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Capillary electrophoresis (CE) has been used to characterize pectin fractions obtained from mother samples of varying degrees (and intramolecular patterns) of methylesterification (DM). These samples have been carefully produced by fractionation, (a) from the original mother samples on the grounds of molar mass, and (b) subsequently from the daughter fractions so obtained, on the grounds of charge. The results confirm theoretical predictions regarding the dependence of electrophoretic mobility on molar mass and DM and give further evidence that CE is able to give realistic information regarding the intermolecular DM of pectins.

Various feruloylated arabinose- and galactose-containing mono- and disaccharides with known linkage configurations (2-O-(trans-feruloyl)-L-arabinopyranose, 5-O-(trans-feruloyl)-L-arabinofuranose, O-[2-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1--5)-L-arabinofuranose, and O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1--4)-D-galactopyranose) were analyzed by electrospray ionization mass spectrometry using an ion trap or a quadrupole time-of-flight (Q-TOF) mass analyzer. Collision-induced dissociation (CID) experiments using the two mass analyzers generated similar tandem mass spectrometric (MS/MS) fragmentation patterns. However, the ester-bond cleavage ions were more abundant using the Q-TOF mass analyzer. Compared with the positive ion mode, the negative ion mode produces simpler and more useful CID product-ion patterns. For arabinose-containing feruloylated compounds, results obtained with both analyzers show that it is possible to assign the location of the feruloyl group to the O-2 or O-5 of arabinosyl residues. In the characterization of the 2-O-feruloyl and 5-O-feruloyl linkages, the relative abundance of the cross-ring fragment ions at m/z 265 (-60 u or -62 u after 18O-labelling) and at m/z 217 (-108 u or -110 u after 18O-labelling) play a relevant role. For galactose-containing feruloylated compounds, losses of 60, 90 and 120 Da observed in MS3 experiment correspond to the production of 0,2A1, 0,3A1 and (0,2A1-60 Da) cross-ring cleavage ions, respectively, fixing the location of feruloyl group at the O-6 of the galactose residue.

Side chains of sugar beet (Beta vulgaris) pectins, which are mainly composed of arabinose (Ara) and galactose (Gal) residues, are esterified by ferulic acid units. Enzymatic hydrolysis of beet cell walls yielded several feruloylated oligosaccharides, which were separated by hydrophobic interaction chromatography. Two new oligomers were isolated in the fraction eluted by 25:75 (v/v) ethanol:water. An arabinotriose and an arabinotetraose esterified by two ferulic acid residues were obtained, and their structure was elucidated by mass spectrometry. It is shown that feruloyl groups are linked to O-5 of Ara residues, in addition to the known O-2 position. This work establishes for the first time, to our knowledge, that two neighboring Ara units may be esterified by two ferulic acid units. This close proximity may have important biochemical implications.

An experimental design was constructed to study the influence of different parameters of acidic extraction on yield, chemical composition and intrinsic viscosity of pectins from fresh sugar-beet roots. pH, and, to a lower extent, time and temperature of extraction, affected greatly the yield and features of extracted pectins whereas the type of acid had no effect. Influences of extraction conditions on pectins characteristics were quantified and second order models were built.

An improved hplc method for the simultaneous determination of the degrees of methylation and acetylation of pectins has been developed. The proposed method involves saponification in heterogeneous medium followed by the separation of methanol, acetic acid and internal standard on a C18 column and quantification by refractometry. The method is reproducible, accurate, and presents several advantages that are discussed in the present paper.

International audience; kThe abilities of native and modified sugar beet pulps to remove Ni(II) and Cu(II) ions from aqueous solutions were compared. Their preparation by chemical treatments (saponification, hot 0.05 M HCl and cold 0.05 M NaOH extractions) is described. The sugar composition, which was strongly affected during these modifications, is discussed in terms of metal sorption efficiencies. ne influence of these modifications was also evaluated by comparing the content of the functional groups determined by potentiometric titration, and the rate and extent of Cu(II) and Ni(II) uptakes onto the raw and modified materials. Nickel and copper sorptions were fast and complete within 30 min and the kinetic parameters were calculated using a second order model. The equilibrium data fitted well with the Langmuir model and showed the affinity order of the materials for the metal ions. The base-extracted pulp and saponified pulp exhibited the highest Ni(II) and Cu(H) ion uptakes among the materials tested. (C) 2002 Elsevier Science Ltd. All rights reserved.

Maize bran heteroxylan samples were extracted in various conditions of severity. Their ferulate and diferulate content was investigated by GC-MS of methyl ester-TMSi derivatives. When extracted by 0.5 M NaOH in mild conditions, the heteroxylan sample contained a low level of ferulic acid (0.032% by wt.) and the main diferulate surviving alkaline extraction was found to be the 8-8' diferulate. On peroxidase treatment, this sample nevertheless produced a firm and brittle gel without any change in the diferulate profile. Typical lignin structures, mainly comprising syringyl units interconnected through beta-O-4, beta-1 and beta-beta interunit bonds, were evidenced in the maize bran sample. More importantly, these lignin structures were found to be tightly associated with the alkali-extracted heteroxylans. Thioacidolysis revealed the occurrence of 0.1-0.5% (by wt.) lignin structures in heteroxylan fractions extracted in mild or severe conditions, before and after purification of the polysaccharides. The gelling potential of the heteroxylan fractions was not only dependent on their ferulate level, but also influenced by associated lignin structures. These results argue for the occurrence of covalent linkages between heteroxylan chains and lignin structures which could participate in the peroxidase-driven gelation of feruloylated polysaccharides. They demonstrate the role of low lignin levels in the organization of native or reconstructed polysaccharide networks.

Pectins : their origin, structure and function

Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean beta-conglycinin alpha subunit. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP. Beta-conglycinin alpha subunit (68 kDa) presents seven consensus sequences, but only 0.5-1 mol P/mol alpha subunit was incorporated by CK2. [32P]Phosphorylated beta-conglycinin alpha subunit was cleaved either by cyanogen bromide or by trypsin. 32P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest. The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS). Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70-89, 116-126, and 116-127 sequences. Over the seven consensus sequences of beta-conglycinin alpha subunit, Ser 75 is the only one which was phosphorylated. Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position. Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites.

2 graph.; International audience

Two phosphorylation methods using soybean storage proteins as model substrates were compared. A chemical reaction was set up using common inexpensive reagents (iron and pyrophosphate), as well as an enzymatic method using cAMP-independent casein kinase II from the yeast Yarrowia lipolytica.

Pea and soybean globulins were phosphorylated by a casein kinase II from Yarrowia lipolytica. Soybean 7S was the best substrate, followed by pea 7S, pea US and soybean US. Phosphate was incorporated into soybean 7S a and α’ subunits, pea 7S 47, 23 and 17 kDa polypeptides, and US acidic subunits.

Some by-products from the food industry contain high proportions of plant cell walls which can be used in human nutrition to produce “dietary fibre” or “functional fibre”, i.e. compounds which can be used for their water-holding/binding properties, oil-binding capacity,… or as a source of polysaccharides such as pectins which are suitable after extraction, as gelling or thickening agents. Extrusion-cooking of cell-wall rich products (e.g. wheat bran, apple pomace, citrus peels, sugar-beet pulp, pea hulls…) led to an important solubilisation of polysaccharides of various types without extensive degradation of the polymeric structure. The possibility of obtaining gelled systems directly with the extruded pectin-rich materials was demonstrated. This treatment has the advantage of avoiding the acid extraction of the pectins; it could be also used to obtain extruded products which could be used in various preparations traditionnally requiring high methoxyl pectins as gelling agents. The final products could be therefore enriched in dietary fibres and devoid of additives.

Cell walls from sugar-beet pulp contain some feruloyl groups linked to the pectic neutral side-chains. Enzymic as well as chemical hydrolysis of the pulp yielded a series of feruloylated oligosaccharides, which have been purified by Sephadex LH-20 and Biogel P-2 chromatography in aqueous solvents. Feruloylated arabinose di-, tri-, hexa-, hepta-, and octa-saccharides as well as feruloylated galactose disaccharides were obtained after hydrolysis of the pulp with a mixture of fungal carbohydrases (Driselase). Feruloylated arabinose and galactose monosaccharides were obtained through mild acid hydrolyses. Both arabinose and galactose residues in the side-chains are feruloylated, 50–55% of the feruloyl groups being linked to arabinose residues and 45–50% to galactose residues. It is concluded that 1 out of 56 arabinose residues and 1 out of 16 galactose residues present as pectic side-chains in sugar-beet pulp carry a feruloyl group.

The swelling capacity, the water-binding capacity and the water-holding capacity were determined on four fibre sources: wheat bran; pea hulls; sugar-beet; and citrus fibre. Each fibre source was previously ground to obtain three different mean particle sizes. Grinding decreased swelling capacity and water-binding capacity of wheat bran, sugar-beet and citrus fibre probably by alteration and collapsing of the fibre matrix. The water-holding capacity of these fibres was little affected at equilibrium. Both water-binding and water-holding capacities of pea hulls tended to increase after grinding, probably because of an increase in surface area and pore volume. In the case of sugar beet fibre, the experimental parameters such as stirring conditions altered the physical structure of the fibres which resulted in a large change in water-binding capacity. These results underline the influence of the physical structure of the fibre on the hydration properties.

1D NMR (1H and 13C) and 2D NMR spectroscopy have been used to determine the structure of feruloylated oligosaccharides obtained by enzymic degradation or mild acid hydrolysis of sugar-beet pulp. Feruloylated oligosaccharides derived from pectic neutral side-chains containing arabinose or galactose residues were identified. In the feruloylated arabinose oligosaccharides, feruloyl groups were linked to O-2 of L-Ara f residues. The structure of the feruloylated arabinose disaccharide was identified as O-[2-O-(transferuloyl)-alpha-L-Ara f]-(1--5)-L-Ara f and that of the feruloylated arabinose trisaccharide as O-alpha-L-Ara f-(1--3)-O-[2-O-(trans-feruloyl)-alpha-L-Ara f]-(1--5)-L- Ara f. The structure of the feruloylated galactose disaccharide was identified as O-[6-O-(trans-feruloyl) -beta-D-Gal p]-(1--4)-D-Gal p. From our results, we suggest that the feruloyl groups present in sugar-beet pulp are linked to the arabinofuranosyl residues of the main core of alpha-(1--5)-linked arabinan chains and to the galactopyranosyl residues of the main core of beta-(1--4)-linked type I galactan chains.

Wheat bran was extruded under various conditions with a Clextral BC 45 twin screw extruder. Water (6·1–17·5 % of dry solid feed rate) and feed rate (20–32 kg/h) were varied in order to obtain treatments of different severities. These were monitored by the specific mechanical energy measured by the electrical consumption of the main motor drive. The water solubility of the extruded products increased from 20 to 40 % and the soluble fibre content from 8 to 16 %. These values were well correlated to the specific mechanical energy (230–380 kWh/t). Analysis of the sugar composition showed a slight increase in the solubility of glucuronoarabinoxylans arising from the external pericarp layer and a marked increase in the solubility of arabinoxylans and β-glucans probably arising from the aleurone and endosperm cell walls. Water absorption capacity, as measured by the Baumann method, increased from 270 to 375 % for the products processed under low severity treatments.

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

National audience; During Arabidopsis seed differentiation the epidermal cells of the testa produce a variety of cell wall structures. Fully differentiated cells are characterised by reinforced proximal and radial cell walls and a central columella of secondary cell wall material, a distal primary cell wall, and mucilage polysaccharides surrounding the columella. The latter are released on imbibition to form a hydrogel that surrounds the seed. We have previously shown that Arabidopsis mucilage is formed of a mixture of polysaccharides in two structurally distinct layers, both of which are mainly composed of the pectin rhamnogalacturonan I [1]. A simple visual screen for the absence of mucilage release has allowed the identification of natural Arabidopsis variants affected in two novel loci that influence polysaccharide properties; the MUM2 beta–galactosidase and the PMEI6 pectin methylesterase [2, 3, 4]. Most Arabidopsis accessions release mucilage when imbibed and we have examined the extent of natural variability in soluble mucilage characteristics. Mucilage composition varied little between accessions; in contrast the amount and physicochemical properties of mucilage vary widely. Analyses have been carried out to link these variations with particular habitats and potential mucilage functions.

Chapitre 16; absent

Selected Papers from the 15. Gums and Stabilisers for the Food Industry Conference. Wrexham (UK), June 2009; International audience

Partially acetylated and methylated oligogalacturonides produced by enzymatic hydrolysis of sugar beet pectin were analysed by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS). The (18)O labelling of the oligomer reducing end allowed the precise assignment of the fragments resulting from glycosidic bond and cross-ring cleavages. The collisional-induced dissociation of the C(i) and Z(j) fragment ions through sequential MS(n) experiments always displayed (0, 2)A-type cross-ring cleavage ions which were related to C(2)H(4)O(2) losses. These (0, 2)A ions appeared to be highly diagnostic ions allowing the precise location of the acetyl groups to the O-2 and/or O-3 of the acetylated galacturonic acid residues.

Beta-conglycinin alpha subunit has been phosphorylated using a cAMP-independent protein kinase (CK2) purified from the yeast Yarrowia lipolytica. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Asp/Glu. Only 0.5 to 1 mol P/mol alpha subunit was incorporated although seven consensus sequences are present. Phosphorylated beta-conglycinin alpha subunit (P-alpha) was digested by trypsin. The resulting peptides were analysed by RP-HPLC coupled to electrospray ionisation mass spectrometry (LC-ESMS). Two phosphopeptides were identified corresponding to 70-89 and 116-127 sequences with Ser 75 and Ser 117 phosphorylated respectively. Ser 75 is one of the predicted phosphorylation sites according to the consensus sequence criteria. Ser 117 is inside a very acidic peptide but does not belong to a previously described consensus sequence.

Ce mémoire décrit une synthèse de mes activités de recherche réalisées ces quinze dernières années ainsi que l’orientation que je souhaite apporter à mes travaux. La première partie est une présentation de mon parcours professionnel, des encadrements effectués ainsi que de la valorisation de mon travail. Le manuscrit continu avec la description de mon activité de recherche qui est basée sur le développement de méthodes en résonance magnétique nucléaire (RMN) pour étudier des milieux complexes. J’ai choisi de focaliser mon mémoire sur les trois thématiques principales sur lesquelles j’ai travaillées. Dans un premier temps, je m’intéresse à l’apport de la mobilité moléculaire pour étudier différents systèmes. Ensuite, je me focalise sur l’application d’un contraste basé sur l’échange chimique pour étudier la production/consommation de glucose dans différents systèmes. Je termine ce paragraphe en montrant la difficulté à extraire des informations quantitatives depuis un spectre RMN acquis à partir de molécules hyperpolarisées. La dernière partie du document décrit mon projet de recherche, constitué autour de deux thématiques : les contrastes métaboliques en imagerie par résonance magnétique (IRM) et l’IRM in situ. Ce projet de recherche est basé sur mes compétences décrites dans la partie précédente ainsi que sur mon environnement de travail.