Search

Pomelo peel, a by-product of fruit processing, is an excellent source of pectin. We aimed to study the effects of thermal treatments on the extraction and in vitro fermentation patterns of pectins from pomelo peel. We used subcritical water extraction and hot water extraction, with or without chelator (citric acid or EDTA-2Na) assistance to obtain the pectins. The pectin fractions were characterized by their constituent monosaccharide composition and molecular weight (Mw) distribution. The degree of methyl esterification of pomelo pectins was determined using FT-IR spectroscopy. We also compared the fermentation patterns of the pectin using an in vitro simulated human gut fermentation model, monitored Mw changes during fermentation by high performance size exclusion chromatography (HPSEC), and analyzed short-chain fatty acid (SCFA) using gas chromatograph. Our results showed that both chelator assistance and subcritical water treatment increased the pectin yield. Chelator-assisted extraction increased the pectin Mw and decreased the degree of methyl esterification (DM) whereas, subcritical water treatment had the opposite effect. Pectin with low rhamnogalacturonan-I (RG-I) and high homogalacturonan (HG) domain can be obtained from the used pomelo peels by citric acid-assisted extraction. Although gut microbiota intensively utilized uronic acid (UA) of pomelo during early fermentation stages, constituent neutral monosaccharides were consumed even faster than UA. Pomelo pectins enhanced the relative abundance of Bacteroides and Prevotella 9, and stimulated the production of SCFA, particularly acetic acid. Generally, pomelo pectin could be considered a new source for modulating gut microbiota towards a healthy pattern in functional foods.

The performance of pigs is affected by the rate of nutrient absorption in the gastrointestinal tract, which depends in turn strongly on the rate of stomach emptying. The 13C breath test provides a non-invasive diagnostic tool to measure gastric emptying patterns. Despite the wide acceptance of this method in human intervention studies, it has not found its way to the domain of animal sciences. In this study, we used the breath test to measure gastric emptying in young growing pigs using [1-13C] octanoic acid to trace digesta solids and [1-13C] glycine to study liquids. Pigs were fed a starch-rich diet, varying in starch source (isolated starch from barley, maize or high-amylose maize) or form (isolated barley starch, ground barley or extruded barley), after which 13CO2 enrichment was frequently measured during 11 h. Outliers in 13CO2 enrichment in the response curve of each pig were identified with a Cook 1/4s distance outlier test in combination with a leave-one-out analysis. Effects of experimental treatments on breath test parameters were tested using a GLM. In general, pigs were easy to train and the tailor-made mask allowed effortless sampling. Gastric emptying of all pigs followed a biphasic pattern, with a higher 13C recovery during the first peak. The first peak in gastric emptying of solids reached its maximum enrichment within 2 h after feeding in all cases. For digesta liquids, this peak was reached earlier for pigs fed ground barley (2.2 h after feeding), compared to pigs fed diets containing isolated starch (2.8 h after feeding). The second peak in gastric emptying of solids was reached later for pigs fed ground barley (5.9 h after feeding), compared with pigs fed extruded barley (4.5 h after feeding) and pigs fed diets containing isolated barley starch (4.8 h after feeding). In conclusion, the 13C breath test is a convenient, non-invasive tool to gain more insights into the gastric emptying pattern of pigs.

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

Three Moroccan citrus peels, such as orange, lemon, and bigarade, were chosen to extract the pectin using Box Behnken Design. At a temperature of 90 °C, pH = 1.5, and extraction time = 120 min, a high pectin yield was recorded with 28.57, 26.86, and 26.55% for lemon (LPP), bigarade (BPP), and orange (OPP) peel pectins respectively. The extracted pectins have a less branched linear structure with high GalA contents for BPP (86.0 mol%), LPP (82.0 mol%), and OPP (79.0 mol%), and a high degree of methyl esterification (>74%), but a low degree of acetylation (

The aim of the present study was to characterize selected by-products from Brazilian food industry and their in vitro digestibility. These by-products (cassava and orange bagasses and passion fruit peels) are potentially rich sources of dietary fibres, but currently they are mostly disposed. Their analysis revealed differences in composition for the same by-product type from different suppliers. Cassava bagasses were mainly composed of starch, with high variability among tested by-products (45–77.5% starch). In vitro experiments indicated that cassava bagasses had ~ 12% of resistant starch. The orange bagasses had free glucose and highly methyl esterified pectin as the main constituents (~23.5% of total pectin). Seventy-seven % of digestible glucose present in the orange bagasse were absorbed within 3 h experimental run. Passion fruit peels were a good source of fibres, especially pectin (~19%) and (hemi)cellulose (~16%). These in vitro experiments indicated that passion fruit peel had slower absorption of glucose than the other by-products, with 80% of digestible glucose absorbed within 5 h. In conclusion, the tested by-products are good sources of diverse types of fibres and have a great potential to be incorporated into different food products, decreasing food waste and contributing to a sustainable food system.

To understand the architecture of the plant cell wall, it is of importance to understand both structural characteristics of cell wall polysaccharides and interactions between these polysaccharides. Interactions between polysaccharides were studied in the residue after water and chelating agent extraction by sequential extractions with H2O and alkali. The 6 M alkali residue still represented 31%, 11% and 5% of all GalA present in carrot, tomato and strawberry, respectively, and these pectin populations were assumed to strongly interact with cellulose. Digestion of the carrot 6 M alkali residue by glucanases released ∼27% of the 6 M residue, mainly representing pectin. In tomato and strawberry alkali residues, glucanases were not able to release pectin populations. The ability of glucanases to release pectin populations suggests that the carrot cell wall contains unique, covalent interactions between pectin and cellulose.

This paper set out to differentiate the Maillard induced glycation reactivity of individual milk proteins using different saccharides under well-defined reaction conditions. α-Lactalbumin, β-lactoglobulin and β-casein were incubated with mono-, di- and trisaccharides in the dry state under standardised buffered conditions and glycation was expressed relative to the available reactive groups per protein (DG). Protein reactivity, described by the DG max and initial speed of glycation (v), followed the same order for each protein-saccharide incubation: α-lactalbumin > β-lactoglobulin ≫ β-casein. Glycation of whey proteins by different monosaccharides was double that of β-casein. Differences in DG between whey proteins and β-casein decreased with increased saccharide size. A two-fold difference was found for glycation in the presence of the dimers lactose and maltose for β-casein but not for the whey proteins. The percentage of glycated lysines increased with increased lysine to protein size ratio.

Infants are known for their high inter-individual variability in gut microbiota composition in the first months of life. This poses difficulties when predicting the fate of non-digestible carbohydrates (NDCs) in the infant gut using in vitro models, since numerous experiments with individual faecal inocula of different infants are required. In this study an in vitro fermentation experiment was performed with galacto-oligosaccharides (GOS) using both individual and pooled faecal inocula of five two-week-old infants. It was shown that pooled faecal inoculum can be used to judge the fermentability of GOS, as a similar trend in total organic acid production and relative increase in Bifidobacterium was observed for the pooled faecal inoculum and four out of five individual faecal inocula. An additional fermentation using pooled faecal inoculum of five other infants of the same age confirmed these findings. Additionally, we provided evidence for both size- and isomer-specific fermentation of GOS by infant microbiota, which reveals the potential for the production of tailored NDC mixtures to meet the needs of specific subgroups of infants. Hence, the use of pooled faecal inocula contributes to increasing our knowledge on structure-specific effects of NDCs in infants efficiently.

Sugar beet pulp (SBP) and lemon peel wastes (LPW) were used to obtain two mixtures of pectic oligosaccharides (denoted as SBPOS and LPOS, respectively). Oligogalacturonides in LPOS showed a larger molecular weight, higher degree of methylation and lower degree of acetylation than the ones in SBPOS. The suitability of pectic oligosaccharides, pectins from SBP and LPW and commercial FOS for causing prebiotic effects were compared by in vitro fermentation and fluorescence in situ hybridization using human faecal inocula and eight different probes. The joint populations of bifidobacteria and lactobacilli increased from 19% up to 29%, 34% and 32% in cultures with LPOS, SBPOS and FOS, respectively. Faecalibacterium and Roseburia also increased their counts with all the substrates (especially with LPOS). The highest concentrations of organic acids were observed in media containing oligosaccharides. This work confirms that pectic oligosaccharides present better prebiotic properties than pectins, and similar or better than FOS.

Replacing dairy components from milk replacer (MR) with vegetable products has been previously associated with decreased protein and fat digestibility in milk-fed calves resulting in lower live weight gain. In this experiment, the major carbohydrate source in MR, lactose, was partly replaced with gelatinized corn starch (GCS) to determine the effect on protein and fat digestibility in milk-fed calves. In total, 16 male Holstein-Friesian calves received either MR with lactose as the carbohydrate source (control) or 18% GCS at the expense of lactose. In the adaptation period, calves were exposed to an increasing dose of GCS for 14 weeks. The indigestible marker cobalt ethylenediaminetetraacetic acid was incorporated into the MR for calculating apparent nutrient digestibility, whereas a pulse dose of chromium (Cr) chloride was fed with the last MR meal 4 h before slaughter as an indicator of passage rates. The calves were anesthetized and exsanguinated at 30 weeks of age. The small intestine was divided in three; small intestine 1 and 2 (SI1 and SI2, respectively) and the terminal ileum (last ~100 cm of small intestine) and samples of digesta were collected. Small intestinal digesta was analysed for α-amylase, lipase and trypsin activity. Digestibility of protein was determined for SI1, SI2, ileum and total tract, whereas digestibility of fat was determined for SI1, SI2 and total tract. Apparent protein digestibility in the small intestine did not differ between treatments but was higher in control calves at total tract level. Apparent crude fat digestibility tended to be increased in SI1 and SI2 for GCS calves, but no difference was found at total tract level. Activity of α-amylase in SI2 and lipase in both SI1 and SI2 was higher in GCS calves. Activity of trypsin tended to be higher in control calves and was higher in SI1 compared with SI2. A lower recovery of Cr in SI2 and a higher recovery of Cr in the large intestine suggest an increased rate of passage for GCS calves. Including 18% of GCS in a milk replacer at the expense of lactose increased passage rate and decreased apparent total tract protein digestibility. In the small intestine, protein digestion did not decrease when feeding GCS and fat digestion even tended to increase. Overall, effects on digestion might be levelled when partially replacing lactose with GCS, because starch digestion is lower than that of lactose but fat digestion may be slightly increased when feeding GCS.

Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC–TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.

A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme ( Bli PME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. Bli PME is able to release up to 100% of the methylesters from lime pectin (DM 34–76 → DM 0) and up to 73% of all methylesters from SBPs (DM 30–73 → DM 14). Bli PME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the Bli PME-modified pectins using HILIC–MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of Bli PME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME.

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10–100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with β-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.

Research was conducted to understand cell wall polysaccharide (CWPs) degradation in chicory root pulp (CRP) during an in vitro fermentation by human faecal microbiota. Enzyme extracts (EEs) from CRP fermentation liquids (2, 12 and 24 h) were tested for enzyme activities towards p-nitrophenyl-glycosides and polysaccharides. The EEs were also incubated for 24 h on polysaccharides and cell wall residues (CWRs) derived from CRP, to determine the mechanism and efficiency of enzyme action. The study showed that the presence of arabinofuranosidase, β-galactosidase, endo-arabinanase, endo-galactanase and exo-polygalacturonase increased for the 24 h EE, whereas the activity of enzymes degrading (hemi)cellulose was highest in the 12 h EE. It was hypothesised that increased levels of arabinofuranosidase, β-galactosidase, endo-arabinanase, endo-galactanase, exo-polygalacturonase, pectin de-esterifying enzymes and endo-polygalacturonase contributed to a synergy in degrading pectin in CRP from 12 to 24 h of fermentation. The higher degradability of arabinan compared to galactan in the network is due to the architecture in CRP involving more accessible arabinan than galactan.

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.

Unprocessed and acid-extruded rapeseed meal (RSM) was fed to broiler chickens, with and without addition of commercial pectolytic enzymes. Nonstarch polysaccharide (NSP) fermentability and unfermented NSP structures from RSM were studied in the excreta in detail. From unprocessed RSM, 24% of the nonglucose polysaccharides could be fermented. Acid treatment did not have a significant effect, but enzyme addition did improve fermentability to 38%. Most likely, the significant increase in NSP fermentability can be ascribed to the addition of pectolytic enzymes, which decreased branchiness of the water-soluble arabinan. Mainly xyloglucan, (glucurono-)xylan, (branched) arabinan, and cellulose remained in the excreta. The proportion of unextractable carbohydrates increased in excreta from broilers fed acid-extruded RSM. Probably, acid extrusion resulted in a less accessible NSP matrix, also decreasing the accessibility for pectolytic enzymes added in the diet. During alkaline extraction of the excreta, 39 to 52% (wt/wt) of the insoluble carbohydrates was released as glucosyl- and uronyl-rich carbohydrates, probably originally present via ester linkages or hydrogen bonding within the cellulose-lignin network. These linkages are expected to hinder complete NSP fermentation and indicate that digestibility of RSM may benefit substantially from an alkaline treatment or addition of esterases.

Replacing part of glucose with galactose in the post-weaning diet beneficially affects later life metabolic health in female mice. The liver is the main site of galactose metabolism, but the direct effects of this dietary intervention on the liver in the post-weaning period are not known. The aim of this study was to elucidate this. Weanling female mice (C57BL/6JRccHsd) were fed a starch containing diet with glucose (32 en%) monosaccharide (GLU), or a diet with glucose and galactose (1:1 both 16 en%) (GLU+GAL). Body weight, body composition, and food intake were determined weekly. After 3 weeks, mice were sacrificed, and serum and liver tissues were collected. Global hepatic mRNA expression was analyzed and hepatic triglyceride (TG) and glycogen contents were determined by enzymatic assays. Body weight and body composition were similar in both groups, despite higher food intake in mice on GLU+GAL diet. Hepatic TG content was lower in GLU+GAL-fed than GLU-fed females, while glycogen levels were unaffected. Analysis of global expression patterns of hepatic mRNA showed that mainly inflammation-related pathways were affected by the diet, which were predominantly downregulated in GLU+GAL-fed females compared to GLU-fed females. This reduction in inflammation in GLU+GAL-fed females was also reflected by decreased serum concentrations of acute phase protein Serum amyloid A 3. In conclusion, replacing part of glucose with galactose in the post-weaning diet reduces hepatic TG content and hepatic inflammation.

Five xanthans were hydrolyzed to their repeating units using cellulases. Hydrophilic interaction chromatography with online electrospray ionization ion trap mass spectrometry and evaporative light scattering detection was used to analyze the oligomers released. It was concluded that six different pentamer repeating units (RUs) exists within a xanthan sample. The most abundant RU shows acetylation on the inner mannose and pyruvylation on the outer mannose. The second most abundant RU shows acetylation on both the inner and the outer mannose. It becomes clear that more variations in the xanthan structure exist than generally recognized. Comparison of five different xanthan samples revealed that, although the molecular composition of xanthan samples can be exactly the same, the ratio in which the RUs occur can differ significantly. It is, therefore, concluded that xanthan samples should be characterized for both, their molecular composition and the relative abundance of the RUs present.

Antibiotic treatments can lead to a disruption of the human microbiota. In this in-vitro study, the impact of antibiotics on adult intestinal microbiota was monitored in a new high-throughput approach: a fermentation screening-platform was coupled with a phylogenetic microarray analysis (Intestinal-chip). Fecal inoculum from healthy adults was exposed in a fermentation screening-platform to seven widely-used antibiotics during 24. h in-vitro fermentation and the microbiota composition was subsequently determined with the Intestinal-chip.Phylogenetic microarray analysis was first verified to be reliable with respect to variations in the total number of bacteria and presence of dead (or inactive) cells. Intestinal-chip analysis was then used to identify and compare shifts in the intestinal microbial composition after exposure to low and high dose (1μgml-1 and 10μgml-1) antibiotics. Observed shifts on family, genus and species level were both antibiotic and dose dependent. Stronger changes in microbiota composition were observed with higher doses. Shifts mainly concerned the bacterial groups Bacteroides, Bifidobacterium, Clostridium, Enterobacteriaceae, and Lactobacillus. Within bacterial groups, specific antibiotics were shown to differentially impact related species.The combination of the in-vitro fermentation screening platform with the phylogenetic microarray read-outs has shown to be reliable to simultaneously analyze the effects of several antibiotics on intestinal microbiota. © 2012 Elsevier B.V.

Research on prebiotics and other novel health-promoting food components has been active for over a decade. Arabinoxylan (AX) derived arabinoxylooligosaccharides (AXOS), which may have various chemical structures, depending on the xylan source and the degradation method used, stand increasingly in the spotlight as potential prebiotics. During the past decade, the studies of the possibilities to produce the AXOS by using biocatalytic conversion have received more attention. In addition, there is an interest in the use of novel cereal biomass for the production of AXOS. The aim of this study was to investigate the influence of various commercial enzyme preparations on the degradability of insoluble arabinoxylans in wheat and rye wholemeal treated by extrusion, identify and quantify xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) in treated media. The enzymatic degradation of rye and wheat cell wall materials was monitored by HPSEC, HPAEC and MALDI-TOF-MS techniques. It was noticed that there is no significant difference between extruded and natural cereals, and type of cereals had not significant influence on XOS and AXOS production. The most effective biocatalysts were hemicellulases expressed in the enzyme preparations from Trichoderma and Aspergillus spp. (Depol 692), Humicola and Bacillus spp. (Ceremix Plus). Degradability of rye and wheat cell wall materials by these enzyme preparations obtained break down percentages of 70–87% and 67–77%, respectively. After enzymatic treatment, only small amounts of xylose, xylobiose, and xylotriose was eluted compare to the amount of more complex oligosaccharides with higher degree of polymerization (DP). The mass spectra of oligosaccharides indicated the presence of a homologous series of pentoses ranging from DP 4 to 15. This indicates that chosen enzyme preparations acted well on wheat and rye biomass, and released quite high amounts of XOS and AXOS.

Filamentous fungi are a good source of hemicellulolytic enzymes for biomass degradation. Enzyme preparations were obtained as culture supernatants from 78 fungal isolates grown on wheat straw as carbon source. These enzyme preparations were utilized in the hydrolysis of insoluble wheat straw and corn fiber xylan rich fractions. Up to 14% of the carbohydrates in wheat straw and 34% of those in corn fiber were hydrolyzed. The degree of hydrolysis by the enzymes depended on the origin of the fungal isolate and on the complexity of the substrate to be degraded. Penicillium, Trichoderma or Aspergillus species, and some non-identified fungi proved to be the best producers of hemicellulolytic enzymes for degradation of xylan rich materials. This study proves that the choice for an enzyme preparation to efficiently degrade a natural xylan rich substrate, is dependent on the xylan characteristics and could not be estimated by using model substrates.

The characterization of oligosaccharides in the feces of breast-fed babies is a valuable tool for monitoring the gastrointestinal fate of human milk oligosaccharides (HMOs). In the present study we monitored fecal oligosaccharide profiles together with the HMO-profiles of the respective breast milks up to six months postpartum, by means of capillary electrophoresis-laser induced fluorescence detection and mass spectrometry. Eleven mother/child pairs were included. Mother's secretor- and Lewis-type included all combinations [Le(a-b+), Le(a+b-), Le(a-b-)]. The fecal HMO-profiles in the first few months of life are either predominantly composed of neutral or acidic HMOs and are possibly effected by the HMO-fingerprint in the respective breast milk. Independent of the initial presence of acidic or neutral fecal HMOs, a gradual change to blood-group specific oligosaccharides was observed. Their presence pointed to a gastrointestinal degradation of the feeding-related HMOs, followed by conjugation with blood group specific antigenic determinants present in the gastrointestinal mucus layer. Eleven of these 'hybrid'-oligosaccharides were annotated in this study. When solid food was introduced, no HMOs and their degradation- and metabolization products were recovered in the fecal samples.

Two β-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K(m)) than BxlB towards para-nitrophenyl β-D-xylopyranoside (pNPX) and β-1,4-xylo-oligosaccharides (degree of polymerisation 2-6). For both enzymes k(cat)/K(m) decreased with increasing β-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different β-xylosyl-oligosaccharides were synthesised in 2-36% (BxlA) and 6-66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides D-mannose, D-lyxose, D-talose, D-xylose, D-arabinose, L-fucose, D-glucose, D-galactose and D-fructose as acceptors, whereas BxlB used the same except for D-lyxose, D-arabinose and L-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved β-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from D-lyxose, D-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, β-D-Xylp-(1→4)-D-Lyxp, β-D-Xylp-(1→6)-D-Galp, β-D-Xylp-(1→4)-α-D-Glcp-(1→3)-β-D-Fruf, β-D-Xylp-(1→4)-α-D-Glcp-(1→2)-β-D-Fruf, and β-D-Xylp-(1→6)-β-D-Fruf-(2→1)-α-D-Glcp, as structure-determined by 2D NMR, indicating that GH3 β-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined.

Correspondance: martijn.noort@tno.nl; International audience; The nature of the adverse effects of wheat bran fractions on bread-making quality was studied. Two fractions of bran, representing different tissue layers and having different compositions, were used. The particle size of the bran fractions was varied by various milling techniques. All fractions were added to white flour and water addition was adjusted to obtain dough with a constant consistency. Both dough-mixing properties and bread-making quality were affected by the addition of bran. The negative influence was enhanced when bran particle size was reduced. The effects on bread quality are strongly correlated to negative effects of bran on gluten network formation. The results show that fibre gluten interactions are the main cause for the negative effects of fibres, rather than dilution of gluten, piercing of gas cells or particles disturbing the gluten network. Two possible explanations for the enhancement of the adverse effects when reducing the particle size of bran fractions are discussed: 1) increased interaction surface 2) liberation of reactive components due to cell breakage

Epoxy starch derivatives were synthesized by epoxidation of allylated starch. The reaction was performed with low substituted 1-allyloxy-2-hydroxypropyl-waxy maize starch (AHP-WMS; degree of substitution (DS) of 0.23) using hydrogen peroxide and acetonitrile Via a two step spectrophotometric assay, it was determined that epoxy-WMS contained 0.13 ± 0.03 mmol epoxy groups per gram dry allylated starch which corresponds to DS value of 0.025. Enzymatic digestibility, swelling capacity and solubility were significantly reduced after epoxidation. The detailed chemical structure of epoxy-WMS was characterized by enzymatic hydrolysis followed by chromatographic and mass spectrometric techniques. Only a small amount of epoxidized oligomers was found in the enzymatic digested products of epoxy-WMS. Apparently, the epoxidation reaction is highly efficient but subsequent reactions of epoxy groups lead to a considerable amount of cross-links and diol groups. Additionally, epoxy starch derivatives were successfully applied as carrier matrix for immobilization of an enzyme.

The cause of discrepancies in the literature regarding the specificity of immobilized Candida antarctica lipase B in the acylation of oligosaccharides was examined. Molecular sieves, generally used to control the water content during acylation reactions, turned out to have an important role in this. It was proven that molecular sieves alone can catalyze the acylation of fructose oligomers using vinyl laurate, leading to multiple substitution of the oligomers. This effect was the most profound at conditions unfavorable for the enzyme, because this resulted in a relatively high concentration of the chemically produced adducts. The enzyme alone catalyzed the formation of monosubstituted oligomers. It was proven that even solvent pre-drying by molecular sieves already causes the release of catalyzing compounds to the liquid, leading to subsequent catalysis. These findings should be taken into account when applying molecular sieves in this type of reactions in the future. Molecular sieves could, moreover, be used as a catalyst when multiple substitution is desired.

Okra pectin obtained by hot buffer extraction (HBSS) consists of an unusual pectic rhamnogalacturonan I structure in which acetyl groups and alpha galactose residues are substituted on rhamnose residues within the backbone. The okra Chelating agent Soluble Solids (CHSS) pectin consists of slightly different structures since relatively more homogalacturonan is present within the macromolecule and the rhamnogalacturonan I segments carry slightly longer side chains. The rheological properties of both okra pectins were examined under various conditions in order to understand the unusual slimy behaviour of okra pectins. The viscosity of the okra HBSS pectin was 5–8 times higher than the viscosity of the okra CHSS pectin. The okra HBSS pectin showed an elastic behaviour (G' > G¿) over a wide range of frequencies (10-1–10 Hz), at a strain of 10%, while okra CHSS and saponified okra HBSS/CHSS pectin showed predominantly viscous responses (G'

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-α- l -arabinan and (1,4)-β- d -galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.

The effect of process conditions used for wheat straw pretreatments on the liquor- and residue-composition was studied. Hereto, the pretreatment conditions were expressed in a `combined severity -factor¿. The higher the combined severity factor () the more xylan was released from the wheat straw, but the more xylan decomposed and furfural formation occurred. The percentage of residual xylan present after pretreatment appeared to be a good indicator concerning cellulose degradability or bio-ethanol production. Namely, cellulose degradation by using commercial enzymes was higher at higher severities corresponding to a lower amount of residual xylan. The xylan release and degradation was studied in more detail by using HPSEC and MALDI-TOF mass spectrometry. The more severe the treatment the more (acetylated) xylose oligomers with a DP lower than nine were analysed. The presence of (acetylated) xylans with a DP of 9¿25 increased slightly from low to medium severity. The quantification of the DP-distribution of the (acetylated) xylans released proved to be a good tool to predict cellulose degradability. Keywords: Wheat straw; Bioethanol; Severity; Heat treatment; Cellulases

The affinity of xylan to cellulose is an important aspect of many industrial processes, e.g. production of cellulose, paper making and bio-ethanol production. However, little is known about the adsorption of structurally different xylans to cellulose. Therefore, the adsorption of various xylans to bacterial cellulose (BC) was studied. Also, the relationship between xylan size and adsorption was analysed. BC was used as cellulosic material, because of its high specific surface area and homogeneous structure. In general, unsubstituted linear xylan parts favoured adsorption to BC. Xylan affinity for BC was also related to xylan size. The presence of arabinosyl and O-acetyl substituents to xylan decreased the adsorption of xylan to BC considerably. Removing substituents resulted in higher amounts of adsorbed material. Most likely, increasing the number of unsubstituted xylosyl residues induced the formation of xylan¿xylan interactions, which contributed to adsorption to BC. Schematic models are proposed showing the adsorption of structurally different xylans to BC. Keywords: Xylan; Bacterial cellulose; Adsorption; Affinity; Structural characteristics

To study the effect of reagent type on the distribution pattern of acetyl groups in acetylated cowpea starch, amylose and amylopectin populations were isolated from the starch granules after modification to a low degree of substitution (DS < 0.1) with acetic anhydride and vinyl acetate, respectively. Slowly reacting reagent vinyl acetate resulted in higher DS values for the amylopectin populations when compared to the rapidly reacting reagent acetic anhydride. The two reagents had similar effects on the acetylation level of amylose, suggesting that the amorphous regions of granules were easily accessible for both reagents. The acetyl substitution patterns were analyzed by enzymatic degradation followed by characterization of the obtained fragments using chromatographic and mass spectrometric techniques. The distributions of acetyl groups along the amylose and amylopectin chains were more clustered for modification with vinyl acetate as compared with modification with acetic anhydride. Between the two acetylation types, pronounced differences in the acetyl substitution patterns were observed for the large fragments obtained after α-amylase digestion; only slight differences were exhibited for the small fragments obtained by exhaustive enzymatic digestion of amylose and amylopectin populations.

Functionality of pectins as a food ingredient is strongly related to their chemical fine structure. Chemical characteristics of pectins are determined by many different parameters in their manufacture (choice of the raw material and extraction conditions). Pectin companies are thus in need of rapid methods to check the performance of extracted pectins. An important factor in the characterisation is the homogeneity of the pectin preparation, which is usually determined by laborious, time consuming, soft gel based chromatographic procedures. An HPLC method with a weak anion exchange column (WAX column) was found to discriminate between commercial pectins efficiently: pectins with similar DM (degree of methyl-esterification) or similar DS (degree of substitution: methyl-esters and amide groups) having different physical properties showed various populations. Furthermore the pectins with a blockwise or random distribution of the substituents showed different elution profiles. This indicates that the WAX column is sensitive not only to the amount of substituents but also to the distribution of these substituents.

Specific enzymatic degradation in combination with chromatographic and spectrometric techniques was used to understand acetyl group distribution over the amylopectin populations of differently sized granule fractions from potato and sweet potato starches. The hydrolysates obtained after α-amylase, s-amylase, pullulanase, and the combination of pullulanase, α-amylase and amyloglucosidase treatment were investigated by high-performance size-exclusion chromatography (HPSEC), high-performance anion-exchange chromatography (HPAEC) and Maldi-Tof-MS (Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry). The acetyl groups were found to be located near the branching point, in the external chain and in the internal chain regions. The acetyl group distributions were different for amylopectin from different granule size fractions. Higher DP (degree of polymerization) fragments were present in the digests of acetylated amylopectin populations of the small size granule starches. Our studies confirmed that acetyl groups were unevenly distributed over the amylopectin populations.

Polysaccharides were extracted from green Coffea arabica beans with water (90 °C, 1 h). Galactomannans were isolated from the water extract using preparative anion-exchange chromatography. Almost all of the galactomannans eluted in two neutral populations, while almost all of the arabinogalactans bound to the column, indicating that these arabinogalactans contain charged groups. Analysis of the molecular weight distribution of the two neutral populations showed that they differ in their molecular weight. Further characterization of these neutral populations by NMR and by MALDI-TOF MS after enzymatic degradation with an endo-mannanase, showed the presence of acetyl groups linked to the galactomannans, a feature not previously described for this type of polysaccharides from coffee beans. It was found that the high molecular weight (ca. 2000 kDa) neutral fraction was highly substituted both with galactose residues and acetyl groups, while the low molecular weight (ca. 20 kDa) population was much less substituted. Based on these results it can be concluded that at least two distinctly different populations of galactomannans are present in green coffee beans. It was also shown that the degradation of the galactomannans from green coffee beans with an endo-mannanase from A. niger is hindered by the presence of acetyl groups.

A new, fast method for the quantitative analysis of methoxyl groups in pectin using head-space gas chromatography (HS-GC) has been developed. With this method, results were obtained which were in reasonable agreement with the conventional HPLC method, and the reproducibility of the measurements is high. The advantages of the HS-GC method are that only a small amount of sample (2 mg) per analysis is needed, the chromatogram shows a nice symmetrically shaped methanol peak which is very easy to integrate, the sample preparation for HS-GC is short and easy, and for soluble pectins the sample in the head space vial can also directly be used for analysis of the galacturonic acid content and the degree of acetylation.

Polysaccharides were sequentially extracted from green and roasted Coffea arabica beans with water (90 °C), EDTA, 0.05, 1, and 4 M NaOH and characterized chemically. Additionally, the beans were subjected to a single extraction with water at 170 °C. Green arabica coffee beans contained large proportions of 1→4-linked mannans, of which on average 1 in every 23 mannopyranose residues was branched with single unit galactose side-chains at O-6. A part of these galactomannans could be extracted relatively easy with water and EDTA. These galactomannans were found to have a relatively high degree of branching (gal:man∼1:8) and a relatively low molecular weight in comparison to the remaining galactomannans (gal:man∼1:15–24). Additionally, 1→3-linked galactans, heavily branched at O-6 with side-chains containing arabinose and galactose residues, were present in the green coffee beans, as well as smaller amounts of pectins, cellulose, and xyloglucans. Roasting resulted in a loss of 8% of the dry weight. This could be partly explained by the relatively high percentage of sugars which was lost during the roasting process, most probably as a result of conversion into, e.g. Maillard and pyrolysis products. After roasting the extractability of polysaccharides was increased significantly. A decrease in the degree of branching as well as a decrease in molecular weight of arabinogalactans, galactomannans, and xyloglucans was observed after roasting.

Hydrolysates from two hydrothermally treated xylan-rich agrobased materials, Eucalyptus wood and brewery's spent grain were fractionated by anion-exchange chromatography and size-exclusion chromatography. Hereby, several pools were obtained and they were characterised by their sugar composition. Additionally, the oligosaccharides in the pools described were further identified by high-performance anion-exchange chromatography and mass spectrometry. The hydrothermally treated brewery's spent grain resulted in three pools of which two contained relatively high molecular weight xylan, singly and doubly branched with arabinose [XnAm], separated from a pool of xylo-oligosaccharides less branched with arabinose. The fractionation of the hydrothermally treated Eucalyptus wood resulted in a neutral pool, mainly consisting of a series of (acetylated) xylo-oligosaccharides [XnAcm], and three acidic pools. Two of these acidic pools contained a series of (acetylated) xylo-oligosaccharides including one 4-O-methylglucuronic acid [Xn(GlcAme)1Acm], while the third acidic pool contained (acetylated) xylo-oligosaccharides substituted with two 4-O-methylglucuronic acids [Xn(GlcAme)2Acm]. Additionally, a series of xylo-oligosaccharides containing both 4-O-methylglucuronic acid and a hexose, most likely galactose, was detected in the acidic Eucalyptus pools [Xn(GlcAme)1 or 2AcmH]. Information was obtained about the number of acetyl-groups linked to the (4-O-methylglucurono-) xylo-oligosaccharides. Finally, it is demonstrated with an example that the different substituents to the xylo-oligosaccharides present are of relevance for the fermentability of the xylo-oligosaccharides by human faecal samples.

Four xylan rich by-products, namely wheat bran, brewery's 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, brewery's 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 brewery's 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.

A simple method was developed that enabled the enzymatic determination of the galactose distribution in galactomannans. endo-Mannanase of Aspergillus niger was used to degrade the galactomannan polymers and the degradation products were determined with high-performance anion-exchange chromatography. A whole range of commercial high-to-low substituted galactomannans was analyzed in this way. It was found that differences in the anion-exchange chromatograms reflected dissimilarities in the distribution of galactose and could be used directly to discern these dissimilarities. The differences among the various elution profiles were used to construct a similarity distance tree. In addition to this approach, the absolute amount of non-substituted mannose released by the enzyme was found to be a good discriminating factor. In this way, galactomannans with regular, blockwise, and randomly distributed galactose could be discerned. All guars and the highly substituted gum of Prosopis juliflora were found to have a blockwise distribution of galactose. For different batches of tara gum both random and blockwise distributions were found. Among batches of locust bean gum the greatest variation was observed: both random, blockwise, and ordered galactose distributions were present. Cassia gum was found to have a highly regular distribution of galactose.

Two galacturonic-acid-containing polysaccharide fractions (ChSS and P) were isolated from soybean meal and subjected to lithium treatment. The fragments obtained were analyzed by using monosaccharide and methylation analyses, and NMR spectroscopy. Lithium degradation of ChSS, followed by sodium borodeuteride reduction, hydrolysis, sodium borohydride reduction, and acetylation afforded alditol acetates, of which the labeled ones reflected residues linked to GalA. As followed from quantifications of the labeled and non-labeled alditols from each constituent monosaccharide by GLC–EIMS, 6 mol% of Ara, 22 mol% of Fuc, 13 mol% of Gal, 53 mol% of Rha, and 57 mol% of Xyl are glycosidically linked to GalA. Analysis of the lithium-treated polymer revealed that it contains arabinogalactan side chains linked to Rha O-4, which consist of a β-(1→4)-linked galactan substituted with highly branched arabinan chains. On average, an arabinogalactan chain contains up to 29 Gal and 25 Ara residues. Surface plasmon resonance was used to determine conditions for affinity chromatography. Furthermore, this technique confirmed the presence of terminal α-Fuc residues in ChSS. Polysaccharide P turned out to be relatively resistant to lithium degradation.

Methyl-esterified and methyl-glycosydated oligogalacturonides (oligoGalA) were produced to be used as substrates for the characterization of pectinolytic enzymes acting on the homogalacturonan backbone. The reactions were monitored with recently developed techniques like high-performance anion-exchange chromatography (HPAEC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allow sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of esterification and or glycosydation. HPAEC at neutral pH separates not only oligogalacturonides with a different degree of esterifcation but seems to separate some isomers of e.g. monomethyl- and dimethyl-triGalA as well. Breakdown products formed by hydrolysis side reactions were revealed and could even be quantified by HPAEC pH 12 analysis. Using these techniques the conditions for each of the reactions were optimized. Esterification was performed best at concentrations of maximal 0.02 N sulfuric acid in anhydrous methanol at 4°C. Hardly any glycosydation occurs and the level of hydrolysis of the oligoGalA was less than 5%. Methyl-glycosydation and simultaneous esterification was performed best in 0.1 N sulfuric acid in anhydrous methanol at room temperature. HPAEC revealed only a limited hydrolysis (

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.

Alcohol insoluble solids from lemon albedo were extracted to obtain a chelating agent soluble pectin fraction (ChSS), a diluted sodium hydroxide soluble pectin fraction (ASS) which was separated into a water soluble and water insoluble part (ASSws and ASSwi), and an insoluble residue (Residue). ChSS, ASSws, ASSwi and Residue represented 64.3%, 10.4%, 0.4% and 8.1% of the galacturonic acid residues present in the lemon albedo, respectively. The pectin fractions and the residue were characterised by their sugar composition, degrees of methylation and acetylation, and molecular weight distribution. After extensive digestion with polygalacturonase and pectinesterase, the remaining high molecular weight fragments, representing the so-called pectic hairy regions (HR), were isolated and further characterised. After cold alkaline treatment (de-esterification), HR shown to be accessible to a further degradation by rhamnogalacturonan hydrolase (RGase) and the digests were analysed by high-performance size-exclusion chromatography (HPSEC) and high-performance anion-exchange chromatography (HPAEC). The various HR fractions differed in their degradation pattern: typically 2–4% of all glycosidic linkages were hydrolysed. The amount of RGase oligomers formed from the various fractions decreased in the order these were extracted while the degree of galactose substitution of the oligomers increased. Indications were found for the presence of small quantities of xylogalacturonan fragments within the pectic hairy regions of lemon albedo. On the basis of these results the structure of lemon pectin is compared with the structure of pectins extracted from other sources.

The mode of action of RG-hydrolase and RG-lyase toward purified linear rhamnogalacturonan (RG) oligomers has been studied. Major tools in the characterization of the degradation products were the exo-acting RG-rhamnohydrolase and RG-galacturonohydrolase. They were used to prepare a series of standards of RG oligomers for HPAEC. 1H NMR spectroscopy confirmed the structure assignment made using HPAEC for a selection of isolated degradation products. Identification of degradation products from purified RG oligomers was then performed by comparing retention times of HPAEC peaks with those of standards. RG-hydrolase was able to cleave RG oligomers which contained five Rha units or more, i.e. DP 9 with a Rha unit at both nonreducing and reducing end. Its preferential cleavage site was at four units from the first nonreducing Rha. RG-lyase was active toward oligomers that contained at least six GalA units, i.e. DP 12 with a GalA at the nonreducing and a Rha at the reducing end. The preferential cleavage site was for the smaller oligomers four residues, and for the largest oligomer six residues from the reducing Rha. From the observed cleavage patterns it can be speculated that in hairy regions, the RG stretches have to be at least 13 residues long for RG-hydrolase and 16 residues long for RG-lyase in order to produce one tetramer.

Cell wall material was isolated as water unextractable solids (WUS) from soybean meal. The isolation of WUS yields a fraction that contains 92% of the polysaccharides present in soybean meal and only a few other components. Arabinose, galactose, uronic acids and glucose (cellulose) were the major constituent sugars. WUS was sequentially extracted with chelating agent (chelating agent soluble solids, ChSS), dilute alkali (dilute alkali soluble solids, DASS), 1 m alkali (1 m alkali soluble solids, 1 m ASS) and 4 m alkali (4 m alkali soluble solids, 4 m ASS) to leave a cellulose-rich residue (RES). ChSS was the major extract, yielding 38% of the polysaccharides present in the WUS. All extracts and the residue were characterised by their sugar composition and their molecular-weight distribution. The extracts ChSS and DASS were fractionated by anion exchange chromatography. They showed identical elution patterns: an unbound fraction, five bound fractions of which one fraction eluted only with alkali. Anion exchange chromatography was also performed after saponification of both pectin-rich extracts, again resulting in identical elution patterns.

Oligosaccharides derived from alkali-extracted sorghum glucuronoarabinoxylan by digestion with a combination of (1--4)-beta-D-arabinoxylan arabinofuranohydrolase (AXH) and endo-(1--4)-beta-D-xylanase (Xyl I), both from Aspergillus awamori, were purified by size-exclusion chromatography followed by preparative high-performance anion-exchange chromatography. Structural studies including monosaccharide analysis, methylation analysis, 1H NMR spectroscopy, and mass spectrometry were carried out, resulting in the characterisation of four novel oligosaccharides, namely, alpha-D-GlcpA-(1--2)-beta-D-Xyl p-(1--4)-beta-D-Xyl p-(1--4) -beta-D-Xyl p, alpha-D-GlcpA-(1--2)-beta-D-Xyl p-(1--4) [alpha-L-Araf-(1--3)]-beta-D-Xyl p-(1--4)-beta-D-Xyl p, alpha-D-GlcpA-(1--2)-beta-D- Xyl p-(1--4)-beta-D-Xyl p-(1--4)[alpha-L-Araf-(1--2)-alpha)-alpha- L-Araf-(1--3)]-beta-D-Xyl p-(1--4)-beta-D-Xyl p, and alpha-D-GlcpA-(1--2) -beta-D-Xyl p-(1--4)[alpha-L-Araf-(1--3] beta-D-Xyl p-(1--4)[alpha-L-Araf-(1--2)-alpha-L-Araf-(1--3)] -beta-D-Xyl p-(1--4)-beta-D-Xyl p. The various oligosaccharides identified provide additional insight into the structure of sorghum glucuronoarabinoxylan. Furthermore, novel data were generated with respect to the substrate specificity of AXH and Xyl I towards glucuronoarabinoxylans in general.

The on-line combination of high-performance anion-exchange chromatography and mass spectrometry via a thermospray interface has proved to be a powerful tool in the characterization of sugar oligomers obtained by enzymatic digestion of plant cell wall polysaccharides. The potential of the method can be improved by the use of a new column material, CarboPac PA100, which requires lower sodium acetate concentrations for the elution of large sugar oligomers. Further, the application of multiple-ion detection optimizes the information obtained from the analysis by improving both the signal-to-noise ratio and the conservation of the chromatographic resolution. Negative-ion instead of positive-ion detection results in significantly better signals, especially for the larger sugar oligomers.

A qualitative study of the characteristics in the mass spectrometric analysis of various neutral and acidic oligosaccharides using electrospray ionization is reported. Experiments were performed in both positive- and negative-ion modes. In the positive-ion mode molecular mass information for neutral non-derivatized oligosaccharides could be obtained up to Mr 4000. The method was applied to the determination of the degree of methylation of methylated β-cyclodextrins and the identification of unknown oligosaccharides enzymatically derived from pear and apple fruit material.

The on-line coupling of high-performance anion-exchange chromatography and thermospray mass spectrometry via an anion-micromembrane suppressor and a booster pump is described. The system was applied to the analysis of homologous series of oligosaccharides. Among others, the mass spectrometric detection of β-1,4-xylose oligomers up to degree of polymerization (DP) 25 is demonstrated. Further, the system was used in the analysis of more complex oligosaccharide samples, containing mixed oligomers of hexoses, pentoses and uronic acids. In such samples oligomers up to DP 10 can be tested. The potential use of this approach in the characterization of oligosaccharides obtained from (enzymic) degradation of plant cell wall oligosaccharides is discussed.

Plant cell walls contain polymers with complex and badly understood interactions that can be studied by enzymic extraction. Apple cell wall material, partially depectinated by a chelating agent, was treated with pure polysaccharidases (pectin-lyase, polygalacturonase, pectinesterase, endo-β-(1,4)-glucanases, exo-β-(1,4)-glucanase, endo-α-(1,5)-arabinanase, arabinofuranosidase, endo-β-(1,4)-galactanase and endo-β-(1,4)-xylanase) and combinations of these enzymes. Glucanases, arabinanases and galactanase extracted neutral oligomers. Enzyme formulations active on highly methylated pectins freed galacturonate oligomers and high-molecular-weight material. Addition of endo-β-(1,4)-glucanase to these enzyme formulations increased the amounts of high-molecular-weight material in the extracts. One of the endo-β-(1,4)-glucanases had a more marked action. The high-molecular-weight material was rich in neutral sugars, notably arabinose and galactose, and was degraded to a large extent by arabinanases and galactanase. The results were analysed by principal component analysis.