Purification Of Oligosaccharides Research Articles

Evaluation of solid-phase extraction sorbents for purification of oligosaccharides and glycans derivatized by positively charged labels followed by capillary electrophoretic analysis.

The sample preparation including labeling and clean-up represents a key analytical step in the analysis of oligosaccharides and glycans by either chromatographic or electrophoretic separation methods. Although the majority of labeling has been performed by neutral and/or negatively charged tags, the introduction of a positive charge into the saccharide molecule can significantly improve the analysis, especially with mass spectrometry detection. In this work, we present the evaluation of five solid-phase extraction sorbents differing in extraction chemistry for the clean-up and concentration of positively labeled maltooligosaccharides from the reaction mixtures. Maltooligosaccharides containing four to seven glucose units were labeled by cationic tags (2-aminoethyl)trimethylammonium chloride and (carboxymethyl)trimethylammonium chloride hydrazide and the extraction conditions were optimized followed by electrophoretic analysis with conductivity detection. The effects of the solid-phase extraction sorbent chemistry, extraction conditions, and sample composition are discussed. All tested sorbents were capable of cleaning up maltooligosaccharides from the reaction mixtures to some extent after optimization of the solid-phase extraction procedure (51.9%-98.9% recovery). The best-rated amide-based sorbent was used to process the sample of N-linked glycans enzymatically released from ribonuclease B.

Capture‐Release Strategy Facilitates Rapid Enzymatic Assembly of Oligosaccharides

Comprehensive SummaryA convenient and highly efficient strategy was developed for the simplification of enzymatic synthesis and purification of oligosaccharides. Glycosyl acceptors terminated with a thiol tag were extended by enzymatic modular assembly (EMA) in the aqueous phase, and the desired products were captured during solid‐phase workup (SPW) using commercially available thiopropyl Sepharose 6B resin through a reversible disulfide linkage. Finally, the products were released from solid‐phase resin in the presence of dithiothreitol (DTT) as reducing agent and the resin could be recovered and reused. This strategy overcomes the uncertain influence of polymer‐ or ionic‐supports commonly used in enzymatic glycosylation, and showed perfect compatibility with all 10 enzyme modules for the rapid assembly of a series of complex oligosaccharides.

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.

Modelling of membrane cascades for the purification of oligosaccharides

The aim of this study was to evaluate the potential of NF membrane cascades for continuous oligosaccharide purification. Three different nanofiltration membranes were evaluated, and the best combination in terms of membrane type and process parameters was determined for two commercial oligosaccharide mixtures of fructooligosaccharides (FOS) and galactooligosaccharides (GOS). To represent the cascade mathematically, a dynamic model was built based on film theory and on measurements performed in single-stage conditions. The model predictability was demonstrated with experiments in a membrane cascade set-up.Considering an initial purity of 84% for FOS and 40.4% for GOS, the model predicted a maximum attainable purity of 94.9% and 46.7% for FOS and GOS respectively. A minimum yield of 90% was used as constraint during the optimisation process, in which the physical limitations of the set up were also taken into account. This paper demonstrates that the trade-off situation between purity and yield can be overcome by using cascade configuration, leading to an efficient separation that cannot be achieved by single-stage membrane systems.

Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization

Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI–MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.

Recovery and purification of oligosaccharides from copra meal by recombinant endo-β-mannanase and deciphering molecular mechanism involved and its role as potent therapeutic agent.

Production of manno-oligosaccharides (MOSs) from pretreated and defatted copra meal (dFCO) hydrolysis was achieved by endo-mannanase. Structural characterization of dFCO by FT-IR and NMR exhibited resemblance with galactomannan. The time-dependent hydrolysis of dFCO by recombinant endo-β-(1 → 4)-mannanase of Clostridium thermocellum by TLC and HPAEC displayed the release of mannose and MOSs mannobiose and mannotriose. Purified MOSs yielded 40 % mannobiose and 18 % mannotriose confirmed by mass spectroscopy which showed mannobiose (m/z = 365) and mannotriose (m/z = 527). The homology based structural analysis of catalytic endo-mannanase (CtManT) showed the catalytic core composed of Glu181 and Glu300 acting as acid/base and Glu288 as a nucleophile during galactomannan hydrolysis. Sub-site mapping of CtManT exhibited two aglycone and four glycone sites at cleavage sites existing on either side of β-(1 → 4)-linkage of galactomannan. Isolated MOSs displayed potential prebiotic characteristics and supported higher growth of probiotic Lactobacillus acidophilus and Bifidobacterium infantis than with standard inulin. Moreover, MOSs displayed over 97 % tolerance to simulated gastric juice, intestinal fluid, and α-amylase proving its potential as a stable prebiotic over inulin. In vitro cytotoxicity assay of MOSs (500 µg/mL) on human epithelial colorectal adenocarcinoma cell line (HT-29) demonstrated 60 % decreased viability of cells after 48 h displaying anti-tumorigenic property.

Towards the chemoenzymatic synthesis of heparan sulfate oligosaccharides: Oxidative cleavage of p-nitrophenyl group with ceric ammonium salts.

We have developed an efficient chemoenzymatic synthesis of heparan sulfate oligosaccharides employing the para-nitrophenyl (p-NP) β-glucuronide as an acceptor compatible with enzymatic elongation and one that significantly simplifies oligosaccharide purification on C-18 resin. Employing ceric ammonium nitrate as oxidative reagent to remove the p-NP group unexpectedly also removed the glucuronic acid residue at the reducing-end, affording a smaller oligosaccharide. The application of ceric ammonium sulfate allowed the removal of the p-NP without concomitant loss of the adjacent glucuronic acid offering a route to longer heparin sulfate oligosaccharide products.

Natural Variability in Bovine Milk Oligosaccharides from Danish Jersey and Holstein-Friesian Breeds

Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities.

膜分離システムの最適化に関する研究

In order to quantitatively estimate the effects of membrane fouling and osmotic pressure on the performance of membrane separation systems, a concentration polarization equation and a membrane transport equation based on nonequilibrium thermodynamics were applied to membrane processes for liquid foods, and new mathematical models were proposed. These models were then applied to several membrane processes in the food industry, including an ultrafiltration process for cheese whey fractionation, a reverse osmosis process for fruit juice concentration, and a nanofiltration process for oligosaccharide purification. The efficiency of the membrane processes could be precisely expressed by the new models, and the validity of the models was demonstrated. Thus, some membrane processes in the food industry can be optimized by using these models, and their feasibility evaluation is described.

Galactosyl oligosaccharide purification by ethanol precipitation

Galactosyl oligosaccharides (GOS) are prebiotics commonly manufactured by β-galactosidase conversion of lactose, producing a mixture containing GOS, lactose, glucose and galactose. Enrichment of GOS in this mixture adds value to the product. This study aimed to determine whether the addition of ethanol to aqueous saccharide solutions could be used to selectively precipitate and enrich GOS from a reaction mixture. High concentrations of ethanol (>70% v/v) were required to induce precipitation. The total saccharide concentration was a significant variable, with higher GOS enrichment occurring at lower total saccharide concentrations. Varying the temperature between 10 and 40 °C had less impact than had changes in the concentration of saccharide or ethanol. GOS was enriched 2.3 (±0.1) fold in the precipitate formed in a solution of 90% (v/v) ethanol with 28 g/L of total saccharide at 40 °C. Performing two such precipitations sequentially reduced the monosaccharides from 48% (w/w) of the total saccharides to 4% (w/w). GOS precipitation has potential for industrial application as it is simple in operation and offers levels of purification similar to those by other techniques.

Purification of oligosaccharides obtained from Pinus pinaster hemicelluloses by diafiltration

The aim of this work was to assess the performance of diafiltration as a method for purification of oligosaccharides obtained by autohydrolysis of Pinus pinaster wood. Pinus pinaster wood hemicelluloses are mainly made up of galactoglucomannans, which were partially hydrolyzed in aqueous treatments (autohydrolysis) performed under suitable operational conditions (isothermal processing at 175°C for 24.5 min). The oligosaccharides present in autohydrolysis liquors obtained under optimal conditions were purified by diafiltration in a dead-end fi ltration device using a regenerated cellulose membrane (1 kDa MWCO). Continuous diafi ltration resulted in increased weight percent of substituted oligosaccharides respect to the total nonvolatile solutes (from 79.2% up to 94.7%), as well as in the selective removal of monosaccharides (the substituted oligosaccharides to monosaccharides ratio increased from 4.3 up to 17.8).

Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins

A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.

Separation of oligoglucuronans of low degrees of polymerization by using a high shear rotating disk filtration module

This paper investigates the purification of oligosaccharides of small degrees of polymerization (DP) obtained by enzymatic degradation of a bacterial polysaccharide produced during fermentation by a Sinorhizobium meliloti mutant strain. We have used a dynamic filtration module consisting in a disk equipped with radial vanes rotating at high speed near a fixed organic membrane. Two processes were tested. The first one consisted in two ultrafiltrations (UF) at 20 and 10 kDa followed by a nanofiltration (NF) at 1 kDa to extract glucuronans of 6 < DP < 55 in the 1 kDa retentate and in the second process we added an initial UF at 50 kDa and a final NF at 0.7 kDa to collect oligomers of 4 ≤ DP ≤ 6 in the last retentate. Due to high shear rates produced at the membrane by the disk, permeate fluxes were very large at all ultrafiltration steps, ranging from 370 L h −1 m −2 with the 20 kDa membrane, 350 with the 50 kDa one (600 kPa) and 280 with the 10 kDa (1500 kPa). They also decayed relatively little with increasing concentration, to, respectively, 360 and 230 L h −1 m −2 for 50 and 20 kDa at a volume reduction factor of 4. In NF, these permeate fluxes were, respectively, 80 L h −1 m −2 at 1 kDa (2000 kPa) and 70 L h −1 m −2 at 0.7 kDa. The addition of an initial UF at 50 kDa reduced the membrane fouling by large solutes in the next ultrafiltrations and raised the oligomers mass collected in the 1 kDa retentate from 3.89 to 6.83 g from the same initial total glucuronans mass of 40 g. These results confirm the high potential of dynamic filtration for fractionation of complex biotechnological solutions due to high shear rates generated at the membrane which, not only increase permeate fluxes by reducing concentration polarization but raise the membrane rejection by reducing diffusive transport through NF membranes.

Modification of Lipopolysaccharide with Colanic Acid (M-antigen) Repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.

One‐Pot Solid‐Phase Glycoblotting and Probing by Transoximization for High‐Throughput Glycomics and Glycoproteomics

The development of rapid and efficient methods for high-throughput protein glycomics is of growing importance because the glycoform-focused reverse proteomics/genomics strategy will greatly contribute to the discovery of novel biomarkers closely related to cellular development, differentiation, growth, and aging as well as a variety of diseases such as cancers and viral infection. Recently, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with aminooxy-functionalized polymers, which we termed "glycoblotting" (see S.-I. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The chemoselective blotting of oligosaccharides present in crude biological materials onto synthetic polymers relies on the unique oxime-bond formation between aminooxy group displayed on the supporting materials and aldehyde/ketone group at the reducing terminal of all oligosaccharides, thus enabling highly selective and rapid oligosaccharide purification. Aiming to improve the detection sensitivity of the released oligosaccharides, we introduce here a novel strategy for one-pot solid-phase glycoblotting and probing by transoximization. We found that oligosaccharides captured by the polymer supports via the oxime bond can be released in the presence of excess O-substituted aminooxy derivatives in a weakly acidic condition. The released oligosaccharides could be recovered as newly formed oxime derivatives of the O-substituted aminooxy compound added, thus demonstrating the simultaneous releasing and probing. In addition, we synthesized a novel aminooxy-functionalized monomer, N-[2-[2-(2-tert-butoxycarbonylaminooxyacetylamino-ethoxy)ethoxy]ethyl]-2-methacrylamide, which allows for the large-scale preparation of a versatile polymer characterized by its high stability, high blotting capacity, and easy use. The one-pot protocol allowed to profile 23 kinds of N-glycan chains of human serum glycoproteins. This concept was further applied for the glycopeptides analysis in a crude mixture followed by galactose oxidase treatment to generate free aldehyde group at the non-reducing terminal of oligosaccharide moiety of glycopeptides. Our technique may be implemented in existing biochemistry and molecular diagnostics laboratories because enriched oligosaccharides and glycopeptides by solid-phase transoximization with high-sensitive labeling reagents are widely applicable in a variety of common analytical methods using two-dimensional HPLC, LC/MS, and capillary electrophoresis as well as modern mass spectrometry.

Purification of oligosaccharides from rice husk autohydrolysis liquors by ultra- and nano-filtration

An ultrafiltration-based process for oligosaccharide and peptide fractionation from a macroalgae subcritical water hydrolysate was studied. A wide range of separation results was obtained depending on the membrane pore. 100 kDa cut-off size was enough for hydrolysate clarification with total retention of colloidal materials. Oligosaccharides present in the hydrolysate showed the highest retention with all membranes, glucans mostly, followed by galactans, and finally arabinans. Peptides obtained after subcritical water treatment were some of the lowest rejected compounds, even using a 5 kDa membrane. The increase in temperature from 20 to 50 °C and feed flow rate from 6.6 to 11.2 L/h enhanced permeate flux for 5 kDa membrane, without perturbing the membrane retention. The Hermia's models identified the cake layer resistance as the major fouling resistance in hydrolysate filtrations at 20 °C, but standard pore blockage was the principal fouling mechanism at 50 °C. A fractionation process with sequential filtration stages at 20 °C and TMP = 1.1 bar was examined. Oligosaccharides were fractionated in the retentates of the sequential filtrations with 100, 5 and 1 kDa membranes. The final permeate collected from the 1 kDa membrane was freeze-dried to obtain a peptide-rich solid (71 wt%) that could be used in different applications.

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-alpha secretion by a Dectin-1-expressing macrophage cell line.

Polymeric composite membranes based on carbon/PSf

Enzymatic membrane reactors were obtained from polymeric membranes and activated carbon, and used for oligosaccharide purification. The activated carbon was used to adsorb the enzyme, directly or via a metal ion as intermediate. We studied the adsorption capacity of two activated carbons (a commercial carbon and a home-made one) and the formation of the complex. In a second step, we studied the activity of the enzymes in batch experiments, and analyzed the synthesis and performance of the membrane reactors. Different kinds of enzymatic membrane reactors were obtained with immobilized solid enzymes. Our results show good agreement between the kinetics of the reactions and the velocity of the flux across the membrane, since both reaction and separation were properly achieved. We also determined optimum amounts of enzyme for obtaining the desired products with a low degree of polymerization and low concentrations of monomer.

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4.

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.

Optimization of the extraction and purification of oligosaccharides from defatted soybean meal

Summary Optimization of the extraction of oligosaccharides from defatted soybean meal (DSM) was investigated under various conditions. The optimal ratio of water to DSM and optimal temperature for oligosaccharide extraction was 5:1 and 50 °C, respectively. The use of a stirring process, without any further grinding, improved the extractability of oligosaccharides from DSM, a 10% ethanol‐water solution was more effective than distilled water alone. To purify the oligosaccharides, ultrafiltration was used. More than 90% of the protein was removed from the extracts at a volume concentration ratio (VCR) of 3–5. The percentages of fructose, sucrose, raffinose and stachyose in permeate for a VCR of 5 were 38.6, 51.4, 54.2 and 52.6%, respectively. A VCR of 5 was the most effective for protein removal and recovery of oligosaccharides.