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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.

Eucalyptus globulus wood samples were subjected to double hydrothermal processing to remove extractives in the first stage, and to cause the selective solubilisation of 4-O-methylglucuronoxylan in the second stage. The hemicellulose-derived products present in the liquors from the second hydrothermal stage (substituted xylooligosaccharides, denoted XOS) were refined by treatments with membranes and ion exchange. The purified XOS product was assayed for composition and characterised by HPLC-RI, HPAEC-PAD, HPSEC, MALDI-TOF-MS and NMR techniques. The results suggested the presence of neutral and acidic XOS with a degree of acetylation of about 0.6. The fermentability of the refined XOS product by faecal inocula was assessed by measuring both substrate consumption and formation of short-chain fatty acids.

Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (alpha/alpha)(6)-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426-Ala431 in TP and Thr419-Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (alpha1, alpha1)- and alpha-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.

The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g.L(-1) culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1-->6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-D-Galp-(1-->6)-D-Galp, alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->beta2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-D-galactopyranoside (40 mm), alpha-(1-->6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, L-arabinose, L-fucose and L-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and L-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. alpha-D-Galp-(1-->6)-D-Manp; alpha-D-Galp-(1-->6)-beta-D-Glcp-(1-->4)-D-Glcp; alpha-D-Galp-(1-->6)-beta-D-Galp-(1-->4)-D-Fruf; alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->alpha1)-D-Glcp; and alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Fruf.

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

Mixtures of the complex human milk oligosaccharides (HMOs) are difficult to analyze and gastrointestinal bioconversion products of HMOs may complicate analysis even more. Their analysis, therefore, requires the combination of a sensitive and high-resolution separation technique with a mass identification tool. This study introduces for the first time the hyphenation of CE with an electrospray mass spectrometer, capable to perform multiple MS analysis (ESI-MS(n)) for the separation and characterization of HMOs in breast milk and feces of breast-fed babies. LIF was used for on- and off-line detections. From the overall 47 peaks detected in off-line CE-LIF electropherograms, 21 peaks could be unambiguously and 11 peaks could be tentatively assigned. The detailed structural characterization of a novel lacto-N-neo-tetraose isomer and a novel lacto-N-fucopentaose isomer was established in baby feces and pointed to gastrointestinal hydrolysis of higher-Mw HMOs. CE-LIF-ESI-MS(n) presents, therefore, a useful tool which contributes to an advanced understanding on the fate of individual HMOs during their gastrointestinal passage.

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.

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of alpha-D-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming beta-glucosyl disaccharides with beta-(1-->4)- regioselectivity from five monosaccharides as well as branched beta-glucosyl trisaccharides with beta-(1-->4)-regioselectivity from three (1-->6)-linked disaccharides. CtCDP showed strict beta-(1-->4)-regioselectivity and catalysed linear chain extension of the three beta-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two beta-glucosyl oligosaccharide product series to represent novel compounds, i.e. beta-D-glucopyranosyl-[(1-->4)-beta-D-glucopyranosyl](n)-(1-->2)-D-gluco pyranose, and beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl](n)-(1-->3)-D-glucop yranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the Cl hydroxyl of beta-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of Cl substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using alpha-D-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.

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'

The filamentous fungus Chrysosporium lucknowense (C1) is a rich source of cell wall degrading enzymes. In the present paper four arabinose releasing enzymes from C1 were characterized, among them one endoarabinanase, two arabinofuranosidases and one exoarabinanase. Combinations of these enzymes released up to 80% of the arabinose present in sugar beet arabinan to fermentable monosugars. Besides the main product arabinobiose, unknown arabinose oligomers are produced from highly branched arabinan when endoarabinanase was combined with exoarabinanase and/or arabinofuranosidase. All described arabinose releasing enzymes are temperature stable up to 50 degrees C and have a broad pH stability. This makes C1 arabinohydrolases suitable for many biotechnical applications, like co-fermentation bioethanol production.

Chemical derivatization is often applied to improve polysaccharide functionality. Primary hydroxyl groups in starch can, for example, be oxidized to aldehydes by using a 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated reaction. The major part of the aldehydo groups is subsequently converted to carboxyl groups by NaOCl. The exact structure of TEMPO-oxidized starch was studied to promote a better understanding of the TEMPO oxidation mechanism and the functionality of oxidized starches. By using weak and strong acidic hydrolysis, and methanolysis, at elevated temperatures, oxidized starches with different degrees of oxidation (DO) were broken down into oligomers and monomers. Analysis of the oligomers by chromatographic and mass spectrometric techniques revealed that blocks of glucuronic acid moieties are present in the oxidized starch polymers. The α(1 → 4) glucuronic acid-glucuronic acid bond was found to be very resistant to breakdown by acids. The α(1 → 4) glucuronic acid-glucose bond also showed increased resistance to acids compared to α(1 → 4) glucose-glucose bonds. The size of the blocks of glucuronic acid moieties increased when DO increased. Furthermore, the presence of clusters of aldehydes close to carboxyl groups directly after oxidation was proven. This implies that TEMPO, which is positively charged in its active state, is apparently attracted by the negatively charged carboxyl groups. Because of this, TEMPO tends to be active in areas where carboxyl groups have already been formed, which leads to a block wise distribution of the glucuronic acid moieties. © 2010 Elsevier Ltd. All rights reserved.

Cell wall materials derived from leaves and hypocotyls of Arabidopsis mutant and wild type plants have been incubated with a mixture of pure and well-defined pectinases, hemicellulases, and cellulases. The resulting oligosaccharides have been subjected to MALDI-TOF MS and CE-LIF analysis. MALDI-TOF MS analysis provided a fast overview of all oligosaccharides released, whereas CE-LIF-measurements enabled separation and characterization of many oligosaccharides under investigation. Both methods have been validated with leaf material of known mutant Arabidopsis plants and were shown to be able to discriminate mutant from wild type plants. Downscaling of the MALDI-TOF MS and CE-LIF approaches toward the hypocotyl level was established, and the performance of MALDI-TOF MS and CE-LIF was shown in the successful recognition of the Arabidopsis mutant gaut13 as an interesting candidate for further analysis.

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG-I) on melanoma cell growth and survival in vitro. We added okra RG-I containing an almost pure RG-I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2-hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin-3 (Gal-3) protein.Incubation with okra RG-I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N-cadherin and alpha5 integrin subunit was reduced and that of the multifunctional carbohydrate-binding protein, Gal-3, at the cell membrane increased.These findings suggest that okra RG-I induces apoptosis in melanoma cells by interacting with Gal-3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occur.

A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing s-glucose 1-phosphate (s-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and s-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mms-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be a-Glcp-(1?4)-Glcp (maltose), a-Glcp-(1?4)-GlcNp (maltosamine), a-Glcp-(1?4)-GlcNAcp (N-acetyl maltosamine), a-Glcp-(1?4)-Manp, a-Glcp-(1?4)-Xylp and a-Glcp-(1?4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

Multiform coated titanium implants are widely used in orthopedic and dental surgery. In this study, we have investigated the reactivity of pectin-coated titanium samples implanted under the latissimus dorsi—muscle fascia of rats. Samples were coated with two enzyme treated apple pectins; modified hairy regions (MHR-A and MHR-B) that differed in chemical structure. Aminated (AMI) and uncoated titanium (Ti) served as controls. The thicknesses of the peri-implant fibrous tissue capsules formed 1 or 3 weeks after implantation were measured as indicative of possible inflammatory reactions toward the biomaterials. After 1 week, the MHR-B implant was surrounded by a thicker fibrous capsule (42.9 µm) than any of the other sample types: MHR-A (33.2 µm), AMI (32.5 µm), and Ti (32.3 µm), the last one being the only statistically significant difference. After 3 weeks, however, this difference disappeared; the capsule thicknesses around MHR-B and Ti implants had decreased to the values found for AMI and MHR-A. Additionally, the capsule formation represents merely a stromal rather than an inflammatory reaction, as indicated by the absence of activated macrophages or foreign body giant cells in the capsules. These results indicate for the first time the in vivo tolerability of covalently linked pectins, and suggest the feasibility of pectin-coated bone and dental implants for clinical use.

The formation of complexes between proteins and polysaccharides is of great importance for many food systems like foams, emulsions, acidified milk drinks, and so on. The complex formation between beta-lactoglobulin (beta-lg) and pectins with a well-defined physicochemical fine structure has been studied to elucidate the influence of overall charge and local charge density of pectin on the complex formation. Binding isotherms of beta-lg to pectin are constructed using fluorescence anisotropy, which is shown to be an excellent technique for this purpose, as it is fast and requires low sample volumes. From the binding isotherms the maximal adsorbed amount, binding constant (k(obs)) and the cooperativity of binding are obtained at different ionic strengths. The Hill model is used to fit the binding isotherms and is shown to be preferable over a Langmuir fit. At pH 4.25, k(obs) shows a maximum at an ionic strength of 10 mM when using a low methyl esterified pectin (LMP) due to the balance of attractive and repulsive electrostatic forces between beta-lg and pectin and beta-lg neighbors. For two high methyl esterified pectins, one with a blockwise distribution of methyl esters (HMP(B)) and one with a random distribution (HMP(R)), this ionic strength maximum is absent and k(obs) decreases with increasing ionic strength. k(obs) is found to be largest for LMP and HMP(B) and considerably lower for HMP(R). A positive cooperativity is observed for both LMP (above an ionic strength of 45 mM) and HMP(R) (above an ionic strength of 15 mM) but not for HMP(B). Positive cooperativity is thought to be caused by a rearrangement of the pectin helix structure caused by binding of beta-lg, thus creating new or binding sites with a higher affinity. To attain strong binding of beta-lg to pectin it is preferable to use a pectin with a blockwise distribution of methyl esters. When complex formation takes place in high ionic strength media an LMP gives the best results, while at low ionic strength a high methyl esterified pectin with blockwise distribution may give better results, due to reduced electrostatic repulsion between both pectin and beta-lg and beta-lg neighbors.

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 complex formation between beta-lactoglobulin (beta-lg) and pectin is studied using pectins with different physicochemical characteristics. Pectin allows for the control of both the overall charge by degree of methyl-esterification as well as local charge density by the degree of blockiness. Varying local charge density, at equal overall charge is a parameter that is not available for synthetic polymers and is of key importance in the complex formation between oppositely charged (bio)polymers. LMP is a pectin with a high overall charge and high local charge density; HMPB arid HMPR are pectins with a low overall charge, but a high and low local charge density, respectively. Dynamic light scattering (DLS) titrations identified pH(c), the pH where soluble complexes of beta-lg and pectin are formed and pH phi, the PH of phase separation, both as a function of ionic strength. pH(c) decreased with increasing ionic strength for all pectins and was used in a theoretical model that showed local charge density of the pectin to control the onset of complex formation. pH phi passed through a maximum with increasing ionic strength for LMP because of shielding of repulsive interactions between beta-lg molecules bound to LMP, while attractive interactions were repressed at higher ionic strength. Potentiometric titrations of homo-molecular solutions and mixtures of beta-lg and pectin showed charge regulation in beta-lg-pectin complexes. Around pH 5.5-5.0 the pK(a)s of beta-lg ionic groups are increased to induce positive charge on the beta-lg molecule; around pH 4.5-3.5 the pK(a) values of the pectin ionic groups are lowered to retain negative charge on the pectin. Since pectins with high local charge density form complexes with beta-lg at higher ionic strength than pectins with low local charge density, pectin with a high local charge density is preferred in food systems where complex formation between protein and pectin is desired. (C) 2008 Elsevier Ltd. All rights reserved.

A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting beta-elimination. These pectin fragments were characterized by their molecular weight distribution, sugar composition, 13C-NMR, and MALDI-TOF-MS analysis. These analyses revealed that the large oligosaccharides released by beta-eliminative treatment were composed of alpha-1,5 linked arabinosyl residues with 2- and 3-linked alpha-arabinosyl side chains, and, or beta-1,4 linked galactosyl side chains. Fractions were tested for complement-fixing activity in order to determine their interaction with the complement system. These results strongly indicated that there was a minimal unit size responsible for the complement-fixing activity. Neutral pectin fragments (8 kDa) obtained from beta-elimination were inactive in the complement system, although they contained a sugar composition previously shown to be highly active. Larger pectin fragments (17 kDa) retained some activity, but much lower than polymers containing rhamnogalacturonan type 1 (RGI) structures isolated from the same source. This implied that structural elements containing multiple side chains is necessary for efficient complement-fixing activity.

The impact of microbial proteases on the release of carbohydrates from BSG was studied. The proteases were able to release the non-cellulosic glucose, a portion of feruloylated arabinoxylan and over 50% of the protein from brewers' spent grain (BSG) after 24 h hydrolysis. The non-cellulosic glucose was derived from residual starch-derived products persisting in BSG after mashing. The proteases did not cleave the hydroxycinnamate ester linkages present on the arabinoxylan backbone, and thus do not behave as feruloyl esterases. However, the material solubilised from spent grain by the proteases contained up to 198 µg bound ferulic acid/g extract, which represented 8.6% of the total ferulic acid present in BSG. These results suggest that a portion of water-extractable feruloylated arabinoxylan and starch is trapped within the BSG matrix by a proteinaceous barrier.

Water-soluble native O-acetyl galactoglucomannan (GGM) from spruce is a polysaccharide that can be produced in an industrial scale. To develop GGM applications, information is needed on its stability, particularly under acidic conditions. Therefore, acid hydrolysis of spruce GGM was investigated at various pH levels and temperatures. The results allow an estimation of the stability of GGM under food processing conditions and in biological systems. Determination of the average molar mass demonstrated that spruce GGM was stable at pH 1 and 37°C, as well as at pH 3 and 70°C. GGM was hydrolysed at pH 1 and 90°C. GGM oligomers and monomers were detected after degradation. Some of the oligomers contained O-acetyl groups. Monosaccharides were the predominant products in the hydrolysates after treatment at pH 1 and 90°C for 48 h. Pentoses, present in GGM samples as impurities, were released more easily than GGM hexoses. Glucose was more difficult to release than mannose. Traces of 6-deoxy-mannose and levoglucosan were found in the hydrolysates, indicating further degradation of hydrolysed monosaccharides.

Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME-catalyzed pectin de-esterification was obtained when combining pressures in the range 200-300 MPa with temperatures in the range 50-55 degrees C. The mode of pectin de-esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 degrees C) on the mode of pectin conversion was detected.

As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials

BACKGROUND: Treatment in aqueous media (hydrothermal or autohydrolysis reactions) is an environmentally friendly technology for fractionating lignocellulosic materials. Rice husks were subjected to hydrothermal processing under a variety of operational conditions to cause the selective breakdown of xylan chains, in order to assess the effects of reaction severity on the distribution of reaction products. RESULTS: The effects of severity (measured by the severity factor, R0) on the concentrations of the major autohydrolysis products (monosaccharides, xylo- and glucooligosaccharides, xylooligosaccharide substituents, acetic acid, acid-soluble lignin and elemental nitrogen) were assessed. The interrelationship between the severity of treatment and molecular weight distribution was established by high-performance size-exclusion chromatography. Selected samples were subjected to refining treatments as ethyl acetate extraction and ion exchange for refining purposes, and the concentrates were assayed by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CONCLUSIONS The protein equivalent of the products present in liquors accounted for 43 to 51% of the protein present in the raw rice husks. The concentrations of glucose (derived from starchy material) and arabinose (split from the xylan backbone) were fairly constant with severity. Even in treatments at low severity, high molecular weight compounds derived from xylan accounted for a limited part of the stoichiometric amount. Operating under harsh conditions, about 50% of the total xylan-derived compounds corresponded to fractions with a degree of polymerization (DP)

Analysis of low molecular weight (LMw) coffee brew melanoidins is challenging due to the presence of many non-melanoidin components that complicate analysis. This study focused on the isolation of LMw coffee brew melanoidins by separation of melanoidins from non-melanoidin components that are present in LMw coffee brew material. LMw coffee fractions differing in polarity were obtained by reversed-phase solid phase extraction and their melanoidin, sugar, nitrogen, caffeine, trigonelline, 5-caffeoylquinic acid, quinic acid, caffeic acid, and phenolic groups contents were determined. The sugar composition, the charge properties, and the absorbance at various wavelengths were investigated as well. The majority of the LMw melanoidins were found to have an apolar character, whereas most non-melanoidins have a polar character. The three isolated melanoidin-rich fractions represented 56% of the LMw coffee melanoidins and were free from non-melanoidin components. Spectroscopic analysis revealed that the melanoidins isolated showed similar features as high molecular weight coffee melanoidins. All three melanoidin fractions contained approximately 3% nitrogen, indicating the presence of incorporated amino acids or proteins. Surprisingly, glucose was the main sugar present in these melanoidins, and it was reasoned that sucrose is the most likely source for this glucose within the melanoidin structure. It was also found that LMw melanoidins exposed a negative charge, and this negative charge was inversely proportional to the apolar character of the melanoidins. Phenolic group levels as high as 47% were found, which could be explained by the incorporation of chlorogenic acids in these melanoidins.

The chemoenzymatic synthesis of feruloylated arabino-oligosaccharides has been achieved, using a feruloyl esterase type C from Sporotrichum thermophile (StFaeC). The structure of the feruloylated products was confirmed by ESI-MS(n).

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.

Pectin is of interest both as cell wall component and as food additive. The precise chemical structure of pectin remains under debate, although the structural elements of pectin are rather well described. In order to get more insight in the inter linkage between the various structural elements, apple pectin modified hairy regions were degraded by controlled acid hydrolysis. From the degradation products oligomeric fragments were selected which could represent interconnection points, and these oligomers were characterized using LC?MS and NMR approaches. It was shown that the oligomers GalA3Rha1, GalA4Rha2, and GalA5Rha3 consisted out of a homogalacturonan and a rhamnogalacturonan type I segment connected via a GalAp?-(1 ? 2) Rhap linkage. In addition, a GalA6Rha3Xyl1 oligomer was identified, which consisted out of a xylogalacturonan and a rhamnogalacturonan type I segment. These oligomers indicated that in apple pectin both homogalacturonan and xylogalacturonan were covalently linked to rhamnogalacturonan type I. With these new insights, currently used pectin models were refined. Keywords: Homogalacturonan; Xylogalacturonan; Rhamnogalacturonan I; Pectin model; Covalent linkage; Pectin structure

Yellow pea starch was fractionated into small and large size granule fractions and then modified with acetic anhydride and vinyl acetate (acetylation after sieving) or first acetylated in the same way and then fractionated into small and large size fractions (acetylation before sieving). Acetylation with different procedures (acetylation before or after sieving) resulted in different degrees of substitution for small size granule fractions when acetic anhydride was used as reagent. Modification with vinyl acetate gave products with much higher peak viscosities than modification with acetic anhydride for the same granule size and same degree of substitution (DS). The location and distribution of acetyl groups was investigated by analyzing the ¿-amylase hydrolysates of isolated amylose and amylopectin with chromatography and mass spectrometry. Mass spectra showed that the substituent distribution mainly depended on the type of reagent and was not affected by the granule size. Both amylose and amylopectin were modified in an heterogeneous way and the reactions took place in different regions of the granule. It is postulated that acetylation occurs more homogeneously throughout the granule when vinyl acetate is used as reagent, while the reaction with acetic anhydride to a large extent takes place in the outer lamellae of granule. Keywords: Yellow pea starch; Amylose; Amylopectin; Acetic anhydride; Vinyl acetate

Cell wall material from whole oat grains was sequentially extracted to study the structural characteristics of individual arabinoxylan (AX) populations. Araf was singly substituted at both O-3 (mainly) and O-2 positions of Xylp, and no disubstitution of Xylp with Araf residues was found in oat AXs. Both highly substituted and sparsely substituted segments were found in AXs in Ba(OH)2 extracts, whereas AXs in 1 and 6 M NaOH extracts were rarely branched and easily aggregated. Both O-2-linked GlcA and 4-O-MeGlcA residues were present in oat AXs. A series of AX oligomers with galactose as a substituent was detected for the first time in oats. The present study suggested that the distribution of Araf was contiguous in oat AXs, different from the homogeneous distribution of Araf in wheat and barley AXs, which might result in different fermentation patterns in humans and animals.

Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100 g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type.

Scope: Main objectives of this study were (1) to demonstrate direct signaling of starch on human dendritic cells (DCs), (2) to study whether this is mediated by the pattern recognition receptors such as Toll-like receptors (TLRs) and (3) to study whether intestinal epithelial cells (IECs) are involved in modulating the starch induced immune activation of DCs.Methods and results: Two different types of resistant starch, High-maize (R) 260 (RS2) and Novelose (R) 330 (RS3) were characterized for their starch content and particle size. Human DCs and reporter cells for TLRs were incubated with starches and analyzed for NF-kB/AP-1 activation. Complex coculture systems were applied to study the cross-talk. High-maize (R) 260 predominantly binds to TLR2 while Novelose (R) 330 binds to TLR2 and TLR5. The strong immune-stimulating effects of High-maize (R) 260 were attenuated by starch-exposed IECs illustrating the regulatory function of IECs. Despite these attenuating effects, DCs kept producing Th1 cytokines.Conclusion: Resistant starch possesses direct signaling capacity on human DCs in a starch-type-dependent manner. IECs regulate these responses. High-maize (R) 260 skews toward a more regulatory phenotype in coculture systems of DCs, IEC, and T cells.

Calf milk replacers contain 40 to 50% lactose. Fluctuating dairy prices are a major economic incentive to replace lactose from milk replacers by alternative energy sources. Our objective was, therefore, to determine the effects of replacement of lactose with glucose, fructose, or glycerol on energy and protein metabolism in veal calves. Forty male Holstein-Friesian calves (114 +/- 2.4 kg) were fed milk replacer containing 46% lactose (CON) or 31% lactose and 15% of glucose (GLUC), fructose (FRUC), or glycerol (GLYC). Solid feed was provided at 10 g of dry matter (DM)/kg of metabolic body weight (BW0.75) per day. After an adaptation of 48 d, individual calves were harnessed, placed in metabolic cages, and housed in pairs in respiration chambers. Apparent total-tract disappearance of DM, energy, and N and complete energy and N balances were measured. The GLUC, FRUC, and GLYC calves received a single dose of 1.5 g of [U-C-13]glucose, [U-C-13]fructose, or [U-C-13]glycerol, respectively, with their milk replacer at 0630 h and exhaled (CO2)-C-13 and C-13 excretion with feces was measured. Apparent total-tract disappearance was decreased by 2.2% for DM, 3.2% for energy, and 4.2% for N in FRUC compared with CON calves. Energy and N retention did not differ between treatments, and averaged 299 16 kJ/kg of BW0.75 per day and 0.79 +/- 0.04 g/kg of BW0.75 per day, respectively, although FRUC calves retained numerically less N (13%) than other calves. Recovery of C-13 isotopes as (CO2)-C-13 did not differ between treatments and averaged 72 +/- 1.6%. The time at which the maximum rate of (CO2)-C-13 production was reached was more than 3 h delayed for FRUC calves, which may be explained by a conversion of fructose into other substrates before being oxidized. Recovery of C-13 in feces was greater for FRUC calves (7.7 +/- 0.59%) than for GLUC (1.0 +/- 0.27%) and GLYC calves (0.5 +/- 0.04%), indicating incomplete absorption of fructose from the small intestine resulting in fructose excretion or fermentation. In conclusion, energy and N retention was not affected when replacing >30% of the lactose with glucose, fructose, or glycerol. Increased fecal losses of DM, energy, and N were found in FRUC calves compared with CON, GLUC, and GLYC calves. Postabsorptive losses occurred with the urine for glucose and glycerol, which caused a lower respiratory quotient for GLUC calves during the night. Fructose was oxidized more slowly than glucose and glycerol, probably as a result of conversion into other substrates before oxidation.

Scope: In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied.Methods and results: The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This was different when DCs and fibers were co-cultured together with supernatants from human epithelial cells (Caco spent medium). Caco spent medium enhanced the production of IL-12, IL-1Ra, IL-6, IL-8, TNF-alpha, MCP-1 (monocyte chemotactic protein), and MIP-1 alpha but this was strongly attenuated by the dietary fibers. This attenuating effect on proinflammatory cytokines was dependent on the interaction of the fibers with Toll-like receptors as it was reduced by Pepinh-myd88. The interaction of galacto-oligosaccharides, chicory inulin, wheat arabinoxylan, barley beta-glucan with epithelial cells and DCs led to changes in the production of the Th1 cytokines in autologous T cells, while chicory inulin, and barley beta-glucan reduced the Th2 cytokine IL-6. The Treg-promoting cytokine IL-10 was induced by galacto-oligosaccharides whereas chicory inulin decreased the IL-10 production.Conclusions: Our results suggest that dietary fibers can modulate the host immune system not only by the recognized mechanism of effects on microbiota but also by direct interaction with the consumer's mucosa. This modulation is dietary fiber type dependent.

Calf milk replacers (MR) commonly contain 40% to 50% lactose. For economic reasons, starch is of interest as a lactose replacer. Compared with lactose, starch digestion is generally low in calves. It is, however, unknown which enzyme limits the rate of starch digestion. The objectives were to determine which enzyme limits starch digestion and to assess the maximum capacity for starch digestion in milk-fed calves. A within-animal titration study was performed, where lactose was exchanged stepwise for one of four starch products (SP). The four corn-based SP differed in size and branching, therefore requiring different ratios of starch-degrading enzymes for their complete hydrolysis to glucose: gelatinised starch (alpha-amylase and (iso) maltase); maltodextrin ((iso)maltase and alpha-amylase); maltodextrin with alpha-1,6-branching (isomaltase, maltase and alpha-amylase) and maltose (maltase). When exceeding the animal's capacity to enzymatically hydrolyse starch, fermentation occurs, leading to a reduced faecal dry matter (DM) content and pH. Forty calves (13 weeks of age) were assigned to either a lactose control diet or one of four titration strategies (n = 8 per treatment), each testing the stepwise exchange of lactose for one SP. Dietary inclusion of each SP was increased weekly by 3% at the expense of lactose and faecal samples were collected from the rectum weekly to determine DM content and pH. The increase in SP inclusion was stopped when faecal DM content dropped below 10.6% (i.e. 75% of the average initial faecal DM content) for 3 consecutive weeks. For control calves, faecal DM content and pH did not change over time. For 87% of the SP-fed calves, faecal DM and pH decreased already at low inclusion levels, and linear regression provided a better fit of the data (faecal DM content or pH v. time) than non-linear regression. For all SP treatments, faecal DM content and pH decreased in time (P

The distributions of phosphorus and hydroxypropyl groups within granules of cross-linked and hydroxypropylated sweet potato starches were investigated. Chemical surface peeling of starch granules was performed after sieving of native and modified starches into large-size (diameter ≥ 20 μm) and small-size (diameter < 20 μm) fractions. Starch granules were surface gelatinized in a 4M calcium chloride solution at different levels. After the peeling step, the remaining starch granules were analysed for the content of phosphorus and hydroxypropyl groups. The phosphorus level of the parental starch gradually decreased from periphery to core of the granules. The increase in phosphorus content after cross-linking in periphery was higher than that in core. The subsequent hydroxypropylation reaction resulted in lower phosphate levels. Hydroxypropylation resulted in a gradient of hydroxypropyl group concentration from periphery to core. Cross-linking prior to the hydroxypropylation resulted in lower levels of hydroxypropyl groups and less pronounced differences between periphery and core.

Sweet potato starch was modified by cross-linking, hydroxypropylation, and combined cross-linking and hydroxypropylation, and the starches were subsequently sieved to obtain differently sized granule fractions. The effects of granule size of native and modified sweet potato starch fractions and all fractions were investigated with respect to their physicochemical properties. The large-size granule fraction (27-30 μm) showed a 16-20% higher chemical phosphorylation and a 4-7% higher hydroxypropylation than the small-size granule fraction (14-16 μm). The large-size granule fractions of native and modified sweet potato starches showed lower transition temperatures (0.7-3.1 °C for peak temperature of gelatinization) and lower enthalpy changes (0.6-1.9 J/g) during gelatinization than the small-size granule fractions, making the sweet potato starch different from cereal starches. The large-size granule fraction of native starch showed a higher paste viscosity (78-244 cP) than the corresponding small-size granule fraction. In addition, cross-linking and hydroxypropylation affected the paste viscosity of the large-size granule fraction significantly more than that of the small-size granule fraction when compared to the corresponding parental starch fractions. The large-size granule fraction of native and dual-modified starches showed a lower syneresis after freeze-thaw treatments than the small-size granule fractions. The difference in swelling power between large- and small-size granule fractions was not significant. In general, the large-size granule fraction of sweet potato starch was more susceptible for cross-linking and hydroxypropylation and the physicochemical properties were changed to a higher extent compared to the corresponding small-size granule fraction.

Purpose To investigate whether breast-milk composition and microbiota differ in healthy mothers and mothers with celiac disease (CD) to ultimately contribute to identify additional factors determining CD risk. Methods Breast-milk samples from healthy mothers (n = 12) and mothers with CD (n = 12) were collected. Cytokines and secretory immunoglobulin A (sIgA) were analyzed by bead-arrays and flow cytometry and human milk oligosaccharides (HMOs) were assessed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Breast-milk microbiota composition was analyzed by conventional and quantitative real-time PCR. Result Breast milk from CD mothers showed significantly lower levels of interleukin (IL) 12p70 (P < 0.042), transforming growth factor (TGF)-beta 1 (P < 0.018) and sIgA (P < 0.003) and almost significantly lower levels of interferon (IFN)-gamma (P < 0.058). Six mothers in each group belonged to the secretor Le(a-b+) type, one to the secretor Le(a-b-) type and five to the non-secretor Le(a+b-) type. CD mothers of non-secretor Le(a+b-) type showed increased Lacto-N-tetraose content (P < 0.042) compared with healthy mothers. CD mothers' milk showed reduced gene copy numbers of Bifidobacterium spp. (P < 0.026) and B. fragilis group (P < 0.044). Conclusion CD mothers' breast milk is characterized by a reduced abundance of immunoprotective compounds (TGF-beta 1 and sIgA) and bifidobacteria. The reduction in these components could theoretically diminish the protective effects of breast-feeding on the child's future risk of developing CD.

Sweet potato starch was cross-linked using sodium trimetaphosphate and hydroxypropylated using propylene oxide. The level and position of phosphorus and hydroxypropyl groups within cross-linked and hydroxypropylated sweet potato starch was investigated by phosphorus and proton nuclear magnetic resonance spectroscopy ((31)P, (1)H NMR). The cross-linking reaction produced monostarch monophosphate and distarch monophosphate in a molar ratio of 1:1.03, indicating that only half of the introduced phosphorus resulted in a possible cross-link. One cross-link per approximately 2900 glucose residues was found. Phosphorylation leading to monostarch monophosphate mainly occurred at O-3 and O-6 (ratio 1:1). It was inferred that the majority of the cross-links formed in distarch monophosphate were between two glucose residues positioned in different starch chains, while a minor part of the cross-links may be formed between two glucose residues within the same starch chain. Hydroxypropylation under alkaline conditions resulted in the formation of intra-molecular phosphorus cross-links, subsequent hydroxypropylation following cross-linking lowered both the level of intra- and inter-molecular cross-linking. Using (1)H NMR the molar substitution of hydroxypropylation was determined to be 0.155-0.165. The hydroxypropylation predominantly occurred at O-2 (61%), and the level of substitution at O-6 (21%) was slightly higher than that at O-3 (17%). In dual modified starch, the preceding cross-linking procedure resulted in a slightly lower level of hydroxypropylation, where the substitution at O-6 decreased more compared to the substitution at O-2 and O-3.

A high-molecular-mass polysaccharide galactan (M 2000 kDa) was isolated from flax at the stage of cell wall thickening of the bast fiber development. The polymer structure was studied by 1H NMR spectroscopy and MALDI TOF mass spectrometry. It is built up of Gal (59%), Rha (15%), GalA (23%), and Ara (3%) residues. The galactan backbone consists of successively alternating monomer disaccharide units (- 4GalA1 - 2Rha1 -)n and is similar in its structure to the backbone of rhamnogalacturonan-1 (RG-I). Rhamnose residues bear in position 4 ß-(1 - 4)-galactose side chains of various lengths with a polymerization degree of up to 28 or higher. A part of the side chains have branchings.

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.

Different separation (HPAEC, RP–HPLC, CE) and identification (MALDI–TOF–MS, ESI–MS n ) techniques were compared to analyse oligosaccharides obtained after incubation of xyloglucan with endo-glucanase. It was possible to analyse xyloglucan oligosaccharides with each technique. Several techniques, including off line (HPAEC–MALDI–TOF–MS) or online (CE–ESI–MS n , RP–HPLC–ESI–MS n ) connection provided complementary information on xyloglucan structure. Online CE–MS and RP–HPLC–MS are described for the first time in xyloglucan analysis. Advantages and disadvantages of the techniques for different purposes such as structural characterisation of oligosaccharides or oligosaccharide profiling are discussed. Black currant xyloglucans had a rather simple XXXG-type structure with galactose and fucose containing side chains. © 2006 Elsevier B.V. All rights reserved.

The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were added to mashed berries, a synergistic effect was observed between treatment with commercial enzymes and HPP. Compared to treatment at atmospheric pressure, pectic polysaccharides were degraded to a larger extent when HPP was used. In contrast, hemicelluloses were hardly affected by the added enzymes when HPP was included, although they were degraded during similar treatment at atmospheric pressure. Additionally, the activity of rhamnose-releasing enzymes present in minor quantities might be enhanced after HPP, resulting in a decrease of rhamnose in the polymeric cell wall material. These results exploring the effect of HPP at representative conditions clearly point out the potential of HPP for polysaccharide modification.

Various plant polysaccharide derived mono- and oligosaccharides were derivatized with the fluorescent 9-aminopyrene-1,4,6-trisulfonate (APTS) and subjected to capillary electrophoresis (CE) in combination with laser induced fluorescence (LIF) detection. CE-LIF was suitable for mol-based quantification of various APTS-monosaccharides. CE-LIF of APTS-oligosaccharides showed high resolutions, while analysis times were at maximum 15 min. The coupling of CE to electrospray-iontrap mass spectrometery (MS) with online UV detection showed to be a powerful technique in the identification of APTS-oligosaccharides. For the first time, various APTS-xylo-oligosaccharides, having either no, O-acetyl, arabinosyl or xylosyl substitutions at varying positions, were identified by using CE-LIF and CE-MS(n).

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.