Involvement of a Hydrophobic Pocket and Helix†11 in Determining the Modes of Action of Prenylated Flavonoids and Isoflavonoids in the Human Estrogen Receptor.

Six prenylated (iso)flavonoids were purified from a licorice root extract and subjected to competition experiments with six commercially available (iso)flavonoids. The agonistic and antagonistic activities of these compounds towards both hERα (human estrogen receptor alpha) and hERß were determined. Differences in the modes of action (agonist or antagonist) were observed for the various compounds tested. In general, each compound had the same mode of action towards both ERs. In silico modeling was performed in order to study the differences in estrogenicity observed between the compounds. It is suggested that prenyl chains fit into a hydrophobic pocket present in the hER, resulting in an increased agonistic activity. In addition, it was shown that an increase in length (≈1.7 Å) of pyran prenylated isoflavonoids resulted in an antagonistic mode of action. This might be caused by collision of the pyran ring with helix 11 in the ligand binding cavity of the hER.

Development and validation of a confirmative LC-MS/MS method for the determination of ß-exotoxin thuringiensin in plant protection products and selected greenhouse crops.

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography-high-resolution mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5-7.7 % and within-lab reproducibility (RSD(WLR)) of 17 %. The method was used for analysis of >100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops.

Production of novel antioxidative phenolic amides through heterologous expression of the plant's chlorogenic acid biosynthesis genes in yeast.

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker's yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.

Profiling human gut bacterial metabolism and its kinetics using [U-13C]glucose and NMR.

This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

An intracellular pH gradient in the anammox bacterium Kuenenia stuttgartiensis as evaluated by 31P NMR.

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

Touching the High Complexity of Prebiotic Vivinal Galacto-oligosaccharides Using Porous Graphitic Carbon Ultra-High-Performance Liquid Chromatography Coupled to Mass Spectrometry.

Galacto-oligosaccharides (GOS) are used in infant formula to replace the health effects of human milk oligosaccharides, which appear to be dependent upon the structure of the individual oligosaccharides present. However, a comprehensive overview of the structure-specific effects is still limited as a result of the high structural complexity of GOS. In this study, porous graphitic carbon (PGC) was used as the stationary phase during ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). This approach resulted in the recognition of more than 100 different GOS structures in one single run, including reducing and non-reducing GOS isomers. Using nuclear magnetic resonance-validated structures of GOS trisaccharides, we discovered MS fragmentation rules to distinguish reducing isomers with a mono- and disubstituted terminal glucose by UHPLC-PGC-MS. UHPLC-PGC-MS enabled effective recognition of structural features of individual GOS components in complex GOS preparations and during, e.g., biological conversion reactions. Hence, this study lays the groundwork for future research into structure-specific health effects of GOS.

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR.

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.

Ethyl tert-butyl ether (EtBE) degradation by an algal-bacterial culture obtained from contaminated groundwater.

EtBE is a fuel oxygenate that is synthesized from (bio)ethanol and fossil-based isobutylene, and replaces the fossil-based MtBE. Biodegradation of EtBE to harmless metabolites or end products can reduce the environmental and human health risks after accidental release. In this study, an algal-bacterial culture enriched from contaminated groundwater was used to (i) assess the potential for EtBE degradation, (ii) resolve the EtBE degradation pathway and (iii) characterize the phylogenetic composition of the bacterial community involved in EtBE degradation in contaminated groundwater. In an unamended microcosm, algal growth was observed after eight weeks when exposed to a day-night light cycle. In the fed-batch reactor, oxygen produced by the algae Scenedesmus and Chlorella was used by bacteria to degrade 50 µM EtBE replenishments with a cumulative total of 1250 µM in a day/night cycle (650 lux), over a period of 913 days. The microbial community in the fed-batch reactor degraded EtBE, using a P450 monooxygenase and 2-hydroxyisobutyryl-CoA mutase, to tert-butyl alcohol (TBA), ethanol and CO2 as determined using 13C nuclear magnetic resonance spectroscopy (NMR) and gas chromatography. Stable isotope probing (SIP) with 13C6 labeled EtBE in a fed-batch vessel showed no significant difference in community profiles of the 13C and 12C enriched DNA fractions, with representatives of the families Halomonadaceae, Shewanellaceae, Rhodocyclaceae, Oxalobacteraceae, Comamonadaceae, Sphingomonadaceae, Hyphomicrobiaceae, Candidatus Moranbacteria, Omnitrophica, Anaerolineaceae, Nocardiaceae, and Blastocatellaceae. This is the first study describing micro-oxic degradation of EtBE by an algal-bacterial culture. This algal-bacterial culture has advantages compared with conventional aerobic treatments: (i) a lower risk of EtBE evaporation and (ii) no need for external oxygen supply in the presence of light. This study provides novel leads towards future possibilities to implement algal-bacterial consortia in field-scale groundwater or wastewater treatment.

Structural Motifs of Wheat Straw Lignin Differ in Susceptibility to Degradation by the White-Rot Fungus Ceriporiopsis subvermispora.

The white-rot fungus Ceriporiopsis subvermispora delignifies plant biomass extensively and selectively and, therefore, has great biotechnological potential. We previously demonstrated that after 7 weeks of fungal growth on wheat straw 70% w/w of lignin was removed and established the underlying degradation mechanisms via selectively extracted diagnostic substructures. In this work, we fractionated the residual (more intact) lignin and comprehensively characterized the obtained isolates to determine the susceptibility of wheat straw lignin's structural motifs to fungal degradation. Using 13C IS pyrolysis gas chromatography-mass spectrometry (py-GC-MS), heteronuclear single quantum coherence (HSQC) and 31P NMR spectroscopy, and size-exclusion chromatography (SEC) analyses, it was shown that ß-O-4' ethers and the more condensed phenylcoumarans and resinols were equally susceptible to fungal breakdown. Interestingly, for ß-O-4' ether substructures, marked cleavage preferences could be observed: ß-O-4'-syringyl substructures were degraded more frequently than their ß-O-4'-guaiacyl and ß-O-4'-tricin analogues. Furthermore, diastereochemistry (threo > erythro) and γ-acylation (γ-OH > γ-acyl) influenced cleavage susceptibility. These results indicate that electron density of the 4'-O-coupled ring and local steric hindrance are important determinants of oxidative ß-O-4' ether degradation. Our findings provide novel insight into the delignification mechanisms of C. subvermispora and contribute to improving the valorization of lignocellulosic biomass.

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Transport and compartmentation of phosphite in higher plant cells--kinetic and P nuclear magnetic resonance studies.

Phosphite (Phi, H(2)PO(3)(-)), being the active part of several fungicides, has been shown to influence not only the fungal metabolism but also the development of phosphate-deficient plants. However, the mechanism of phosphite effects on plants is still widely unknown. In this paper we analysed uptake, subcellular distribution and metabolic effects of Phi in tobacco BY-2 cells using in vivo(31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy. Based on the kinetic properties of the phosphate transport system of tobacco BY-2 cells, it was demonstrated that phosphite inhibited phosphate uptake in a competitive manner. To directly follow the fate of phosphate and phosphite in cytoplasmic and vacuolar pools of tobacco cells, we took advantage of the pH-sensitive chemical shift of the Phi anion. The NMR studies showed a distinct cytoplasmic accumulation of Phi in Pi-deprived cells, whereas Pi resupply resulted in a rapid efflux of Phi. Pi-preloaded cells shifted Phi directly into vacuoles. These studies allowed for the first time to follow Phi flux processes in an in vivo setting in plants. On the other hand, the external Pi nutrition status and the metabolic state of the cells had a strong influence on the intracellular compartmentalization of xenobiotic Phi.

Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing.

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.

Comparison of analytical and semi-preparative columns for high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance.

The application of analytical and semi-preparative columns in reversed-phase liquid chromatography-solid-phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR) was compared. The work was aiming at separating a higher sample amount in a single run and in this way to reduce the necessary NMR measurement time of separated compounds. Several parameters for compound separation and trapping procedures were optimised: flow rate of HPLC and make-up water pumps, choice of stationary phase cartridges and drying time. The separation and loadability of nine model compounds on analytical and semi-preparative columns was determined, as well as the focussing capacity of SH-type SPE cartridges. It was found that a semi-preparative column--or multiple peak trapping on analytical columns--gave better results than a standard 4.6mm analytical column for non-polar compounds (e.g. flavonoid aglycones, sesquiterpene lactones, non-polar terpenes, logP>2), but for polar compounds (logP<-2) did not offer any advantage over an analytical column, or was even disadvantageous. For intermediately polar compounds (-2

Glyceollins and dehydroglyceollins isolated from soybean act as SERMs and ER subtype-selective phytoestrogens.

Seven prenylated 6a-hydroxy-pterocapans and five prenylated 6a,11a-pterocarpenes with different kinds of prenylation were purified from an ethanolic extract of fungus-treated soybean sprouts. The activity of these compounds toward both human estrogen receptors (hERα and hERß) was determined in a yeast bioassay and the activity toward hERα was additionally tested in an U2-OS based hERα CALUX bioassay. In the yeast bioassay, compounds with chain prenylation showed in general an agonistic mode of action toward hERα, whereas furan and pyran prenylation led to an antagonistic mode of action. Five of these antagonistic compounds had an agonistic mode of action in the U2-OS based hERα CALUX bioassay, implying that these compounds can act as SERMs. The yeast bioassay also identified 8 ER subtype-selective compounds, with either an antagonistic mode of action or no response toward hERα and an agonistic mode of action toward hERß. The ER subtype-selective compounds were characterized by 6a-hydroxy-pterocarpan or 6a,11a-pterocarpene backbone structure. It is suggested that either the extra D-ring or the increase in length to 12-13.5Å of these compounds is responsible for an agonistic mode of action toward hERß and, thereby, inducing ER subtype-selective behavior.

Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts.

A rapid method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining an HPLC with on-line radical scavenging using DPPH* as a model radical and an HPLC-DAD-SPE-NMR system. Using this method a commercial rosemary extract was investigated. All major compounds present in the extract were collected on SPE cartridges after their separation. Advantages of on-line SPE peak trapping are the possibility to perform HPLC with non-deuterated solvents, a concentration effect and being able to record NMR spectra in pure 100% deuterated solvents. After comparing DAD and DPPH scavenging chromatograms, 1H NMR spectra of compounds having radical scavenging activities were recorded. Afterwards all compounds were collected and infused into an ESI-MS. The five main active compounds - carnosol, carnosic acid carnosaldehyde, 12-methoxycarnosic acid and epiisorosmanol could be identified from the combined UV, NMR and mass spectral data without actually isolating them. It was possible to record on-line an HMBC spectrum of carnosic acid. Also one compound was tentatively identified as epirosmanol methyl ether.

Level and position of substituents in cross-linked and hydroxypropylated sweet potato starches using nuclear magnetic resonance spectroscopy.

Sweet potato starch was cross-linked using sodium trimetaphosphate and hydroxypropylated using propylene oxide. The level and position of phosphorus and hydroxypropyl groups within cross-linked and hydroxypropylated sweet potato starch was investigated by phosphorus and proton nuclear magnetic resonance spectroscopy ((31)P, (1)H NMR). The cross-linking reaction produced monostarch monophosphate and distarch monophosphate in a molar ratio of 1:1.03, indicating that only half of the introduced phosphorus resulted in a possible cross-link. One cross-link per approximately 2900 glucose residues was found. Phosphorylation leading to monostarch monophosphate mainly occurred at O-3 and O-6 (ratio 1:1). It was inferred that the majority of the cross-links formed in distarch monophosphate were between two glucose residues positioned in different starch chains, while a minor part of the cross-links may be formed between two glucose residues within the same starch chain. Hydroxypropylation under alkaline conditions resulted in the formation of intra-molecular phosphorus cross-links, subsequent hydroxypropylation following cross-linking lowered both the level of intra- and inter-molecular cross-linking. Using (1)H NMR the molar substitution of hydroxypropylation was determined to be 0.155-0.165. The hydroxypropylation predominantly occurred at O-2 (61%), and the level of substitution at O-6 (21%) was slightly higher than that at O-3 (17%). In dual modified starch, the preceding cross-linking procedure resulted in a slightly lower level of hydroxypropylation, where the substitution at O-6 decreased more compared to the substitution at O-2 and O-3.

Production of butyrate from lysine and the Amadori product fructoselysine by a human gut commensal.

Human intestinal bacteria produce butyrate, which has signalling properties and can be used as energy source by enterocytes thus influencing colonic health. However, the pathways and the identity of bacteria involved in this process remain unclear. Here we describe the isolation from the human intestine of Intestinimonas strain AF211, a bacterium that can convert lysine stoichiometrically into butyrate and acetate when grown in a synthetic medium. Intestinimonas AF211 also converts the Amadori product fructoselysine, which is abundantly formed in heated foods via the Maillard reaction, into butyrate. The butyrogenic pathway includes a specific CoA transferase that is overproduced during growth on lysine. Bacteria related to Intestinimonas AF211 as well as the genetic coding capacity for fructoselysine conversion are abundantly present in colonic samples from some healthy human subjects. Our results indicate that protein can serve as a source of butyrate in the human colon, and its conversion by Intestinimonas AF211 and related butyrogens may protect the host from the undesired side effects of Amadori reaction products.

Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood.

A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)-(1)H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC-NMR was performed in order to study the structural features of the acetylated oligomers 'on-line' avoiding O-acetyl migration. Finally, the precise location of the 2-O- or 3-O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.

Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide.

The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine.

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.