Enzymatic analysis of levan produced by lactic acid bacteria in fermented doughs

Levans and inulins are fructans with mainly beta-(2 -> 6) and beta-(2 -> 1) linkages, respectively. Levans are produced by many lactic acid bacteria, e.g. during sourdough fermentation. Levans have shown prebiotic properties and may also function as in situ-produced hydrocolloids. So far, levan contents have been measured by acid hydrolysis, which cannot distinguish levans from e.g. inulins. In order to develop a specific analysis for levan in food matrices, a Paenibacillus amylolyticus endolevanase was combined with exoinulinase for levan hydrolysis. A separate endoinulinase treatment was used to detect the possible presence of inulin. Interfering sugars were removed by a pre-wash with aqueous ethanol. Levan content was estimated from fructose and glucose released in the hydrolysis, with a correction made for the residual fructose and glucose-containing sugars. The method was validated using wheat model doughs spiked with commercial Erwinia levan, and tested by analyzing levan content in Leuconostoc mesenteroides DSM 20343-fermented fava bean doughs.Peer reviewe

Identification and structural analysis of cereal arabinoxylan-derived oligosaccharides by negative ionization HILIC-MS/MS

Recent works provide evidence of the prebiotic potential of arabinoxylan-derived oligosaccharides (A)XOS. In this study, we developed a structural analysis for cereal-derived (A)XOS by negative ionization HILIC-MS/MS. Initially, we assessed twelve (A)XOS samples of known structures with different linkage positions and branching points by direct-infusion negative ESI-MSn. We subsequently developed the negative ion HILIC-MS/MS with a post-column addition of ammonium chloride. The selected (A)XOS represented both linear (arabinofuranosyl residue linked to the non-reducing end of xylooligosaccharide) and branched structures. Each (A)XOS sample produced a specific spectrum in negative ion ESI-MSn. By analyzing cross-ring fragment ions, we determined the linkage positions of linear (A)XOS. The presence or absence of diagnostic ions in the MS3 allowed us to detect different branches (O-2- or/and O-3-linked arabinofuranosyl with/or without O-4-linked xylopyranosyl at the non-reducing end). Furthermore, we could identify all analyzed samples by HILIC-MS/MS, based on the formed spectral library and chromatographic retention times.Peer reviewe

Laccase/TEMPO oxidation in the production of mechanically strong arabinoxylan and glucomannan aerogels

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Active fungal GH115 alpha-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans

Background Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans’ structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. Results The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. Conclusions Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall–targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.Peer reviewe

Tissue-specific study across the stem reveals the chemistry and transcriptome dynamics of birch bark.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications.peerReviewe