Comparative analysis of sulfated galactans from red algae by reductive hydrolysis and mild methanolysis coupled to two different HPLC techniques

International audienceThe sugar determination of the sulfated galactans, agars and carrageenans of various red algae was performed using two different techniques of depolymerisation with subsequent HPLC analysis: 1) reductive hydrolysis/ HPAEC-PAD; 2) mild methanolysis/ RPLC-DR. Both techniques were optimized to release quantitatively the composite sugars (galactose, 6-O-methyl-galactose, the labile 3,6-anhydrogalactose and 2-O-methyl-3,6-anhydrogalactose residues) and precise relative response factors of authentic 3,6-anhydrogalactose were determined. The methanolysate neutralisation step, performed subsequently to methanolysis depolymerisation, was demonstrated as a key step for the quantitative recovery of the anhydrogalactose residues. The yield of the main sugars released by the two techniques were in good agreement for the commercial agarose and iota and kappa carrageenans studied

Sugar determination in ulvans by a chemical-enzymatic method coupled to high performance anion exchange chromatography

International audienceThe sugar determination of ulvans, the water-soluble polysaccharides from Ulva sp. and Enteromorpha sp., was optimized by combining partial acid prehydrolysis (2 mol L-1 trifluoroacetic acid, 120°C) with enzymic hydrolysis (with β-D-glucuronidase). The different constitutive sugars (rhamnose, galactose, glucose, xylose, glucuronic acid), released after hydrolysis, were separated by high performance anion-exchange chromatography and determined by pulsed amperometric detection. The ulvanobiouronic acid, β-D-GlcA-(1,4)-L-Rha, which is the main constituent of ulvans was always present after 3 h of trifluoroacetic acid hydrolysis (approx. 2% D.M.) when acid hydrolysis was performed alone but the xylose amount fell to 75% of its maximum value at this time. The optimal duration of 2 mol L−1 trifluoroacetic acid hydrolysis of ulvans (i.e. without any degradation of xylose, rhamnose and glucuronic acid) was 45 min. Additionnal treatment of the partial acid hydrolysate by purified β-D-glucuronidase allowed the hydrolysis of the residual ulvanobiouronic acid in rhamnose and glucuronic acid. High performance anion exchange chromatography coupled to this chemical-enzymic hydrolysis revealed to be a high resolution chromatographic technique for monitoring the hydrolysis of the aldobiouronic acid by β-D-glucuronidase. This method allowed the simultaneous quantitative determination of neutral and acidic sugars and revealed the presence of iduronic acid inulvans

Studies on the simultaneous determination of acidic and neutral sugars of plant cell wall materials by HPLC of their methyl glycosides after combined methanolysis and enzymic prehydrolysis

Studies on the Simultaneous Determination of Acidic and Neutral Sugars of Plant-Cell Wall Materials by Hplc of Their Methyl Glycosides after Combined Methanolysis and Enzymatic PrehydrolysisInternational audienceA method which involves enzymic hydrolysis followed by methanolysis and separation of their methyl glycosides by HPLC was applied to complex polysaccharides from three fibre preparations (pea hulls, sugar-beet pulp and wheat bran). The results were compared to those obtained by (1) methanolysis without enzymic prehydrolysis, (2) gas chromatography of the alditol acetates of the neutral sugars released by acid hydrolysis, and (3) colorimetric determination of the uronic acids. Methanolysis alone allows the estimation of non- cellulosic polysaccharides (pectins and hemicelluloses), whereas combined methanolysis and enzyme prehydrolysis also leads to the determination of cellulose, except for wheat bran which is a highly lignified plant cell wall material

Fractionation of xyloglucans and glucomannans according to their fine structure

National audienc

Structural characterization of underivatized arabino-xylo-oligosaccharides by negative-ion electrospray mass spectrometry

International audienceVarious arabino-xylo-oligosaccharides with known substitution patterns were assessed by negative ESI-Q-TOFMS and ESI-ITMS. The CID spectra of linear xylo-oligosaccharides and of nine isomeric mono- and disubstituted arabino-xylo-oligosaccharides established that structures differing in their substitution pattern can be differentiated by this approach. The negative-ion fragmentation spectra of the deprotonated quasi-molecular ions are mainly characterized by glycosidic cleavage ions from the C-series, which provide sequence informations, and by cross-ring cleavage 0,2Ai ions, which provide partial linkage information. When the collision energy increased, the cross-ring cleavage 0,2Ai ions underwent consecutive loss of water to produce 0,2Ai − 18 fragment ions and glycosidic cleavage ions of the B-series are also produced besides the Ci ions. Contrary to linear xylo-oligosaccharides, Ci ions, which originate from C-3 monosubstituted xylosyl residues never produce the related cross-ring cleavage 0,2Ai ions. Disubstitution at O-2 and O-3 of xylosyl residues appears to enhance the production of the 0,2Ai ions compared to monosubstitution. For the differentiation of the mono- and disubstitution patterns of the penultimate xylosyl residue, the relative abundance of the glycosidic cleavage ions at m/z 263 and 299 found on Q-TOF CID spectra plays a relevant role and appears to be more informative than MSn spectra obtained on a ion trap instrument

Mass spectrometry for pectin structure analysis

Pectin are extremely complex biopolymers made up of different structural domains. Enzymatic degradation followed by purification and structural analysis of the degradation products proved to be efficienttools for the understanding of pectin fine structure, including covalent interactions between pectic structural domains or with other cell wall polysaccharides. Due to its high sensitivity, high throughput andcapacity to analyze mixtures, mass spectrometry has gained more and more importance as a tool for oligosaccharides structural characterization in the past 10 years. This review will focus on the combined useof mass spectrometry and enzymatic digestion for pectins structural characterization

Hemicellulose fine structure is affected differently during ripening of tomato lines with contrasted texture

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceThe impact of genetic and fruit ripening on hemicelluloses fine structure was studied in twelve near isogenic lines of tomato fruits harboring firmness QTL. The sugar composition and the MALDI-TOF MS oligosaccharides profile after glucanase hydrolysis of the cell walls were determined from all green and red fruits pericarp tissue. MS profiles showed two major series of oligomers attributed to xyloglucan (XG) and glucomannan (GM) with minor peaks for xylan and ions attributed to galacto-oligomers. The oligosaccharides MS intensity varied significantly with the fruit genetic and ripening status. Correlations between MS intensity indicated structural regulations of both XG and GM structures with genetics and ripening. These results point to a region on the tomato chromosome 9 controlling cell wall galactose metabolism

Des précurseurs de polymères aux carbonitrures de silicium modifiés à façon, vers des fibres céramiques fonctionnalisées

International audienc

Fractionation and structural characterization of LiCl-DMSO soluble hemicelluloses from tomato.

To prepare and explore the structure of native hemicellulose from tomato, extraction of the natively acetylated polysaccharides was achieved from partially depectinated cell walls by DMSO doped with LiCl. DEAE anion exchange chromatography of the LiCl-DMSO extract allowed the removal of residual acidic pectin and the isolation of acetylated glucuronoxylan. The hemicellulose neutral fraction from the anion exchanger was fractionnated by size exclusion chromatography into xyloglucan (XyG) and galactoglucomannan (GgM) either as single major constituents or as mixtures of both. Residual hemicellulose in the cell wall was extracted by 4.0 M and not 1.0 M KOH. The fine structure of all LiCl-DMSO fractions and alkali extracts was assessed by coupling β-glucanase, β-mannanase and β-xylanase enzymatic degradations to the analysis of the resulting fragments by HPAEC and MALDI-TOF mass spectrometry. This approach revealed substitutions in part of the GgM fractions by pentose residues, presumably arabinose and/or xylose occuring in highly substituted block domains. It also demonstrated a different glucanase hydrolysis profile from 4.0 M KOH compared to LiCl-DMSO soluble fractions. The present extraction and purification scheme allow the recovery of several populations of acetylated hemicellulose families which emphasize the structural diversity and complexity of these polysaccharides