Ian J. Colquhoun

Structure identification of feruloylated oligosaccharides from sugar-beet pulp by NMR spectroscopy *

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.

Metabolomics of Fecal Extracts Detects Altered Metabolic Activity of Gut Microbiota in Ulcerative Colitis and Irritable Bowel Syndrome

(1)H NMR spectroscopy of aqueous fecal extracts has been used to investigate differences in metabolic activity of gut microbiota in patients with ulcerative colitis (UC) (n = 13), irritable bowel syndrome (IBS) (n = 10), and healthy controls (C) (n = 22). Up to four samples per individual were collected over 2 years giving a total of 124 samples. Multivariate discriminant analysis, based on NMR data from all three groups, was able to predict UC and C group membership with good sensitivity and specificity; classification of IBS samples was less successful and could not be used for diagnosis. Trends were detected toward increased taurine and cadaverine levels in UC with increased bile acid and decreased branched chain fatty acids in IBS relative to controls; changes in short chain fatty acids and amino acids were not significant. Previous PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the same fecal material had shown alterations of the gut microbiota when comparing UC and IBS groups with controls. Hierarchical cluster analysis showed that DGGE profiles from the same individual were stable over time, but NMR spectra were more variable; canonical correlation analysis of NMR and DGGE data partly separated the three groups and revealed a correlation between the gut microbiota profile and metabolite composition.

Gene and Metabolite Regulatory Network Analysis of Early Developing Fruit Tissues Highlights New Candidate Genes for the Control of Tomato Fruit Composition and Development

Variations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum) fruit. To get insights into the networks involved in these coordinated processes and to identify key regulatory genes, we explored the transcriptional and metabolic changes in expanding tomato fruit tissues using multivariate analysis and gene-metabolite correlation networks. To this end, we demonstrated and took advantage of the existence of clear structural and compositional differences between expanding mesocarp and locular tissue during fruit development (12–35 d postanthesis). Transcriptome and metabolome analyses were carried out with tomato microarrays and analytical methods including proton nuclear magnetic resonance and liquid chromatography-mass spectrometry, respectively. Pairwise comparisons of metabolite contents and gene expression profiles detected up to 37 direct gene-metabolite correlations involving regulatory genes (e.g. the correlations between glutamine, bZIP, and MYB transcription factors). Correlation network analyses revealed the existence of major hub genes correlated with 10 or more regulatory transcripts and embedded in a large regulatory network. This approach proved to be a valuable strategy for identifying specific subsets of genes implicated in key processes of fruit development and metabolism, which are therefore potential targets for genetic improvement of tomato fruit quality.

Application of chemometrics to the 1H NMR spectra of apple juices : discrimination between apple varieties

Discrimination between apple juices produced from different varieties (Spartan, Bramley, Russet) has been achieved by applying principal components analysis (PCA) and linear discriminant analysis to 1H NMR spectra of the juices. The use of covariance and correlation matrix PCA methods was investigated and different regions of the spectrum were analysed in view of the large range of signal intensities. All the methods gave a high success rate of classification, with at least 24 out of 26 samples being correctly assigned when five principal components were used. Under optimum conditions a 100% success rate was achieved. Examination of the principal component loadings showed that the levels of malic acid and sucrose were two important chemical variables, but variations in the composition of the minor constituents were also found to make a significant contribution to the discrimination.

Composition and content of flavonol glycosides in broccoli florets (Brassica olearacea) and their fate during cooking

The two main flavonol glycosides present in broccoli florets were identified as quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside. Three minor glucosides of quercetin and kaempferol were also detected, namely isoquercitrin, kaempferol 3-O-glucoside and a kaempferol diglucoside. The sophorosides of quercetin and kaempferol were present in raw florets at a level of 65 mg kg−1 and 166 mg kg−1 fresh weight, respectively. The total content of quercetin and kaempferol glycosides expressed as aglycone was 43 and 94 μg g−1 fresh weight, respectively, and these agree with other recently published data. During the cooking process only 14–28% of the individual glucosides were retained in the cooked tissue, the remainder being largely leached into the cooking water with only a small loss attributed to the formation of the respective aglycones.

Isolation and characterization of rhamnogalacturonan oligomers, liberated during degradation of pectic hairy regions by rhamnogalacturonase.

Digests of modified hairy regions of apple pectin (MHR) obtained after degradation by rhamnogalacturonase (RGase) were analyzed for oligomer composition using high-performance anion-exchange chromatography and pulsed amperometric detection. A series of oligomers which appear to be characteristic of RGase degradation could be recognized. These oligomers were isolated on a preparative scale by size-exclusion chromatography and preparative anion-exchange chromatography and analyzed for sugar composition. 1H NMR spectroscopy showed that the oligomers consisted of between 4 and 9 sugar units with a backbone of alternating rhamnose and galacturonic acid residues, partly substituted with galactose residues linked to C-4 of the rhamnose moiety. The HPLC elution pattern showed that higher oligomers were also formed during incubation with RGase. These have the same basic structure but may contain other sugar units in addition to those given above. The oligomer composition of RGase digests of MHR isolated from apple, pear, leek, onion, carrot, and potato was very similar. Using anion-exchange chromatography to monitor the degradation of MHR at increasing incubation times, it was found that all the oligomers were present from the initial stages of the enzyme reaction and that the ratio between the different oligomers remained constant with time. Implications of these results for the structure of MHR and the mechanism of RGase action are discussed.

Identification by n.m.r. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase

2D-N.m.r. methods have been used to determine the composition of a mixture of oligosaccharides obtained by enzymic degradation of the modified hairy (ramified) regions of apple pectin with a new rhamnogalacturonase. The structures of the oligosaccharides were based on the unit alpha-Rhap-(1----4)-alpha-GalA-(1----2)-alpha-Rhap-(1----4)- GalA. A-beta-Galp unit was 4-linked to approximately half of the terminal Rhap residues and to half of the (1----2)-linked Rhap residues. The sample contained a mixture of a tetrasaccharide, two pentasaccharides, and one hexasaccharide.

FTIR and NMR studies on the hydration of a high-Mr subunit of glutenin

The hydration behaviour of a purified high-M(r) subunit of glutenin has been studied using Fourier transform infra-red (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. The water-insoluble protein was examined in an unalkylated form with intermolecular disulfide bonds, and in a reduced and alkylated (unpolymerized) form. Hydration produced a marked increase in chain mobility, especially above a threshold water content of about 37% w/w. NMR experiments also showed that some parts of the chain were held in a much less mobile state, even at higher water contents. Little difference could be seen between alkylated and unalkylated subunits, implying that NMR is sensitive to localized motions, but not to any restrictions imposed by disulfide bridges close to the chain ends. FTIR spectra of the protein films have shown that increasing hydration enables changes to occur in favour of a more extended and beta-sheet-type structure. The changes in secondary structure are very noticeable at water contents corresponding to the NMR mobility threshold. The behaviour is influenced by intermolecular interactions. beta-sheet formation is enhanced by the presence of disulfide bonds in the unalkylated samples. There is little evidence of beta-structure (sheet or extended chain) either in the dry state, where protein-protein interactions are strongest, or in dilute acetic acid solution, where the interactions are weakest. The balance between protein-protein and protein-water hydrogen-bonding interactions therefore appears to influence the formation of beta-sheet and extended chain structures, and these may in turn affect the elasticity of high M(r) subunits.

Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan alpha-L-rhamnopyranosyl-(1-->4)-alpha-D-galactopyranosyluronide lyase.

The recently described rhamnogalacturonase B, which is able to degrade ramified hairy regions of pectin, was found to be a rhamnogalacturonan [alpha]-L-rhamnopyranosyl-(1->4)-[alpha]-D-galactopyranosyluronide lyase. The cleavage site and mechanism differ from that of the previously described rhamnogalacturonase A, which is a hydrolase and can now be termed rhamnogalacturonan [alpha]-D-galactopyranosyluronide-(1->2)-[alpha]-L-rhamnopyranosyl hydrolase.

Analysis of structure and function of gellans with different substitution patterns

Abstract Chemical mutagenesis or exposure to antibiotic stress of Sphingomonas paucimobilis ATCC 31461 and R40 have been used to isolate mutants producing modified gellan gum polysaccharides. N.m.r. and conventional carbohydrate analysis methods have been used to characterise these polysaccharides. The 1 H and 13 C n.m.r. spectra of gellan gum have been fully assigned and the anomeric regions have been shown to be very sensitive to the type and location of non-carbohydrate substituents. Analysis of the gellan gum mutants suggests that they differ in the nature of acetate and glycerate substitution. Such gellan-related polysaccharides have been used to test the selective effect of acyl substituents on the gelation of gellan gum.