Separation Of Oligosaccharides Research Articles

Membrane technologies for the separation and purification of functional oligosaccharides: A review

Functional oligosaccharides have numerous health benefits. They hold considerable promise in the food processing and healthcare sectors. However, oligosaccharides derived from microbial fermentation, chemical decomposition, and enzymatic hydrolysis often contain impurities, necessitating further purification to enhance the quality and economic value of the final product. Among various purification techniques, membrane separation stands out owing to its operational simplicity, low energy requirements, and scalability, making it ideal for industrial applications. Recently, this method has increasingly been used to separate and purify functional oligosaccharides. Microfiltration and ultrafiltration membranes are typically used in the early stages of filtration to remove proteins, bacterial contaminants, and particulates. In contrast, nanofiltration membranes offer higher separation precision, making them preferable for the detailed purification, concentration, and classification of functional oligosaccharides. This review article mainly focuses on the applications of nanofiltration membrane technologies in the processing of typical functional oligosaccharides, such as fructooligosaccharides, galactooligosaccharides, and xylooligosaccharides. Recent advancements in nanofiltration are discussed, and the properties of the feed liquid, operational parameters, and challenges related to membrane fouling are analyzed. This review aims to serve as a comprehensive guide for the efficient separation and purification of functional oligosaccharide membranes, and advocates for the broader implementation of membrane technologies in the food and medical industries.

Highly-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples

Alterations of protein glycosylation can serve as sensitive and specific disease biomarkers. Labeling procedures for improved separation and detectability of oligosaccharides have several drawbacks, including incomplete derivatization, side-products, noticeable desialylation/defucosylation, sample loss, and interference with downstream analyses. Here, we develop a label-free workflow based on high sensitivity capillary zone electrophoresis-mass spectrometry (CZE-MS) for profiling of native underivatized released N-glycans. Our workflow provides a >45-fold increase in signal intensity compared to the conventional CZE-MS approaches used for N-glycan analysis. Qualitative and quantitative N-glycan profiling of purified human serum IgG, bovine serum fetuin, bovine pancreas ribonuclease B, blood-derived extracellular vesicle isolates, and total plasma results in the detection of >250, >400, >150, >310, and >520 N-glycans, respectively, using injected amounts equivalent to <25 ng of model protein and nL-levels of plasma-derived samples. Compared to reported results for biological samples of similar amounts and complexity, the number of identified N-glycans is increased up to ~15-fold, enabling highly sensitive analysis of sample amounts as low as sub-0.2 nL of plasma volume equivalents. Furthermore, highly sialylated N-glycans are identified and structurally characterized, and untreated sialic acid-linkage isomers are resolved in a single CZE-MS analysis.

Development and application of a sensitive phosphonium-hydrazide oligosaccharide labelling reagent in capillary electrophoresis- electrospray ionization- mass spectrometry

Glycosylation is one of the most ubiquitous post-translational modifications (PTM) of proteins. Although the ionization efficiency of native glycans is fairly low, with the assistance of chemical derivation strategies, mass spectrometry (MS) has been extensively used in glycomics because of its high sensitivity, accuracy, and speed. In this study, a novel glycan labelling reagent, (4-hydrazidebutyl) triphenylphosphonium bromide (P4HZD), with a permanent positive charge was developed. The comprehensive capabilities of P4HZD for MS analysis of oligosaccharides were evaluated in detail using maltodextrin as a standard. This labelling reagent can be used in common biological MS techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. The MS signal intensity of maltodextrin species could be enhanced up to 96-fold in MALDI-MS by labelling with P4HZD, making P4HZD favorable for MALDI-MS-based high-throughput screening of oligosaccharides. Moreover, P4HZD-labelled oligosaccharides with a degree of polymerization (DP) from 1 to 18 could be separated and analysed by capillary electrophoresis (CE) combined with positive ion mode ESI-MS. In comparison with a commercialized oligosaccharide tag, Girard's reagent P (GirP), P4HZD was more effective for enhancing the signal of oligosaccharides in the middle or higher mass range using both ESI and MALDI ion sources. Two biologics, immunoglobulin G 2 (IgG 2) and fusion protein (FP), were chosen as model complex biological samples to test the efficacy of detection and separation of oligosaccharides by MALDI-MS and CE-ESI-MS analysis with P4HZD labelling. The results indicated that P4HZD is a promising labelling reagent for the detection of oligosaccharides in complex biological samples. The tandem workflow combines the strengths of MALDI-MS and CE-ESI-MS to fulfil the analytical demands of high-throughput screening, while affording good separation.

Review on Preparation, Separation and Characterization of Alginate Oligosaccharide

Review on Preparation, Separation and Characterization of Alginate Oligosaccharide

Maltose-functionalized HILIC stationary phase silica gel based on self-assembled oligopeptides and its application for the separation of polar compounds.

In this study, carbonyldiimidazole was used to bond maltose-modified oligopeptides (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys) to the surface of silica spheres for hydrophilic interaction liquid chromatography (HILIC). Attenuated total reflectance-Fourier transform infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, thermogravimetric analysis, BET technique, and water contact angle measurement results confirmed the successful immobilization of the obtained material. Compared with the conventional method for preparing carbohydrate stationary phases, this method involves simpler steps and less time-consuming processes. The experimental results proved that the retention mechanism of the maltose-based HILIC column matched the typical HILIC retention mechanism. The column showed high separation efficiency and stability toward the separation of polar compounds such as amino acids, bases, nucleosides, water-soluble vitamins, and salicylic acid and its analogs. The column achieved high selectivity toward oligosaccharide separation. In addition, this efficient analysis demonstrates the applicability of the as-prepared material in the field of food inspection.

Separation of Fructosyl Oligosaccharides in Maple Syrup by Using Charged Aerosol Detection.

Fructosyl oligosaccharides, including fructo-oligosaccharide (FOS), are gaining popularity as functional oligosaccharides and have been found in various natural products. Our previous study suggested that maple syrup contains an unidentified fructosyl oligosaccharide. Because these saccharides cannot be detected with high sensitivity using derivatization methods, they must be detected directly. As a result, an analytical method based on charged aerosol detection (CAD) that can detect saccharides directly was optimized in order to avoid relying on these structures and physical properties to clarify the profile of fructosyl oligosaccharides in maple syrup. This analytical method is simple and can analyze up to hepta-saccharides in 30 min. This analytical method was also reliable and reproducible with high validation values. It was used to determine the content of saccharides in maple syrup, which revealed that it contained not only fructose, glucose, and sucrose but also FOS such as 1-kestose and nystose. Furthermore, we discovered a fructosyl oligosaccharide called neokestose in maple syrup, which has only been found in a few natural foods. These findings help to shed light on the saccharides profile of maple syrup.

Highly thermostable and promiscuous β-1,3-xylanasen designed by optimized ancestral sequence reconstruction

The ancestor of β-1,3-xylanases (AncXyl09) were reconstructed by the optimized ancestral sequences reconstruction strategy to solve the poor catalytic performances of existing β-1,3-xylanases. The results showed that the half-life at 50 °C was 65.08 h, indicating good thermostability. The large number of hydrogen bonds and the disulfide bonds were the major attributes related with the thermal stability of Anxyl09. Interestingly, AncXyl09 could hydrolyze lichen besides the original substrate of β-1, 3-xylan, which is the first reported β-1,3-xylanase with substrate promiscuity. Moreover, the hydrolytic products are mainly disaccharides, the content of β-1,3-xylobiose and lichoridiose more than 70% as determined by high performance liquid chromatography (HPLC), which could significantly facilitate the separation and purification of oligosaccharides. The successful design of AncXyl09 was the representative of the semi-rationally engineered β-1, 3-xylanase, which will shield a new light on the β-1,3-xylanase engineering, active oligosaccharide preparation and marine algae resource utilization.

Separation and identification of neutral oligosaccharides with prebiotic activities from apple pectin

Plant-derived neutral oligosaccharides and pectic-oligosaccharides (POS) have been suggested as prebiotics based on their degree of polymerization (DP) and structural complexity. In the present study, neutral oligosaccharides without galacturonic acid were produced from commercial apple pectin using an optimized concentration of pectinase and degradation time. The crude oligosaccharide solution was refined by membrane separation using a DEAE-Sepharose CL-6B column, a graphite carbon column and an Xbridge C18 column, which resulted in the neutral oligosaccharides with DP 3–13. The monosaccharide composition of oligosaccharides primarily included glucose, arabinose, galactose and xylose without galacturonic acid. The structure of the oligosaccharides was analyzed using mass spectrometry with an electrospray ionization source by UPLC on an HILIC column and 1H NMR. The MS/MS spectra in positive ion mode and 1H NMR showed a main oligosaccharide structure corresponding to the connection of arabinogalactan with Galβ1→4Galβ1→4Galβ1→4Galβ1→α3Araf. The growth of the selected Lactobacillus. strains was improved by adding refined neutral oligosaccharides with DP = 3–13 compared with commercial inulin, and growth of Escherichia coli had not basically grown. Therefore, neutral oligosaccharides (DP = 3–13) can be considered effective prebiotics for potential application in functional food.

Comparison of the steric selectivity on hydrophilic interaction chromatography columns modified with poly(acrylamide) possessing different morphology

Poly(acrylamide) (PAAm)-modified hydrophilic interaction chromatography (HILIC) columns were prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) and free radical polymerization (FRP) to generate brush-like and mushroom-like polymer chains on silica particles, respectively. The maltose homologues (MHs) and cyclodextrins (CDs) were chosen as analytes to evaluate steric selectivity by the different polymer morphologies in the ATRP-PAAm and the FRP-PAAm columns. The ATRP-PAAm exhibited superior retention than the FRP-PAAm and three commercial HILIC columns. The house-made PAAm columns provided significant hydrophilicity that enabled to analysis the oligosaccharides even in 60:40 mixture of acetonitrile–aqueous buffer. In the case of three ATRP-PAAm columns characterized by different polymer lengths and the density on the silica particles, those are different thickness of the water-enriched layer, and phase ratio φ, based on hydrophilicity of them columns. The logarithm of the retention factor (ln k) displayed a non-linear dependence on the inverse of the temperature (1/T, T = 278–333 K). Notably, a similar correlation was observed to exist between the logarithm of the phase ratio (ln φ), and 1/T. A van't Hoff plot was used to determine the thermodynamic parameters of the partition process for each MH. The values of the Gibbs free energy (ΔG°) for the analytes partition on the ATRP-PAAm columns were smaller than their counterparts measured for the FRP-PAAm columns; by contrast, the opposite trend was observed for the ΔG° values measured for CDs. The standard entropy ΔS° for MHs and CDs were comparable for the two types PAAm columns, while, the standard enthalpy, ΔH° displays significant difference between the ATRP and the FRP PAAm columns. These findings indicate that the differences between PAAm morphology and polymer densities on the stationary phase surface affect analyte differentiation on the basis of molecular steric factors. The higher selectivity for MHs and CDs displayed by ATRP-PAAm columns with respect to their FRP-PAAm and commercial amide columns will be useful for the fine separation of oligosaccharides.

Separation of chitosan oligosaccharides by precipitation adding hydrochloric acid

Separation of chitosan oligosaccharides by precipitation adding hydrochloric acid

Research Progress on Separation and Purification of Mannan Oligosaccharide

Research Progress on Separation and Purification of Mannan Oligosaccharide

Enhancement of fucosylated N-glycan isomer separation with an ultrahigh column temperature in porous graphitic carbon liquid chromatography-mass spectrometry

Due to the heterogeneous and isomeric nature of glycans, the development of an advanced separation of distinct glycan isomers is essential for glycan research and application. In this study, we utilized porous graphite carbon (PGC) chromatography for the separation of isomeric oligosaccharides without reduction or chemical derivatization at 190 °C in a custom-built heating oven. Furthermore, the fine structures of glycan isomers could be identified by using ultrahigh temperature PGC liquid chromatography mass spectrometry (UHT-PGC-LCMS). A nonreduced hydrolyzed dextran was applied to verify the performance of UHT-PGC. When the temperature of the PGC column was increased from 25 to 190 °C, the liquid chromatography separation power of the nonreduced dextran ladder significantly increased. The advantage of the UHT-PGC column was its high peak capacity with gradient elution in 10 min at 190 °C, 6700 psi, and a 250 μL/min flow rate for native glycan analysis. Four synthetic Lewis antigen isomers were used to elucidate the separation effectiveness in UHT-PGC. Moreover, mass spectrometry-based sequencing to generate specific diagnostic ions from the four synthetic Lewis antigens was used to predict isomeric glycans based on the relative intensity ratio (RIR) of diagnostic ions. The intensities of the diagnostic ions of synthetic isomers were used to identify each isomer of the fucosylated glycan. The results clearly showed that terminal Lewis A and X residues were in the 3- and 6-arms of N-glycan, respectively.

Modular instrumentation for capillary electrophoresis with laser induced fluorescence detection using plug-and-play microfluidic, electrophoretic and optic modules

This study reports on the development of a novel instrument for capillary electrophoresis (CE) coupled with laser induced fluorescence (LIF) detection that is inspired by the Lego-toy concept. The Lego CE-LIF design is an evolution of purpose-made CE instrumentation, allowing the users to construct their own analytical device with a high degree of standardization (i.e. a “standard” setup) without requirement of mechanical and electronic workshop facilities. To allow instrument reproduction outside the original fabrication laboratory, which is not trivial for in-house-built CE systems, the new design is based on unprecedent ‘plugging’ hyphenation of various off-the-shelf parts available for microfluidics, optics and electrophoresis. To render the operation with Lego CE-LIF optimal, we developed a new background electrolyte (BGE), using for the first time extremely high concentrations of zwitterionic and large weakly charged species for much improvement of detection sensitivity. The Lego CE-LIF was demonstrated for separation and detection of oligosaccharides labelled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). The new gel-free BGE for oligosaccharide analysis also allowed simplification of the conventional CE-LIF protocol used with commercial instruments while keeping satisfactory separation performances. Furthermore, the new BGE is fully compatible with a non-thermostatted Lego CE instrument thanks to low current and therefore low heat generation under application of a high voltage.

Modeling and optimal operation of intermittent feed diafiltration for refining oligodextran using nanoporous ceramic membranes

Developing a facile and efficient model for the prediction and optimization of diafiltration processes is desired in the separation and purification of functional oligosaccharides. Herein, a data-driven model was proposed to describe the intermittent feed diafiltration process. To evaluate this model, oligodextran was applied as a representative oligosaccharide, and a nanoporous ZrO2 ceramic membrane was employed to narrow the molecular weight distribution of oligodextran. The role of concentration polarization effect in affecting the flux and solute rejection was well considered in the modeling of the diafiltration process. The permeation flux and yields predicted by this model agreed well with the experimental data. More importantly, the maximum productivity could be determined by this model, which takes the diluent consumption and the operation time into account, simultaneously, in optimizing of the diafiltration process. The proposed diafiltration model performs a good potential to be a candidate in the auxiliary design for the separation and purification processes of functional oligosaccharides.

A novel imidazolium bonding stationary phase derived from N-(3-aminopropyl)-imidazole for hydrophilic interaction liquid chromatography

In this work, a novel imidazolium bonding method was proposed for the synthesis of hydrophilic interaction liquid chromatography (HILIC) stationary phases. One obtained stationary phase (SilprAprImCl) was derived from direct reaction between N-(3-aminopropyl)-imidazole and 3-chloropropylated silica gel. Other two materials (SilprAprImBF4 and SilprAprImTf2N) were obtained from SilprAprImCl by ion exchange reaction, respectively. Fourier-transform infrared spectroscopy and elemental analysis afforded the proofs of successful imidazolium immobilization and satisfied bonding efficiency. Various polar compounds such as saccharides, nucleosides, and nucleobases were utilized to evaluate the retention behaviours of these materials in HILIC mode. Different effects from mobile composition, column temperature, imidazolium unite and paired anions (Cl−, BF4−, and Tf2N−) in imidazolium were proved and discussed. Separation mechanism and the role of the imidazolium ions were also investigated in mobile phases with different pH. Moreover, chromatographic stability was evaluated by consecutive injections. Finally, the reliability of these stationary phases was demonstrated by the separation of oligosaccharides in real fructooligosaccharides samples.

Direct Oligosaccharide Profiling Using Thin-Layer Chromatography Coupled with Ionic Liquid-Stabilized Nanomatrix-Assisted Laser Desorption-Ionization Mass Spectrometry.

The in-depth characterization of glycan structures is crucial to understanding their structure-function relationships and their effects on health and various diseases. Despite advances in rapid analysis, the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is limited for complex mixtures of carbohydrates due to their low ionization efficiency and the difficulty in separating oligosaccharides because of their high structural similarity. In this study, we developed an ionic liquid (IL)-stabilized, nanomatrix-decorated, thin-layer chromatography (TLC)-MALDI MS method for simultaneous and rapid separation, detection, and identification of oligosaccharides to achieve efficient profiling. The IL demonstrated good dispersion and stabilization for the spin coating of dihydroxybenzoic acid-functionalized magnetic nanoparticles (DHB@MNPs) on the TLC plate with spot homogeneity, which contributed to the observed high reproducibility (<20% CV) and 12- and 28-fold signal enhancement. Although the TLC was not able to separate isomeric glycans, the DHB@MNPs generate diagnostic glycosidic and cross-ring cleavage ions, enabling on-spot structural elucidation of composition, sequence, branching, and linkage of glycans in each separated spot. Without chemical derivatization of glycan samples, glycan visualization by TLC and tandem MS, our integrated platform, allowed the identification of 25 oligosaccharides from human milk, and heatmap analysis revealed the variability in the oligosaccharide abundance in samples from individual donors at different lactation times, which may provide insight into the microbiota and immunity of infants. With the demonstrated simplicity of our sample preparation method along with the achieved separation and in-depth structural characterization, our approach can be used for the rapid screening of other oligosaccharide-rich samples.

Separation of Isomeric O-Glycans by Ion Mobility and Liquid Chromatography-Mass Spectrometry.

Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (PGCLC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and N-glycans. Here, we present a direct comparison of the IM- and LC-separation of O-glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by PGCLC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.

Selective binding of heparin oligosaccharides in a magnetic thermoresponsive molecularly imprinted polymer

Heparin is a highly sulfated polysaccharide, applied in clinic for treatment of thrombotic diseases. The biological activity is closely related to its molecular structure e.g. compositions of disaccharides and oligosaccharides units. The classical method to isolate the oligosaccharides after depolymerization by heparinases or nitrous acid I s by size exclusion chromatography which is a time-consuming process. In this study, we explored the possibility for rapid separation of oligosaccharides using a novel polymer material. The magnetic thermoresponsive molecularly imprinted polymers (MIPs) were synthesized using heparin disaccharide as a template, AEM, NIPAAm, and AAm as functional monomer, and MBAA as crosslinker by surface radical polymerization in an aqueous media. Incubation of the MIP with hepairn oligosaccharides demonstrated specific binding to the template molecule. This binding to the targeted molecule was affected by reaction temperature with regard to binding capacity and specificity. The recognition specificity and selectivity can be modulated by varying the compositions of multi-functional monomers. The pseudo-second-order kinetic model and Langmuir isotherm model provide the best fit to the equilibrium adsorption of heparin disaccharides by MIPs. The results suggest that the new material can be used for rapid separation of di- and tetra-saccharides of heparin, which can also be adapted to the applications for isolation of oligosaccharides from other polysaccharides, e.g. heparan sulfate and chondoriting sulfate.

Preparation of glutathione-functionalized zwitterionic silica material for efficient enrichment of sialylated N-glycopeptides.

A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides' enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples. Graphical Abstract A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry, and it shows remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment.

Microfabricated liquid junction hybrid capillary electrophoresis-mass spectrometry interface for fully automated operation.

One of the challenging instrumental aspects in coupling an automated CE instrument with ESI mass spectrometry (CE-MS) is finding the balance between the stability, reproducibility and sensitivity of the analysis and compatibility with the standard CE instrumentation. Here, we present a development of a new liquid junction based electrospray interface for automated CE-MS, with a focus on the technical design followed by computer modeling of transport conditions as well as characterization of basic performance of the interface. This hybrid arrangement designed as a microfabricated unit attachable to the automated CE instrument allows using of a wide range of separation capillaries with respect to their diameter, length or internal coating (e.g., for suppressed electroosmotic flow). Different compositions of the ESI liquid and background electrolyte solutions can be used if needed. The microfabricated part, prepared by laser machining from polyimide, includes a self-aligning liquid junction, a short transport channel, and a pointed sprayer for the electrospray ionization. This microfabricated part is positioned in a plastic connection block securing the separation capillary and flushing ports. Transport conditions were modelled using computer simulation and the real life performance of the interface was compared to that of a commercial sheath liquid interface. The basic performance of the interface was demonstrated by separations of peptides, proteins, and oligosaccharides.