Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Residual Carbohydrates from in Vitro Digested Processed Rapeseed (Brassica napus) Meal
Pustjens, A.M. ; Vries, S. de; Gerrits, W.J.J. ; Kabel, M.A. ; Schols, H.A. ; Gruppen, H. - \ 2012
Journal of Agricultural and Food Chemistry 60 (2012)34. - ISSN 0021-8561 - p. 8257 - 8263.
nutrient digestibility - growth-performance - pectic substances - dehulled rapeseed - feed ingredients - particle-size - dietary fiber - soybean-meal - pigs - polysaccharides
Rapeseed meal (RSM) was subjected to different physical or chemical pretreatments to decrease residual, hard to degrade carbohydrates and to improve fermentability of RSM polysaccharides. Next, these pretreated samples were in vitro digested and fermented, with or without the addition of commercial pectinolytic enzymes. Remaining carbohydrates were quantified, and two physical characteristics were analyzed: (1) water-binding capacity (WBC) of the insoluble residue and (2) viscosity of the soluble fraction. Mild acid pretreatment in combination with commercial pectinolytic enzyme mixtures showed best digestion of RSM carbohydrates; only 32% of the total carbohydrate content remained. For most pretreatments, addition of commercial pectinolytic enzymes had the strongest effect on lowering the WBC of the in vitro incubated RSM. In the cases that less carbohydrate remained after in vitro digestion, the WBC of the residue decreased, and less gas seems to be produced during fermentation.
In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans
Jonathan, M.C. ; Borne, J.J.G.C. van den; Wiechen, P. van; Souza Da Silva, C. ; Schols, H.A. ; Gruppen, H. - \ 2012
Food Chemistry 133 (2012)3. - ISSN 0308-8146 - p. 889 - 897.
gastrointestinal-tract - colonic function - fatty-acids - polysaccharides - kinetics - starch - gas - fermentability - chromatography - degradation
In vitro fermentation of twelve dietary fibers by fecal inocula from pigs and humans were performed. The fibers included homoglucans, mannans, fructans, polyuronides, and complex heteroglycans. Gas production, short chain fatty acid production and fiber degradation products were monitored during fermentation. Human inoculum has more ability to ferment resistant starch and fibers containing uronic acids. In contrast, pig inoculum is able to ferment cellulose, which is hardly fermented by human inoculum. The sugar and linkage composition of the fibers has an important influence on fiber fermentation patterns. Fibers containing uronic acids induced the production of acetate, whereas fibers containing neutral sugars induced the production of propionate or butyrate. Fermentation of the fructans showed that molecular size could be an influential factor, and fermentation of complex heteroglycans showed that the arrangement of sugars in the molecules may also affect the fermentation patterns. This experiment also shows that monitoring of fiber degradation products is important for understanding how fibers are degraded during fermentation.
Characterization of substituents in xylans from corn cobs and stover
Dongen, F.E.M. van; Eylen, D. van; Kabel, M.A. - \ 2011
Carbohydrate Polymers 86 (2011)2. - ISSN 0144-8617 - p. 722 - 731.
xylo-oligosaccharides - polysaccharides - arabinoxylans - pretreatment - acetylation - extraction - products - enzymes
Structural knowledge on hemicellulose from corn cobs and stover is helpful to better understand their position within the plant cell wall architecture as well as their enzymatic saccharification. In this research different extracts were prepared with water, 1 M and 4 M alkali. Most of the xylans were yielded in the 1 M alkali fractions. The xylans were substituted with arabinose and glucuronic acid; 9 and 5 (cobs) and 14 and 9 (stover) per 100 xylosyl residues, respectively. Also esterlinked groups were present, like acetic, ferulic and coumaric acids; 49, 4 and 6 (cobs) and 39, 2 and 5 per 100 xylosyl residues (stover), respectively. Incubation with a well-characterized endoxylanase showed a more blockwise distribution of side-groups for stover xylans compared to cobs. Analysis of soluble fractions of dilute acid treated cobs and stover showed that especially feruloylated oligomers were resistant.
Determination of pectin content of eucalyptus wood
Coetzee, B. ; Schols, H.A. ; Wolfaardt, F. - \ 2011
Holzforschung 65 (2011)3. - ISSN 0018-3830 - p. 327 - 331.
quince chaenomeles-japonica - cell - polysaccharides - degradation - substances - responses - fruits - plants - hplc - wall
Very little is known about the occurrence of pectin in wood and it is speculated that between 10 mg g-1 and 40 mg g-1 of wood consists of pectin. The present study aimed to quantify pectin in eucalyptus wood and to determine the influence of tree species, yield potential of the site, tree age class and wood tissue type on its occurrence. Wood was hydrolysed using the Saeman procedure and the neutral and acidic monosaccharides quantified with high-performance liquid chromatography (HPLC). The d-galacturonic acid was the predominant pectic monosaccharide followed by d-galactose, l-arabinose and l-rhamnose. Through the addition of all pectic monosaccharides, it was determined that eucalyptus wood contained between 15.2 mg g-1 and 25.8 mg g-1 pectin. Wood tissue type had a major influence on the total pectin content of the samples. The cambium contained the highest concentration of pectin, reflecting more active growth. These results contribute to the understanding of wood biochemistry and can be useful in the industries producing biofuels or paper.
New insights on the formation of colloidal whey protein particles
Riemsdijk, L.E. van; Snoeren, J.P.M. ; Goot, A.J. van der; Boom, R.M. ; Hamer, R.J. - \ 2011
Food Hydrocolloids 25 (2011)3. - ISSN 0268-005X - p. 333 - 339.
phase-separation - globular-proteins - cold gelation - rich foods - microstructure - mixtures - gels - polysaccharides - temperature - morphology
This paper describes the formation and properties of whey protein particle suspensions having different particle sizes and different abilities to form S–S bridges. Simple shear flow was used to control the protein particles size. The ability to form S–S bridges was steered by blocking the reactive thiol groups of the whey proteins with N-ethylmaleimide. Microscopy and light scattering showed that simple shear flow applied during the formation of whey protein particles give irregularly shaped particles. Especially small particles aggregated into particle clusters. Microscopy and rheological measurements (strain and shear rate sweeps) showed that the protein particle clustering was favoured by the ability of the protein to form S–S bridges and to a lesser extend by a smaller particle size. From the study, it can be concluded that the formation of S–S bridges has no effect on the formation process of protein particles, but S–S bridges are important for the ability of the whey protein particles to form particle clusters
Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols
Abzac, P. D'; Bordas, F. ; Hullebusch, E. ; Lens, P.N.L. ; Guibaud, G. - \ 2010
Applied Microbiology and Biotechnology 85 (2010)5. - ISSN 0175-7598 - p. 1589 - 1599.
activated-sludge - waste-water - polysaccharides - complexation - flocs - acid
The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g(-1) of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.
Performance and energy metabolism in restrictively fed weanling pigs are not affected by feedinig either fermented cereals or their end-products
Bruininx, E.M.A.M. ; Binnendijk, G.P. ; Zandstra, T. ; Heetkamp, M.J.W. ; Peet-Schwering, C.M.C. van der; Gerrits, W.J.J. - \ 2010
Journal of Animal Physiology and Animal Nutrition 94 (2010)6. - ISSN 0931-2439 - p. e355 - e365.
epithelial-cell proliferation - growing pigs - physical-activity - organic-acids - polysaccharides - digestibility - diets - fiber
To study the effects of feeding fermented cereals or just fermentation end-products on performance and energy metabolism, 18 restrictedly fed groups of eight pigs each were assigned to one of three dietary treatments: (i) a liquid control diet (C) containing 40% of a mixture of barley and wheat; or (ii) a liquid diet (F) containing 40% fermented barley and wheat; or (iii) a liquid diet as C with the addition of some important fermentation end-products (FP; organic acids and ethanol) in concentrations similar to those in the fermented F-diet. Energy and nitrogen balances, heat production, and performance traits were measured during two consecutive periods (days 1–5 and days 6–14). There was a considerable increase in average dry matter intake that tended (p = 0.06) to be higher in the FP-group than in the other groups. Apparent fecal digestibility of dry matter, ash, nitrogen and energy during period 2 were not affected (p > 0.1). Averaged over both periods, none of the energy metabolism parameters were affected by the diets (p > 0.1). However, there were diet × period interactions for metabolizable energy-intake (p = 0.07), energy retention (p <0.05), the respiratory quotient (RQ; p <0.01) and activity-related heat production (HACT, p = 0.05). Additionally, there were some differences between the diets in the average hourly patterns in RQ and HACT. In conclusion, restricted feeding of either 40% fermented cereals nor their fermentation end-products affected performance and energy metabolism traits in weanling pigs. Nevertheless, lower postprandial activity-related heat production by pigs given the fermented cereals suggest a stimulating effect of fermented cereals on short term satiety that was not seen in pigs given fermentation end-products only
Characterization of Oligomeric Xylan Structures from Corn Fiber Resistant to Pretreatment and Simultaneous Saccharification and Fermentation
Appeldoorn, M.M. ; Kabel, M.A. ; Eylen, D. van; Gruppen, H. ; Schols, H.A. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)21. - ISSN 0021-8561 - p. 11294 - 11301.
grass cell-walls - maize bran - ferulic acid - liquid-chromatography - oligosaccharides - identification - heteroxylans - hydrolysis - polysaccharides - arabinoxylan
Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of corn fiber. Oligosaccharides that resisted a mild acid pretreatment and subsequent enzymatic hydrolysis, representing 50% of the starting material, were fractionated on reversed phase and size exclusion material and characterized. The oligosaccharides within each fraction were highly substituted by various compounds. Oligosaccharides containing uronic acid were accumulated in two polar fractions unless also a feruloyl group was present. Feruloylated oligosaccharides, containing mono- and/or diferulic acid, were accumulated within four more apolar fractions. All fractions contained high amounts of acetyl substituents. The data show that complex xylan oligomers are present in which ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acids were combined within an oligomer. Hypothetical structures are discussed, demonstrating which enzyme activities are lacking to fully degrade corn glucuronoarabinoxylans.
Biofilm formation on reverse osmosis membranes is initiated and dominated by Sphingomonas spp
Bereschenko, L.A. ; Stams, A.J.M. ; Euverink, G.J.W. ; Loosdrecht, M.C.M. - \ 2010
Applied and Environmental Microbiology 76 (2010)8. - ISSN 0099-2240 - p. 2623 - 2632.
targeted oligonucleotide probes - pseudomonas-aeruginosa - community structure - microbial biofilms - bacteria - identification - adhesion - ultramicrobacterium - polysaccharides - hybridization
The initial formation and spatiotemporal development of microbial biofilm layers on surfaces of new and clean reverse osmosis (RO) membranes and feed-side spacers were monitored in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The feed water of the RO system had been treated by the sequential application of coagulation, flocculation, sand filtration, ultrafiltration, and cartridge filtration processes. The design of the flow cells permitted the production of permeate under cross-flow conditions similar to those in spiral-wound RO membrane elements of the full-scale system. Membrane autopsies were done after 4, 8, 16, and 32 days of flow-cell operation. A combination of molecular (fluorescence in situ hybridization [FISH], denaturing gradient gel electrophoresis [DGGE], and cloning) and microscopic (field emission scanning electron, epifluorescence, and confocal laser scanning microscopy) techniques was applied to analyze the abundance, composition, architecture, and three-dimensional structure of biofilm communities. The results of the study point out the unique role of Sphingomonas spp. in the initial formation and subsequent maturation of biofilms on the RO membrane and feed-side spacer surfaces
First characterization of bioactive components in soybean tempe that protect human and animal intestinal cells against enterotoxigenic Escherichia coli (ETEC) infection
Roubos-van den Hil, P.J. ; Schols, H.A. ; Nout, M.J.R. ; Zwietering, M.H. ; Gruppen, H. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)13. - ISSN 0021-8561 - p. 7649 - 7656.
soya bean tempe - aspergillus-niger - adhesion - fermentation - inhibition - diarrhea - piglets - k88 - polysaccharides - arabinanases
Tempe extracts can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells and thereby can play a role in controlling ETEC-induced diarrhea. The component responsible for this adhesion inhibition activity is still unknown. This research describes the purification and partial characterization of this bioactive component of tempe. After heating, defatting, and protease treatment, the extracts were found to remain active. However, after treatment with polysaccharide-degrading enzyme mixtures the bioactivity was lost. Ultrafiltration revealed the active component to be >30 kDa. Further purification of the bioactive tempe extracts yielded an active fraction with an increased carbohydrate content of higher arabinose content than the nonactive fractions. In conclusion, the bioactive component contains arabinose and originates from the arabinan or arabinogalactan side chain of the pectic cell wall polysaccharides of the soybeans, which is probably released or formed during fermentation by enzymatic modifications
Analytical profiling of plant cell wall polysaccharides
Westphal, Y. - \ 2010
Wageningen University. Promotor(en): Harry Gruppen; Fons Voragen, co-promotor(en): Henk Schols. - [S.l.] : S.n. - ISBN 9789085857068 - 168
polysacchariden - celwandstoffen - chemische analyse - analytische methoden - polysaccharides - cell wall components - chemical analysis - analytical methods
Keywords: High performance liquid chromatography, capillary electrophoresis, MALDI-TOF
mass spectrometry, NMR, cell wall oligosaccharides, arabinan, Arabidopsis thaliana, cell wall degrading enzymes, profiling

The plant cell wall polysaccharides cellulose, hemicelluloses and pectins are very heterogeneous and complex structures consisting of at least 20 different sugars and
30 different linkage types. Additionally, hemicelluloses and pectins might be acetylated and/or feruloylated. Furthermore, pectins carry methyl esters. The degree and distribution of these modifications may vary significantly depending on source and developmental stage. In this research several analytical tools were developed for the analysis of complex mixtures of cell wall derived oligomers.
The combination of 1) the use of pure and well-defined cellulases, hemicellulases and pectinases, and 2) the detection of the oligosaccharides released by MALDI-TOF MS and CE-LIF resulted in a screening method for Arabidopsis cell wall mutants, which addresses all enzyme-accessible polysaccharides in the cell wall.
Porous graphitized carbon (PGC)-HPLC with evaporative light scattering and mass
detection was introduced to a broad range of neutral and acidic cell wall polysaccharide derived oligosaccharides and separation of almost all oligosaccharides under investigation was achieved. The used gradient ensured 1) a sufficient separation of many oligosaccharides and 2) a sequential elution of first the neutral and then the acidic oligosaccharides. This elution behavior in combination with online-recorded MSn analysis facilitates the identification of (unknown) peaks.
A wide range of branched arabino-oligosaccharides was isolated from sugar beet arabinan and characterized with NMR. HPAEC was demonstrated to have insufficient
resolution to separate all linear and branched arabino-oligosaccharides. Therefore, PGC-HPLC-ELSD/MS and CE-LIF-MS were explored for the separation and detection of isomeric arabino-oligomers and were demonstrated to perform well. The combination of the controlled enzyme treatment, the predictive retention behavior on PGC-material, and the LC/CE-MS2 fragmentation patterns led to the prediction of the structures of unknown branched arabino-oligosaccharides in a complex mixture.
Branched arabino-oligosaccharides isolated from sugar beet arabinan
Westphal, Y. ; Kuhnel, S. ; Waard, P. de; Hinz, S.W.A. ; Schols, H.A. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Carbohydrate Research : an international journal 345 (2010)9. - ISSN 0008-6215 - p. 1180 - 1189.
cell-walls - structural characterization - feruloyl oligosaccharides - polysaccharides - acid - nmr - pectins - pulp
Sugar beet arabinan consists of an a-(1,5)-linked backbone of l-arabinosyl residues, which can be either single or double substituted with a-(1,2)- and/or a-(1,3)-linked l-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an a-(1,5)-linked backbone of l-arabinosyl residues. One series carries single substituted a-(1,3)-linked l-arabinosyl residues at the backbone, whereas the other series consists of a double substituted a-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.
Introducing Capillary Electrophoresis with Laser-Induced Fluorescence (CE-LIF) as a Potential Analysis and Quantification Tool for Galactooligosaccharides Extracted from Complex Food Matrices
Albrecht, S.A. ; Schols, H.A. ; Klarenbeek, B. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)5. - ISSN 0021-8561 - p. 2787 - 2794.
gastrointestinal symptoms - galacto-oligosaccharide - polysaccharides - bifidobacteria - metabolism - prebiotics - ingestion - products - sugars
The analysis and quantification of (galacto)oligosaccharides from food matrices demands both a reproducible extraction method as well as a sensitive and accurate analytical method. Three typical matrices, namely, infant formula, fruit juice, and a maltodextrin-rich preparation, to which a commercial galactooligosaccharide mixture was added in a product concentration range from 1.25 to 30%, served as model substrates. Solid-phase extraction on graphitized carbon material upon enzymatic amyloglucosidase pretreatment enabled a good recovery and a selective purification of the different galactooligosaccharide structures from the exceeding amounts of particularly lactose and maltodextrins. With the implementation of capillary electrophoresis in combination with laser-induced fluorescence (CE-LIF) detection, a new possibility facilitating a sensitive qualitative and quantitative determination of the galactooligosaccharide contents in the different food matrices is outlined. Simultaneous monitoring and quantifying prebiotic oligosaccharides embedded in food matrices presents a promising and important step toward an efficient monitoring of individual oligosaccharides and is of interest for research areas dealing with small quantities of oligosaccharides embedded in complex matrices, e.g., body liquids.
TEMPO oxidation of gelatinized potato starch results in acid resistant blocks of glucuronic acid moieties
Haar, R. ter; Timmermans, J.W. ; Slaghek, T.M. ; Dongen, F.E.M. van; Schols, H.A. ; Gruppen, H. - \ 2010
Carbohydrate Polymers 81 (2010)4. - ISSN 0144-8617 - p. 830 - 838.
physicochemical properties - selective oxidation - mediated oxidation - polysaccharides - pectin
Chemical derivatization is often applied to improve polysaccharide functionality. Primary hydroxyl groups in starch can, for example, be oxidized to aldehydes by using a 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated reaction. The major part of the aldehydo groups is subsequently converted to carboxyl groups by NaOCl. The exact structure of TEMPO-oxidized starch was studied to promote a better understanding of the TEMPO oxidation mechanism and the functionality of oxidized starches. By using weak and strong acidic hydrolysis, and methanolysis, at elevated temperatures, oxidized starches with different degrees of oxidation (DO) were broken down into oligomers and monomers. Analysis of the oligomers by chromatographic and mass spectrometric techniques revealed that blocks of glucuronic acid moieties are present in the oxidized starch polymers. The a(1 ¿ 4) glucuronic acid–glucuronic acid bond was found to be very resistant to breakdown by acids. The a(1 ¿ 4) glucuronic acid–glucose bond also showed increased resistance to acids compared to a(1 ¿ 4) glucose–glucose bonds. The size of the blocks of glucuronic acid moieties increased when DO increased. Furthermore, the presence of clusters of aldehydes close to carboxyl groups directly after oxidation was proven. This implies that TEMPO, which is positively charged in its active state, is apparently attracted by the negatively charged carboxyl groups. Because of this, TEMPO tends to be active in areas where carboxyl groups have already been formed, which leads to a block wise distribution of the glucuronic acid moieties.
Enzymatic production of hyaluronan oligo- and polysaccharides
Kooy, F.K. - \ 2010
Wageningen University. Promotor(en): Gerrit Eggink; Hans Tramper, co-promotor(en): Carmen Boeriu. - [S.l. : S.n. - ISBN 9789085856481 - 174
hyaluronzuur - derivaten - oligosacchariden - polysacchariden - industriële microbiologie - industriële enzymen - hyaluronic acid - derivatives - oligosaccharides - polysaccharides - industrial microbiology - industrial enzymes
Hyaluronan oligo- and polysaccharides are abundant in the human body. Depending on the chain length, hyaluronan is an important structural component or is involved in influencing cell responses during embryonic development, healing processes, inflammation and cancer. Due to these diverse roles of hyaluronan, there are multiple applications already in use or in development, such as supplementation of fluid in eyes and joints, cosmetic tissue augmentation, enhancing wound healing, tissue engineering, cancer treatment, controlled drug release and targeted drug delivery. State-of-the-art hyaluronan production techniques include bacterial fermentation to produce long hyaluronan polymers with a small chain length distribution and in vitro enzymatic systems to produce hyaluronan oligosaccharides of one chain length. Both production strategies make use of hyaluronan synthase (HAS), an enzyme that elongates UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) into hyaluronan.

The main question in hyaluronan production today is how the chain length of the products can be controlled. Since most production processes use hyaluronan synthases, the aim of this thesis was to elucidate the polymerization mechanism of Pasteurella multocida hyaluronan synthase (PmHAS) from a biochemical point of view. In addition, the acquired knowledge is used for improving the control on hyaluronan chain length in polymerization reactions using PmHAS. Valuable information important for production processes on the intrinsic properties of the enzyme, such as substrate affinity, can be obtained by kinetic studies using single-step elongations. Kinetic studies also provide insights on how polymerization is achieved and, combined with structural studies, the identification of amino acid residues that are important for polymerization. This knowledge can be used for improving the hyaluronan synthesis performance of the enzyme.

Kinetic studies require purified substrates in quantities of mg-scale. Hyaluronan (HA) oligosaccharides were obtained through stepwise hyaluronan cleavage using hyaluronidase and consecutive separation of the reaction mixture by flash-chromatography (Chapter 2). The enzymatic hydrolysis was optimized by experimental design studies with pH, enzyme concentration and reaction time as parameters. Empirical models were developed for the yield of each individual target HA oligosaccharide using the results from a central composite design. Selective production of short HA oligomers (HA ≤ 10) or longer oligosaccharides (HA > 10) was made possible through implementation of the reaction conditions indicated by the empirical models. Separated HA oligomers were characterized by a combination of anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry with time-off-flight analysis. Using these techniques, the desired quantities of purified target HA oligosaccharides (n = 4, 6, 8 and 10) were obtained and used in further studies.

Besides the single-step elongations assessed in kinetic studies, full polymerization studies with both UDP-sugars available were used to investigate the influence of substrate concentrations on the chain length distribution of the hyaluronan products. In order to quantify all oligosaccharides formed during PmHAS polymerization in μl-scale reactions, HA templates consisting of a fluorophore-labeled HA tetrasaccharide (HA4) were generated (Chapter 3). A fast, simple and sensitive assay was developed based on fluorophore-assisted carbohydrate electrophoresis (FACE) that was used for quantification and characterization of PmHAS polymerization products.

The individual β1,3-glucuronyl-transferase (UA-transferase) and β1,4-N-acetylglucosamine-transferase (NAc-transferase) activities of PmHAS were investigated separately using kinetic studies, where the reaction of an HA oligosaccharide was followed with, respectively, UDP-GlcUA or UDP-GlcNAc in single-step elongations. In Chapter 4, the influence of HA oligosaccharide length (n = 4, 5, 6, 7, 8 and 9) on the polymerization reaction was investigated by one-substrate kinetics, varying only the HA oligosaccharide concentration at saturating UDP-sugar concentration. These reactions followed Michaelis Menten kinetics, although HA oligosaccharides may become inhibiting at elevated concentrations above 6 mM. The observed kcat values increased with increasing HA oligosaccharide length to a constant value at HA6 and HA7. The specificity constant kcat/Km values for HA oligosaccharides in the UA-transferase domain increased at increasing oligosaccharide length, whereas in the NAc-transferase domain kcat/Km values were constant at a low value. This indicates that there are two separate oligosaccharide binding sites of different lengths, one in each transferase domain of PmHAS. In Chapter 4, it was demonstrated that the chain-lenght distribution in PmHAS polymerization reactions can be decreased, and thus improved, by using saturating concentrations of both HA oligosaccharides and UDP-sugars.

Chapter 5 describes two-substrate kinetic studies, where in single-step elongations both HA oligosaccharide and one of the UDP-sugars were varied, to investigate the polymerization mechanism of each individual transferase domain in PmHAS. Dead-end inhibition studies and goodness-of-fit parameters were used to distinguish between two-substrate models. From this analysis follows that both transferase domains elongate the UDP-sugar through a sequential mechanism, which is most likely an ordered one. In this proposed mechanism, the UDP-sugar is first bound followed by binding of the HA oligosaccharide, after which first the elongated HA oligosaccharide and then UDP is released. Large differences between Km values for UDP-GlcNAc and UDP-GlcUA, also found in Class I HAS enzymes, suggest that UDP-GlcNAc concentration is involved in the regulation of HAS activity and thus the chain length of hyaluronan products.

Structural studies were used to evaluate the results obtained with kinetic studies. In Chapter 4, a structural homology model of PmHAS was built based on crystal structure K4CP chondroitin polymerase in E. coli, which has a high sequence identity of 62% and high sequence homology of 78% with PmHAS. The active sites of PmHAS are structurally related to other glycosyltransferases and this provided information on where the oligosaccharide binding sites could be located. These putative oligosaccharide binding sites differ in size, as was predicted by kinetic studies (Chapter 4). Furthermore, structural similarities between PmHAS, α1,3-galactosyltransferase (α3GT) and β1,4-galactosyltransferase (β4Gal-T1) demonstrated that PmHAS contains in each transferase domain one flexible loop that forms a bridge over the active site. In crystal structures of α3GT and β4Gal-T1, these flexible loops have been shown to change conformation upon binding the UDP-sugar. Based on similarities in kinetic mechanisms and structures between PmHAS, α3GT and β4Gal-T1, it is likely that the flexible loops in PmHAS follow a similar conformational change, which makes the proposed ordered mechanism the only possible mechanism (Chapter 5).

In Chapter 6, the knowledge on the PmHAS polymerization mechanism gained in earlier chapters is reviewed and used to create new insights in the polymerization mechanism of Class I HAS enzymes. Both Class I HASs and PmHAS are used in hyaluronan production, and, therefore, the differences and similarities are discussed in Chapter 6. During hyaluronan production, there are many different aspects, such as intrinsic properties of the enzyme, cell metabolism and fermentation reaction conditions, that influence hyaluronan chain length and yield (Chapter 6). Moreover, hyaluronan production systems that are able to produce hyaluronan of desired length are discussed in Chapter 6 and a personal view of how these systems can be improved is presented.
Production, Refining, Structural Characterization and Fermentability of Rice Husk Xylooligosaccharides
Gullon, P. ; Gonzales-Munoz, M.J. ; Gool, M.P. van; Schols, H.A. ; Hirsch, J. ; Ebringerova, A. ; Rarajo, J.C. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)6. - ISSN 0021-8561 - p. 3632 - 3641.
substituted xylo-oligosaccharides - human colonic microbiota - treated eucalyptus wood - in-vitro - dietary modulation - ft-ir - autohydrolysis - fermentation - liquors - polysaccharides
Oligosaccharides produced by hydrothermal processing of rice husks (xylooligosaccharides and glucooligosaccharides) were refined by membrane processing (operating in diafiltration and concentration modes), subjected to xylanase treatment to reduce the average molar mass, and subjected to further purification by ultrafiltration (operating in concentration mode) and ion exchange. The purified products were assayed for composition, molar mass distribution and structural characterization by HPLC, HPAEC-PAD, HPSEC, MALDI-TOF-MS and NMR (1H and 13C). The fermentability of the purified product by fecal inocula was assessed on the basis of the time courses of pH and oligosaccharide concentrations. Succinate, lactate, formiate, acetate, propionate and butyrate were the major products resulting from fermentation experiments.
Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis
Westphal, Y. ; Schols, H.A. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Journal of Chromatography. A, Including electrophoresis and other separation methods 1217 (2010)5. - ISSN 0021-9673 - p. 689 - 695.
treated eucalyptus wood - hairy ramified regions - capillary-electrophoresis - black-currants - polysaccharides - pectin - xylogalacturonan - quantification - nomenclature - ionization
Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSn-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar ß-(1,4)-xylo- and ß-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of ß-(1,4)-galacto-, ß-(1,4)-manno- and a-(1,4)-gluco-oligosaccharides. Charged oligomers such as a-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.
Polysaccharide Expertise Netwerk (EPNOE)
Dam, J.E.G. van; Boeriu, C.G. - \ 2010
polysacchariden - biomassa - interdisciplinair onderzoek - netwerken (activiteit) - innovaties - duurzaamheid (sustainability) - biobased economy - polysaccharides - biomass - interdisciplinary research - networking - innovations - sustainability
Korte informatie over het Polysaccharide Expertise Netwerk (EPNOE).
Physicochemical properties of pectins from okra (Abelmoschus esculentus (L.) Moench)
Sengkhamparn, N. ; Sagis, L.M.C. ; Vries, R.J. de; Schols, H.A. ; Sajjaanantakul, T. ; Voragen, A.G.J. - \ 2010
Food Hydrocolloids 24 (2010). - ISSN 0268-005X - p. 35 - 41.
rheological properties - flow - polysaccharides
Okra pectin obtained by hot buffer extraction (HBSS) consists of an unusual pectic rhamnogalacturonan I structure in which acetyl groups and alpha galactose residues are substituted on rhamnose residues within the backbone. The okra Chelating agent Soluble Solids (CHSS) pectin consists of slightly different structures since relatively more homogalacturonan is present within the macromolecule and the rhamnogalacturonan I segments carry slightly longer side chains. The rheological properties of both okra pectins were examined under various conditions in order to understand the unusual slimy behaviour of okra pectins. The viscosity of the okra HBSS pectin was 5–8 times higher than the viscosity of the okra CHSS pectin. The okra HBSS pectin showed an elastic behaviour (G' > G¿) over a wide range of frequencies (10-1–10 Hz), at a strain of 10%, while okra CHSS and saponified okra HBSS/CHSS pectin showed predominantly viscous responses (G'
Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges
Simon, S. ; Pairo, B. ; Villain, M. ; Abzac, P. D'; Hullebusch, E. ; Lens, P.N.L. ; Guibaud, G. - \ 2009
Bioresource Technology 100 (2009)24. - ISSN 0960-8524 - p. 6258 - 6268.
activated-sludge - extraction methods - part i - exopolymers - biofilms - polysaccharides - complexation - separation - protocols - hplc
The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1-7 kDa) and Superdex 20010/300GL (10-600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.
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