Henk A. Schols

If Homogalacturonan Were a Side Chain of Rhamnogalacturonan I. Implications for Cell Wall Architecture

Pectin, an important cell wall component of dicotyledonous plants, is probably the most complex macromolecule in nature. Here, we critically summarize the large amount of data on pectin structure. An alternative model for the macromolecular structure of pectin is put forward, together with ideas on

Determination of the degree of methylation and acetylation of pectins by h.p.l.c.

Abstract A method for the simultaneous quantitative analysis of methoxyl and acetate groups in pectin has been developed, using h.p.l.c. on a cation exchange resin in the protonated form and refractive index detection. Saponification conditions for release of methoxyl and acetate from pectins for direct injection on the column are described. With this h.p.l.c. method results were obtained which were in reasonable agreement with conventional methods.

Effects of dietary fibre on subjective appetite, energy intake and body weight: a systematic review of randomized controlled trials.

Dietary fibres are believed to reduce subjective appetite, energy intake and body weight. However, different types of dietary fibre may affect these outcomes differently. The aim of this review was to systematically investigate the available literature on the relationship between dietary fibre types, appetite, acute and long‐term energy intake, and body weight. Fibres were grouped according to chemical structure and physicochemical properties (viscosity, solubility and fermentability). Effect rates were calculated as the proportion of all fibre–control comparisons that reduced appetite (n = 58 comparisons), acute energy intake (n = 26), long‐term energy intake (n = 38) or body weight (n = 66). For appetite, acute energy intake, long‐term energy intake and body weight, there were clear differences in effect rates depending on chemical structure. Interestingly, fibres characterized as being more viscous (e.g. pectins, β‐glucans and guar gum) reduced appetite more often than those less viscous fibres (59% vs. 14%), which also applied to acute energy intake (69% vs. 30%). Overall, effects on energy intake and body weight were relatively small, and distinct dose–response relationships were not observed. Short‐ and long‐term effects of dietary fibres appear to differ and multiple mechanisms relating to their different physicochemical properties seem to interplay. This warrants further exploration.

Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process.

Abstract A high-molecular-weight pectic fraction, released from the cell walls of apple tissue by pectolytic, hemicellulolytic, and cellulolytic enzymes, was isolated from apple juice prepared by the liquefaction process. The fraction, termed modified hairy regions (MHR), was characterized as a highly branched rhamnogalacturonan with arabinose-rich side chains and represented 0.26% of the fresh apple. Arabinose was the most abundant sugar (55 mol%) in MHR, which had a high rhamnose-galacturonic acid ratio (0.29) and degrees of methylation and acetylation of 42% and 60%, respectively. The sugar and linkage compositions, distribution of molecular weights, susceptibility to β-elimination mediated by alkali and 4-methylmorpholine N-oxide, and the effect of various technical and pure enzymes on MHR and chemically and enzymically modified MHR have been studied. From the results and 13C-n.m.r. data, a tentative structure for MHR is proposed.

Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.

Complex Pectins: Structure elucidation using enzymes

Abstract A pectic fraction, retained by ultrafiltration of the juice from enzyme treated apple tissue and resistant to further enzymic degradation, was isolated and characterized using chemical and enzymic methods. The fraction was termed MHR (modified hairy regions) and this fraction was characterized by a high arabinose content, next to a high rhamnose to galacturonic acid ratio and a high acetyl content and smaller proportions of xylose and galactose. Rhamnogalacturonase (RGase), an enzyme able to hydrolyze galacturonic acid-(1→2)-rhamnosyl linkages within the rhamnogalacturonan backbone of MHR was used to obtain both oligomeric and polymeric degradation products. These RGase-oligomers consist of a tetrameric or hexameric backbone of alternating rhamnose and galacturonic acid residues with a galactose residue substituted at C-4 of part of the rhamnose moieties. Next to the subunit from which these oligomers were released, two other subunits were recognized: a highly methyl esterified xylogalacturonan segment and residual stubs of the backbone rich in branched arabinose side chains. Comparison of the MHR with non-modified pectic hairy regions of apple cell wall, isolated in a mild and defined way, revealed great resemblance indicating that the modifications of the MHR during enzymic liquefaction were only minor. Analogous MHR fractions could be isolated from potato fibre, pear, carrot, leek, and onion tissue. Finally, an adapted model is presented for the prevailing population of apple MHR having the highest molecular weight. The universal validity of this model for pectic hairy regions from other plant sources is discussed.

Hydrothermally treated xylan rich by-products yield different classes of xylo-oligosaccharides

Four xylan rich by-products, namely wheat bran, brewerys spent grain, corn cobs and Eucalyptus wood, were characterised and subjected to a mild hydrothermal treatment in order to release and degrade the xylan from the starting materials. The chemical characterisation of the feedstock materials, with emphasis on the extracted xylan fractions and using enzymatic degradation of these xylans, resulted in rather detailed pictures of the xylans present. Depending on the feedstock material studied, the xylan present was substituted with arabinose, 4-O-methylglucuronic acid and acetyl groups. During the hydrothermal treatment, arabinose was rather easily removed from the xylan-backbone (wheat bran, brewerys spent grain and corn cobs). The acetyl groups were partly released from the feedstocks, becoming available to catalyse the depolymerisation of the xylan. Also, part of the uronic acids were released, mainly during the treatment of Eucalyptus wood. Due to the partial release of the substituents and cleavage of the xylan by the treatment performed, a wide variety of xylo-oligosaccharides with different structural features corresponding to the xylan-structure of the original feedstock were obtained. Xylo-oligosaccharides branched with arabinose were identified in the hydrolysate from brewerys spent grain, while in the hydrolysate of corn cobs and Eucalyptus wood xylo-oligosaccharides substituted with 4-O-methylglucuronic acid were present as well. Additionally, a series of partially acetylated (acidic) xylo-oligosaccharides was identified in the Eucalyptus wood hydrolysate.

Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins

A rhamnogalacturonase (RGase), that could degrade the modified hairy regions (MHR) prepared from apple cell walls, was isolated and purified from a technical preparation of Aspergillus aculeatus. The RGase cleaved galactopyranosyluronic-rhamnopyranosyl linkages. No activity was observed towards other cell-wall polysaccharides or p-nitrophenyl glycosides. The optimal conditions for RGase were pH 3–4 and 40–50°. MHR was degraded by RGase, and methylation analysis and 13C-n.m.r. spectroscopy indicated the products to have a tetrasaccharide backbone of alternating rhamnose and galacturonic acid residues. Some oligomers had a galactose residue 4-linked to rhamnose. The potential value of this new enzyme is considered.

A xylogalacturonan subunit present in the modified hairy regions of apple pectin.

Abstract The high molecular weight fraction (fraction A) of the modified hairy regions (MHR) from apple cell walls was treated by rhamnogalacturonase (RGase) after saponification and after deacetylation by rhamnogalacturonan acetylesterase (RGAEase). Three fractions could be recognized by size-exclusion chromatography: rhamnogalacturonan oligomers, residual stubs of the rhamnogalacturonan backbone rich in arabinan side-chains, and a fraction rich in xylose and galacturonic acid. The “xylogalacturonans” obtained after saponification and after enzymic deacetylation had rather similar sugar compositions (xylose:galacturonic acid ratios of 0.4–0.9) and molecular weights. After saponification, the xylogalacturonan was eluted as a single peak on anion-exchange chromatography whereas three peaks were obtained when the MHR was deacetylated by RGAEase, indicating variations in the degrees of methylation. One of the methyl-esterified xylogalacturonan fractions was characterized by NMR spectroscopy: the xylose residues were β-(1 → 3)-linked to some of the galacturonic acid residues within a rather high molecular weight xylo-(1 → 4)-α-galacturonan and a degree of methylation of 39 was calculated. The methyl esters were found to be equally divided between the substituted and unsubstituted galacturonosyl residues.

Occurrence of pectic hairy regions in various plant cell wall materials and their degradability by rhamnogalacturonase

Pectic polysaccharide fractions of high molecular weight, resistant to further degradation by pectolytic, hemicellulolytic, and cellulolytic enzymes, were isolated from potato fibre and from pear, carrot, leek, and onion tissue by the liquefaction process. The fractions, referred to as modified hairy regions (MHR), were characterized by the determination of their sugar composition, linkage type composition, degree of esterification (methyl ester and O-acetyl groups), and molecular weight distribution. Galacturonic acid, galactose, and rhamnose were found to be the major sugar residues in most of the MHR preparations, while arabinose was the main sugar in pear MHR. The rhamnose-galacturonic acid ranged between 0.44 for pear MHR to 0.63 for MHR from leek. High degrees of acetylation (da) were calculated assuming that acetyl groups were only attached to galacturonic acid residues. All MHR fractions had a similar molecular weight distribution which was rather heterogeneous. It was observed that all MHR preparations were degraded by RGase in a similar fashion. In all digests, a characteristic population of reaction products having a molecular weight of ca. 1000-2000, representing rhamnogalacturonan oligomers, was present. It was concluded that pectic hairy regions with comparable structural features are common to a variety of fruit and vegetable tissues.