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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Non-digestible polysaccharides to support the intestinal immune barrier: in vitro models to unravel molecular mechanisms
Tang, Yongfu - \ 2017
Wageningen University. Promotor(en): H.J. Wichers, co-promotor(en): J.J. Mes; C.C.F.M. Govers. - Wageningen : Wageningen University - ISBN 9789463437134 - 166
polysaccharides - health - immunomodulatory properties - homeostasis - intestinal diseases - human nutrition research - polysacchariden - gezondheid - immunomodulerende eigenschappen - homeostase - darmziekten - voedingsonderzoek bij de mens

Non-digestible polysaccharides (NDPs) are considered as important ingredients to support health. Among these health effects, immunomodulatory effects raised interests in the past decade. The intestine is the primary organ that interact with NDPs. The intestinal epithelial cells (IECs) form a dynamic physical barrier and together with associated immune cells determine for a large part our immune homeostasis. Studying the direct interaction between NDPs and intestinal and immune cells could help us to uncover the mechanism by which NDPs exert immunomodulatory effects and how NDPs can differ in this activity. In this thesis, we investigated the immunomodulatory effects of NDPs through interaction with intestinal immune cells using in vitro methods in order to characterise the NDPs and preselect NDPs with differential activity for further in vivo evaluations.

The intestinal immune barrier is formed by various IECs and immune cells, which are introduced and their specific functions discussed in Chapter 1. NDPs could interact directly with both IECs and immune cells that sample in or from the lumen. The majority of IECs are enterocytes and most relevant immune cells responsible for sampling in the lumen have been characterised as macrophages, which leads us to focus on these cell types by in vitro approaches. In addition, basic information on NDPs and current status on health effects of NDPs both in vitro and in vivo are discussed.

In Chapter 2, the direct response of IEC to NDPs stimulation was investigated. IECs form the largest surface of the body that, with a crucial role as barrier also, perform a role in signalling towards immune cells. We used 21-day transwell cultured Caco-2 to resemble the small intestinal enterocytes that form largest part of this intestinal layer. We first characterized the chemical composition of five NDPs which revealed different mono sugar composition, linkages of backbone and side chains and a wide range of MW (from 17 KDa to 2100 KDa). The NDPs could reduce translocation of FITC-Dextran of 4 kDa across the epithelial layer, potentially through physical interference. Gene expression analysis indicated the induction of unique gene expression characteristics in Caco-2 cells upon exposure to different NDPs. An arabinoxylan preparation from wheat and a lentinan-containing extract from shiitake mushrooms showed upregulation of gene expression of the NF-κB family and chemokines CCL20 and CXCL10. Besides these immune related changes by some NDPs, we also observed changes in receptor expression (like TLR2, CD14 and GPCRs) and other pathways, amongst which the cholesterol biosynthesis pathway.

Macrophages, as the resident population of immune cells penetrating between or associating with close contact with the IECs, are generally classified as inflammatory (M1) or as tolerant (M2) macrophages. In Chapter 3, we set up a macrophage differentiation method based on primary blood cells and selected and validated M1 and M2 specific gene expression markers. Next, we analysed the effect when macrophages are exposed to NDPs and compared the resulting macrophages with M1 and M2 macrophages. Based on M1 and M2 markers we identified an alternative subset that we named MNDP. This MNDP was further studied by microarray analysis and revealed a commonly modulated set of genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function.

In Chapter 4, we further functionally characterize these MNDP in comparison to M1 and M2 macrophages based on a set of functional assays. NDP-treated macrophages showed no IDO activity and showed an inhibited antigen uptake and processing capacity compared to M1 and M2 macrophages. Also their phagocytic capacity was reduced compared to both M1 and M2 macrophages. Furthermore, the alternative expression pattern for NDP-treated macrophages, as demonstrated by gene expression, was confirmed by protein measurements. The signature mix of the chemokines CCL1, CCL5, CCL20, CCL24, CXCL8, and IL1β secreted by MNDP, and in particular when macrophages were treated with Naxus, was shown to induce a recruitment of monocytes.

As macrophage plasticity could be essential for intestinal immune homeostasis, resolving activity of inflammatory responses upon a challenge is important. Besides, redirecting differentiation and function of tolerant macrophages can also be beneficial to the intestinal immune status. In Chapter 5, we analysed plasticity of M1 and M2 macrophages to NDPs exposure. Macrophage plasticity was demonstrated as M1 and M2 could be skewed to an alternative subset indicated by a dedicated set of gene expression markers, selected to characterize M1, M2 and MNDP macrophages. In addition, phagocytosis and antigen processing capacity of both M1 and M2 were decreased by the NDP Naxus. Besides, Naxus could change the secretion of cytokines by macrophages that previously were differentiated towards M1 and M2. For M2, this resulted in an increase of recruitment of monocytes by M2 macrophages.

In Chapter 6, we discussed the important findings in each chapter of this thesis together with current literature, and gave a general perspective on this research line focussing on the immunomodulating activity of NDPs and the direction for future research. We suggested NDPs in terms of Naxus as candidate for guiding investigations in ex vivo and in vivo studies for immunomodulation of intestinal disease.

Targeted and non-targeted effects in cell wall polysaccharides from transgenetically modified potato tubers
Huang, J.H. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576292 - 126
potatoes - cell walls - polysaccharides - transgenic plants - pectins - tubers - xyloglucans - genetically engineered foods - galactans - characteristics - nontarget effects - effects - aardappelen - celwanden - polysacchariden - transgene planten - pectinen - knollen - xyloglucanen - genetisch gemanipuleerde voedingsmiddelen - galactanen - karakteristieken - onbedoelde effecten - effecten

The plant cell wall is a chemically complex network composed mainly of polysaccharides. Cell wall polysaccharides surround and protect plant cells and are responsible for the stability and rigidity of plant tissue. Pectin is a major component of primary cell wall and the middle lamella of plants. However, pectin biosynthesis in planta and the mechanisms underlying the influence of structural differences arising from a modified biosynthesis machinery on functional properties remain poorly understood. In our research, the changes in the chemical structures of cell wall polysaccharides after transgenic modification of potato tuber polysaccharides were examined. The cell wall material from potato wild-type varieties, from known and from new potato transgenic lines targeting changes of the homogalacturonan or rhamnogalacturonan I backbone were isolated and characterized. The modified cell wall polysaccharides were examined by determining their individual monosaccharide levels on fresh weight base and their cell wall characteristic parameters, and levels of acetylation and methyl esterification of cell wall pectin. Data for both targeted and non-targeted structures of cell wall polysaccharides from wild-type and transgenic potatoes were obtained. A shorter galactan side chain was found from the buffer soluble pectin and calcium bound pectin of β-galactosidase (β-Gal) transgenic lines. All pectin fractions from rhamnogalacturonan lyase (RGL) transgenic lines had less galactan chains attached to their rhamnogalacturonan I backbones. Two uridine diphosphate-glucose 4-epimerase (UGE) transgenic lines, UGE 45 and UGE 51, had diverse effects on length of the galactan side chain. The xyloglucans from the RGL and UGE transgenic lines retained its XXGG building blocks but differed in the proportion of repeating units compared to the respective wild-type varieties. In contrast, the β-Gal transgenic lines predominantly consisted of XXXG-type xyloglucan in the 4 M alkali extract, but showed XXGG-type building blocks in 1 M alkali extract. In addition, a quick-screening method was validated and used to analyze 31 transgenic lines and their respective wild-type potato varieties. An overall comparison of pectin backbone, pectin side chains, acetylation and methyl-esterification of pectin, pectin content and (hemi)cellulose content of cell wall polysaccharides from these transgenic lines provided a better insight in the frequency, level and combination of both targeted and non-targeted structural changes compared to that of their respective wild-type varieties. The same evaluation method was used to correlate cell wall composition in wild-type and selected transgenic lines and their established gene expression with the texture of corresponding cooked potato cubes. Changed physical properties for the genetically modified tubers could be connected to specific cell wall characteristics. Tubers from transgenic lines containing cell wall pectin with short galactan side chains were less firm after cold processing compared to wild-type tubers. The enhanced understanding of transgenic modifications of potato tubers resulting into significant targeted and non-targeted modifications in cell wall polysaccharides will lead to a better selection of potato lines with tailored cell wall characteristics and desired properties of the tubers during processing.

Potato cell walls are composed of pectin, hemicellulose and cellulose. Cell wall polysaccharides are responsible for the stability, rigidity and flexibility of plant tissue. Pectin, a major component of primary plant cell walls, primarily consists of homogalacturonan (HG) and rhamnogalacturonan I (RG-I). To understand the structure–function relationships of potato cell wall pectin, this study aimed to identify the characteristics of both pectin and other polysaccharides as present in cell wall material (CWM) and of individual polysaccharide populations from wild-type potato varieties and their respective transgenic potato lines.

Chapter 1 gives a general introduction to the fine chemical structures of potato cell wall polysaccharides, the main models of cell wall architecture and the cell wall-degrading enzymes, which include pectinases, hemicellulases and cellulases. In addition, transgenic modification of the cell wall through the heterologous expression of various enzymes from fungal or plant origin that could modify potato cell wall polysaccharides in planta is addressed. Transgenic modifications of potato cell wall polysaccharides that targeted pectin structures and cellulose levels are summarised. However, due to unsuccessful starch removal during CWM isolation and incomplete analysis of CWM yield and composition, characteristics regarding the different cell wall polysaccharides from previously-studied transgenic potato lines are hardly available.

CWMs were extracted from the Karnico (wild-type) potato and its transgenic lines that expressed either β-galactosidase or rhamnogalacturonan lyase (Chapter 2). Improved starch removal procedures proved to be successful. Pectic polysaccharides were fractionated from CWMs of wild-type potato and its transgenic lines β-Gal-14 and RGL-18. Most β-Gal-14 pectin populations had less galactose (Gal) than wild-type, indicating that the transgenic line had shorter galactan side chains, although the side chain length differed for individual pectin populations. The ratio of HG:RG-I was introduced to evaluate the pectin backbone structure. High HG:RG-I ratios were consistently found in RGL-18 pectic polysaccharide populations. A low level of RG-I segments in combination with lower Gal contents indicated the removal of the galactan-rich RG-I segments in all pectin populations of RGL transgenic lines. In addition, RGL-18 transgenic modification increased the methyl-esterification and lowered the acetylation of pectins present in hot buffer extracts, when compared to wild-type. No effect on pectin esterification was found for β-Gal transgenic lines. Side effects of the mutation generated unexpected changes in the various pectin populations.

The xyloglucan structure was extensively modified after transgenic modification of the pectin structure. Two xyloglucan extracts were obtained from the Karnico and its β-Gal-14 and RGL-18 transgenic lines (Chapter 3). The extracts of the Karnico and RGL-18 lines were mainly comprised of the XXGG-type xyloglucan as represented by XXGG and XSGG as predominant repeating units. In contrast, the XXXG-type xyloglucan was primarily present in the β-Gal-14 4 M alkali extract built up by LLUG repeats, although XXGG type of xyloglucan was present in the 1M alkali extract. Both the RGL and β-Gal transgenic lines had different proportions of xyloglucan building blocks (XSGG/XXGG ratios) than wild-type. After transgenic modification of pectin backbone or pectin side chains, the xyloglucan structures has been biosynthetically modified by plant itself.

Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyses the conversion of UDP-glucose into UDP-galactose, which hypothetically should lead to more galactose being built into the cell wall polysaccharides. CWMs from the Kardal (wild-type) potato and its UGE45-1 and UGE51-16 transgenic lines were isolated, fractionated and characterised (Chapter 4). Both the UGE45 and UGE51 genes encoded for UGE enzymes, but the corresponding transgenic lines exhibited different modifications of the galactan side chains and of other cell wall structures. The Gal content of CWM from the UGE45-1 transgenic line was 38% higher than that of the wild-type and resulted in longer pectin side chains. The Gal content present in CWM from UGE51-16 was 17% lower than that of wild-type, which resulted in a slightly shorter galactan side chains for most pectin populations. Both UGE transgenic lines showed a decreased acetylation and an increased methyl-esterification of the cell wall pectin. Side effects were found in the xyloglucan structures of the transgenes as reflected by different proportions of XSGG/XXGG repeating units in comparison to wild-type. Pectin side chain biosynthesis had not only a varying level of galactan side chain modification, but also influenced the structure and possibly the interaction of other cell wall polysaccharides.

In Chapter 5, a new screening strategy is introduced to evaluate higher numbers of transgenic potato tubers via CWM yield and sugar composition. A total of four wild-type potato varieties and 31 transgenic lines were evaluated to determine the effects on targeted structures including RG-I or HG pectin backbone elements, galactan or arabinogalactan side chains, acetyl groups of pectin and cellulose levels. Modification of the pectin backbone or pectin side chains in the transgenic lines has either a simultaneous increase or simultaneous decrease of HG:RG-I ratio, side chain length and methyl-esterification of pectin. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level targeted lines had also high HG:RG-I ratios, longer galactan chains and similar pectin content compared to the wild-type, indicative for a less branched pectin backbone with longer side chains. From the monosaccharide composition data, various pectin and cell wall characteristics parameters are suggested as powerful indicators of cell wall polysaccharide structure.

In Chapter 6, the achievements of this research are summarised and discussed in the context of potato cell wall architecture. The strategy and outcome of a quick screening method for multiple transgenic lines and an in-depth analysis of individual pectin and xyloglucan populations for the evaluation of potato CWMs is discussed. Furthermore, the texture of steam-cooked potatoes and the stability of potato cubes after freeze-thaw cycles are correlated with gene expression and cell wall composition in wild-type and selected transgenetically modified potato tubers. CWMs from transgenetically modified potatoes showed different physical properties during processing. In isolated CWMs, acetylation of cell wall pectin, molar Gal levels and starch content were the main parameters that could be related to the texture or firmness of tubers. Tubers from transgenic lines that resulted in shorter pectin side chains felt apart more easily after several freeze-thaw cycles than wild-type tubers and tubers with an increased length of pectin side chains. The modification of both targeted as well as non-targeted structures have now been shown to occur in many different potato transgenic lines, but precise mechanisms and consequences for the cell wall architecture remain unclear. Research performed so far, as well as research needed for getting a better understanding of plant cell wall architecture, is discussed.

Plants4Cosmetics : perspectives for plant ingredients in cosmetics
Boeriu, C.G. - \ 2015
Wageningen : Wageningen UR - Food & Biobased Research (Report / Wageningen UR Food & Biobased Research 1603) - 38 p.
cosmetics - plants - flavonoids - phenols - pigments - plant pigments - polysaccharides - geranium - hyacinthus - chrysanthemum - orchidaceae - skin - hair - oil plants - medicinal plants - natural products - biobased chemicals - biobased economy - cosmetica - planten - flavonoïden - fenolen - pigmenten - plantenpigmenten - polysacchariden - huid - haar - olieleverende planten - medicinale planten - natuurlijke producten - chemicaliën uit biologische grondstoffen
In opdracht van Bio Base Westland en de TKI Tuinbouw Koepel PPS Plantenstoffen, heeft Wageningen UR – Food & Biobased Research een exploratieve desktop studie uitgevoerd gericht op de identificatie van veelbelovende routes voor de valorisatie van plantinhoudstoffen - waaronder ook reststromen uit de tuinbouw - voor de cosmetische industrie. Een uitgebreide analyse van de beschikbare informatie werd uitgevoerd om de mogelijkheden voor de Nederlandse tuinbouwsector te bepalen. Er is gekeken naar marktkansen in de cosmetische industrie met inbegrip van natuurlijke en biologische ingrediënten.
Replacing lactose from calf milk replacers : effects on digestion and post-absorptive metabolism
Gilbert, M.S. - \ 2015
Wageningen University. Promotor(en): Wouter Hendriks, co-promotor(en): Walter Gerrits; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576032 - 171
vleeskalveren - lactose - kunstmelk - polysacchariden - glucose - fructose - glycerol - zetmeelvertering - metabolisme - fermentatie - kalvervoeding - diervoeding - voedingsfysiologie - veal calves - lactose - filled milk - polysaccharides - glucose - fructose - glycerol - starch digestion - metabolism - fermentation - calf feeding - animal nutrition - nutrition physiology

Summary PhD thesis Myrthe S. Gilbert

Replacing lactose from calf milk replacers – Effects on digestion and post-absorptive metabolism

Veal calves are fed milk replacer (MR) and solid feed. The largest part of the energy provided to veal calves originates from the MR. Calf MR contains 40 to 50% lactose, originating from whey, a by-product from cheese production. High and strongly fluctuating dairy prices are a major economic incentive to replace lactose from the calf MR by alternative energy sources. The objective of this thesis was to study the effects of replacing lactose from calf MR on nutrient digestion and fermentation and post-absorptive metabolism.

In Chapter 2 and 3, four starch products (SP) were evaluated for replacing lactose. The four SP differed in size and branching, and consequently required different ratios of starch-degrading enzymes for their complete hydrolysis to glucose. Gelatinized starch required α-amylase and (iso)maltase; maltodextrin required (iso)maltase and α-amylase; maltodextrin with α-1,6-branching required isomaltase, maltase and α-amylase and maltose required maltase. In Chapter 2, adaptation to these SP was assessed during 14 weeks, using a within-animal titration study. Forty male Holstein-Friesian calves (n = 8 per treatment) were assigned to either a lactose control MR or one of four titration strategies, each testing the stepwise exchange of lactose for one of the SP. For control calves, fecal dry matter (DM) content and fecal pH did not change over time. The response in fecal DM content and fecal pH in time did not differ between SP treatments and decreased linearly with 0.57% and 0.32 per week, respectively, where one week corresponded to an increase in SP inclusion of 3%. This indicates that the capacity for starch digestion was already exceeded at low inclusion levels, resulting in SP fermentation. All SP required maltase to achieve complete hydrolysis to glucose and it was, therefore, suggested that maltase is the rate-limiting enzyme in starch digestion in milk-fed calves.

Following the titration, a fixed inclusion level of 18% of the SP in the MR was applied. Effects on starch-degrading enzyme activity, nutrient disappearance, SP fermentation and jugular glucose appearance were measured (Chapter 3). Lactase activity in the brush border was high in the proximal small intestine of all calves, resulting in a high apparent ileal disappearance of lactose (≥ 99% of intake). Maltase and isomaltase activities in the brush border were not increased for any of the SP treatments. Luminal α-amylase activity was lower in the proximal small intestine but greater in the distal small intestine of SP-fed calves compared to control calves. This amylase activity in the distal small intestine of SP-fed calves might have been of microbial origin. Apparent SP disappearance did not differ between SP treatments. The difference between apparent ileal (62%) and total tract (99%) SP disappearance indicated substantial SP fermentation in the large intestine (37% of intake). In addition, total tract SP fermentation was quantified using fecal 13C excretion which originated from the naturally 13C-enriched corn SP. Total tract SP fermentation averaged 89% of intake, regardless of SP treatment. MR leaking into the reticulorumen was measured as the recovery of Cr in the reticulorumen at slaughter after feeding MR pulse-dosed with Cr 4h prior to slaughter. MR leaking into the reticulorumen averaged 11% for SP-fed calves. By difference, this leaves 41% of the SP intake fermented in the small intestine. This coincided with increased fecal nitrogen (N) and DM losses for SP-fed calves. However, apparent total tract crude fat disappearance tended to increase when replacing lactose with SP. The substantial SP fermentation indicates that only 10% of the SP intake was enzymatically hydrolyzed and absorbed as glucose. This was in agreement with the marginal increase in 13C enrichment in peripheral plasma glucose after feeding naturally 13C-enriched gelatinized starch and maltose, compared to a clear increase after feeding naturally 13C-enriched lactose to control calves. It was concluded that fermentation, rather than enzymatic digestion, is the main reason for small intestinal starch disappearance in milk-fed calves. The expected decrease in growth performance with such extensive SP fermentation is partially compensated by the greater crude fat digestion and possibly by a reduced urinary glucose excretion when replacing lactose with SP.

Glucose, fructose and glycerol do not require enzymatic hydrolysis and can be absorbed directly from the small intestine. However, these lactose replacers might differentially affect glucose and insulin metabolism and with that energy partitioning. The effects of partly replacing lactose with glucose, fructose or glycerol on energy and N partitioning and glucose homeostasis and insulin sensitivity were, therefore, studied in Chapter 4 and 5. Forty male Holstein-Friesian calves either received a lactose control MR or a MR in which one third of the lactose was replaced with glucose, fructose or glycerol (n = 10 per treatment). Energy and N retention were not affected by MR composition. Fructose absorption from the small intestine was incomplete resulting in fructose fermentation. This resulted in fecal losses of DM, energy and N and the lowest numerical energy and N retention for fructose-fed calves. Postprandial plasma concentrations of glucose exceeded the renal threshold for glucose in glucose-fed calves and control calves, which resulted in urinary glucose excretion. Glycerol was likely excreted with the urine of glycerol-fed calves. Oxidation of glucose, fructose and glycerol was quantified by feeding a single dose of [U-13C]glucose, [U-13C]fructose or [U-13C]glycerol with the MR and subsequently measuring 13CO2 production. Oxidation of lactose replacers did not differ between lactose replacers and averaged 72% of intake. However, the time at which the maximum rate of oxidation was reached was delayed for fructose-fed compared to glucose-fed and glycerol-fed calves, indicating that fructose was converted into other substrates before being oxidized. Conversion of fructose and glycerol into glucose was confirmed by an increase in 13C enrichment of peripheral plasma glucose after feeding [U-13C]fructose and [U-13C]glycerol, respectively. Insulin sensitivity did not differ between MR treatments, but was already low at the start of the experiment at 15 weeks of age and remained low throughout the experiment. It was concluded that glucose and glycerol can replace one third of the lactose from the calf MR, but that inclusion of fructose should be lower to prevent incomplete absorption from the small intestine.

In literature and the studies in this thesis, high inter-individual variation in growth performance was found in veal calves. The experiment described in Chapter 6 was, therefore, designed to assess the predictability of later life growth performance by charactering calves in early life. In addition, it was examined whether the ability of calves to cope with MR in which lactose is partially replaced by alternative energy sources can be predicted. From 2 to 11 weeks of age, male Holstein-Friesian calves were fed a lactose control MR and solid feed according to a practical feeding scheme and were characterized individually using targeted challenges related to feeding motivation, digestion, post-absorptive metabolism, immunology, behavior and stress. Based on the results in Chapter 4, a combination of glucose, fructose and glycerol in a 2:1:2 ratio was used to replace half of the lactose from the MR (GFG). From 11 to 27 weeks of age, calves received a lactose control MR or the GFG MR (n = 65 per treatment). Growth performance from 11 to 27 weeks of age tended to be lower for GFG-fed than for control calves (-25 g/d). Measurements in early life explained 12% of the variation in growth performance in later life. However, this was mainly related to variation in solid feed refusals. When growth performance was adjusted to equal solid feed intake, only 4% of the variation in standardized growth performance in later life, reflecting feed efficiency, could be explained by early life measurements. This indicates that > 95% of the variation in feed efficiency in later life could not be explained by early life characterization. It is hypothesized that variation in health status explains substantial variation in feed efficiency in veal calves. Significant relations between fasting plasma glucose concentrations, fecal dry matter and fecal pH in early life and feed efficiency in later life depended on MR composition. These measurements are, therefore, potential tools for screening calves in early life on their ability to cope with a MR in which half of the lactose is replaced by glucose, fructose and glycerol (in a 2:1:2 ratio).

The studies reported in this thesis demonstrate that glycerol, glucose and a combination of glucose, fructose and glycerol in a 2:1:2 ratio are promising lactose replacers. The effects of replacing lactose by other carbohydrate or energy sources described in this thesis are required to evaluate the potential of lactose replacers for inclusion in calf milk replacers and provide input for feed evaluation for calves and ruminants.

The impact of dietary fibers on dendritic cell responses in vitro is dependent on the differential effects of the fibers on intestinal epithelial cells
Bermudez-Brito, M. ; Sahasrabudhe, N.M. ; Rösch, C. ; Schols, H.A. ; Faas, M.M. ; Vos, P. de - \ 2015
Molecular Nutrition & Food Research 59 (2015)4. - ISSN 1613-4125 - p. 698 - 710.
immune function - receptor 2 - health - homeostasis - modulation - mortality - polysaccharides - activation - mechanisms - prebiotics
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-a, MCP-1 (monocyte chemotactic protein), and MIP-1a 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 ß-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 ß-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.
Theory of brushes formed by psi-shaped macromolecules at solid-liquid interfaces
Zhulina, E.B. ; Leermakers, F.A.M. ; Borisov, O.V. - \ 2015
Langmuir 31 (2015)23. - ISSN 0743-7463 - p. 6514 - 6522.
starlike polymer brushes - dendronized polymers - gold nanoparticles - good solvent - surface - polysaccharides - adsorption - dendrimers - coatings
We present a theoretical analysis targeted to describe the structural properties of brushes formed by ¿-shaped macromolecules tethered by terminal segment of stem to planar surface while exposing multiple free branches to the surrounding solution. We use an analytical self-consistent field approach based on the strong stretching approximation, and the assumption of Gaussian elasticity for linear chain fragments of the tethered macromolecules. The effect of weak and strong polydispersity of branches is analyzed. In the case of weakly polydisperse macromolecules, variations in length of branches lead to a more uniform polymer density distribution with slight increase in the brush thickness compared to the case of monodisperse chains with the same degree of polymerization. We demonstrate that in contrast to linear chains, strong polydispersity of ¿-shaped macromolecules does not necessarily lead to strong perturbations in polymer density distribution. In particular, mixed brushes of the so-called “mirror” dendrons (in which number of stem monomers in one component coincides with number of monomers in a branch of the other component, and vice versa) give rise to a unified polymer density distribution with shape independent of the brush composition. The predictions of analytical theory are systematically compared to the results of numerical self-consistent field modeling based on the Scheutjens–Fleer approach
Aqueous fractionation yields chemically stable lupin protein isolates
Berghout, J.A.M. ; Marmolejo-Garcia, C. ; Berton-Carabin, C.C. ; Nikiforidis, C.V. ; Boom, R.M. ; Goot, A.J. van der - \ 2015
Food Research International 72 (2015). - ISSN 0963-9969 - p. 82 - 90.
in-water emulsions - seed oil bodies - oxidative stability - antioxidant properties - lipid oxidation - physicochemical properties - functional-properties - quality - acids - polysaccharides
The chemical stability of lupin protein isolates (LPIs) obtained through aqueous fractionation (AF, i.e. fractionation without the use of an organic solvent) at 4 °C or 20 °C was assessed. AF of lupin seeds results in LPIs containing 2 wt.% oil. This oil is composed of mono- and poly-unsaturated fatty acids and the isolate may thus be prone to lipid and protein oxidation. Lipid and protein oxidation marker values of LPIs obtained at 4 °C and at 20 °C were below the acceptability limit for edible vegetable oils and meat tissue protein; the level of lipid oxidation markers was lower at 20 °C than at 4 °C. The fibre-rich pellet and the protein-rich supernatant obtained after AF also had lower levels of oxidation markers at 20 °C than at 4 °C. This is probably the result of a higher solubility of oxygen in water at lower temperature, which could promote lipid oxidation. The differences between fractions can be explained by the differences in their composition; the fibre-rich pellet contains polysaccharides that potentially have an anti-oxidative effect, while the protein-rich supernatant is rich in sulphur-rich proteins that may scavenge metal ions and free radicals from the aqueous phase. Additionally, the differences in solubility of metal ions and metal-chelating properties of protein at pH 4.5 and pH 7.0 explain the higher level of oxidation in the LPI at pH 4.5 compared with the LPI at pH 7.0. The application of a heat treatment to reduce oxidation decreased the protein and oil recovery values, and increased oxidation values above the acceptability limit. Therefore, AF at 20 °C is the most suitable process to obtain chemically stable LPIs.
Assessing the immunomodulatory potential of high-molecular-weight extracts from mushrooms; an assay based on THP-1 macrophages
Velde, J. van de; Wilbers, R.H.P. ; Westerhof, L.B. ; Raaij, D.R. van; Stavrakaki, I. ; Sonnenberg, A.S.M. ; Bakker, J. ; Schots, A. - \ 2015
Journal of the Science of Food and Agriculture 95 (2015)2. - ISSN 0022-5142 - p. 344 - 350.
monocytic leukemia-cells - agaricus-blazei murill - toll-like receptors - in-vitro - induction - responses - line - polysaccharides - differentiation - expression
BACKGROUND Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. RESULTS Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5–5)¿×¿105 cells mL-1. CONCLUSION An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-a, IL-1ß, IL-6 and IL-10. © 2014 Society of Chemical Industry
Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1¿3)-ß-D-glucan.
Smiderle, F.R. ; Baggio, C.H. ; Borato, D.G. ; Santana-Filho, A.P. ; Sassaki, G.L. ; Iacomini, M. ; Griensven, L.J.L.D. van - \ 2014
PLoS ONE 9 (2014)10. - ISSN 1932-6203
formalin test - in-vivo - fruiting bodies - beta-glucans - congo-red - polysaccharides - mice - macrophages - extract - complex
The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a ß-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of ß-D-Glcp (1¿3)-linked. The six main signals in the 13C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1ß, TNF-a, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified ß-(1¿3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, ß-(1¿3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of ß-(1¿3)-D-glucan.
Purification, Characterization, and Prebiotic Properties of Pectic Oligosaccharides from Orange Peel Wastes
Gómez, B. ; Gullón, B. ; Remoroza, C.A. ; Schols, H.A. ; Parajó, J.C. ; Alonso, J.L. - \ 2014
Journal of Agricultural and Food Chemistry 62 (2014)40. - ISSN 0021-8561 - p. 9769 - 9782.
in-vitro fermentation - butyrate-producing bacteria - human fecal microbiota - human gut - polysaccharides - fermentability - pretreatment - hydrolysis - product - acid
Pectic oligosaccharides (POS) were obtained by hydrothermal treatment of orange peel wastes (OPW) and purified by membrane filtration to yield a refined product containing 90 wt % of the target products. AraOS (DP 3–21), GalOS (DP 5–12), and OGalA (DP 2–12, with variable DM) were identified in POS mixtures, but long-chain products were also present. The prebiotic potential of the concentrate was assessed by in vitro fermentation using human fecal inocula. For comparative purposes, similar experiments were performed using orange pectin and commercial fructo-oligosaccharides (FOS) as substrates for fermentation. The dynamics of selected microbial populations was assessed by fluorescent in situ hybridization (FISH). Gas generation, pH, and short-chain fatty acid (SCFA) production were also measured. Under the tested conditions, all of the considered substrates were utilized by the microbiota, and fermentation resulted in increased numbers of all the bacterial groups, but the final profile of the microbial population depended on the considered carbon source. POS boosted particularly the numbers of bifidobacteria and lactobacilli, so that the ratio between the joint counts of both genera and the total cell number increased from 17% in the inocula to 27% upon fermentation. SCFA generation from POS fermentation was similar to that observed with FOS, but pectin fermentation resulted in reduced butyrate generation.
Effects of inclusion of hydrolyzed yeast on the immune response and performance of piglets after weaning
Molist, F. ; Eerden, E. van; Parmentier, H.K. ; Vuorenmaa, J. - \ 2014
Animal Feed Science and Technology 195 (2014). - ISSN 0377-8401 - p. 136 - 141.
growth-performance - saccharomyces-cerevisiae - natural antibodies - weanling pigs - nutrient digestibility - weaned piglets - supplementation - polysaccharides - challenge - chickens
The aim of this study was to examine whether yeast derivative (YD) based on brewery yeast hydrolyzate added to a post-weaning diet affected performance and immune responses in weaning pigs. One hundred and twenty pigs were allocated to 20 pens, taking initial body weight into account, and were distributed into two groups as follows: a negative control diet and the same diet supplemented with 2 g YD/kg. The YD used was Progut® (Hankkija Oy/Suomen Rehu, Hyvinkää, Finland). At days 7 and 21 of the experiment, half of the piglets per group were challenged intramuscularly with 1 mL of a solution of 20% sheep red blood cells (SRBC) in sterile phosphate buffered saline (PBS). At days 0, 14, 21 and 28 of the experiment, blood samples from the challenged piglets were obtained and acute-phase proteins (Pig-MAP), natural antibodies of the IgM- and IgG-isotype binding to keyhole limpet hemocyanin (KLH), and agglutinating antibody titers to SRBC were measured. Yeast derivative inclusion improved feed conversion ratio (P=0.025) for the overall period, tended to increase IgG (P=0.087) and IgM (P=0.061) antibodies in serum-binding KLH, and increased (P=0.037) SRBC agglutination titers. Collectively, these data suggest that YD supplementation as 2 g Progut®/kg to weanling pigs triggered the immune system to a more responsive state without penalizing the animal performance which could potentially be beneficial for overcoming disease challenges. Piglets fed with 2 g Progut®/kg for 28 days after weaning also showed an improvement in feed conversion ratio.
the role of soluble and insoluble fibers during fermentation of Chicory root pulp
Ramasamy, U. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789461739650 - 152
cichorei - pulp - vezels - fermentatie - celwandstoffen - polysacchariden - chicory - pulps - fibres - fermentation - cell wall components - polysaccharides

This thesis was aimed at understanding the in vitro fermentability of soluble and insoluble fibers in chicory root pulp (CRP). First, CRP and ensiled chicory root pulp (ECRP) were characterized for cell wall polysaccharides (CWPs). Both CRP and ECRP were rich in CWPs (56-58 w/w (%)) and had rather similar sugar compositions. The CWPs consist of 62 % pectin, 11% hemicellulose and 27% cellulose. Pectin and xyloglucan were acetylated and the rhamnogalacturonan-I segments of pectin were branched mostly with arabinan. Compared to CRP, ECRP has four times more soluble pectin.

In vitrofermentability in a batch model for 24 h using human faecal inoculum, showed that fibers in both CRP (51% carbohydrate utilisation) and ECRP (59% carbohydrate utilisation) were fermentable, especially pectin (80-87%). The increased levels of soluble pectin (arabinan, homogalacturonan and galactan) and the hypothesized open cell wall structure in ECRP contributed to a quicker fermentation and a higher level of carbohydrate utilization compared to CRP. In contrast to batch fermentation, fermentation in the dynamic TNO In vitro model of the colon (TIM-2) was rapid (57% carbohydrate utilisation in 2 h). ECRP carbohydrates (85%) were less fermented in 24 h compared to CRP carbohydrates (92%) due to lower utilisation of ECRP insoluble fibers than CRP insoluble fibers. It was hypothesized that soluble fibers that are readily fermentable and dominantly present in ECRP, programmed the microbiota in TIM-2 to fully adapt to these soluble fibers. After their utilization, the microbiota was not able to adapt towards the fermentation of insoluble fibers.

Analysis of enzyme activities during batch fermentation of CRP showed increased levels of arabinofuranosidase, β-galactosidase, endo-arabinanase, endo-galactanase, exo-polygalacturonase, pectin de-esterifying enzymes and endo-polygalacturonase. They synergistically contributed to degrading pectin in CRP from 12 to 24 h of fermentation.

Water holding capacity and enzymatic modification of pressed potato fibres
Ramasamy, U. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Mirjam Kabel. - Wageningen : Wageningen University - ISBN 9789461739643 - 156
aardappelpulp - aardappelen - vezels - celwandstoffen - polysacchariden - waterbergend vermogen - hydrolyse - enzymen - potato pulp - potatoes - fibres - cell wall components - polysaccharides - water holding capacity - hydrolysis - enzymes

Cell wall polysaccharides (CWPs) contribute to the water holding capacity (WHC) of fibre rich feeds, such as pressed potato fibres (PPF). However, the role of CWPs on the WHC of PPF was unidentified so far.

PPF was characterized to be abundant in arabinogalactan (AG) linked rhamnogalacturonan-I (RG-I), homogalacturonan (HG) and cellulose, next to which xyloglucan (XG) contributed the most of the hemicellulosic CWPs. The CWP network in potatoes was loosened upon starch extraction of potatoes and solubilized HG-RG-I-AG.

Analyses of the WHCs upon enzyme treatments indicated that the WHC of PPF was mainly caused by a network of insoluble, non-cellulosic CWPs such as pectic CWPs (HG-RG-I-AG) and XG. Findings in this thesis showed that AGs were better degraded than xyloglucans (XGs). Since XGs were found to be equally important in contributing to the WHC as AGs, the substantial removal of AGs, as well as XGs, should be advantageous to lower the WHC.

Other than lowering the WHC, the use of a pectinase-rich preparation improved the recovery of starch from potatoes by the degradation of mainly pectic CWPs, in particular pectic AG side chains and HG. The degradation of arabinan was observed to be inhibited by components in potato juice (PJ).

Carbohydrate composition of compost during composting and mycelium growth of Agaricus bisporus
Jurak, E. ; Kabel, M.A. ; Gruppen, H. - \ 2014
Carbohydrate Polymers 101 (2014). - ISSN 0144-8617 - p. 281 - 288.
xylan-degrading enzymes - mushroom compost - polysaccharides - biodegradation - degradation - biomass - alkali
Changes of plant cell wall carbohydrate structures occurring during the process to make suitable compost for growth of Agaricus bisporus are unknown. In this paper, composition and carbohydrate structures in compost samples collected during composting and mycelium growth were analyzed. Furthermore, different extracts of compost samples were prepared with water, 1 M and 4 M alkali and analyzed. At the beginning of composting, 34% and after 16 days of mycelium growth 27% of dry matter was carbohydrates. Carbohydrate composition analysis showed that mainly cellulose and poorly substituted xylan chains with similar amounts and ratios of xylan building blocks were present in all phases studied. Nevertheless, xylan solubility increased 20% over the period of mycelium growth indicating partial degradation of xylan backbone. Apparently, degradation of carbohydrates occurred over the process studied by both bacteria and fungi, mainly having an effect on xylan-chain length and solubility.
A detailed comparative study between chemical and bioactive properties of Ganoderma lucidum from different origins
Stojkovic, D.S. ; Barros, L. ; Calhelha, R.C. ; Glamoclija, J. ; Ciric, A. ; Griensven, L.J.L.D. van; Sokovic, M. ; Ferreira, I.C.F.R. - \ 2014
International Journal of Food Sciences and Nutrition 65 (2014)1. - ISSN 0963-7486 - p. 42 - 47.
medicinal mushrooms - antioxidant properties - wild mushrooms - liquid-chromatography - fruiting body - fr. karst - portugal - polysaccharides - molecules - nutrients
A detailed comparative study on chemical and bioactive properties of wild and cultivated Ganoderma lucidum from Serbia (GS) and China (GCN) was performed. This species was chosen because of its worldwide use as medicinal mushroom. Higher amounts of sugars were found in GS, while higher amounts of organic acids were recorded in GCN. Unsaturated fatty acids predominated over saturated fatty acids. GCN revealed higher antioxidant activity, while GS exhibited inhibitory potential against human breast and cervical carcinoma cell lines. No cytotoxicity in non-tumour liver primary cell culture was observed for the different samples. Both samples possessed antibacterial and antifungal activities, in some cases even better than the standard antimicrobial drugs. This is the first study reporting a comparison of chemical compounds and bioactivity of G. lucidum samples from different origins.
Crystal structure of endo-xylogalacturonan hydrolase from Aspergillus tubingensis
Rozeboom, H.J. ; Beldman, G. ; Schols, H.A. ; Dijkstra, B.W. - \ 2013
FEBS Journal 280 (2013)23. - ISSN 1742-464X - p. 6061 - 6069.
site-directed mutagenesis - endopolygalacturonase ii - sequence alignments - features - polysaccharides - processivity - degradation - pectin - niger - polygalacturonase
Endo-xylogalacturonan hydrolase is a member of glycoside hydrolase family 28 (GH28) that hydrolyzes the glycosidic bond between two ß-xylose-substituted galacturonic acid residues in pectin. Presented here is the X-ray crystal structure of the endo-xylogalacturonan hydrolase from Aspergillus tubingensis (XghA) at 1.75 Å resolution. The high degree of structural conservation in the active site and catalytic apparatus compared with polygalacturonases indicates that cleavage of the substrate proceeds in essentially the same way as found for the other GH28 enzymes. Molecular modeling of a xylosylated tri-galacturonate in the active site identified the amino acid residues involved in substrate binding. They border a substrate-binding cleft that is much wider than in other polygalacturonases, and can accommodate xylosylated substrates. The most extensive interactions appear to occur at subsite +2, in agreement with the enzyme kinetics results, which showed enhanced activity on substrates with a xylose attached to the galacturonic acid bound at subsite +2
Fate of rapeseed meal polysaccharides during digestion in pigs and poultry : effect of processing and enzyme addition
Pustjens, A.M. - \ 2013
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Mirjam Kabel; Walter Gerrits. - S.l. : s.n. - ISBN 9789461736604 - 184
raapzaad - raapzaadmeel - polysacchariden - spijsvertering - varkens - pluimvee - voedermiddelbewerking - enzymen - rapeseed - rapeseed oilmeal - polysaccharides - digestion - pigs - poultry - feed processing - enzymes

In this thesis, the fate of non-starch polysaccharides (NSP) from rapeseed meal (RSM) during fermentation in vitro and in vivo was studied. The aim was to understand and improve the fermentation of NSP from RSM in poultry and pigs, by processing and enzyme addition. First, the NSP-structures in RSM were characterized as being branched arabinan, arabinogalactan type II, homogalacturonan, glucurono-xylan, XXGG- and XXXG-type xyloglucan, and cellulose. Second, RSM was processed using shear, heat, and acid prior to in vitro incubation, in the presence or absence of pectolytic enzymes. Acid-treatment combined with pectolytic enzymes was the best option to improve NSP-solubilization in vitro. Unprocessed and acid-extruded RSM with or without addition of enzymes were fed to broilers. In broilers, 22% of the NSP in unprocessed RSM could be fermented, which only significantly improved to 38% by addition of commercial pectolytic enzymes. In broilers’ excreta, XXXG-type xyloglucan, (glucurono-)xylan, arabinan, and cellulose remained unfermented. Unprocessed and acid-extruded RSM was also fed to growing pigs and NSP-fermentation was followed along the digestive tract. In pigs, at the terminal ileum 22% of the NSP was cumulatively fermented and total tract around 70% was fermented. Acid-extrusion improved total tract NSP-fermentability in pigs numerically by 4% points. Water-soluble carbohydrates were nearly completely fermented. In the feces some rhamnogalacturonan, (branched) arabinan, linear xylan, XXXG-type xyloglucan, galactomannan, and cellulose remained. Surprisingly, during alkaline extraction of the broilers’ excreta and pigs’ feces, around 40% (w/w) of the insoluble carbohydrates was released as glucosyl- and/or 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.

Enzymatic saccharification of sugar beet pulp for the production of galacturonic acid and arabinose; a study on the impact of the formation of recalcitrant oligosaccharides
Leijdekkers, A.G.M. ; Bink, J.P.M. ; Geutjes, S. ; Schols, H.A. ; Gruppen, H. - \ 2013
Bioresource Technology 128 (2013). - ISSN 0960-8524 - p. 518 - 525.
rhamnogalacturonan regions - ethanol-production - pectin - fermentation - hydrolysis - polysaccharides - pretreatment - cellulose - enzymes
Enzymatic saccharification of sugar beet pulp was optimized on kg-scale to release the maximum amounts of monomeric galacturonic acid and arabinose with limited concomitant degradation of cellulose, using conditions that are feasible for industrial upscaling. A selected mixture of pectinases released 79% of the galacturonic acid and 82% of the arabinose as monomers from sugar beet pulp while simultaneously degrading only 17% of the cellulose. The recalcitrant structures that were obtained after hydrolysis were characterized using mass spectrometry. The most abundant structures had an average degree of polymerization of 4–5. They were identified as partially acetylated rhamnogalacturonan-oligosaccharides, mostly containing a terminal galacturonosyl residue on both reducing and non-reducing end, partially methyl esterified/acetylated homogalacturonan-oligosaccharides, mostly containing methyl and acetyl esters at contiguous galacturonosyl residues and arabinan-oligosaccharides, hypothesized to be mainly branched. It could be concluded that especially rhamnogalacturonan-galacturonohydrolase, arabinofuranosidase and pectin acetylesterase are lacking for further degradation of recalcitrant oligosaccharides
Agaricus bisporus and Agaricus brasiliensis (1 ¿ 6)-ß-d-glucans show immunostimulatory activity on human THP-1 derived macrophages
Smiderle, F.R. ; Alquini, G. ; Tadra-Sfeir, M.Z. ; Lacomini, M. ; Wichers, H.J. ; Griensven, L.J.L.D. van - \ 2013
Carbohydrate Polymers 94 (2013)1. - ISSN 0144-8617 - p. 91 - 99.
beta-glucans - fungal metabolites - molecular-weight - polysaccharides - lipopolysaccharide - mushroom - activation - receptor - rats - expression
The (1 ¿ 6)-ß-d-glucans from Agaricus bisporus and Agaricus brasiliensis were purified to evaluate their effects on the innate immune system. THP-1 macrophages were used to investigate the induction of the expression of TNF-a, IL1ß, and COX-2 by RT-PCR. The purification of the polysaccharides gave rise to fractions containing 96–98% of glucose. The samples were analyzed by GC–MS, HPSEC and 13C NMR, which confirmed the presence of homogeneous (1 ¿ 6)-ß-d-glucans. The ß-glucans were incubated with THP-1 derived macrophages, for 3 h and 6 h to evaluate their effects on the expression of pro-inflammatory genes. Both ß-glucans stimulated the expression of such genes as much as the pro-inflammatory control (LPS). When the cells were incubated with LPS + ß-glucan, a significant inhibition of the expression of IL-1ß and COX-2 was observed for both treatments after 3 h of incubation. By the results, we conclude that the (1 ¿ 6)-ß-d-glucans present an immunostimulatory activity when administered to THP-1 derived macrophages
Production of acetone, butanol, and ethanol from biomass of the green seaweed Ulva lactuca
Wal, H. van der; Sperber, B.L.H.M. ; Houweling-Tan, G.B.N. ; Bakker, R.R.C. ; Brandenburg, W.A. ; Lopez Contreras, Ana - \ 2013
Bioresource Technology 128 (2013)2013. - ISSN 0960-8524 - p. 431 - 437.
clostridium-acetobutylicum - dietary fiber - amino-acid - marine - polysaccharides - 1,2-propanediol - fermentation - macroalgae - strains - rigida
Green seaweed Ulva lactuca harvested from the North Sea near Zeeland (The Netherlands) was characterized as feedstock for acetone, ethanol and ethanol fermentation. Solubilization of over 90% of sugars was achieved by hot-water treatment followed by hydrolysis using commercial cellulases. A hydrolysate was used for the production of acetone, butanol and ethanol (ABE) by Clostridium acetobutylicum and Clostridium beijerinckii. Hydrolysate-based media were fermentable without nutrient supplementation. C. beijerinckii utilized all sugars in the hydrolysate and produced ABE at high yields (0.35 g ABE/g sugar consumed), while C. acetobutylicum produced mostly organic acids (acetic and butyric acids). These results demonstrate the great potential of U. lactuca as feedstock for fermentation. Interestingly, in control cultures of C. beijerinckii on rhamnose and glucose, 1,2 propanediol was the main fermentation product (9.7 g/L).
Residual Carbohydrates from in Vitro Digested Processed Rapeseed (Brassica napus) Meal
Pustjens, A.M. ; Vries, S. de; Gerrits, W.J.J. ; Kabel, M.A. ; Schols, H.A. ; Gruppen, H. - \ 2012
Journal of Agricultural and Food Chemistry 60 (2012)34. - ISSN 0021-8561 - p. 8257 - 8263.
nutrient digestibility - growth-performance - pectic substances - dehulled rapeseed - feed ingredients - particle-size - dietary fiber - soybean-meal - pigs - polysaccharides
Rapeseed meal (RSM) was subjected to different physical or chemical pretreatments to decrease residual, hard to degrade carbohydrates and to improve fermentability of RSM polysaccharides. Next, these pretreated samples were in vitro digested and fermented, with or without the addition of commercial pectinolytic enzymes. Remaining carbohydrates were quantified, and two physical characteristics were analyzed: (1) water-binding capacity (WBC) of the insoluble residue and (2) viscosity of the soluble fraction. Mild acid pretreatment in combination with commercial pectinolytic enzyme mixtures showed best digestion of RSM carbohydrates; only 32% of the total carbohydrate content remained. For most pretreatments, addition of commercial pectinolytic enzymes had the strongest effect on lowering the WBC of the in vitro incubated RSM. In the cases that less carbohydrate remained after in vitro digestion, the WBC of the residue decreased, and less gas seems to be produced during fermentation.
In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans
Jonathan, M.C. ; Borne, J.J.G.C. van den; Wiechen, P. van; Souza Da Silva, C. ; Schols, H.A. ; Gruppen, H. - \ 2012
Food Chemistry 133 (2012)3. - ISSN 0308-8146 - p. 889 - 897.
gastrointestinal-tract - colonic function - fatty-acids - polysaccharides - kinetics - starch - gas - fermentability - chromatography - degradation
In vitro fermentation of twelve dietary fibers by fecal inocula from pigs and humans were performed. The fibers included homoglucans, mannans, fructans, polyuronides, and complex heteroglycans. Gas production, short chain fatty acid production and fiber degradation products were monitored during fermentation. Human inoculum has more ability to ferment resistant starch and fibers containing uronic acids. In contrast, pig inoculum is able to ferment cellulose, which is hardly fermented by human inoculum. The sugar and linkage composition of the fibers has an important influence on fiber fermentation patterns. Fibers containing uronic acids induced the production of acetate, whereas fibers containing neutral sugars induced the production of propionate or butyrate. Fermentation of the fructans showed that molecular size could be an influential factor, and fermentation of complex heteroglycans showed that the arrangement of sugars in the molecules may also affect the fermentation patterns. This experiment also shows that monitoring of fiber degradation products is important for understanding how fibers are degraded during fermentation.
Characterization of substituents in xylans from corn cobs and stover
Dongen, F.E.M. van; Eylen, D. van; Kabel, M.A. - \ 2011
Carbohydrate Polymers 86 (2011)2. - ISSN 0144-8617 - p. 722 - 731.
xylo-oligosaccharides - polysaccharides - arabinoxylans - pretreatment - acetylation - extraction - products - enzymes
Structural knowledge on hemicellulose from corn cobs and stover is helpful to better understand their position within the plant cell wall architecture as well as their enzymatic saccharification. In this research different extracts were prepared with water, 1 M and 4 M alkali. Most of the xylans were yielded in the 1 M alkali fractions. The xylans were substituted with arabinose and glucuronic acid; 9 and 5 (cobs) and 14 and 9 (stover) per 100 xylosyl residues, respectively. Also esterlinked groups were present, like acetic, ferulic and coumaric acids; 49, 4 and 6 (cobs) and 39, 2 and 5 per 100 xylosyl residues (stover), respectively. Incubation with a well-characterized endoxylanase showed a more blockwise distribution of side-groups for stover xylans compared to cobs. Analysis of soluble fractions of dilute acid treated cobs and stover showed that especially feruloylated oligomers were resistant.
Determination of pectin content of eucalyptus wood
Coetzee, B. ; Schols, H.A. ; Wolfaardt, F. - \ 2011
Holzforschung 65 (2011)3. - ISSN 0018-3830 - p. 327 - 331.
quince chaenomeles-japonica - cell - polysaccharides - degradation - substances - responses - fruits - plants - hplc - wall
Very little is known about the occurrence of pectin in wood and it is speculated that between 10 mg g-1 and 40 mg g-1 of wood consists of pectin. The present study aimed to quantify pectin in eucalyptus wood and to determine the influence of tree species, yield potential of the site, tree age class and wood tissue type on its occurrence. Wood was hydrolysed using the Saeman procedure and the neutral and acidic monosaccharides quantified with high-performance liquid chromatography (HPLC). The d-galacturonic acid was the predominant pectic monosaccharide followed by d-galactose, l-arabinose and l-rhamnose. Through the addition of all pectic monosaccharides, it was determined that eucalyptus wood contained between 15.2 mg g-1 and 25.8 mg g-1 pectin. Wood tissue type had a major influence on the total pectin content of the samples. The cambium contained the highest concentration of pectin, reflecting more active growth. These results contribute to the understanding of wood biochemistry and can be useful in the industries producing biofuels or paper.
New insights on the formation of colloidal whey protein particles
Riemsdijk, L.E. van; Snoeren, J.P.M. ; Goot, A.J. van der; Boom, R.M. ; Hamer, R.J. - \ 2011
Food Hydrocolloids 25 (2011)3. - ISSN 0268-005X - p. 333 - 339.
phase-separation - globular-proteins - cold gelation - rich foods - microstructure - mixtures - gels - polysaccharides - temperature - morphology
This paper describes the formation and properties of whey protein particle suspensions having different particle sizes and different abilities to form S–S bridges. Simple shear flow was used to control the protein particles size. The ability to form S–S bridges was steered by blocking the reactive thiol groups of the whey proteins with N-ethylmaleimide. Microscopy and light scattering showed that simple shear flow applied during the formation of whey protein particles give irregularly shaped particles. Especially small particles aggregated into particle clusters. Microscopy and rheological measurements (strain and shear rate sweeps) showed that the protein particle clustering was favoured by the ability of the protein to form S–S bridges and to a lesser extend by a smaller particle size. From the study, it can be concluded that the formation of S–S bridges has no effect on the formation process of protein particles, but S–S bridges are important for the ability of the whey protein particles to form particle clusters
Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols
Abzac, P. D'; Bordas, F. ; Hullebusch, E. ; Lens, P.N.L. ; Guibaud, G. - \ 2010
Applied Microbiology and Biotechnology 85 (2010)5. - ISSN 0175-7598 - p. 1589 - 1599.
activated-sludge - waste-water - polysaccharides - complexation - flocs - acid
The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g(-1) of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.
Performance and energy metabolism in restrictively fed weanling pigs are not affected by feedinig either fermented cereals or their end-products
Bruininx, E.M.A.M. ; Binnendijk, G.P. ; Zandstra, T. ; Heetkamp, M.J.W. ; Peet-Schwering, C.M.C. van der; Gerrits, W.J.J. - \ 2010
Journal of Animal Physiology and Animal Nutrition 94 (2010)6. - ISSN 0931-2439 - p. e355 - e365.
epithelial-cell proliferation - growing pigs - physical-activity - organic-acids - polysaccharides - digestibility - diets - fiber
To study the effects of feeding fermented cereals or just fermentation end-products on performance and energy metabolism, 18 restrictedly fed groups of eight pigs each were assigned to one of three dietary treatments: (i) a liquid control diet (C) containing 40% of a mixture of barley and wheat; or (ii) a liquid diet (F) containing 40% fermented barley and wheat; or (iii) a liquid diet as C with the addition of some important fermentation end-products (FP; organic acids and ethanol) in concentrations similar to those in the fermented F-diet. Energy and nitrogen balances, heat production, and performance traits were measured during two consecutive periods (days 1–5 and days 6–14). There was a considerable increase in average dry matter intake that tended (p = 0.06) to be higher in the FP-group than in the other groups. Apparent fecal digestibility of dry matter, ash, nitrogen and energy during period 2 were not affected (p > 0.1). Averaged over both periods, none of the energy metabolism parameters were affected by the diets (p > 0.1). However, there were diet × period interactions for metabolizable energy-intake (p = 0.07), energy retention (p <0.05), the respiratory quotient (RQ; p <0.01) and activity-related heat production (HACT, p = 0.05). Additionally, there were some differences between the diets in the average hourly patterns in RQ and HACT. In conclusion, restricted feeding of either 40% fermented cereals nor their fermentation end-products affected performance and energy metabolism traits in weanling pigs. Nevertheless, lower postprandial activity-related heat production by pigs given the fermented cereals suggest a stimulating effect of fermented cereals on short term satiety that was not seen in pigs given fermentation end-products only
Characterization of Oligomeric Xylan Structures from Corn Fiber Resistant to Pretreatment and Simultaneous Saccharification and Fermentation
Appeldoorn, M.M. ; Kabel, M.A. ; Eylen, D. van; Gruppen, H. ; Schols, H.A. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)21. - ISSN 0021-8561 - p. 11294 - 11301.
grass cell-walls - maize bran - ferulic acid - liquid-chromatography - oligosaccharides - identification - heteroxylans - hydrolysis - polysaccharides - arabinoxylan
Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of corn fiber. Oligosaccharides that resisted a mild acid pretreatment and subsequent enzymatic hydrolysis, representing 50% of the starting material, were fractionated on reversed phase and size exclusion material and characterized. The oligosaccharides within each fraction were highly substituted by various compounds. Oligosaccharides containing uronic acid were accumulated in two polar fractions unless also a feruloyl group was present. Feruloylated oligosaccharides, containing mono- and/or diferulic acid, were accumulated within four more apolar fractions. All fractions contained high amounts of acetyl substituents. The data show that complex xylan oligomers are present in which ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acids were combined within an oligomer. Hypothetical structures are discussed, demonstrating which enzyme activities are lacking to fully degrade corn glucuronoarabinoxylans.
Biofilm formation on reverse osmosis membranes is initiated and dominated by Sphingomonas spp
Bereschenko, L.A. ; Stams, A.J.M. ; Euverink, G.J.W. ; Loosdrecht, M.C.M. - \ 2010
Applied and Environmental Microbiology 76 (2010)8. - ISSN 0099-2240 - p. 2623 - 2632.
targeted oligonucleotide probes - pseudomonas-aeruginosa - community structure - microbial biofilms - bacteria - identification - adhesion - ultramicrobacterium - polysaccharides - hybridization
The initial formation and spatiotemporal development of microbial biofilm layers on surfaces of new and clean reverse osmosis (RO) membranes and feed-side spacers were monitored in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The feed water of the RO system had been treated by the sequential application of coagulation, flocculation, sand filtration, ultrafiltration, and cartridge filtration processes. The design of the flow cells permitted the production of permeate under cross-flow conditions similar to those in spiral-wound RO membrane elements of the full-scale system. Membrane autopsies were done after 4, 8, 16, and 32 days of flow-cell operation. A combination of molecular (fluorescence in situ hybridization [FISH], denaturing gradient gel electrophoresis [DGGE], and cloning) and microscopic (field emission scanning electron, epifluorescence, and confocal laser scanning microscopy) techniques was applied to analyze the abundance, composition, architecture, and three-dimensional structure of biofilm communities. The results of the study point out the unique role of Sphingomonas spp. in the initial formation and subsequent maturation of biofilms on the RO membrane and feed-side spacer surfaces
First characterization of bioactive components in soybean tempe that protect human and animal intestinal cells against enterotoxigenic Escherichia coli (ETEC) infection
Roubos-van den Hil, P.J. ; Schols, H.A. ; Nout, M.J.R. ; Zwietering, M.H. ; Gruppen, H. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)13. - ISSN 0021-8561 - p. 7649 - 7656.
soya bean tempe - aspergillus-niger - adhesion - fermentation - inhibition - diarrhea - piglets - k88 - polysaccharides - arabinanases
Tempe extracts can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells and thereby can play a role in controlling ETEC-induced diarrhea. The component responsible for this adhesion inhibition activity is still unknown. This research describes the purification and partial characterization of this bioactive component of tempe. After heating, defatting, and protease treatment, the extracts were found to remain active. However, after treatment with polysaccharide-degrading enzyme mixtures the bioactivity was lost. Ultrafiltration revealed the active component to be >30 kDa. Further purification of the bioactive tempe extracts yielded an active fraction with an increased carbohydrate content of higher arabinose content than the nonactive fractions. In conclusion, the bioactive component contains arabinose and originates from the arabinan or arabinogalactan side chain of the pectic cell wall polysaccharides of the soybeans, which is probably released or formed during fermentation by enzymatic modifications
Analytical profiling of plant cell wall polysaccharides
Westphal, Y. - \ 2010
Wageningen University. Promotor(en): Harry Gruppen; Fons Voragen, co-promotor(en): Henk Schols. - [S.l.] : S.n. - ISBN 9789085857068 - 168
polysacchariden - celwandstoffen - chemische analyse - analytische methoden - polysaccharides - cell wall components - chemical analysis - analytical methods
Keywords: High performance liquid chromatography, capillary electrophoresis, MALDI-TOF
mass spectrometry, NMR, cell wall oligosaccharides, arabinan, Arabidopsis thaliana, cell wall degrading enzymes, profiling

The plant cell wall polysaccharides cellulose, hemicelluloses and pectins are very heterogeneous and complex structures consisting of at least 20 different sugars and
30 different linkage types. Additionally, hemicelluloses and pectins might be acetylated and/or feruloylated. Furthermore, pectins carry methyl esters. The degree and distribution of these modifications may vary significantly depending on source and developmental stage. In this research several analytical tools were developed for the analysis of complex mixtures of cell wall derived oligomers.
The combination of 1) the use of pure and well-defined cellulases, hemicellulases and pectinases, and 2) the detection of the oligosaccharides released by MALDI-TOF MS and CE-LIF resulted in a screening method for Arabidopsis cell wall mutants, which addresses all enzyme-accessible polysaccharides in the cell wall.
Porous graphitized carbon (PGC)-HPLC with evaporative light scattering and mass
detection was introduced to a broad range of neutral and acidic cell wall polysaccharide derived oligosaccharides and separation of almost all oligosaccharides under investigation was achieved. The used gradient ensured 1) a sufficient separation of many oligosaccharides and 2) a sequential elution of first the neutral and then the acidic oligosaccharides. This elution behavior in combination with online-recorded MSn analysis facilitates the identification of (unknown) peaks.
A wide range of branched arabino-oligosaccharides was isolated from sugar beet arabinan and characterized with NMR. HPAEC was demonstrated to have insufficient
resolution to separate all linear and branched arabino-oligosaccharides. Therefore, PGC-HPLC-ELSD/MS and CE-LIF-MS were explored for the separation and detection of isomeric arabino-oligomers and were demonstrated to perform well. The combination of the controlled enzyme treatment, the predictive retention behavior on PGC-material, and the LC/CE-MS2 fragmentation patterns led to the prediction of the structures of unknown branched arabino-oligosaccharides in a complex mixture.
Branched arabino-oligosaccharides isolated from sugar beet arabinan
Westphal, Y. ; Kuhnel, S. ; Waard, P. de; Hinz, S.W.A. ; Schols, H.A. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Carbohydrate Research : an international journal 345 (2010)9. - ISSN 0008-6215 - p. 1180 - 1189.
cell-walls - structural characterization - feruloyl oligosaccharides - polysaccharides - acid - nmr - pectins - pulp
Sugar beet arabinan consists of an a-(1,5)-linked backbone of l-arabinosyl residues, which can be either single or double substituted with a-(1,2)- and/or a-(1,3)-linked l-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an a-(1,5)-linked backbone of l-arabinosyl residues. One series carries single substituted a-(1,3)-linked l-arabinosyl residues at the backbone, whereas the other series consists of a double substituted a-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.
Introducing Capillary Electrophoresis with Laser-Induced Fluorescence (CE-LIF) as a Potential Analysis and Quantification Tool for Galactooligosaccharides Extracted from Complex Food Matrices
Albrecht, S.A. ; Schols, H.A. ; Klarenbeek, B. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)5. - ISSN 0021-8561 - p. 2787 - 2794.
gastrointestinal symptoms - galacto-oligosaccharide - polysaccharides - bifidobacteria - metabolism - prebiotics - ingestion - products - sugars
The analysis and quantification of (galacto)oligosaccharides from food matrices demands both a reproducible extraction method as well as a sensitive and accurate analytical method. Three typical matrices, namely, infant formula, fruit juice, and a maltodextrin-rich preparation, to which a commercial galactooligosaccharide mixture was added in a product concentration range from 1.25 to 30%, served as model substrates. Solid-phase extraction on graphitized carbon material upon enzymatic amyloglucosidase pretreatment enabled a good recovery and a selective purification of the different galactooligosaccharide structures from the exceeding amounts of particularly lactose and maltodextrins. With the implementation of capillary electrophoresis in combination with laser-induced fluorescence (CE-LIF) detection, a new possibility facilitating a sensitive qualitative and quantitative determination of the galactooligosaccharide contents in the different food matrices is outlined. Simultaneous monitoring and quantifying prebiotic oligosaccharides embedded in food matrices presents a promising and important step toward an efficient monitoring of individual oligosaccharides and is of interest for research areas dealing with small quantities of oligosaccharides embedded in complex matrices, e.g., body liquids.
TEMPO oxidation of gelatinized potato starch results in acid resistant blocks of glucuronic acid moieties
Haar, R. ter; Timmermans, J.W. ; Slaghek, T.M. ; Dongen, F.E.M. van; Schols, H.A. ; Gruppen, H. - \ 2010
Carbohydrate Polymers 81 (2010)4. - ISSN 0144-8617 - p. 830 - 838.
physicochemical properties - selective oxidation - mediated oxidation - polysaccharides - pectin
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 a(1 ¿ 4) glucuronic acid–glucuronic acid bond was found to be very resistant to breakdown by acids. The a(1 ¿ 4) glucuronic acid–glucose bond also showed increased resistance to acids compared to a(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.
Enzymatic production of hyaluronan oligo- and polysaccharides
Kooy, F.K. - \ 2010
Wageningen University. Promotor(en): Gerrit Eggink; Hans Tramper, co-promotor(en): Carmen Boeriu. - [S.l. : S.n. - ISBN 9789085856481 - 174
hyaluronzuur - derivaten - oligosacchariden - polysacchariden - industriële microbiologie - industriële enzymen - hyaluronic acid - derivatives - oligosaccharides - polysaccharides - industrial microbiology - industrial enzymes
Hyaluronan oligo- and polysaccharides are abundant in the human body. Depending on the chain length, hyaluronan is an important structural component or is involved in influencing cell responses during embryonic development, healing processes, inflammation and cancer. Due to these diverse roles of hyaluronan, there are multiple applications already in use or in development, such as supplementation of fluid in eyes and joints, cosmetic tissue augmentation, enhancing wound healing, tissue engineering, cancer treatment, controlled drug release and targeted drug delivery. State-of-the-art hyaluronan production techniques include bacterial fermentation to produce long hyaluronan polymers with a small chain length distribution and in vitro enzymatic systems to produce hyaluronan oligosaccharides of one chain length. Both production strategies make use of hyaluronan synthase (HAS), an enzyme that elongates UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) into hyaluronan.

The main question in hyaluronan production today is how the chain length of the products can be controlled. Since most production processes use hyaluronan synthases, the aim of this thesis was to elucidate the polymerization mechanism of Pasteurella multocida hyaluronan synthase (PmHAS) from a biochemical point of view. In addition, the acquired knowledge is used for improving the control on hyaluronan chain length in polymerization reactions using PmHAS. Valuable information important for production processes on the intrinsic properties of the enzyme, such as substrate affinity, can be obtained by kinetic studies using single-step elongations. Kinetic studies also provide insights on how polymerization is achieved and, combined with structural studies, the identification of amino acid residues that are important for polymerization. This knowledge can be used for improving the hyaluronan synthesis performance of the enzyme.

Kinetic studies require purified substrates in quantities of mg-scale. Hyaluronan (HA) oligosaccharides were obtained through stepwise hyaluronan cleavage using hyaluronidase and consecutive separation of the reaction mixture by flash-chromatography (Chapter 2). The enzymatic hydrolysis was optimized by experimental design studies with pH, enzyme concentration and reaction time as parameters. Empirical models were developed for the yield of each individual target HA oligosaccharide using the results from a central composite design. Selective production of short HA oligomers (HA ≤ 10) or longer oligosaccharides (HA > 10) was made possible through implementation of the reaction conditions indicated by the empirical models. Separated HA oligomers were characterized by a combination of anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry with time-off-flight analysis. Using these techniques, the desired quantities of purified target HA oligosaccharides (n = 4, 6, 8 and 10) were obtained and used in further studies.

Besides the single-step elongations assessed in kinetic studies, full polymerization studies with both UDP-sugars available were used to investigate the influence of substrate concentrations on the chain length distribution of the hyaluronan products. In order to quantify all oligosaccharides formed during PmHAS polymerization in μl-scale reactions, HA templates consisting of a fluorophore-labeled HA tetrasaccharide (HA4) were generated (Chapter 3). A fast, simple and sensitive assay was developed based on fluorophore-assisted carbohydrate electrophoresis (FACE) that was used for quantification and characterization of PmHAS polymerization products.

The individual β1,3-glucuronyl-transferase (UA-transferase) and β1,4-N-acetylglucosamine-transferase (NAc-transferase) activities of PmHAS were investigated separately using kinetic studies, where the reaction of an HA oligosaccharide was followed with, respectively, UDP-GlcUA or UDP-GlcNAc in single-step elongations. In Chapter 4, the influence of HA oligosaccharide length (n = 4, 5, 6, 7, 8 and 9) on the polymerization reaction was investigated by one-substrate kinetics, varying only the HA oligosaccharide concentration at saturating UDP-sugar concentration. These reactions followed Michaelis Menten kinetics, although HA oligosaccharides may become inhibiting at elevated concentrations above 6 mM. The observed kcat values increased with increasing HA oligosaccharide length to a constant value at HA6 and HA7. The specificity constant kcat/Km values for HA oligosaccharides in the UA-transferase domain increased at increasing oligosaccharide length, whereas in the NAc-transferase domain kcat/Km values were constant at a low value. This indicates that there are two separate oligosaccharide binding sites of different lengths, one in each transferase domain of PmHAS. In Chapter 4, it was demonstrated that the chain-lenght distribution in PmHAS polymerization reactions can be decreased, and thus improved, by using saturating concentrations of both HA oligosaccharides and UDP-sugars.

Chapter 5 describes two-substrate kinetic studies, where in single-step elongations both HA oligosaccharide and one of the UDP-sugars were varied, to investigate the polymerization mechanism of each individual transferase domain in PmHAS. Dead-end inhibition studies and goodness-of-fit parameters were used to distinguish between two-substrate models. From this analysis follows that both transferase domains elongate the UDP-sugar through a sequential mechanism, which is most likely an ordered one. In this proposed mechanism, the UDP-sugar is first bound followed by binding of the HA oligosaccharide, after which first the elongated HA oligosaccharide and then UDP is released. Large differences between Km values for UDP-GlcNAc and UDP-GlcUA, also found in Class I HAS enzymes, suggest that UDP-GlcNAc concentration is involved in the regulation of HAS activity and thus the chain length of hyaluronan products.

Structural studies were used to evaluate the results obtained with kinetic studies. In Chapter 4, a structural homology model of PmHAS was built based on crystal structure K4CP chondroitin polymerase in E. coli, which has a high sequence identity of 62% and high sequence homology of 78% with PmHAS. The active sites of PmHAS are structurally related to other glycosyltransferases and this provided information on where the oligosaccharide binding sites could be located. These putative oligosaccharide binding sites differ in size, as was predicted by kinetic studies (Chapter 4). Furthermore, structural similarities between PmHAS, α1,3-galactosyltransferase (α3GT) and β1,4-galactosyltransferase (β4Gal-T1) demonstrated that PmHAS contains in each transferase domain one flexible loop that forms a bridge over the active site. In crystal structures of α3GT and β4Gal-T1, these flexible loops have been shown to change conformation upon binding the UDP-sugar. Based on similarities in kinetic mechanisms and structures between PmHAS, α3GT and β4Gal-T1, it is likely that the flexible loops in PmHAS follow a similar conformational change, which makes the proposed ordered mechanism the only possible mechanism (Chapter 5).

In Chapter 6, the knowledge on the PmHAS polymerization mechanism gained in earlier chapters is reviewed and used to create new insights in the polymerization mechanism of Class I HAS enzymes. Both Class I HASs and PmHAS are used in hyaluronan production, and, therefore, the differences and similarities are discussed in Chapter 6. During hyaluronan production, there are many different aspects, such as intrinsic properties of the enzyme, cell metabolism and fermentation reaction conditions, that influence hyaluronan chain length and yield (Chapter 6). Moreover, hyaluronan production systems that are able to produce hyaluronan of desired length are discussed in Chapter 6 and a personal view of how these systems can be improved is presented.
Production, Refining, Structural Characterization and Fermentability of Rice Husk Xylooligosaccharides
Gullon, P. ; Gonzales-Munoz, M.J. ; Gool, M.P. van; Schols, H.A. ; Hirsch, J. ; Ebringerova, A. ; Rarajo, J.C. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)6. - ISSN 0021-8561 - p. 3632 - 3641.
substituted xylo-oligosaccharides - human colonic microbiota - treated eucalyptus wood - in-vitro - dietary modulation - ft-ir - autohydrolysis - fermentation - liquors - polysaccharides
Oligosaccharides produced by hydrothermal processing of rice husks (xylooligosaccharides and glucooligosaccharides) were refined by membrane processing (operating in diafiltration and concentration modes), subjected to xylanase treatment to reduce the average molar mass, and subjected to further purification by ultrafiltration (operating in concentration mode) and ion exchange. The purified products were assayed for composition, molar mass distribution and structural characterization by HPLC, HPAEC-PAD, HPSEC, MALDI-TOF-MS and NMR (1H and 13C). The fermentability of the purified product by fecal inocula was assessed on the basis of the time courses of pH and oligosaccharide concentrations. Succinate, lactate, formiate, acetate, propionate and butyrate were the major products resulting from fermentation experiments.
Introducing porous graphitized carbon liquid chromatography with evaporative light scattering and mass spectrometry detection into cell wall oligosaccharide analysis
Westphal, Y. ; Schols, H.A. ; Voragen, A.G.J. ; Gruppen, H. - \ 2010
Journal of Chromatography. A, Including electrophoresis and other separation methods 1217 (2010)5. - ISSN 0021-9673 - p. 689 - 695.
treated eucalyptus wood - hairy ramified regions - capillary-electrophoresis - black-currants - polysaccharides - pectin - xylogalacturonan - quantification - nomenclature - ionization
Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSn-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar ß-(1,4)-xylo- and ß-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of ß-(1,4)-galacto-, ß-(1,4)-manno- and a-(1,4)-gluco-oligosaccharides. Charged oligomers such as a-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.
Polysaccharide Expertise Netwerk (EPNOE)
Dam, J.E.G. van; Boeriu, C.G. - \ 2010
polysacchariden - biomassa - interdisciplinair onderzoek - netwerken (activiteit) - innovaties - duurzaamheid (sustainability) - biobased economy - polysaccharides - biomass - interdisciplinary research - networking - innovations - sustainability
Korte informatie over het Polysaccharide Expertise Netwerk (EPNOE).
Physicochemical properties of pectins from okra (Abelmoschus esculentus (L.) Moench)
Sengkhamparn, N. ; Sagis, L.M.C. ; Vries, R.J. de; Schols, H.A. ; Sajjaanantakul, T. ; Voragen, A.G.J. - \ 2010
Food Hydrocolloids 24 (2010). - ISSN 0268-005X - p. 35 - 41.
rheological properties - flow - polysaccharides
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'
Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges
Simon, S. ; Pairo, B. ; Villain, M. ; Abzac, P. D'; Hullebusch, E. ; Lens, P.N.L. ; Guibaud, G. - \ 2009
Bioresource Technology 100 (2009)24. - ISSN 0960-8524 - p. 6258 - 6268.
activated-sludge - extraction methods - part i - exopolymers - biofilms - polysaccharides - complexation - separation - protocols - hplc
The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1-7 kDa) and Superdex 20010/300GL (10-600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.
Culinary-medicinal mushrooms: must action be taken?
Griensven, L.J.L.D. van - \ 2009
International Journal of Medicinal Mushrooms 11 (2009)3. - ISSN 1521-9437 - p. 281 - 286.
agaricus-bisporus - fungal metabolites - cancer-therapy - extracts - immunochemotherapy - polysaccharides
In the Western world, the mushroom industry suffers from overproduction. Expectations are stronger than reality, and as a result, production is too high and prices are too low. Because bulk production has taken the lead, which not only happens in the West, overproduction occurs regularly. Low pricing influences the quality concept of consumers and hence their appreciation of mushrooms. This cannot continue without doing great harm to the socioeconomic structure of the industry. Therefore, measures have to be taken to introduce mushrooms as a true health food in the Western world. This may form a first step in the acceptance of mushroom extracts and mushroom-derived compounds as medicine in the prevention and cure of disease. The present review discusses the acceptance of mushrooms as health food and medicine and suggests pathways for necessary action
Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by immobilized enzymatically tailored pectins
Gallet, M. ; Vayssade, M. ; Morra, M. ; Verhoef, R.P. ; Perrone, S. ; Cascardo, G. ; Vigneron, P. ; Schols, H.A. ; Nagel, M.D. - \ 2009
Acta Biomaterialia 5 (2009). - ISSN 1742-7061 - p. 2618 - 2622.
hairy ramified regions - glinus-oppositifolius - rhamnogalacturonan-i - cell - polysaccharides - adhesion - activation - polymer - plant - vitro
The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-a) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24 h on MHR-a-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-a secretion were studied. The results indicate that MHR-a coating inhibits the LPS-induced activation of macrophages.
Release and characterization of single side chains of white cabbage pectin and their complement-fixing activity
Westereng, B. ; Coenen, G.J. ; Michaelsen, T.E. ; Voragen, A.G.J. ; Samuelsen, A.B. ; Schols, H.A. ; Knutsen, S.H. - \ 2009
Molecular Nutrition & Food Research 53 (2009). - ISSN 1613-4125 - p. 780 - 789.
plantago-major l - structural features - biological-activity - cell-walls - polysaccharides - arabinogalactan - residues - acid - xylogalacturonan - homogalacturonan
A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting -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 -eliminative treatment were composed of -1,5 linked arabinosyl residues with 2- and 3-linked -arabinosyl side chains, and, or -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 -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.
Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups
Sengkhamparn, N. ; Bakx, E.J. ; Verhoef, R.P. ; Schols, H.A. ; Sajjaanantakul, T. ; Voragen, A.G.J. - \ 2009
Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1842 - 1851.
hairy ramified regions - fat ingredient substitute - rhamnogalacturonan oligomers - structural-characterization - galacturonic acid - oligosaccharides - polysaccharides - degradation - substances - products
The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20 kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal a-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I.
Modulating in vitro bone cell and macrophage behavior by immobilized enzymatically tailored pectins
Bussy, C. ; Verhoef, R.P. ; Haeger, A. ; Morra, M. ; Duval, J.L. ; Vigneron, P. ; Bensoussan, A. ; Velzenberger, E. ; Cascardo, G. ; Cassinelli, C. ; Schols, H.A. ; Knox, J.P. ; Nagel, M.D. - \ 2008
Journal of Biomedical Materials Research Part A 86a (2008)3. - ISSN 1549-3296 - p. 597 - 606.
hairy ramified regions - osteoblast adhesion - extracellular-matrix - surface modification - synthetic peptides - rhamnogalacturonan - growth - polysaccharides - biomaterials - activation
Previous work has reported the results of a multidisciplinary effort producing a proof-of-concept on the use of pectic polysaccharides in the surface modification of medical devices. This study was designed to learn more about the capability of engineered rhamnogalacturonan-I (RG-I) fractions of apple pectin to control bone cell and macrophage behavior. Thermanox® or polystyrene Petri dishes were surface modified with two different modified hairy regions (MHRs) obtained by different enzymatic liquefaction processes of apples differing in relative amounts and lengths of their neutral side chains: (long-haired) MHR- and (short-haired) MHR-B. Bone explants from 14-day-old chick embryos were cultured for 14 days on both pectic substrata. MHR-B promoted cell migration and differentiation, MHR- did not. On MHR-, J774.2 macrophages grew well, their percentage in G1 phase was decreased and in S phase increased, and they did not secrete either proinflammatory-cytokines or nitrites. Contrasting results were gained from macrophages on MHR-B, except for nitrite secretion. Thus, we conclude that coatings from tailored pectins show different biological activities in vitro and are potential innovative candidates for improving the biocompatibility of medical devices in various applications. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008
Effect of wheat cultivar and enzyme addition to broiler chicken diets on nutrient digestibility, performance, and apparent metabolizable energy content.
Gutierrez del Alamo Oms, A. ; Verstegen, M.W.A. ; Hartog, L.A. den; Villamide, M.J. - \ 2008
Poultry Science 87 (2008)4. - ISSN 0032-5791 - p. 759 - 767.
chemical-composition - supplementation - polysaccharides - variety - poultry - starch - barley - assay
A total of 5,000 one-day-old male broiler chickens were assigned to 8 different treatments in a 4 x 2 factorial design. Four wheat cultivars (Amiro, Guadalupe, Isengrain, and Horzal) and 2 levels (0 or 1 kg/t of feed) of an enzyme cocktail (Avizyme 1300, xylanase, 2,500 U/kg and protease, 800 U/kg) were used. Nutritionally complete mash diets contained 65 and 70% of the test wheat for the starter and grower period, respectively. Test wheats were used in diets for broilers, and growth performance and AME contents were measured. Broiler performance was measured in 4,800 broilers allocated to floor pens with 75 birds each and fed from 1 to 42 d of age. Digestibilities and AME contents of diets were measured in 200 broilers from 6 to 27 d of age individually allocated to battery cages. Chromic oxide (Cr2O3) at an inclusion rate of 0.5% in the diet was used as an indigestible marker. Apparent metabolizable energy was corrected by zero N balance to obtain AMEn. Wheat cultivar strongly influenced animal performance during the starter period (1 to 21 d of age). During the grower period (21 to 42 d of age), only BW and daily feed intake were influenced by wheat cultivar. Differences in daily feed intake were associated with differences in AMEn intake during the starter period, but not during the grower period. Nutrient digestibility was higher with the use of enzyme. Animal performance was not affected (i.e., wheat cultivar differences were not eliminated by using enzymes). During the grower period, significant interactions were detected with regard to nutrient digestibility and AMEn. Differences in AMEn content of wheat could not be explained by digestible starch.
Bifidobacterium glycoside hydrolases and (potential) prebiotics
Broek, L.A.M. van den; Voragen, A.G.J. - \ 2008
Innovative Food Science and Emerging Technologies 9 (2008)4. - ISSN 1466-8564 - p. 401 - 407.
adolescentis dsm20083 - in-vitro - probiotics - oligosaccharides - arabinoxylan - longum - fermentation - bacteria - cloning - polysaccharides
Carbohydrates occur in food as natural constituents or are added as ingredients. In the last decade a number of novel dietary carbohydrates have been introduced as ingredients for food applications, responding to the growing awareness among consumers of the link between health and diet. One important group is formed by the non-digestible oligosaccharides (NDOs) which may function as prebiotics. In many studies it has been shown that prebiotics in the diet positively influence the human intestinal microbiota. It is envisaged that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. In this review we will focus on the degradation of (potential) prebiotics, derived from plant (poly)saccharides, by bifidobacterial glycoside hydrolases. In addition the possibilities to use or produce new classes of NDOs using the most important glycoside hydrolases from Bifidobacterium sp. will be discussed.
Intrahepatic CD8+ lymphocyte trapping during tolerance induction using mushroom derived formulations: A possible role for liver in tolerance induction
Shuvy, M. ; Hershcovici, T. ; Lull-Noguera, C. ; Wichers, H.J. - \ 2008
World Journal of Gastroenterology 14 (2008)24. - ISSN 1007-9327 - p. 3872 - 3878.
inflammatory-bowel-disease - lentinus-edodes mycelia - t-cells - nkt cells - experimental colitis - medicinal mushrooms - immune-reactions - polysaccharides - antitumor - immunomodulation
AIM: To determine the immunomodulatory effect of Shiitake (a mushroom extract), we tested its effect on liver-mediated immune regulation in a model of immune-mediated colitis. METHODS: Four groups of mice were studied. Colitis was induced by intracolonic instillation of TNBS in groups A and B. Groups A and C were treated daily with Shiitake extract, while groups B and D received bovine serum albumin. Mice were evaluated for development of macroscopic and microscopic. The immune effects of Shiitake were determined by FACS analysis of intra-hepatic and intrasplenic lymphocytes and IFN-¿ ELISPOT assay. RESULTS: Administration of Shiitake resulted in by an increased intrasplenic/intrahepatic CD4/CD8 lymphocyte ratio. These effects were accompanied by a 17% increase in the number of intrahepatic natural killer T (NKT) cells. A similar effect was observed when Shiitake was administered to animals without disease induction. CONCLUSION: Shiitake extract affected livermediated immune regulation by altering the NKT lymphocyte distribution and increasing intrahepatic CD8+ T lymphocyte trapping, thereby leading to alleviation of immune-mediated colitis marked alleviation of colitis, manifested by significant improvement in the macroscopic and microscopic scores, and by reduction in IFN-¿-producing colonies in group A, compared to group B mice (1.5 pfu/mL vs 3.7 pfu/mL, respectively). This beneficial effect was associated with a significant increase in the intrahepatic CD8+ lymphocyte trapping, demonstrated
Effect of enzyme extracts isolated from white-rot fungi on chemical composition and in vitro digestibility of wheat straw
Rodrigues, M.A.M. ; Pinto, P. ; Bezerra, R.M.F. ; Dias, A.A. ; Guedes, C.M. ; Cone, J.W. - \ 2008
Animal Feed Science and Technology 141 (2008)3-4. - ISSN 0377-8401 - p. 326 - 338.
volatile fatty-acids - rumen microorganisms - manganese peroxidase - trametes-versicolor - lignin degradation - phenolic-acids - rice straw - fermentation - fiber - polysaccharides
A series of in vitro experiments were completed to evaluate the potential of enzyme extracts, obtained from the white-rot fungi Trametes versicolor (TV1, TV2), Bjerkandera adusta (BA) and Fomes fomentarius (FF), to increase degradation of cell wall components of wheat straw. The studies were conducted as a completely randomized design and analysed using one-way ANOVA. Enzyme activities of the extracts, previously obtained from a liquid culture medium, were characterized in terms of laccase and peroxidase for ligninolytic activity. Carboxymethyl cellulase (CMCase) and avicell digesting cellulase (Avicelase) were used for cellulolytic enzyme assays. Wheat straw samples were incubated with enzyme extracts in a citrate buffer (pH 5.0) in a forced air oven at 25 °C for 6 days. In vitro NDF digestibility (IVNDFD), and the rate and extent of NDF fermentation, without and after incubation with the white-rot enzyme extracts, were determined using a gravimetric microbiological method and a gas production technique, respectively. Results from cell wall chemical composition showed that TV2 and BA enzyme extracts decreased NDF concentration (P
Texture of food gels explained by combining structure and large deformation properties
Berg, L. van den - \ 2008
Wageningen University. Promotor(en): Tiny van Boekel; Erik van der Linden, co-promotor(en): F. van de Velde; Ton van Vliet. - S.l. : S.n. - ISBN 9789085049432 - 193
gels - wei-eiwit - polysacchariden - textuur - structuur - mechanische eigenschappen - reologische eigenschappen - confocale microscopie - gels - whey protein - polysaccharides - texture - structure - mechanical properties - rheological properties - confocal microscopy
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