have long been known to provide fuel. However, many carbohydrates possess intrinsic biological activities that are dictated by their structures. Carbohydrates are the most abundant biopolymers in nature and are also the most structurally complicated and diverse. Consequently, the structural analysis of carbohydrates remains severely limited. To further understand their biological activities, we need new analytical tools to analyze the different classes of carbohydrates that range in size from monosaccharides to polysaccharides. These tools must be capable of rapid throughput with highly sensitive quantitation for use in clinical studies that probe their fate in human and animal fluids and tissues.

The structural analysis of carbohydrates remains challenging mainly due to the lack of rapid analytical methods able to determine and quantitate glycosidic linkages between the diverse monosaccharides found in natural oligosaccharides and polysaccharides. In this research, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS) analysis performed in multiple reaction monitoring (MRM) mode. A library of 22 glycosidic linkages was built using commercial oligosaccharide standards. Permethylation and hydrolysis conditions along with LC-MS/MS parameters were optimized resulting in a workflow requiring only 50 μg of substrate for the analysis. Samples were homogenized, permethylated, hydrolyzed, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis by UHPLC/MRM-MS. Separation by C18 reversed-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15 min run time. Reproducibility, efficacy, and robustness of the method was demonstrated with galactan ( Lupin) and polysaccharides within food such as whole carrots. The speed and specificity of the method enables its application toward the rapid glycosidic linkage analysis of oligosaccharides and polysaccharides.

A rapid method for the quantitation of sixteen neutral and acidic monosaccharides, from both animal and plant sources was developed using ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC/QqQ-MS) in dynamic multiple reaction monitoring (dMRM) mode. Monosaccharides including three pentoses (ribose, xylose, arabinose), two deoxyhexoses (rhamnose, fucose), five hexoses (fructose, mannose, allose, glucose, galactose), two hexuronic acids (glucuronic acid, galacturonic acid), and two N-acetyl-hexosamines (GlcNAc, GalNAc), were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), while underivatized sialic acids, Neu5Ac and Neu5Gc, were simultaneously analyzed with a 10-minute run. With the optimized UHPLC conditions, baseline separations of the isomers were achieved. The sensitivity and calibration ranges of this method were determined. The limits of detection were between femtomoles and attomoles with linear ranges spanning four to six orders of magnitude and coefficients of variation (CVs) ≤7.2%. Spiking experiments performed on a pooled fecal sample demonstrated the high accuracy of this method even when applied to samples with complicated matrices. The validated method was applied to fecal samples from an infant transitioning from breast milk to weaning foods. Major milk monosaccharides including galactose, fucose, glucose, GlcNAc, and Neu5Ac were found to be the most abundant components in the feces of milk-fed infants. PMP-derivatives of nine other monosaccharides including apiose, lyxose, altrose, talose, gulose, glucosamine, galactosamine, mannosamine, and N-acetylmannosamine (ManNAc) were also tested and could be added to the quantitation method depending on the need. The speed and sensitivity of the method makes it readily adaptable to rapid throughput analysis of monosaccharides in biological samples.