Daniel Wefers

Characterization of diferuloylated pectic polysaccharides from quinoa (Chenopodium quinoa WILLD.)

In plants belonging to the order of Caryophyllales, pectic neutral side chains can be substituted with ferulic acid. The ability of ferulic acid to form intra- and/or intermolecular polysaccharide cross-links by dimerization was shown by the isolation and characterization of diferulic acid oligosaccharides from monocotyledonous plants. In this study, two diferulic acid oligosaccharides were isolated from the enzymatic hydrolyzate of seeds of the dicotyledonous pseudocereal quinoa by gel permeation chromatography and preparative HPLC and unambiguously identified by LC-MS(2) and 1D/2D NMR spectroscopy. The isolated oligosaccharides are comprised of 5-5- and 8-O-4-diferulic acid linked to the O2-position of the nonreducing residue of two (1→5)-linked arabinobioses. To get insight into the structure and the degree of phenolic acid substitution of the diferuloylated polysaccharides, polymeric sugar composition, glycosidic linkages, and polysaccharide-bound monomeric phenolic acids and diferulic acids were analyzed. This study demonstrates that diferulic acids are involved into intramolecular and/or intermolecular cross-linking of arabinan chains and may have a major impact on cell wall architecture of quinoa and other dicotyledonous plants of the order of Caryophyllales.

Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase

The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.

Novel arabinan and galactan oligosaccharides from dicotyledonous plants

Arabinans and galactans are neutral pectic side chains and an important part of the cell walls of dicotyledonous plants. To get a detailed insight into their fine structure, various oligosaccharides were isolated from quinoa, potato galactan, and sugar beet pulp after enzymatic treatment. LC-MS2 and one- and two-dimensional NMR spectroscopy were used for unambiguous structural characterization. It was demonstrated that arabinans contain β-(1→3)-linked arabinobiose as a side chain in quinoa seeds, while potato galactan was comprised of β-(1→4)-linked galactopyranoses which are interspersed with α-(1→4)-linked arabinopyranoses. Additionally, an oligosaccharide with two adjacent arabinofuranose units O2-substituted with two ferulic acid monomers was characterized. The isolated oligosaccharides gave further insight into the structures of pectic side chains and may have an impact on plant physiology and dietary fiber fermentation.

Neutral Pectin Side Chains of Amaranth (Amaranthus hypochondriacus) Contain Long, Partially Branched Arabinans and Short Galactans, Both with Terminal Arabinopyranoses

Amaranth is a pseudocereal of high nutritional value, including a high dietary fiber content. Amaranth dietary fiber was suggested to contain large amounts of neutral rhamnogalacturonan I side chains. In this study, endo-arabinanase and endo-galactanase were used to liberate arabinan and galactan oligosaccharides from amaranth fiber. The liberated oligosaccharides were identified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and HPLC-MS(n) using standard compounds, which were isolated from amaranth, sugar beet, potato, and red clover sprouts and characterized by one- and two-dimensional NMR spectroscopy. It was demonstrated that insoluble amaranth arabinans have linear and branched areas, with the O-3 position being the dominant branching point. Minor amounts of branches at position O-2 and double substitution were also found. Amaranth arabinans were also demonstrated to contain terminal α-(1→5)-linked l-arabinopyranose units. In addition, it was evidenced that galactans from amaranth seeds are composed of β-(1→4)-linked d-galactopyranose units, which can also be terminated with l-arabinopyranose units. In direct comparison to structural elucidation of amaranth fiber by using methylation analysis, the advantage of the enzymatic approach over methylation analysis was demonstrated.

Characterization of Dietary Fiber Polysaccharides from Dehulled Common Buckwheat (Fagopyrum esculentum) Seeds

Buckwheat is a pseudocereal that has gained increasing interest of industry and consumers over the past decade. Little, however, is known about its dietary fiber composition and nonstarch polysaccharide structures. Analysis of the monosaccharide composition indicated large amounts of pectic polysaccharides in both insoluble and soluble fiber from buckwheat. Methylation analysis gave further insights into the structures of the polysaccharides. The corresponding partially methylated alditol acetates suggested only low amounts of galactans. Xyloglucans were the main hemicellulosic polysaccharides in the insoluble fiber fraction. Highly branched arabinans, exclusively substituted at position O3, were of higher abundance. These results were confirmed by screening endo-arabinanase and endo-galactanase liberated oligosaccharides with high-performance anion-exchange chromatography with pulsed amperometric detection and LC-MS2. Application of this method also demonstrated highly branched arabinan areas within the ...

Arabinan and Galactan Oligosaccharide Profiling by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

Arabinans and galactans are complex pectic polysaccharides, which greatly influence the physicochemical and physiological properties of plants and plant-based foods. Conventional methods to characterize these challenging polymers are based on derivatization and/or unselective chemical cleavage of the glycosidic bonds of the polysaccharides, resulting in partial loss of essential information such as anomeric configuration. Here, endo-arabinanase and endo-galactanase were used to selectively cleave pectic arabinans and galactans. The liberated oligosaccharides were purified and characterized by LC-MS and one- and two-dimensional NMR spectroscopy resulting in known but also several previously unknown pectic structural elements. For the routine analysis of pectin hydrolysates by HPAEC-PAD, incubation conditions, chromatographic parameters, and relative response factors of the isolated pectic oligosaccharides against an internal standard were determined. The applicability of the method was demonstrated by analyzing different well-characterized plant cell wall materials. It was demonstrated that the developed method yields additional information about pectic arabinan and galactan structures that is not obtained from conventional methods such as methylation analysis.

Specific substrate-driven changes in human faecal microbiota composition contrast with functional redundancy in short-chain fatty acid production

The diet provides carbohydrates that are non-digestible in the upper gut and are major carbon and energy sources for the microbial community in the lower intestine, supporting a complex metabolic network. Fermentation produces the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, which have health-promoting effects for the human host. Here we investigated microbial community changes and SCFA production during in vitro batch incubations of 15 different non-digestible carbohydrates, at two initial pH values with faecal microbiota from three different human donors. To investigate temporal stability and reproducibility, a further experiment was performed 1 year later with four of the carbohydrates. The lower pH (5.5) led to higher butyrate and the higher pH (6.5) to more propionate production. The strongest propionigenic effect was found with rhamnose, followed by galactomannans, whereas fructans and several α- and β-glucans led to higher butyrate production. 16S ribosomal RNA gene-based quantitative PCR analysis of 22 different microbial groups together with 454 sequencing revealed significant stimulation of specific bacteria in response to particular carbohydrates. Some changes were ascribed to metabolite cross-feeding, for example, utilisation by Eubacterium hallii of 1,2-propanediol produced from fermentation of rhamnose by Blautia spp. Despite marked inter-individual differences in microbiota composition, SCFA production was surprisingly reproducible for different carbohydrates, indicating a level of functional redundancy. Interestingly, butyrate formation was influenced not only by the overall % butyrate-producing bacteria in the community but also by the initial pH, consistent with a pH-dependent shift in the stoichiometry of butyrate production.

Characterization of β-glucan formation by Lactobacillus brevis TMW 1.2112 isolated from slimy spoiled beer

Despite several hurdles, which hinder bacterial growth in beer, certain bacteria are still able to spoil beer. One type of spoilage is characterized by an increased viscosity and slimy texture caused by exopolysaccharide (EPS) formation of lactic acid bacteria (LAB). In this study, we characterize for the first time EPS production in a beer-spoiling strain (TMW 1.2112) of Lactobacillus brevis, a species commonly involved in beer spoilage. The strains growth dynamics were assessed and we found an increased viscosity or ropiness in liquid or on solid media, respectively. Capsular polysaccharides (CPS) and released EPS from the cells or supernatant, respectively, were analyzed via NMR spectroscopy and methylation analysis. Both are identical β-(1→3)-glucans, which are ramified with β-glucose residues at position O2. Therefore, we assume that this EPS is mainly produced as CPS and partially released into the surrounding medium, causing viscosity of e.g. beer. CPS formation was confirmed via an agglutination test. A plasmid-located glycosyltransferase-2 was found as responsible for excess β-glucan formation, chromosomal glucanases were proposed for its degradation. The glycosyltransferase-2 gene could also be specifically identified in beer-spoiling, slime-producing Lactobacillus rossiae and Lactobacillus parabuchneri strains, suggesting it as promising marker gene for the early detection of β-glucan-producing Lactobacilli in breweries.

NMR Spectroscopic Profiling of Arabinan and Galactan Structural Elements

Pectic arabinans and galactans presumably affect the physiological and technological properties of plant cell walls and dietary fiber. Their complex structures are usually analyzed by time-consuming methods, which are based on chemical cleavage to monomers. To gain more detailed insights into the arabinan and galactan structures, a time-efficient approach based on enzymatic cleavage and two-dimensional NMR spectroscopy was developed. Heteronuclear single quantum coherence spectroscopy (HSQC) marker signals were evaluated for various structural elements, and relative response factors were determined, allowing a semiquantitative estimation of the structural composition. The method was applied to analyze different insoluble plant materials and soluble polysaccharides. It was demonstrated that the developed approach yielded comparable information about various structural elements that can also be detected by using the conventional methylation analysis. However, by using the NMR method, additional structural information, such as the anomeric configuration of the monomers, is obtained, demonstrating the value of this novel approach.

Structural characterization of the exopolysaccharides from water kefir

Water kefir is a beverage which is produced by initiating fermentation of a fruit extract/sucrose solution with insoluble kefir grains. Exopolysaccharides that are formed from sucrose play a major role in the kefir grain formation, but the exopolysaccharides in the kefir beverage and the detailed structural composition of the whole kefir grains have not been studied yet. Therefore, kefir grains and the corresponding kefir beverage were analyzed for exopolysaccharides by multiple chromatographic approaches and two-dimensional NMR spectroscopy. Furthermore, different fractionation techniques were applied to obtain further information about the exopolysaccharides. The exopolysaccharide-fraction of the investigated kefir beverage was predominantly composed of O3- and O2-branched dextrans as well as lower amounts of levans. The insoluble dextrans from the kefir grains were mostly O3-branched and contained an elevated portion of 1,3-linked glucose units compared to the soluble dextrans. The structurally different exopolysaccharides in water kefir suggest the involvement of multiple bacteria.