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
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 - geranium - hyacinthus - chrysanthemum - orchidaceae - huid - haar - olieleverende planten - medicinale planten - natuurlijke producten - chemicaliën uit biologische grondstoffen - biobased economy
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 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).
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.
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.
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.
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 - biobased economy
Korte informatie over het Polysaccharide Expertise Netwerk (EPNOE).
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
Protein/polysaccharide complexes at air/water interfaces
Ganzevles, R.A. - \ 2007
Wageningen University. Promotor(en): Martien Cohen Stuart; Fons Voragen, co-promotor(en): Harmen de Jongh; Ton van Vliet. - [S.l.] : S.n. - ISBN 9789085046141 - 151
polysacchariden - eiwitten - adsorptie - oppervlaktechemie - oppervlakteverschijnselen - polysaccharides - proteins - adsorption - surface chemistry - surface phenomena
KEYWORDS:protein, polysaccharide,b‑lactoglobulin, pectin, electrostatic interaction, complex coacervation, adsorption, air/water interface, oil/water interface, surface pressure, surface rheology, spectroscopy
Proteins are often used to create and stabilise foams and emulsions and therefore their adsorption behaviour to air/water and oil/water interfaces is extensively studied. Interaction of protein and polysaccharides in bulk solution can lead to the formation of soluble or insoluble complexes. The aim of this thesis was to understand the influence of (attractive and non-covalent) protein/polysaccharide interaction on adsorption behaviour at air/water interfaces (and oil/water interfaces) in terms of adsorption kinetics, and rheological and spectroscopic characterisation of the adsorbed layers. The approach was to first identify the relevant parameters (like charge density, charge distribution or molecular weight of the ingredients) in the mixed protein/polysaccharide adsorption process. Subsequently, for each parameter a range of ingredients was selected/prepared allowing variation of only this single parameter. After investigation of the phase behaviour in bulk solution of the different protein/polysaccharide mixtures to be used, the role of each parameter in mixed protein/polysaccharide adsorption was studied. The parameters most thoroughly assessed were: protein/polysaccharide mixing ratio, polysaccharide charge density and molecular weight and the sequence of adsorption. The majority of the measurements were performed withb‑lactoglobulin (in combination with various polysaccharides e.g. pectin or carboxylated pullulan) at air/water interfaces, at standard conditions of pH 4.5 and low ionic strength (< 10 mM). In addition, experiments were performed at higher ionic strengths, different pH's, with different proteins or at an oil/water interface, to extend the insight in mixed protein/polysaccharide adsorption. This results obtained lead to a generic mechanistic model of mixed protein/polysaccharide adsorption.In conclusion, protein/polysaccharide interaction can be exploited to control protein adsorption at air/water interfaces. Any parameter affecting protein/polysaccharide interaction (e.g. ingredient parameters like polysaccharide molecular weight, charge density and distribution or system parameters like charge ratio, pH and ionic strength) may be varied to obtain the desired adsorption kinetics, surface rheological behaviour, or net charge of the surface layer. The choice of simultaneous protein/polysaccharide adsorption (in the form of complexes) versus sequential adsorption (first the protein, than the polysaccharide) provides an extra control parameter regarding the functionality of mixed adsorbed layers.
Structural Characterisation and Enzymatic Degradation of Exopolysaccharides involved in Paper Mill Slime deposition
Verhoef, R.P. - \ 2005
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Henk Schols. - Wageningen : Wageningen University - ISBN 9789085041412 - 154
polysacchariden - enzymen - degradatie - pulp- en papierwarenindustrie - bacteriën - bacteriële producten - chemische structuur - biofilms - polysaccharides - enzymes - degradation - pulp and paper industry - bacteria - bacterial products - chemical structure - biofilms
Bacteria that adhere to the surface of a paper machine form a biofilm that entraps the fibres and additives used as raw material to form a slime deposit. The formation of these slime deposits can result in serious problems with respect to the papermaking process itself and the end product. Traditionally more or less toxic biocides are used to prevent this problem. However, due to more strict environmental legislation there is growing interest in alternative methods for slime control. One of these methods could be the use of enzymes that degrade the exopolysaccharides (EPSs) that form the major structural element of a biofilm. To obtain these enzymes it is important to know which EPSs to target for enzymatic degradation. Therefore the EPSs produced by several species isolated from different paper mills within Spain, Finland or France were subjected to a (detailed) chemical structure elucidation. From these studies several EPSs were selected as target substrates for enzymatic degradation. The enzyme able to degrade one of these substrates was subjected to purification and characterisation studies.
Long-term performance and behavior of sows fed high levels of non-starch polysaccharides
Peet-Schwering, C.M.C. van der - \ 2004
Wageningen University. Promotor(en): Martin Verstegen; Bas Kemp, co-promotor(en): Leonard den Hartog. - [S.I.] : S.n. - ISBN 9789085040682 - 141
zeugen - varkens - zwangerschap - lactatie - polysacchariden - voer - samenstelling - voortplantingsvermogen - abnormaal gedrag - diergedrag - zeugenvoeding - diervoeding - dierenwelzijn - sows - pigs - pregnancy - lactation - polysaccharides - feeds - composition - reproductive performance - abnormal behaviour - animal behaviour - sow feeding - animal nutrition - animal welfare
The main objective of this thesis was to investigate the long-term effects of feeding sows high levels of dietary fermentable non-starch polysaccharides CNSP) (i.e., NSP from sugar beet pulp) restrictedly or ad libitum during gestation or ad libitum during lactation on behavior, reproductive performance, and development in body weight and backfat thickness. During gestation, sows were group-housed. Feeding gestating sows a high level of dietary fermentable NSP restrictedly reduced the frequency of total non-feeding oral activities in gestation compared with a starch diet. Feeding sows a high level of dietary fermentable NSP during lactation reduced the frequency of total non-feeding oral activities during subsequent gestation compared with a starch diet. Body weight and backfat gains during gestation were lower in sows fed a high level of dietary fermentable NSP restrictedly during gestation over three successive parities than in sows fed a starch diet restrictedly. These results indicate an overestimation of the energy value of fermentable NSP. Body weight and backfat losses during lactation were less in sows fed a high level of dietary fermentable NSP during gestation than in sows fed a starch diet. Sows fed a high level of dietary fermentable NSP during lactation lost more backfat during lactation than sows fed a starch diet. The number of live born piglets was 0.5 piglet higher in sows fed a high level of dietary fermentable NSP from weaning until mating and during subsequent gestation than in sows fed a starch diet. It may be that this effect can be attributed to feeding sows a high level of dietary fermentable NSP from weaning until mating. Lactation diet did not affect the number of live born piglets in the following parity. Gestating sows that were fed a high level of dietary fermentable NSP ad libitum during three successive parities ate 1.3 kg/d more during gestation than sows that were fed a starch diet restrictedly (4.2 versus 2.9 kg/d), resulting in higher body weight and backfat gains during gestation and greater losses in body weight and backfat during lactation. Feed intake during lactation was similar in sows that were fed restrictedly or ad libitum during gestation. Reproductive performance was not affected by feeding gestating sows a high level of dietary fermentable NSP ad libitum. Ad libitum fed sows spent 90 min/d eating whereas restrictedly fed sows spent 24 min/d eating. An increase in time spent eating is associated with a reduction in feeding motivation and in stereotypic behaviors. In conclusion, feeding gestating sows a high level of dietary fermentable NSP reduces the level of stereotypic behavior in gestation compared to a starch diet. Feeding sows a diet with a high level of fermentable NSP during lactation has an additional reducing effect on the development of stereotypic behavior in subsequent gestation. Reproductive performance is not negatively affected by feeding gestating sows a diet with a high level of fermentable NSP (i.e. NSP from sugar beet puIp) restrictediy or ad libitum during three successive parities compared to feeding gestating sows a starch diet restrictedly.
Microstructure, rheology and demixing in emulsions flocculated by polysaccharides
Blijdenstein, T.B.J. - \ 2004
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): G.A. van Aken; Ton van Vliet. - Wageningen : S.n. - ISBN 9789058089588 - 121
emulsies - uitvlokking - polysacchariden - reologie - structuur - emulsions - flocculation - polysaccharides - rheology - structure
keywords: Emulsion, b-lactoglobulin, polysaccharides, salt, sucrose, depletion, bridging, percolation, microstructure, micro-rheology, rheology, demixing, creaming, network compression. Abstract In this thesis, a study is presented on gravity-induced demixing behaviour of oil-in-water emulsions, stabilised by b-lactoglobulin and flocculated by various polysaccharides. Flocculation by polysaccharides mainly results in formation of emulsion droplet networks and can proceed via depletion and via bridging. Structural and rheological properties of these different networks were investigated and compared on a micro-and macroscopic level. These properties were related to the demixing behaviour of the emulsions. For emulsion droplet networks, gravity-induced compression of the network leads to separation of a serum layer. For depletion-induced networks, the initial rate of demixing by network compression is usually low and at high polysaccharide concentrations, usually a delay-time is observed before substantial demixing occurs. This delay-time scales with the permeability of the network, the viscosity and the density difference between oil and water. Once demixing has started, the network quickly collapses until the emulsion droplets are packed rather closely together. In bridging-flocculated emulsions, the initial demixing rate is higher, but more water was retained at longer times. The effects of protein, sugar and salts on demixing of depletion-flocculated networks were investigated as well. Protein affected the rate of flocculation and counteracted network formation. Sucrose affected the demixing rate via the viscosity and density of the aqueous phase, but it did not affect the droplet-droplet interactions. Salt affected the electrostatic droplet-droplet interactions. As a result, depletion-flocculation by dextran was inhibited at low salt concentrations. Addition of Ca2+ ions led to a decrease in repulsion between the protein layers, resulting in stronger droplet-droplet bonds, reinforcing a droplet network and retarding network compression.
Mushroom and herb polysachariides as alternative for antimicrobial growth promotors in poultry
Guo, F. - \ 2003
Wageningen University. Promotor(en): Martin Verstegen; Rene Kwakkel; B.A. Williams; Z.Q. Yang. - [S.l.] : S.n. - ISBN 9789058088260
pluimvee - paddestoelen - medicinale planten - polysacchariden - anti-infectieuze middelen - voedertoevoegingen - groeibevorderaars - immuniteitsreactie - ziekteresistentie - pluimveevoeding - diervoeding - diergezondheid - poultry - mushrooms - medicinal plants - polysaccharides - antiinfective agents - feed additives - growth promoters - immune response - disease resistance - poultry feeding - animal nutrition - animal health
Keywords: mushroom and herb polysaccharides, antimicrobial growth promoters, chickens
Antibiotics are widely used as therapeutics agents and also as growth promoters in poultry production. The possibility of developing resistant populations of bacteria and the side effects of using antibiotics as growth promoters in the farm animals has led to the recent EU-ban on the use of several antibiotics as growth promoters in poultry diets. Therefore, there is an intensive search for alternatives such as probiotics, prebiotics and other feed additives. Immuno-active polysaccharides derived from two mushrooms, Tremella fuciformis ( TreS ) and Lentinus edodes ( LenS ), and the herb Astragalus membranacea Radix ( AstS ), seem to be potential alternatives for antimicrobial growth and health promoters. These products were considered to play an important role in strengthening the animals' defense system by improving the physical conditions of gut ecosystem and enhancing functions of the immune system of chickens. The results presented in this dissertation demonstrated that intact mushroom and herb materials and their polysaccharide extracts showed differences in their physico-chemical properties, therefore, these products showed differences in fermentability and led to significant shifts in the bacterial community when fermented in vitro . These medicinal mushroom and herb materials, particularly their polysaccharide extracts, show promise in altering microbial activities and composition in chicken ceca. The polysaccharide extracts showed a slightly significant effect on growth performance and had no effects on weights of immune and GIT organs in normal broilers. However, the polysaccharide extracts significantly enhanced body growth and manipulated cecal microbial ecosystem such as viscosity and microbial species in Avian mycoplasma Gallisepticum infected chickens. And potential beneficial bacteria were significantly increased by the polysaccharide extracts. The polysaccharide extracts showed significant effects on body growth, immune responses as well as growth of immune organs and development of GIT fragments in coccidian-infected chickens, and particularly when they were used in conjunction with vaccine. The use of the mushroom and herb polysaccharide extracts might enhance T-cell immune responses, characterized by IFN- and IL-2 secretion, against coccidiosis in chickens. Supplementation of mushroom and herb extracts resulted in enhancement of resistance toE. tenella probably by enhancing both cellular and humoral immune responses against E.tenella in chickens.
Schols, H.A. ; Voragen, A.G.J. - \ 2003
In: Handbook of food enzymology / Whitaker, J.R., Voragen, A.G.J., Wong, D.W.S., New York : Dekker - ISBN 9780824706869 - p. 829 - 843.
enzymen - polysacchariden - pectinen - celwanden - enzymes - polysaccharides - pectins - cell walls
Pectins or pectic substances are names of a group of closely associated polysaccharides present in plant cell walls. Due to their anionic nature, pectic polysaccharides are considered to be involved in the regulation of ion transport and may control the permeability of the walls for enzymes. Also they determine the water-holding capacity of the wall
Ruwecelstofrijke voeders voor zeugen: effect op reproductie en gedrag
Peet-Schwering, C.M.C. van der - \ 2002
Lelystad : Praktijkonderzoek Veehouderij (Praktijkrapport. Varkens 10) - 24
zeugen - varkensvoeding - voer - ruwvoer (roughage) - polysacchariden - zetmeel - zwangerschap - lactatie - diergedrag - abnormaal gedrag - dierenwelzijn - voortplantingsvermogen - sows - pig feeding - feeds - roughage - polysaccharides - starch - pregnancy - lactation - animal behaviour - abnormal behaviour - animal welfare - reproductive performance
Op het Praktijkcentrum Rosmalen is onderzoek gedaan naar het effect van het verstrekken van voeders met een hoog zetmeelgehalte of een hoog gehalte aan verteerbare overige organische stof (= VOOS) tijdens de dracht, lactatie of dracht en lactatie op de ontwikkeling van gewicht en spekdikte en reproductieresultaten bij zeugen.
Structural characteristics of polysaccharides from olive fruit cell walls in relation to ripening and processing
Vierhuis, E. - \ 2002
Wageningen University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058085948 - 135
voedselchemie - olijven - celwandstoffen - polysacchariden - rijp worden - voedselverwerking - food chemistry - olives - cell wall components - polysaccharides - ripening - food processing
Key words: Olive fruit; olive oil; pectic polysaccharides; xyloglucans; xylans;
enzyme preparations; phenolic compounds; processing; ripening
Technical enzyme preparations can be used as processing aids in the olive oil industry to obtain a higher yield and a better quality of the oil. These technical enzyme preparations degrade the plant cell wall, thus enhancing the permeability for oil. However, still very little is known about the specific role of the various constituent enzymes present in the preparations towards the polysaccharides in the plant cell wall. This study deals with this subject and describes the structural characteristics of the polysaccharides in the cell wall of olive fruit in relation to processing for oil extraction.
The major polysaccharides in the cell wall of olive fruit were found to be the pectic polysaccharides and the hemicellulosic polysaccharides xyloglucan and xylan. The pectic polysaccharides were highly methyl esterified and rich in arabinose. The xyloglucans had a backbone with three out of four glucose residues substituted with xylose residues and had a very specific substitution pattern, because galactose as well as arabinose residues could be linked to the xylose residues. The presence of arabinose residues linked to xylose residues is a common feature of xyloglucans produced by solanaceous plants, but has not been demonstrated for other dicotylodonous plants. Incubation of the cell wall material from olive fruit with pectin degrading enzymes in combination with endo-glucanases revealed that O -acetyl groups were not only linked to pectic material in olive fruit but also to xyloglucan. It was established that the arabinose residues of the xyloglucan could carry one or two O -acetyl groups. The cell wall material of olive fruit contained besides xyloglucan also considerable amounts of the hemicellulose xylan. These xylans appeared to be very low in substitution. Less than 10% of the xylose residues were mono substituted, mainly with 4- O -methyl-glucuronic acid residues.
The use of technical enzyme preparations during processing affected only a relatively small part of the polysaccharides in the cell wall. About 8-10% of the cell wall polysaccharides were extracted with cold and hot buffer and appeared to be modified during enzymatic processing. The changes of the buffer soluble pectic material were reflected by an increase in yield, a change in molecular weight distribution, a decrease in methyl esterification and a degradation of the (1®4)-linked galactan chains. No differences were observed in the composition of the arabinan chains during enzymatic processing. Analysis of the olive oil revealed that the use of enzyme preparations increased the concentration of phenolic compounds in the oil. Especially, the contents of secoiridoid derivatives such as the dialdehydic form of elenolic acid linked to 3,4-dihydroxyphenylethanol and an isomer of oleuropein aglycon increased significantly. These two phenolic compounds have high antioxidant activities.
The effect of enzyme treatment during the mechanical extraction of olive oil depends on the stage of maturity of the olive fruit. From this perspective the structural characteristics of the cell wall polysaccharides during ripening have also been investigated. During ripening the degree of methyl esterification and acetylation decreased and the solubility of the pectic polymers in buffer markedly increased. No distinct differences could be noticed with regard to the sugar composition and the profile of the molecular weight distribution of the pectic and hemicellulosic polymers during fruit development.
Metabolic engineering of exopolysaccharide production in Lactococcus lactis
Boels, I.C. - \ 2002
Wageningen University. Promotor(en): W.M. de Vos; M. Kleerebezem. - S.l. : S.n. - ISBN 9789058086938 - 144
polysacchariden - melkzuurbacteriën - lactococcus lactis - productie - biosynthese - metabolisme - polysaccharides - lactic acid bacteria - lactococcus lactis - production - biosynthesis - metabolism
Exopolysaccharides (EPS) produced by lactic acid bacteria are important structural components in fermented foods. In addition, they may confer health benefits to the consumer, as mouse model studies have indicated that EPS may have immunostimulatory, anti-tumoral, or cholesterol-lowering activity. Lactococcus lactis strain NIZO B40 produces a phosphorylated EPS with a branched repeating unit containing glucose, galactose and rhamnose. The biosynthesis of this polymer depends on both the specific eps genes organized in a plasmid-located gene cluster and on several household genes that are involved in biosynthesis of the primary EPS precursors, the nucleotide-sugars. This work focused on the household genes to induce overproduction and/or structural modification of EPS. Therefore, we cloned, characterized, and controlled expression of the genes that encode enzymes involved (i) in primary sugar metabolism ( glk , pfk , fbp , pgm ), (ii) the biosynthetic pathway from glucose-1P to the EPS precursors UDP-glucose ( galU ), UDP-galactose ( galU and galE ) and dTDP-rhamnose ( rfbACBD) , and (iii) in the specific pathway ( epsA-K ) for the assembly of the repeating unit, export and polymerization of the NIZO B40 EPS. We provide evidence for metabolic control of the gal and rfb genes in EPS precursor and EPS production. Overexpression of the galU , pgm or the rfb genes resulted in a significant increase of EPS-precursors. Moreover, overexpression of the eps genes led to four-fold increased NIZO B40 EPS production. In addition, reduction of the UDP-galactose level by galE disruption abolished EPS production while a rfb conditional knock out yielded an EPS with altered sugar composition and different physical characteristics.
The research described in this thesis contributes to the understanding of exopolysaccharide biosynthesis in lactic acid bacteria and provides a starting point for applications in the dairy industry, especially with respect to the texture and health benefits of fermented products.
Enzymatic modification of bacterial exopolysaccharides : xanthan lyase as a tool for structural and functional modification of xanthan
Ruijssenaars, H.J. - \ 2001
Wageningen University. Promotor(en): J.A.M. de Bont; S. Hartmans. - S.l. : S.n. - ISBN 9789058083739 - 95
polysacchariden - xanthan - lyasen - biodegradatie - polysaccharides - xanthan - lyases - biodegradation
Bacterial extracellular polysaccharides (EPSs) can be applied, e.g., in foods, as a thickener or stabilizer. The functional properties that make a polysaccharide suitable for such applications are largely determined by the primary structure, i.e., the sugar composition, the linkage types between the sugar units, and the presence of side chains and non-sugar substituents. The aim of this research was to obtain EPS-modifying enzymes that could be used as tools both for studying structure-function relationships of (food-grade) EPSs and for the production of tailor-made EPSs with a specific, desired functionality. EPS-degrading microorganisms could serve as a source of such enzymes.
To get an idea of the probability of finding EPS-degrading microorganisms, a comparative biodegradability study was carried out on eight EPSs, six of which were produced by lactic acid bacteria (Chapter 2). Human faeces or soil were used as inocula. Xanthan, clavan, and the EPSs of Streptococcus thermophilus strains SFi39 and SFi12 were readily degraded. The four other EPSs, produced by Lactococcus lactis ssp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi20, and Lactobacillus helveticus Lh59, were not. Xanthan, the most relevant food-grade EPS, was chosen as the target for further studies.
For efficient screening of polysaccharide-degrading microorganisms, plate methods are required that discriminate between intact and degraded polysaccharide. Such methods can make use of specific physicochemical properties of the polysaccharide, such as complex formation with dyes and gelling capacity. Alternatively, dye-labelled polysaccharides can be applied. Chapter 3 presents a survey of plate methods based on the above principles.
A mixed xanthan-degrading culture was obtained from soil by enrichment on xanthan. From this culture, Paenibacillus alginolyticus XL-1 was isolated. This strain degraded 28% of the xanthan molecule and appeared to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including a xanthan lyase. Xanthan lyase removes the terminal mannosyl residue of the trisaccharide xanthan side chain by aβ-eliminative mechanism, resulting in a double bond in the side chain glucuronyl residue. Xanthan lyase is the only polysaccharide lyase that is exo-acting, releasing residues from the outside of a polysaccharide molecule. All other polysaccharide lyases described to date are endo-acting, attacking the polysaccharide backbone. In P. alginolyticus XL-1, xanthan lyase production is induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A 97-kDa xanthan lyase was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55°C (Chapter 4).
The gene encoding the pyruvated mannose-specific xanthan lyase of P . alginolyticus XL-1, designated xalA , was isolated. The xalA gene encodes a 936-amino acid protein, including a 36-amino acid signal sequence. The XalA protein belongs to polysaccharide lyase family 8, which until now only contained chondroitinases and hyaluronate lyases. The part of the xalA gene encoding the 900-amino acid, 96,887-Da mature enzyme was expressed functionally in Escherichia coli . Like the native enzyme, the recombinant enzyme is specific for pyruvated xanthan. Heterologous production of XalA in E. coli increased the volumetric productivity by a factor 30, compared to production by P. alginolyticus . The recombinant xanthan lyase was used as a tool to modify xanthan, which resulted in a dramatic loss of the capacity to form gels with locust bean gum.
Besides xanthan lyase, P. alginolyticus XL-1 produces other enzymes that could be useful for xanthan modification, such as a xanthan deacetylase and an enzyme releasing uronic acid, or uronic acid-containing oligosaccharides, from xanthan lyase-modified xanthan. Since these enzymes were produced at very low titers, P. alginolyticus XL-1 is not a suitable production organism for xanthan-modifying enzymes. Strain XL-1 may be very useful, however, as a source of genes for heterologous production of xanthan-modifying enzymes.
Structural characterisation and enzymic modification of exopolysaccharides from Lactococcus lactis
Casteren, W. van - \ 2000
Agricultural University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058082206 - 114
lactococcus lactis - polysacchariden - lactococcus lactis - polysaccharides
Since ancient times, lactic acid bacteria have been used for the preservation of food. Some of these bacteria are able to produce exopolysaccharides (EPSs), which may contribute to the peculiar rheology and texture of, for example, milk-derived products. Insight into the relationship between the chemical structure of EPSs and their physical properties can lead to tailor-made polysaccharides, which meet particular requirements in terms of structure and function. In this thesis, the elucidation of the chemical structures of three exopolysaccharides from Lactococcus lactis subsp. cremoris is described. Enzymes are used as a tool during the structural characterisation and modification of EPS and the action of three enzymes having activity towards (chemically modified) EPSs is investigated as well. Finally, a start has been made within this project to study the effect of structural changes of EPSs on the physical properties.
In chapter 1, a brief general introduction into the research subject is given. Besides background information about the use of bacterial EPSs in food and the biosynthesis of EPS, attention is paid to common features in EPS structures from lactic acid bacteria. Different ways to obtain structurally related EPSs are presented and the use of enzymes in polysaccharide research is outlined.
Chapter 2 describes the study of the chemical structure of EPS B40, explaining earlier reported analytical discrepancies.The EPS contains rhamnose:galactose:glucose:phosphate in a molar ratio of 1:1.3:2:1.1. 31P NMR indicated that a single phosphate group is present as a phosphodiester. EPS B40 has chemically been modified using 0.3 M H 2 SO 4 , 28 M HF or 2 M NaOH. From these modifications it is concluded that during the hydrolysis step prior to sugar composition analysis the galactose 3-phosphate linkages are split only partially and that, consequently, the amount of galactose is underestimated. The backbone of HF-modified EPS B40 can be degraded by a crude cellulase preparation from Trichoderma viride . Purification and characterisation of the obtained oligomers (chapter 3), together with the characterisation of the polymer (chapter 2), has resulted in a chemical structure for EPS B40 identical to the repeating unit already described for EPS SBT 0495:
In chapter 3, the enzyme activity responsible for the degradation of HF-modified EPS B40 is identified as an endoglucanase (endoV). Thus, after complete removal of galactosyl residues and phosphate and partial removal of rhamnosyl residues, endo glucanase is able to cleave the backbone consisting of glucosyl and galactosyl residues. Characterisation of the resulting homologous series of oligomers by MS and NMR unequivocally demonstrated that endoV is able to cleave theβ-(1→4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-modified EPS B40 is discussed on the basis of the subsite model for endoV, described in literature. The crude cellulase preparation from T. viride has also been shown to contain a phosphatase able to act on EPS B40 after removal of rhamnosyl and galactosyl residues by mild CF 3 CO 2 H treatment.
In chapter 4, the structural elucidation of EPS B39 is outlined. This novel exopolysaccharide structure contains l-Rha, d-Gal and d-Glc in a molar ratio of 2:3:2. Enzymic modification, methylation analysis and 1D/2D NMR experiments (both 1H- 1H and 1H- 13C) revealed that EPS B39 consists of a branched heptasaccharide repeating unit with the following structure:
Chapter 5 describes the chemical structure of EPS B891, which contains d-Gal and d-Glc in a molar ratio of 2:3. The polysaccharide is partially O -acetylated. By means of HF solvolysis, O -deacetylation, enzymic modification, methylation analysis and 1D/2D NMR studies the novel exopolysaccharide is shown to be composed of repeating units with the following structure:
EPS B39 and O -deacetylated EPS B891 both contain lactosyl side chains and it is demonstrated that the terminally linked galactosyl residues can be removed by using a crude commercial enzyme preparation from Aspergillus aculeatus . The purification and characterisation of theβ-galactosidase responsible for this modification is described in chapter 6. The enzyme has a molecular mass of approximately 120 kDa, a pI between 5.3-5.7 and is optimally active at pH 5.4 and 55-60 oC. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism is shown to be retaining and transglycosylation products are demonstrated using lactose as a substrate. Theβ-galactosidase is able to release terminally linked galactosyl residues from EPS B891 in presence of acetyl groups, but the hydrolysing rate after O -deacetylation is higher. Furthermore, O -deacetylated EPS B891 is degalactosylated faster than EPS B39.
In chapter 7, the results of this thesis are discussed. Emphasis is placed on the approach of using enzymes in structure (-function) studies of exopolysaccharides. Furthermore, the use of (modified) exopolysaccharides for characterising enzyme activities is outlined. Finally, the influence of various structural modifications on the physical properties of EPSs is briefly discussed.
Elucidation of the chemical fine structure of polysaccharides from soybean and maize kernel cell walls
Huisman, M.M.H. - \ 2000
Agricultural University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058081872 - 159
celwanden - polysacchariden - pectinen - galactanen - galacturonzuur - zea mays - glycine max - cell walls - polysaccharides - pectins - galactans - galacturonic acid - zea mays - glycine max
The subject of this thesis was the elucidation of the chemical fine structure of polysaccharides from cell walls of soybean and maize kernel. The two species investigated represent different taxonomic groups, soybean belonging to the dicotyledonous and maize to the monocotyledonous plants. Besides representing the most important structures present in cell wall material, these raw materials are of great importance in food and feed industry.
The characterisation of the soybean cell wall polysaccharides started with the isolation of the cell wall material as Water-Unextractable Solids (WUS) from soybean meal (chapter 2). The isolation procedure yielded a WUS fraction containing almost all polysaccharides present in the meal and only few other components. WUS was sequentially extracted with chelating agent (Chelating agent Soluble Solids, ChSS), dilute alkali (Dilute Alkali Soluble Solids, DASS), 1 m alkali (1 m Alkali Soluble Solids, 1 MASS) and 4 m alkali (4 m Alkali Soluble Solids, 4 MASS) to leave a cellulose-rich residue (RES). The pectin-rich extracts (ChSS and DASS) were found to have identical sugar compositions and contained predominantly galactose, arabinose, and uronic acid residues. The 1 MASS fraction contained xylose in addition to the former three sugars. The hemicellulose-rich fraction (4 MASS) contained mainly xylose and glucose. No indications were found that ChSS and DASS were structurally different, although it is obvious that their arrangement in the cell wall was not identical.
The intact cell wall polysaccharides in the meal and WUS were hardly degradable by enzymes. Once extracted, the polysaccharides from WUS were degraded more easily (chapter 3). The arabinogalactan side chains in the pectin-rich ChSS fraction could to a large extent be removed by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining polymer (fraction P) was isolated and represented 30% of the polysaccharides in the ChSS fraction (12% of the polysaccharides in the WUS). This polymer still contained some remaining arabinose and galactose residues, which could not be removed by the enzyme mixture used.
The pectic backbone (fraction P) appeared to be resistant to enzymatic degradation by both established (like polygalacturonase) and novel pectic enzymes (like RG-hydrolase). After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these two polymeric populations showed that the pectic substances in the original extract (ChSS) contained both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan was absent (chapter 4). The absence of homogalacturonan distinguishes the pectic substances from soybean from pectic polysaccharides extracted from other sources, which contain homogalacturonan and rhamnogalacturonan regions and can be degraded with polygalacturonase and RG-hydrolase, respectively. Acid hydrolysis of fraction P improves the susceptibility of the remaining polymers for RG hydrolase and exo-galacturonase.
The xylogalacturonan present in the ChSS fraction distinguishes itself from xylogalacturonan from other sources known so far. A part of the xylose residues in the xylogalacturonan is substituted with fucose and the xylogalacturonan is resistant to degradation with XGH.
The arabinogalactan side chains, which were removed from the ChSS fraction to obtain fraction P, were the subjects of investigation in chapter 5. Fractionation, monosaccharide and linkage analyses, enzymatic degradation, and mass spectrometry of the oligosaccharides in the digest of ChSS after enzymatic digestion with arabinogalactan degrading enzymes indicated the presence of common linear (1,4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1,4)-linked galactose residues bearing an arabino pyranose residue at the non-reducing terminus, and a mixture of linear oligosaccharides constructed of (1,4)-linked galactose residues interspersed with one internal (1,5)-linked arabinofuranose residue. The presence of an internal arabinofuranose residue in a pectic arabinogalactan chain in cell wall polysacchairdes has not been reported previously, not in soybean, nor in other fruit or vegetable cell walls. Another uncommon feature is the presence of arabinopyranose residues in pectic arabinogalactan.
The pectic substances form only one network of the plant cell wall, the other is the cellulose/hemicellulose network. The hemicelluloses were solubilised from the residue with 1 and 4 m KOH solutions (chapter 6). The polysaccharides extracted with 1 m KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucan fragments only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG), so three out of four glucose residues carry a side chain.
In chapter 7, the structural features of glucuronoarabinoxylans from maize kernels are described. First of all, maize kernel cell wall material was isolated as Water-Unextractable Solids (WUS). As expected the non-starch polysaccharides (NSP) had concentrated in the WUS (57%). These NSP were composed mainly of glucose, xylose, arabinose, and glucuronic acid. Sequential extractions with a saturated Ba(OH) 2 -solution (BE1 extract), and distilled water (BE2 extract) were used to solubilise glucuronoarabinoxylans from maize WUS. The glycosidic linkage composition of the extracts and their resistance to endo-xylanase treatment indicated that the extracted glucuronoarabinoxylans were highly substituted. In the maize BE1 extract 25% of the xylose was unsubstituted, 38% was monosubstituted and 15% was disubstituted. The glucuronoarabinoxylans in maize BE1 appeared to be resistant to endo-xylanase treatment, but could be degraded by a sub-fraction of Ultraflo, a commercial enzyme preparation from Humicola insolens . The digest contained a number of series of oligomers: pentose n , pentose n GlcA, pentose n hexose, and pentose n GlcA 2 . The presence of these glucuronic acid-containing series of oligomers showed that the glucuronic acids in the glucuronoarabinoxylancan can be very close to each other, but are not distributed blockwise. Finally, a new measure for the degree of substitution of glucuronoarabinoxylans was defined. It turned out that the degree of substitution in maize BE1 is much higher (87%) than in sorghum (70%) and wheat flour BE1 (56%). This indicates that the glucuronoarabinoxylans in maize BE1 are more complex than those in sorghum BE1 and explains their resistance to endo-xylanase treatment.
From this research, it can be concluded that both soybean and maize kernel cell wall polysaccharides distinguish themselves in a number of respects from other plant cell walls polysaccharides. The absence of homogalacturonan, but also the presence of internal (1,5)-linked arabinofuranose and terminal arabinopyranose in the pectic arabinogalactan side chains from soybean cell walls and the complexity of the glucuronoarabinoxylan from maize kernel cell walls are discussed in chapter 8. In addition, it was shown that techniques like mass spectrometry and NMR spectroscopy are powerfull techniques to be used after (enzymatic) fragmentation, for chemical characterisation of the original polysaccharides.
An exocellular polysaccharide and its interactions with proteins
Tuinier, R. - \ 1999
Agricultural University. Promotor(en): M.A. Cohen Stuart; G.J. Fleer; C.G. de Kruif; P. Zoon. - S.l. : S.n. - ISBN 9789058080493 - 184
polysacchariden - lactococcus lactis subsp. cremoris - fysische eigenschappen - polysaccharides - lactococcus lactis subsp. cremoris - physical properties
In the food industry polysaccharides are used as thickening or gelling agents. Polysaccharides are usually extracted from plants. Micro-organisms are also capable of excreting polysaccharides: exocellular polysaccharides (EPSs). In some cases EPSs are produced in-situ in food products, notably in acidified milk products. These EPSs function effectively as food thickeners but do not need to be declared in the food label.
Systematic physical analysis of an exocellular polysaccharide produced by a lactic acid bacterium has hardly been performed until now. In order to obtain a better understanding of the role of EPS in (acidified) milk products the physical properties of an EPS from the lactic acid bacterium strain Lactococcus lactis subsp. cremoris B40 were studied (Chapters 2-4) as well as its interactions with milk proteins (Chapters 5-8). The ionic strength of the EPS solutions was always set at 0.10 M, about the ionic strength in milk.
In Chapter 2 the isolation, purification and analysis of the molecular properties of EPS from L. lactis B40, our 'model' EPS, are investigated. The polysaccharide was separated from most low molar mass compounds in the culture broth by filtration processes. Gel permeation chromatography (GPC) was used to size-fractionate the polysaccharide. Fractions were analyzed by multi-angle static light scattering in aqueous solutions from which a number- (M n ) and weight-averaged (M w ) molar mass of (1.47 ± 0.06)·10 3and (1.62 ± 0.07)·10 3kg/mol, respectively, were calculated so that M w /M n1.13. The number-averaged radius of gyration was found to be 86 ± 2 nm. The hydrodynamic radius as determined from dynamic light scattering was consistent with the radius of gyration.
The viscosity of the EPS solutions was studied in simple shear flow as described in Chapter 3. Firstly, the zero-shear viscosity was determined as a function of the concentration. The intrinsic viscosity was determined from the data in the low concentration range. The intrinsic viscosity and the concentration dependence of the (zero-shear) viscosity of the B40 EPS could be predicted from the molar mass and the hydrodynamic radius. In addition the shear-thinning behavior was measured at several concentrations. The shear rate at which the viscosity starts to decrease scales with polymer concentration in accordance with the Rouse theory. By combining existing theories (Rouse and Bueche) it is possible to predict the intrinsic viscosity, concentration dependence of the viscosity, and shear-thinning behavior in terms of the molar mass and the hydrodynamic radius.
The measurements and theoretical description of the dynamic rheological properties of the EPS are presented in Chapter 4. Dynamic rheological measurements were performed as a function of frequency and EPS concentration. The dynamic properties could be described by the bead-spring model of Rouse. Concentrated EPS solutions have a significant elasticity at high concentrations and high frequencies, which is indicative of the presence of significant normal stress differences. It is suggested that these normal stresses may explain the contribution of the EPSs to the ropy behavior of yogurts.
Having characterized the EPS in aqueous solution, its interaction with the most relevant colloidal (protein) particles present in milk products was studied. As the polysaccharide studied in this thesis occurs in dairy products our focus was on the interactions and phase behavior of EPS with the colloidal components in milk. There are three distinctly different types of particles in the colloidal size range in milk: fat globules, casein micelles and whey proteins. Smaller molecular species (over 100,000 in milk) are considered as part of the continuous phase.
In Chapter 5 the interactions with whey proteins are described. Native whey proteins and EPS were co-soluble; they could be mixed in all proportions. However, an effective attraction (a depletion interaction) is induced between aggregated-whey-protein colloid (AWC) particles when they are mixed with the EPS. This depletion interaction originates from a loss of conformational entropy of the EPSs near the surface of neighboring AWC particles and leads to a phase separation at high enough EPS and/or AWC concentrations. The effect of the depletion interaction on the properties of the mixtures of EPS and AWC particles was first studied in the stable, i.e. one-phase region. The strength of attractions was characterized by small-angle neutron scattering (SANS) and dynamic light scattering (DLS). The SANS results could be described quantitatively by the Vrij theory and integral theory (Ornstein-Zernike with HNC closure) in combination with the Schaink-Smit theory and allowed a determination of the position of the spinodal. The DLS results could be described reasonably well by using a theory of Dhont and Kawasaki.
Furthermore, the experimental phase boundary was determined and compared with the Schaink-Smit theory, a mean-field theory which evaluates the free energy of a mixture of colloids and large non-adsorbing polymers. The spinodal so calculated was found to be consistent with the experimentally determined position of the phase boundary.
Spinodal phase separation kinetics was investigated by small-angle light scattering (SALS). At low Q a scattering peak was detected which shifted to lower Q's with time, in agreement with other experimental data and theoretical predictions for spinodal decomposition. Both the scaling of the scattered intensity with Q and the scaling of the Q-position of the peak with time agree with theoretical predictions of Furukawa and Siggia.
The interactions between EPS B40 and casein micelles are treated in in Chapter 6. Casein micelles become mutually attractive when the EPS is added to skim milk. The attraction can be explained as a depletion interaction between the casein micelles induced by the non-adsorbing EPS. We used three scattering techniques (SANS, turbidity measurements and DLS) to measure the attraction. The Vrij theory in combination with integral theory and all the experiments showed that casein micelles became more attractive upon increasing the EPS concentration.
The phase separation arising from depletion interaction in mixtures of casein micelles and EPS is described in Chapter 7. We have determined a phase diagram that describes the separation of skim milk with EPS into a casein-micelle-rich phase and an EPS-rich phase. We compared the phase diagrams with those calculated from theories developed by Vrij, and by Lekkerkerker and co-workers, showing that the experimental phase boundary can be predicted quite well. From measurements of the self-diffusion of the casein micelles in the presence of EPS the spinodal was calculated, which corresponds to the visual observations.
The effect of adding the EPS to an oil-in-water emulsion, stabilized with whey proteins, is reported in Chapter 8. Even at low EPS concentrations the emulsion phase separates. The phase line could be described by depletion interaction theory of Vrij. At high EPS concentrations and dispersed phase volume fractions above 10% we found a stable 'gel'-like region in the phase diagram. In that region the oil droplets attract one another so strongly that a space-filling network is formed at sufficient oil volume fractions.
A kinetic study showed that the rate of creaming/demixing decreases with volume fraction of oil of the system (hydrodynamics) and strongly depends on the concentration of EPS (strength of depletion interaction and continuous-phase viscosity). At low EPS concentration the creaming rate strongly increased with EPS concentration since attractions enhance creaming. At higher EPS concentrations creaming was slowed down by the viscosity increase of the continuous phase and the particle network which was created. This network became so strong at high EPS concentrations that creaming was absent in the 'gel' region. The rheological behavior of the 'gel' was studied by measuring flow curves which could be interpreted by the Potanin model, which describes the rheology of a dispersion of weakly aggregating particles.
In Chapter 9 the practical implications of this work are described. In order to understand the thickening effect of EPSs the molar mass, radius of gyration, and their interrelation are very important. It is indicated how the effectivity of a polysaccharide can be analyzed on the basis of the molar mass and the radius of gyration. The relation between the radius of gyration and the molar mass depends on the kind of monosaccharide residues, the linkage type, and the solvent. Further it is addressed how a fundamental understanding of the interactions between polysaccharides and proteins leads to predictions of the phase line and interpretation of the measured phase behavior. The unwanted effect of phase separation can then be suppressed by using only biopolymer concentrations at which the system is still stable. An understanding of the biopolymer interactions may thus make it possible to adjust the properties of food dispersions. Finally, some suggestions for further research are given.
Exopolysaccharide biosynthesis in Lactococcus lactis : a molecular characterisation
Kranenburg, R. van - \ 1999
Agricultural University. Promotor(en): W.M. de Vos. - S.l. : S.n. - ISBN 9789058081353 - 122
lactococcus lactis - biosynthese - polysacchariden - lactococcus lactis - biosynthesis - polysaccharides
Lactic acid bacteria are Gram-positive bacteria which are used for industrial food fermentation processes. Some have the ability to form exopolysaccharides (EPSs) and these bacteria or the produced EPSs can be used to enhance the structural properties of food products. Furthermore, these EPSs are claimed to be health beneficial. This thesis describes the results of a study on the biosynthesis of these polymers in Lactococcus lactis strains.
Chapter 1 provides an overview of the current knowledge of cell-surface polysaccharide biosynthesis, the glycosyltransferases involved, and export and polymerisation processes. Special attention is paid to genetics, regulation, and EPSs produced by LAB.
Chapter 2 describes the characterisation of EPS production by L. lactis NIZO B40. The strain produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. The EPS production is encoded on a 40-kb plasmid, which was isolated after conjugation and subsequent plasmid curing. On this plasmid, a 12-kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified encoding putative gene products which shared sequence homologies with gene products involved in cell-surface polysaccharide biosynthesis of other bacteria. Based on these homologies, predicted functions as regulation ( epsR ), polymerisation and export ( epsA , epsB , epsI , epsK ), or biosynthesis of the repeating unit ( epsD , epsE / epsF , epsG , epsH ) could be assigned. The eps genes are co-ordinately expressed and transcribed as a single 12-kb mRNA from a promoter upstream of epsR . Heterologous expression of epsD in Escherichia coli showed that its gene product is the so-called priming glucosyltransferase, linking the first sugar of the repeating unit to the lipid carrier.
Chapter 3 describes the functional analysis of the glycosyltransferase genes of the NIZO B40 eps gene cluster. The genes were cloned and expressed in E. coli and L. lactis to determine their function and the sugar-specificity of the encoded enzymes. The EPS consists of repeating units containing a trisaccharide backbone of two glucose and one galactose moieties. The epsDEFG gene products are involved in the synthesis of this trisaccharide, linking glucose to a lipid carrier in the membrane (EpsD), glucose to lipid-linked glucose (EpsE/EpsF), and galactose to lipid-linked cellobiose (EpsG), respectively. The epsJ gene product was found to be involved in the biosynthesis of EPS and is likely to act either as a galactosyl phosphotransferase or as an enzyme which releases the backbone oligosaccharide from the lipid carrier.
Chapter 4 describes the variety of EPS production by L. lactis . Sixteen EPS-producing L. lactis strains were analysed and based on the chemical composition of the EPSs they formed and the genotype of their eps genes, they were grouped in three major groups and two unique strains. Representatives of the three major groups were studied in detail. Group I comprises strain NIZO B40 which was characterised in the previous chapters. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating unit synthesis.
First successful attempts for genetic engineering of the EPS production were achieved by replacing the NIZO B40 priming glucosyltransferase gene, epsD , by an erythromycin resistance gene which resulted in the loss of EPS production and the complementation of the EPS-producing phenotype by controlled expression of priming glycosyltransferase genes from Gram-positive organisms with known function and substrate specificity.
In Chapter 5 the regions involved in replication and mobilisation of the NIZO B40 EPS-plasmid pNZ4000 were characterised. The plasmid contains four highly conserved replication regions that belong to the lactococcal theta replicon family and all are functional and compatible in L. lactis . Plasmid pNZ4000 was shown to be a mobilisation plasmid and two regions involved in mobilisation were identified. Both regions contained a functional origin of transfer ( oriT ). One oriT sequence was followed by a mobA gene, coding for a trans -acting protein involved in conjugative transfer and likely to be the relaxase nicking the nic sites of the oriT sequences.
Chapter 6 describes the complete nucleotide sequence of the EPS-plasmid pNZ4000, which amounts to 42810 bp and represents one of the largest sequenced plasmids in LAB to date. Apart from the regions involved in EPS biosynthesis, replication, and mobilisation, described in Chapters 2 and 5, two regions potentially involved in transport of divalent cations were localised on pNZ4000.
In Chapter 7 the results of the previous chapters are discussed and their implications on practical applications and in particular the perspectives for polysaccharide engineering are described.
Prebiotic effects of non-digestible oligo- and polysaccharides
Hartemink, R. - \ 1999
Agricultural University. Promotor(en): F.M. Rombouts; M.J.R. Nout. - S.l. : S.n. - ISBN 9789058080516 - 205
koolhydraten - oligosacchariden - polysacchariden - darmmicro-organismen - carbohydrates - oligosaccharides - polysaccharides - intestinal microorganisms
This thesis is the result of work carried out within a four-year multi-disciplined program, entitled ' Non-digestible oligosaccharides in foods and feed'. Within the project, four Ph.D. students were employed at the Food Chemistry, Food Microbiology, Human Nutrition and Animal Nutrition groups of the Wageningen Agricultural University. This thesis describes the studies carried out at the Food Microbiology group.
Oligosaccharides are carbohydrates, with an average chain length of 2-10 sugar residues. Most oligosaccharides cannot be digested by the enzymes in the upper gut, nor can these compounds be absorbed. These oligosaccharides are considered non-digestible, and reach the large intestine unaltered. Non-digestible oligosaccharides (NDOs) are mainly of vegetable origin and are a normal part of the human diet. Some of the natural NDOs are now produced commercially using enzymatic methods.
Most NDOs are completely or partially degraded and fermented by the bacterial populations in the large intestine. Some of the NDOs are considered to have a beneficial effect on the health of the host, due to the specific fermentation by two groups of intestinal bacteria, the bifidobacteria and lactobacilli. Commercial NDOs are marketed as a healthy ingredient, due to this selective fermentation, in several Western countries.
Chapter 1 describes the gastrointestinal tract and the bacterial composition in each part thereof. The same chapter gives an overview of the current knowledge of the fermentation of NDOs by intestinal bacteria and the effects on the host health, as far as known.
Chapters 2 and 3 describe the effect of the two types of NDOs, currently available on the Dutch market, on the etiology of dental caries. When consumed, residues of NDOs in foods may remain in the oral cavity. In the oral cavity many different bacteria are capable of degrading and fermenting carbohydrates, which results in the formation of acid and, possibly, dental lesions and caries. NDOs, being carbohydrates, may thus be fermented and are, in theory, a risk factor for dental caries.
In Chapter 2 the degradation and fermentation of fructooligosaccharides (FOS) by the oral microflora is described. It can be concluded that this class of NDOs can be fermented by the most common bacterial species. These NDOs can be considered cariogenic, in vitro , but in vivo studies have to be carried out to determine the actual risk for dental caries.
In Chapter 3, the degradation and fermentation of transgalactosyl-oligosaccharides (TOS) by the oral microflora is described. It was concluded that this class of NDOs is not, or very slowly, degraded and fermented. These NDOs are not considered a risk factor for dental caries.
Within the framework of the project, the Food Chemistry group synthesized and purified a large number of oligosaccharide mixtures from plant cell walls. As these purifications are laborious and the total quantities of pure oligosaccharides are very small, it was decided to determine the fermentation of plant cell wall compounds by intestinal bacteria. These plant cell wall compounds are available in large quantities and thus could be used for screening studies.
Chapter 4 describes the degradation and fermentation of such a plant cell wall polysaccharide, xyloglucan. Xyloglucan is present in many edible plants, but it is commercially prepared from tamarind seeds. The more (chemically) complex the compound the more enzymes are necessary for degradation, and the less bacteria are capable of fermenting the compound. Xyloglucan has a relatively simple chemical structure, but, surprisingly, only very few intestinal bacteria were capable of degrading this compound. The second remarkable conclusion was that most of the bacteria capable of degrading xyloglucan, belonged to the genus Clostridium . Previously, this genus has not been considered of major importance for polysaccharide degradation in the intestine.
Chapter 5 describes the degradation of a second plant cell wall polysaccharide, guar gum, a galactomannan. Like xyloglucan, galactomannans are part of the cell wall of many plants. Guar is commercially produced from the seeds of the Cyamopsis tetragonoloba tree and used as a thickening agent in many foods. Guar also has a relatively simple structure. Nevertheless, only three different bacterial species, capable of degrading guar, could be isolated from human and animal faeces. One of these, Bifidobacterium dentium was considered to be mainly an oral species, but, using guar, could also be isolated from faeces. The same species could also be isolated from samples of saliva from 19 out of 20 volunteers. A second species, Streptococcus bovis could only be isolated from animal faeces, whereas the third species, Clostridium butyricum was present in human and animal faeces. The latter species produced large amounts of gas, and can thus be considered responsible for the increased flatulence observed after the ingestion of guar.
Chapter 6 describes the differences in the fermentation of different oligosaccharides by human faecal inocula. In addition three polysaccharides were used in these studies. All donors had received the same diet and four samples were taken from each volunteer. The results show large differences between test compounds within the same volunteer, and large differences between volunteers on the same test compound. It can be concluded that the fermentation is largely dependent on host (genetic) factors, and not on dietary factors. It was also concluded that formation of gas is correlated with the formation of butyric acid. Butyric acid is considered to be important for the health of the intestinal wall. Gas production can thus be used as a simple screening method for butyrate production.
Within the project the variations in the bacterial composition of human and pig faeces have been studied. It was concluded in the early stages of the project that no methods existed for the reliable quantification of two major intestinal bacterial groups, bifidobacteria and lactobacilli. Two new methods had to be developed for the quantification of these bacterial groups.
Chapter 7 describes the development of a new medium for bifidobacteria, the RB medium. Selectivity is based on raffinose, propionate and lithiumchloride. The medium is not yet an ideal medium for the isolation and quantification for bifidobacteria but, compared with media currently used, it is more selective.
Chapter 8 describes the development of a new medium for lactobacilli, the LAMVAB medium. Selectivity is based on vancomycin and a low pH (5.0). The combination of vancomycin and low pH inhibits practically all other intestinal bacteria. LAMVAB has successfully been employed to isolate lactobacilli from faeces from a large number of animals.
The two newly developed media were compared with two other media, that are used regularly. The results of this comparison is described in Chapter 9 . The media were used to isolate bifidobacteria and lactobacilli in human and cat faeces and pig small intestinal contents. The three media for bifidobacteria performed equally well for human faeces, but for the other two kinds of samples, the RB medium performed better. For lactobacilli, LAMVAB performed better for all three types of samples tested.
Chapter 10 discusses the results of this thesis and some recommendations for further research are given.
As conclusion it can be stated that very few NDOs are degraded and fermented selectively by bifidobacteria. This was confirmed in Chapters 2 and 6, in which degradation and fermentation of FOS by other bacterial groups is described.
Although FOS and TOS are found to be possibly cariogenic, it is not likely that either oligosaccharide will cause caries under normal conditions.
Xyloglucan and guar are degraded only by a limited number of bacteria. Unexpectedly, clostridia played a major role in the degradation of both substrates. Both substrates may be a good substrate for the production of new NDOs, but considering the results it is unlikely that these oligosaccharides are a good substrate for lactobacilli or bifidobacteria.
RB and LAMVAB are new media, which are suitable for the quantitative isolation of bifidobacteria and lactobacilli from human faeces. LAMVAB is also suitable for animal faeces. Both media are more selective than the media used at present.
The role of the intestinal flora as affected by non-starch polysaccharides in broiler chicks
Langhout, D.J. - \ 1998
Agricultural University. Promotor(en): M.W.A. Verstegen; S. Tamminga; J.B. Schutte. - S.l. : S.n. - ISBN 9789054859123 - 173
vleeskuikens - kippen - voer - viscositeit - polysacchariden - antinutritionele factoren - darmmicro-organismen - darmen - ileum - dunne darm - verteerbaarheid - pectinen - rogge - tarwe - broilers - fowls - feeds - viscosity - polysaccharides - antinutritional factors - intestinal microorganisms - intestines - ileum - small intestine - digestibility - pectins - rye - wheat
It is well established that the non-starch polysaccharides (NSP) of wheat, rye and barley have anti-nutritive properties in broiler chicks. The water-soluble fraction of these NSP are assumed to be primary responsible for the anti-nutritive activity, producing depression in digestibility of nutrients and performance.
This study was undertaken to investigate the role of the intestinal microflora on the anti-nutritive properties of water-soluble NSP by using citrus pectin as model substrate. Citrus pectin is a highly fermentable NSP fraction of a viscous nature. Inclusion of high-methylated citrus pectin (HMC) in diets of young chicks resulted in a decreased nutrient digestibility and performance. This was associated with an increase of the microbial activity in the small intestine. In addition, ileal composition of the microflora and the morphology of the small intestinal wall was changed. Furthermore, degradation of conjugated bile acids was increased significantly by including HMC in the diet.
The effect of low-methylated citrus pectin (LMC) on these parameters was less pronounced than with HMC. When the HMC diet was fed to germ-free chicks, digestibility of nutrients and performance were hardly affected. It was concluded that the intestinal microflora mediates the magnitude of the anti-nutritive properties of water-soluble NSP in broiler chicks.
Wheat bran glucuronoarabinoxylans : biochemical and physical aspects
Schooneveld - Bergmans, M.E.F. - \ 1997
Agricultural University. Promotor(en): A.G.J. Voragen; G. Beldman. - S.l. : Schooneveld-Bergmans - ISBN 9789054857167 - 125
graansoorten - maling - Triticum aestivum - tarwe - hexaploïdie - voedsel - voedingsmiddelen - koolhydraten - zetmeel - vezel - polysacchariden - structuur - chemische reacties - cereals - milling - Triticum aestivum - wheat - hexaploidy - food - foods - carbohydrates - starch - fibre - polysaccharides - structure - chemical reactions
Arabinoxylans are present in cereal cell walls and in vitro they have interesting physicochemical properties, such as viscosity and gelation. Although many studies on these properties were reported for wheat flour arabinoxylan, not much research has been directed towards exploitation of these polysaccharides as food gum. For that purpose glucuronoarabinoxylans of wheat bran, a cheap by-product of the cereal industry, were studied with regard to their extractability, their structural and physicochemical properties.
Approximately 50% of the glucuronoarabinoxylans of wheat bran cell wall material were recovered in high purity by barium hydroxide extraction at 70 to 95°C. Delignification or other treatments to open up the cell wall structure were not effective in increasing the yield. The extracted glucuronoarabinoxylans were very diverse in chemical structure and physicochemical properties. About 30% of them had a low degree of substitution, were easily degradable by xylanolytic enzymes and hardly influenced the viscosity of the solvent as a result of extensive aggregation. Over 50% of them had a high degree of substitution, were supposed to contain dimeric branches of arabinose and xylose, were scarcely degradable by xylanolytic enzymes, gave moderate viscosity to solutions and were very effective in stabilizing emulsions. The structure of these glucuronoarabinoxylans could only be speculated upon and it could not be enzymatically modified as a consequence of its complexity and the lack of appropriate enzymes. The remaining glucuronoarabinoxylans either had an intermediate or very high degree of substitution, of which the latter was presumed to be connected to lignin-fragments.
Gel-forming glucuronoarabinoxylans were recovered only in low yield by dilute alkali extraction and subsequent purification was necessary. These feruloylated glucuronoarabinoxylans gelled upon addition of oxidative agents, of which peroxide - peroxidase, glucose - glucoseoxidase - peroxidase and ammonium persulphate were investigated. In comparison with wheat flour arabinoxylans, those of wheat bran appeared to give less flexible networks at high concentration, which was ascribed to their high degree of substitution and high ferulic acid content. Of the dimers formed upon cross-linking, the generally known diferulic acid, being a 5-5 coupled dimer, was only present in relatively low amounts. Dimers, in which the 8-position of the ferulic acid residue is involved were preponderant. The distribution of the dimers was not affected by the type of cross- linking agent or the type of arabinoxylan. However, the presence of lignin fragments in the bran extract was presumed to cause a low ferulic acid recovery upon cross-linking.
Isolation and characterisation of starch biosynthesis genes from cassava (Manihot esculenta Crantz)
Munyikwa, T.R.I. - \ 1997
Agricultural University. Promotor(en): E. Jacobsen; R.G.F. Visser. - S.l. : Munyikwa - ISBN 9789054858416 - 128
koolhydraten - polysacchariden - biosynthese - genen - genomen - manihot esculenta - cassave - carbohydrates - polysaccharides - biosynthesis - genes - genomes - manihot esculenta - cassava
Cassava (Manihot esculenta Crantz) is a tropical crop grown for its starchy thickened roots, mainly by peasant farmers, in the tropics, for whom it is a staple food. There is an increasing demand for the use of cassava in processed food and feed products, and in the paper and textile industries amongst others. This thesis describes research on the cloning of the genes encoding ADP-glucose pyrophosphorylase small and large subunits (AGPase B and S, respectively) and granule bound starch synthase II (GBSSII). These genes and their products were extensively characterised to determine their role in starch biosynthesis in cassava. Functional verification of the genes was carried out by transforming potato and cassava followed by analysis of the starch produced by the transgenic plants.
In Chapter 1 cassava production in the world in general and in Zimbabwe in particular is examined against the backdrop of new cloning and transformation strategies to improve starch quality and quantity. The development of cassava cultivars whose starches have novel physico-chemical properties by genetic modification of the process of starch biosynthesis is examined therein. The main criteria for these new cultivars to emerge are set forth as being: the availability of cloned and characterised starch biosynthesis genes, a universally applicable transformation and regeneration procedure for cassava, transfer to appropriate cassava cultivars, and biosafety analysis of transgenic cassava plants before disbursement to farmers.
The cloning of the cassava starch biosynthesis genes encoding granule bound starch synthase II (GBSSII) and the large and small subunits of ADP-glucose pyrophosphorylase (AGPase) is described in Chapters 2 and 3. The cloning of GBSSII reveals that there is indeed a second isoform of this enzyme in cassava as in other plants species. While sharing very little amino acid sequence homology with cassava GBSSI the GBSSII isophorm shares high amino acid sequence homology to other GBSSII genes from pea and potato. Cassava GBSSII seems to be more important in leaf tissue where it is more highly expressed than in tuber tissue where GBSSI predominates. Mapping of GBSSII revealed that this is a single copy gene located on the male derived linkage group T of the cassava mapping population.
Cloning of the cassava genes coding for the small (B) and large subunit (S) of AGPase revealed interesting aspects about the cassava enzyme. The cassava AGPase is likely to be heterotetrameric in constitution as had been found in other plant species. Comparison of the cassava AGPase sequences with those of already cloned AGPases revealed that AGPase B is more similar to small subunit genes from other plants than to cassava AGPase S coding for the large subunit (Chapter 3). Segregation analysis of a cassava mapping population revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. Both genes are expressed in all cassava tissues but AGPase B was shown to have a higher steady state mRNA level than AGPase S especially in leaf and tuber tissue. Post-transcriptional control of small subunit polypeptide levels could be inferred from the discrepancy between AGPase B mRNA and polypeptide levels. The AGPase enzyme activity was much higher in young cassava leaves than older leaves and tubers. Cassava leaf AGPase activity was increased 3 fold by the addition of 3-PGA (3-phospho-glycerate) and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA, but was highly inhibited by Pi.
In order to verify the biological role of the AGPase B gene antisense constructs were made of the cassava AGPase B behind a CaMV35S promoter (chapter 3). This was transferred into potato plants by Agrobacterium tumefaciens. While the 224 transgenic antisense AGPase B potato plants did not differ in appearance from normal potato plants, 45 transgenic plants, however, had more numerous and smaller tubers than control plants. Antisense plants with reduced AGPase B mRNA levels had 1.5 to 3 times less starch than tubers from the control plants. The levels of the soluble sugars in the antisense plants increased significantly (up to 10 times more glucose, 6 times the amount of fructose, and 5 times the amount of sucrose) when compared to those found in control plants. These results show that a heterologous gene from cassava can have an antisense effect in potato, but that the number of plants required to find plants exhibiting maximum antisense effect has to be very large. This is probably due to sequence homology differences between the cassava AGPase B and potato AGPase B genes which share only 68% amino acid sequence homology.
Chapter 5 describes the further development of an efficient, time and labour saving protocol for transforming cassava based on stringent selection of the luciferase (firefly) marker gene. In addition the first reported transformation of cassava with a gene (AGPase B) other than a marker gene is described. An antisense construct was made for transforming cassava. This consisted of the cassava AGPase B gene which was placed in antisense orientation behind the CaMV35S promoter. This was then coupled to the luciferase gene driven by another CaMV35S promoter. After particle bombardment of cassava FEC transgenic tissue was selected using three different selection regimes: non stringent luciferase selection, stringent luciferase selection and combined chemical (phosphinothrycin) and luciferase selection. Stringent luciferase selection whereby luciferase positive FEC units were precisely pinpointed, isolated and cultured was found to be the most effective and time saving method. It was possible to generate cultures having more than 90% luciferase positive FEC tissue after 12 weeks of stringent LUC selection, compared to 45% and <1 % for combined selection and non stringent selection respectively. The number of luciferase positive mature embryos generated was directly proportional to the percentage of luciferase positive tissue in the original FEC culture. Stringent luciferase selection enabled the time taken for production of transgenic cassava plants to be reduced to 28-36 weeks as compared to 8 months to a year with no stringent selection or LUC/PPT selection.
Cassava plants carrying the AGPase B antisense gene had extremely low levels of starch, compared to control plants, as shown by iodine staining of in vitro induced thick stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer. These results functionally confirm the identity of cassava AGPase B as well as emphasising the critical role of AGPase in starch formation in cassava.
A discussion about the significance and implications of cloning cassava genes and producing transgenic cassava for culture in developing countries is carried out in Chapter 6. While there are clearly many economic and nutritional benefits to producing transgenic cassava, for resource poor farmers, many people in the South are not aware of the biosafety implications of growing transgenic crops. It is further emphasised that discussions and debate should be initiated to make local communities aware of the issues surrounding transgenic crops and their products. In addition it is recommended that some form of international legal framework be set up to ensure that resource poor farmers are not disadvantaged by the patenting of material originating from their communities by individuals and companies in the North. This thesis clearly demonstrates how it will be possible in the near future to produce new cassava cultivars carrying the appropriate genes to affect pronounced changes on tuber productivity and starch quality.
Glucuronoarabinoxylans from sorghum grain
Verbruggen, M.A. - \ 1996
Agricultural University. Promotor(en): A.G.J. Voragen; G. Beldman. - S.l. : Verbruggen - ISBN 9789054855026 - 131
sorghum bicolor - polysacchariden - structuur - chemische reacties - sorghum - sorghum bicolor - polysaccharides - structure - chemical reactions - sorghum
Water-unextractable cell wall materials (WUS) were prepared from raw, polished, and malted sorghum ( Sorghum vulgare cv. Fara Fara). Except for the amounts, hardly any difference could be observed between the WUS of these three raw materials. This means that cell wall materials of the endosperm cell walls are basically the same as those of the outer endosperm and pericarp layers, and that the cell walls largely persist, during malting. These preparations were further fractionated by a sequential extraction procedure using aqueous solutions of saturated Ba(OH) 2 , 1M KOH and 4M KOH. The WUS preparations were composed of glucuronoarabinoxylans (GAX), (1→3),(1→4)-β-D-glucans, cellulose, and some protein. GAX was primarily extracted by Ba(OH) 2 solutions. All GAX fractions were composed of a highly substituted (1→4)-β-D-xylan backbone, substituted by arabinose and uronic acid. It was concluded that sorghum GAX populations were characterized by a reasonable homogeneity, since they could not be separated further by several chromatographic and precipitation techniques.
Degradation studies using purified xylanases, arabinofuranosidases and a glucuronidase alone or in combination, showed that the GAX populations were hardly broken down. Some oligomers were formed by digesting Ba(OH) 2 extracted GAX with a combination of endoxylanase I and (1→4)-β-D-arabinoxylan arabinofuranohydrolase, both purified from Aspergillus awamori . These oligomers were found to have a main chain of three or four xylose units, and to contain α-glucuronic acid linked to O -2 of the non-reducing terminal xylose unit. Two oligomers were found to have a dimeric (1→2)-linked arabinose side, chain linked at O -3 of an internal xylose unit. Also single arabinose substitution occured at O -3 of an internal xylose unit. There are strong indications that these side groups can also be linked at O -2 of an internal xylose residue. The reducing xylose units were unsubstituted. A model for the GAX populations from sorghum was proposed combining the results of the degradation studies, the identification of the oligomers, and knowledge about the mode of action of the enzymes used.
Finally, the developed techniques to investigate GAX in particular, were used to study the behaviour of GAX in the brewing process. Worts and spent grains of mashes, supplemented with commercial enzyme preparations containing xylanases among others were studied. Except for the amount of solubilized GAX, the GAX hardly changed with respect to the sugar composition and molecular weight distribution. A direct relationship between GAX, xylanases, and filtration behaviour of worts prepared from malted sorghum, could therefore not be established.
A molecular analysis of L-arabinan degradation in Aspergillus niger and Aspergillus nidulans
Flipphi, M.J.A. - \ 1995
Agricultural University. Promotor(en): A.J.J. van Ooyen; J. Visser. - S.l. : Flipphi - ISBN 9789054853923 - 165
aspergillus - celwanden - koolhydraten - cellulose - celmembranen - fermentatie - voedselbiotechnologie - glycosidasen - polysacchariden - genexpressie - pleiotropie - moleculaire genetica - aspergillus - cell walls - carbohydrates - cellulose - cell membranes - fermentation - food biotechnology - glycosidases - polysaccharides - gene expression - pleiotropy - molecular genetics
This thesis describes a molecular study of the genetics ofL-arabinan degradation in Aspergillus niger and Aspergillus nidulans. These saprophytic hyphal fungi produce an extracellular hydrolytic enzyme system to depolymerize the plant cell wall polysaccharideL-arabinan. Chapter 1 surveys the occurrence, properties and applications ofL-arabinanolytic enzymes (arabinases). The A.niger system, which constitutes an endolytic endo-1,5-α-L-arabinase (ABN A) and two distinct α-L-arabinofuranosidases (ABF A and ABF B), has been a frequent subject of investigation in the past and represents the best characterizedL-arabinanolytic system to date. These three enzymes are all glycosylated. Current knowledge on the induction of fungal arabinase expression is summarized in this Chapter. Furthermore, the structure of the polysaccharide substrate and its function in the plant cell wall matrix are introduced.
In Chapters 2 to 5, the cloning and characterization of the structural genes coding for the three glycosyl hydrolases from the A. nigerL-arabinan-degrading complex are described. A. niger abf A and abf B ar e the first eukaryotic ABF-encoding genes to be isolated and sequenced, abn A is the first ABN-encoding gene published. Chapter 2 reports on the isolation of the abf A gene encoding ABF A, the minor extracellular ABF. This gene could be cloned by utilizing ABF Aspecific cDNA as the probe. This cDNA was immunochemically identified from a cDNA library generated fromL-arabitol-induced myceliurn of an A. nigerD-xylulose kinase mutant. This mutant is unable to grow onL-arabitol and features enhanced expression of all three arabinases when transferred to medium containing this pentitol as sole carbon source. In Chapter 3 , the cloning of the ABN A-encoding gene (abn A) is described. This gene was isolated following the same strategy as with abf A, although a second cDNA library had to be generated first. The induction process was immunochemically monitored in order to establish the proper induction conditions for the new library. The abn A gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and the cDN A. The N-terminal amino acid sequences of ABN A and a CNBr-derived peptide were determined. Several transcription initiation sites and one polyadenylation site could be identified. The structural region codes for a protein of 321 amino acids and is interrupted by three introns. Extracellular ABN A consists of 302 amino acid residues with a deduced molecular weight of 32.5 kDa and a theoretical pl of 3.5. For the protein, an apparent pl of 3.0 and an apparent molecular weight of 43 kDa, determined upon SDS-PAGE, were previously reported. Chapter 4 documents the isolation and characterization of the abf B gene, coding for the major extracellular ABF. The determination of N-terminal amino acid sequences from ABF B and CNBr-generated peptides allowed the design of deoxyoligonucleotide mixtures which enabled the cloning of abf B. When utilized as primers in a polymerase chain reaction (PCR), ABF B-specific amplification products emerged, one of which was used to probe the gene. The abf B gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and of ABF B- specific cDNA isolated from the library described in Chapter 3. Several transcription initiation sites and one polyadenylation site could be identified. The structural region is a single open reading frame and codes for a protein of 499 amino acids. The mature enzyme consists of 481 amino acid residues with a deduced molecular weight of 50.7 kDa and a theoretical pl of 3.8. An apparent pl of 3.5 and an apparent molecular weight of 67 kDa, determined upon SDS-PAGE, were previously reported. The abf B gene product was suggested to be identical to the ABF purified and characterized by Kaji and Tagawa (Biochim Biophys Acta 207 : 456-464 (1970)). Considering the non-amino acid content of the latter protein, a molecular weight of 64 kDa could be deduced for ABF B. In Chapter 5 , the abf A gene and its gene product were characterized by DNA sequence analyses of the genomic DNA and of the cDNA for which the isolation was described in Chapter 2. The N-terminal amino acid sequences of ABF A and a CNBr-derived peptide were determined. One transcription initiation site and two polyadenylation sites could be identified. The structural region is interrupted by seven introns and codes for a protein of 628 amino acids. Mature ABF A consists of 603 amino acid residues with a deduced molecular weight of 65.4 kDa and a theoretical pl of 3.7. For this ABF, an apparent pi of 3.3 and an apparent molecular weight of 83 kDa, determined upon SDS-PAGE, were previously documented.
Although the three enzymes are all active against (1->5)-α-glycosidic bonds betweenL-arabinofuranosides, ABF A, ABF B and ABN A are genetically unrelated. ABF A was found to be N -glycosylated whereas ABF B and ABN A were not - these enzymes are only O -glycosylated. For each gene, arabinaseoverproducing strains were generated by introducing multiple gene copies in A.niger or in A.nidulans uridine auxotrophic strains through co-transformation. Transformants were isolated upon primary selection for uridine prototrophy. Subsequent overproduction of the genes introduced was demonstrated in these recombinant strains upon growth on sugar beet pulp, both immunochemically and by assaying enzyme activity. abf A was shown to be expressed in the heterologous host A.nidulans, despite the absence of an abf A gene equivalent in this organism. High-copy number A.niger abf B transformants featured impaired secretion of other extracellular proteins upon growth on sugar beet pulp. ABN A overproduction was found to be limited to approximately five times the wild-type level in A.niger abn A transformants, but not in A.nidulans transformants. Such a limitation was not observed in case of the ABFs.
In Chapters 5 and 6, the regulation ofL-arabinan degradation is addressed. The structural genes seem to be regulated mainly at the transcriptional level. Additional copies of either A13F-encoding gene in A.niger were shown to result in a reduction, but not in total silencing of the expression of the wild-type ABN Aencoding gene upon induction with either sugar beet pulp orL-arabitol ( Chapter 5 ). The reduction of the expression level of abn A correlated with the abf gene dosage. The repression effected by extra abf B gene copies was more stringent and more persistent than that elicited by additional abf A copies. Although observed with both inducers, these phenomena were more outspoken and more persistent on sugar beet pulp. Similar, but more moderate effects were observed towards the expression of the other abf gene in multiple copy abf A- and abf B-transformants. It was proposed that the abf genes titrate two distinct gene activators both involved in coordination of arabinase gene expression. However, the three genes were shown to respond differently upon a mycelial transfer toL-arabitol-containing medium, indicating that gene-specific factors are also involved. Four distinct sequence motifs were found in common in the promoter regions of the three genes. One of these elements is identical to the A.nidulans CREA-motif, which has been shown to mediate carbon catabolite repression on several A.nidulans enzyme systems. Arabinase expression in A.niger is known to be repressed in the presence ofD-glucose. Two other motifs are highly homologous to cAMP-responsive elements described in other organisms. For the fourth motif no functional analogues could be found, but the element was found to be present in several other fungal genes which are not involved inL-arabinan degradation at all. It is therefore likely that none of these common elements confer system-specific regulation.
The presumed involvement ofL-arabitol in the induction process of fungal arabinases was further emphasized by the induction characteristics of an A. nidulans mutant unable to grow on the end-product ofL-arabinan degradation,L-arabinose, nor onL-arabitol ( Chapter 6).L-Arabitol is an intermediate ofL-arabinose catabolism in Aspergilli. This mutant was shown to lack NAD +-dependentL-arabitol dehydrogenase activity resulting inL-arabitol accumulation, both intracellularly and in the culture medium, wheneverL-arabinose is present. Upon submerged growth on various carbon sources in the presence ofL-arabinose, the mutant featured enhanced expression of the enzymes involved in extracellularL-arabinan degradation, and of those of the intracellularL-arabinose catabolism. The co-substrates on which the mutant secreted large amounts of arabitol simultaneously exhibited high arabinase expression and featured reduced growth.L-Arabitol secretion and enzyme production were also observed on a mixed carbon source ofD-glucose andL-arabinose, resulting in normal growth. Hence, in the presence ofL- arabinose, the carbon catabolite repression conferred byD-glucose in the wild-type, is overruled in the mutant.
In Chapter 7 , ABN A is shown to have remote sequence similarity with four bacterial xylanolytic glycosyl hydrolases (three β-D-xylosidases and an endo-1,4-β-D-xylanase), three of which feature activity against para -nitrophenyl-α-L-arabinofuranoside. This synthetic compound is commonly utilized to assay potential ABF activity, whereas it is known to be an inhibitor of the fourth enzyme. The homology became evident only after multi pie-sequence alignments and hydrophobic cluster analysis. It was proposed that these enzymes share a binding site for a terminal non-reducing α-linkedL-arabinofuranosyl residue and that they all belong to glycosyl hydrolase family 43. Implications from these suggestions were discussed. The ABFs could not be assigned to an established glycosyl hydrolase family.
Based on theL-arabinolytic system of the brown-rot fungus Monilinia fructigena, the sequence similarity found amongst ABF A and bacterial pullulan-degrading enzymes, and ABF expression levels under carbon starvation conditions and onD-glucose as the carbon source, distinct functions inL-arabinan and plant cell-wall degradation were proposed for ABF A and ABF B. ABF A would be essentially cell-wall associated and act to degradeL-arabinan fragments generated by ABN A. ABF B activity would be important for the primary release of small amounts ofL-arabinose which initiate induction of various endolytic systems to degrade plant cell walls, and thus function in substrate sensing. In line with these considerations, the involvement of other, not yet identified glycosyl hydrolases inL-arabinan degradation by A.niger was suggested.
Induction and repression of arabinase gene expression are further discussed in Chapter 7 . The results of the studies in A.niger (Chapter 5) and A.nidulans (Chapter 6) were interpreted in a mutual context. The identity of the lowmolecular-weight compound directly responsible for induction of arabinase gene expression, was addressed. BothL-arabinose andL-arabitol are likely candidates to fulfil such a role. However, it was not possible to weigh the actual inductive capacities ofL-arabinose andL-arabitol due to their in vivo convertibility and the carbon catabolite repression elicited by the pentose. Competition for such a compound provides an alternative explanation for the phenomena observed in Chapter 5. The involvement of the transcriptional repressor CREA in arabinase gene expression is not limited to the direct repression of structural and regulatory genes of theL-arabinan-degrading system. It also plays a role in inducer exclusion and end-product repression, two processes shown to be eminently involved in the regulation ofL-arabinan degradation in wild-type A.nidulans. Fungal growth rate was suggested to be related to derepression of theL-arabinan-degrading system. The possible involvement of cAMP in arabinase gene expression, as suggested by the presence of potential cis -acting cAMP-responsive elements in the structural genes, was considered. Various ways by which cAMP might modulate arabinase synthesis were surveyed.
|Oligosacchariden als bifidogene factoren.
Hartemink, R. ; Nout, M.J.R. ; Rombouts, F.M. - \ 1994
Voedingsmiddelentechnologie 27 (1994)20. - ISSN 0042-7934 - p. 27 - 29.
bifidobacterium - koolhydraten - chemische reacties - microbiële afbraak - polysacchariden - structuur - bifidobacterium - carbohydrates - chemical reactions - microbial degradation - polysaccharides - structure
Levende bacterien die een gunstige invloed hebben op de darmflora. Ze worden probiotica genoemd en ze worden toegepast in zowel de humane voeding als de veevoeding
|Tailor-made produktie van oligosacchariden.
Laere, K.M.J. van; Schols, H.A. ; Voragen, A.G.J. - \ 1994
Voedingsmiddelentechnologie 27 (1994)20. - ISSN 0042-7934 - p. 33 - 35.
koolhydraten - chemische reacties - chemische structuur - chemicaliën - voedselindustrie - voedseltechnologie - polysacchariden - eigenschappen - structuur - invloeden - carbohydrates - chemical reactions - chemical structure - chemicals - food industry - food technology - polysaccharides - properties - structure - influences
De gewenste oligosacchariden kunnen met behulp van specifieke enzymen en chromatografische technieken 'op maat' worden gemaakt
Immunological and biochemical characterization of extracellular polysaccharides of mucoralean moulds
Ruiter, G.A. de - \ 1993
Agricultural University. Promotor(en): F.M. Rombouts; J.H. van Boom. - S.l. : De Ruiter - ISBN 9789054850861 - 170
mucorales - voedselbesmetting - polysacchariden - immunologische technieken - elisa - mucorales - food contamination - polysaccharides - immunological techniques - elisa - cum laude
In this thesis the characterization is described of the antigenic determinants (epitopes) of the extracellular polysaccharides (EPSs) from moulds belonging to the order of Mucorales. Detailed knowledge of the structure of these epitopes allows for further development of a new generation of methods for reliable detection of moulds in food. These immunoassays, such as the ELISA (Enzyme-linked Immunosorbent Assay), the latex agglutination assay and the dot- blot assay, are based on the specific recognition of antigenic EPSs by IgG antibodies raised against these polysaccharides.
As described in Chapter 2, the water-soluble extracellular polysaccharides which are excreted under various conditions of growth by the mucoralean moulds tested, including the genera Mucor, Rhizopus, Rhizomucor, Absidia, Syncephalastrum and Thamnidium, consist mainly of carbohydrate residues and some protein. The polyclonal IgG antibodies raised in rabbits against EPS of Mucor racemosus were very specific for species of Mucorales; as no cross-reactivity with other moulds was observed. In Chapter 10, the production and characterization of mouse-monoclonal IgG antibodies against the same EPS is described. These antibodies are also very specific for mucoralean moulds but based on different epitopes. However, as shown in Chapter 10, monoclonal antibodies are not necessarily more specific and may be less sensitive than polyclonal antibodies. The immunogenic specificity provides a taxonomic value to EPS as was shown by analysis of EPS preparations of species belonging to the Mortierella isabellina group, which pointed to a classification of this group into the Mucoraceae and not to the Mortierellaceae (Chapter 9).
A new method was developed (Chapter 4) for accurate sugar analysis of complex carbohydrate structures with acid sugar residues. This method includes the successive use of methanolysis and TFA hydrolysis followed by high-performance anion-exchange chromatography (HPAEC) analysis of the monosaccharides. This method provided a rapid, accurate and sensitive assay to determine the exact carbohydrate composition of the uronic-acid containing mucoralean EPSs on the microgram scale without any derivatisation. With this method the presence of fucose, mannose, glucose, galactose, and glucuronic acid residues in the mucoralean EPSs was established as well as their relative amounts.
A rapid method was developed in which high-performance size-exclusion chromatography (HPSEC) was combined with ELISA detection (Chapter 3). This method allowed the rapid screening and determination of fractions for the presence of antigenic polysaccharides, and enabled an optimal choice for column material to be used for isolation of the antigenic fractions. With this method a common rabbit antigenic fraction, characteristic for moulds of Mucorales, was found. It had an apparent molecular mass of approx. 30 kDa. This fraction accounted for only a minor part of the total EPSs and was mainly composed of mannose residues. The major part, a fraction containing approx.
50% glucuronic acid which is known in the literature as mucoran, was found to be antigenic in mice (monoclonal IgG) but not antigenic in rabbits (polyclonal IgG). A β(1-4)-linked D- glucuronan polymer isolated from the mucoralean EPS preparations did not react with the antibodies raised in mice and rabbits (Chapter 6). The antigenic polysaccharides could also be separated from the non-antigenic polysaccharides with immobilized antibodies. As demonstrated for polyclonal rabbit IgG tile antigenic polysaccharides could be separated in one step with an immunoaffinity column with covalently linked IgG antibodies (Chapter 5).
A valuable approach to reveal the structure of fungal carbohydrate epitopes, is the use of purified enzymes which are able to degrade the epitopes, which can then be screened with ELISA. In Chapter 7, such an enzyme was purified from a commercial enzyme preparation from Trichoderma harzianum. It appeared to be an exo-α-D-mannanase. EPSs were treated with this enzyme and the products were analysed by highperformance anion-exchange chromatography and by gas-liquid chromatography/mass spectrometry after derivatisation to alditol acetates. The exo-α-D-mannanase removed the ELISA activity of polysaccharides from Mucorales by hydrolysing the terminal α-D-mannose residues from α(1-2)-linked chains of D-mannose and terminal 2- O -methylmannose residues. This 2- O -methyl-mannose residue was identified in all mucoralean EPS preparations. It represents less than 0.5% (w/w) in all samples tested, and was initially overlooked with all other methods. In particular, determination of glycosidic linkages of carbohydrate residues is often performed by methylation analysis, a method which leaves 2- O -methyl-mannose residues undetected. To our knowledge, this compound has never been reported to occur in fungi. It was proved that the epitopes reactive with rabbit-IgG of Mucorales carry this 2- O -methyl-mannose residue at the non- reducing terminal.
As shown in Chapter 8, the antigenic activity of the mucoralean moulds with polyclonal IgG antibodies is mainly based on the 2- O -methyl-mannose residues. Therefore, it can be assumed that the results of the methylation analyses on antigenic mucoralean oligosaccharides performed by Miyazaki and coworkers were erroneously interpreted as proof of α(1-6)-linked mannose residues. It is most likely, that the oligomers they isolated indeed carried 2- O -methyl- mannose residues at the non-reducing end which were not recognised. Finally, the structure of the epitopes reactive with rabbit-IgG raised against mucoralean EPSs was consolidated by hapten-inhibition experiments with synthetic oligosaccharides, which unequivocally proved that 2- O -methyl-mannose residues play a vital role in this immunochemical reaction (Chapter 8).
This thesis is a contribution to the basic knowledge of the structure of extracellular polysaccharides from moulds. This knowledge can be used in the development of immunochemical methods for moulds, particularly in food products. A protocol was developed to elucidate the antigenic determinant of fungal polysaccharides. This approach appeared fruitful in discovering that the immunochemical reactivity of EPSs from Mucorales moulds reside mainly in non-reducing terminal 2- O -methyl-mannose residues.
Furthermore, this thesis can be considered as a contribution to the development of the knowledge of immunochemistry of sugars in general. This approach may be used to investigate structural features of any polysaccharide or glycoconjugate which is or can be made immunologically active. The methodology is particularly suitable in detecting minor sugars with unusual structure which occur at non-reducing terminals of biopolymers and may have specific biological functions. Specific enzymatic removal of such sugars may uncover structures of which the biological function or specificity (e.g. immunochemical reactivity) may be studied subsequently.
Downstream processing of polysaccharide degrading enzymes by affinity chromatography
Somers, W.A.C. - \ 1992
Agricultural University. Promotor(en): K. van 't Riet; F.M. Rombouts. - S.l. : Somers - ISBN 9789054850540 - 163
fermentatie - voedselbiotechnologie - glycosidasen - chromatografie - enzymen - polysacchariden - affiniteitschromatografie - fermentation - food biotechnology - glycosidases - chromatography - enzymes - polysaccharides - affinity chromatography
The objective of this study was the development of affinity matrices to isolate and purify a number of polysaccharide degrading enzymes and the application of these adsorbents in the large- scale purification of the enzymes from fermentation broths. Affinity adsorbents were developed for endo-polygalacturonase and α-amylase.
The isolation of two of these enzymes was realized using the specific affinity of the enzymes for the corresponding substrates, viz. pectate and starch. Normally interaction between an enzyme and its substrate is accompanied by hydrolysis of the polymeric substrate, resulting in total biodegradation. By specific modification of the substrate it is possible to obtain adsorbents which are capable of binding the enzyme while being resistant against biodegradation.
Pectate is the natural substrate for endo-polygalacturonase. Alginate, a substrate analogue for pectate, is able to bind endo-polygalacturonase while it is not hydrolyzed by the enzyme. Rigid beads can be obtained by calcium complexation of the alginate. The pH and ionic strength of the incubation medium influence the strength of the interaction between endo-polygalacturonase and alginate beads. Adsorption end desorption can be controlled by these two parameters. In this way the enzyme can be isolated and purified from complex mixtures. The adsorbent can be regenerated at least a hundred times in a continuous process (Chapter 2).
The adsorption of the enzyme to the matrix was subject of further study. By determining relevant mass transport parameters such as adsorption equilibrium parameters, diffusion coefficients and rate parameters it appeared to be possible to describe the adsorption process in mathematical terms. The velocity of adsorption is determined by the diffusion velocity of the enzyme in the beads and not by the reaction kinetics of the complex formation. The velocity of the desorption process is also determined by the diffusion velocity of the enzyme out of the bead (Chapter 3).
The most important substrate for α-amylase is starch. Alpha-amylase is used on a large scale for the enzymic conversion of starch into limit dextrins and other oligosaccharides. By means of a chemical crosslinking procedure of starch an adsorbent is obtained which is capable of binding the enzyme while it is degraded only to a limited extent. The adsorption and desorption characteristics of the interaction between enzyme and adsorbent were studied. It appears that the enzyme has the highest affinity for the adsorbent at the pH where it has its maximum catalytic activity. The interaction is biospecific and this principle allows a very selective isolation of the enzyme. The interaction between enzyme and adsorbent is essentially insensitive to changes in ionic strength of the medium. Desorption can be accomplished by a shift of pH or a raise in temperature of the incubation medium (Chapter 4).
The adsorption characteristics were further evaluated. Continuous use of the adsorbent in an isolation process of α-amylase results in a slow biodegradation of the matrix. This effect is accompanied by an increase of capacity of the adsorbent for the enzyme. It appears that the adsorbent can be used repeatedly for the isolation of the enzyme, the biodegradation is made up for by an improved mass transfer of the enzyme into the matrix combined with the increased capacity. The rate of adsorption is determined by the diffusion rate of the enzyme into the porous gel (Chapter 5).
For the direct application of adsorbents in fermentation broths a number of techniques have been proposed. One of these is the use of a fluid bed column. This imposes a few demands on the density and the diameter of the adsorbent. Particles, suitable for use in fluid bed columns were developed by inclusion of crosslinked starch in alginate particles and by the preparation of an alginate/starch copolymer bead. The adsorption characteristics of these adsorbents are comparable with those of crosslinked starch (Chapter 6).
In conclusion it can be stated that affinity separations for endo-polygalacturonase and α-amylase prove to be a selective process with good potential for a one step purification of these enzymes from a fermentation broth. In addition the procedure of adsorbent preparation offers good opportunities to prepare affinity adsorbents for other hydrolases.
This study was performed in a partnership between the Department of Food Science (Food and Bioprocess Engineering Group and Food Chemistry and Food Microbiology Group) and the Department of Genetics. The project was financed by the Netherlands Technology Foundation (STW).
Oligo- and polysaccharide synthesis by Rhizobium leguminosarum and Rhizobium meliloti
Breedveld, M.W. - \ 1992
Agricultural University. Promotor(en): A.J.B. Zehnder; L.P.T.M. Zevenhuizen. - S.l. : Breedveld - 127
rhizobium - micro-organismen - biochemie - metabolisme - synthese - genetica - polysacchariden - rhizobium - microorganisms - biochemistry - metabolism - synthesis - genetics - polysaccharides
Rhizobium and Agrobacterium species are capable of synthesizing a variety of extracellular and cellular oligo- and polysaccharides. Changes in environmental conditions may all affect the composition, physical properties, and relative amounts of oligo- and polysaccharides. Interest in the field of Rhizobium polys accharides has resulted from a development in two distinct areas, (i) the role of oligo- and polysaccharides in the microbe- plant interaction, and (ii) studies on the physico- chemical properties of microbial polysaccharides with a potential technical application (CHAPTER 1).
In this thesis two model strains, Rhizobium leguminosarum bv trifolii TA-1 and Rhizobium meliloti SU-47, were used to study the extent of polysaccharide-production as influenced by cultural conditions. Although this study was aimed at polysaccharide-production in general, most research was focused on the regulation of cyclic (1,2)-β-glucan synthesis. Therefore, inCHAPTER2 a summary on the chemistry, occurrence, biological function and potential applications of these compounds is given.
CHAPTER3 describes the enhanced excretion of cyclic (1,2)-β-glucan by Rhizobium leguminosarum bv trifolii TA-1 as the result of (i) incubation at superoptimal temperature for growth (30-33°C) and (ii) growth at high cell densities. At 33°C, EPS and CPS production was inhibited and up to 3.9 g of cyclic (1,2)-β-glucan/l was produced in the production medium (10 g mannitol and 1 g glutamic acid/l medium) with a rate of 400 mg glucans/g protein/day by a biomass of 540 mg protein/l. At 25°C, the optimal temperature for growth of strain TA-1, EPS and CPS were the main carbohydrate products synthesized, while hardly any glucans were detected in the medium. Other R. leguminosarum strains showed a comparable "temperature-effect". In a medium containing 50 g mannitol and 10 g glutamic acid per liter high cell densities of strain TA-I (3.95 g protein/l) were obtained and 10.9 g/l of cyclic (1,2)-β-glucan within 10 days at 25°C, were excreted, while CPS production was strongly suppressed. The cyclic (1,2)-β-glucans were neutral and had degrees of polymerization (DP) ranging from 17 to 25 with DP=19 as the major glucan cycle.
In production processes, aimed at high volumetric yields, high concentrations of organic nutrients and mineral salts in liquid media may lead to a considerable high osmotic pressure. Therefore, and because synthesis of cyclic glucans were found to be osmo-regulated in some members of the family of Rhizobiaceae, the influence of the osmotic pressure of the medium on the growth of and polysaccharide synthesis by R. meliloti SU-47 and R. leguminosarum bv trifolii TA-1 was studied (CHAPTERS4 and 5). The ability of members of the Rhizobiaceae to grow at high osmotic pressures of the medium, and their tolerance towards several ionic and non-ionic osmolytes, depend strongly on the species and the type of osmolyte. Strains of R. meliloti and A. tumefaciens could grow well in media up to 1 M NaCl while R. leguminosarum could only tolerate up to 0.35 M NaCl. In standard- or production medium with low osmotic pressure cells of strain SU-47 accumulated up to 350 mg cyclic (1,2)-β-glucans/g protein, of which 36% were glycerol-1-phosphate substituted and 64% were neutral. By increasing the osmotic pressure of the medium by the addition of NaCl or other ionic and non-ionic osmolytes, succinoglycan production could be stimulated (up to 2.4 g/l at 0.2 M NaCl), at the expense of the repeating units. Furthermore, the amount of cellular cyclic (1,2)-β-glucans was lowered, to 150 mg/g protein at 0.6 M NaCl, of which the glycerol-1-phosphate substituted glucan fraction was reduced to 15%. Instead, oligosaccharides up to 250 mg/g protein were synthesized with trehalose as the major component. Glycogen synthesis was fully suppressed at this salt concentration. No cyclic glucans were found in the medium (CHAPTER4). By increasing the osmotic pressure of the medium, the synthesis of EPS and CPS by R . leguminosarum bv trifolii TA-1 was suppressed, and cyclic glucans were excreted instead (1500- 2000 mg of glucans/l). This proceeded with a rate of 220 mg glucans/g protein/day by a biomass of 520 mg protein/l. at 0.2 M NaCl. Intracellular cyclic (1,2)-β-glucan concentrations remained at 45-100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells, up to 130 mg/g protein (CHAPTER5).
The response to a NaCl-shock on cellular carbohydrates of NaCl-free grown cells of R. leguminosarum bv trifolii TA-1 and R. meliloti SU- 47 was investigated in non-growing cultures in a batch-fermenter and in cell suspensions using in vivo NMR (CHAPTER6). In a glutamic acid-free medium containing NaCl TA-1 cells but especially SU-47 cells responded immediately by synthesizing trehalose, while glycogen and the external substrate mannitol were metabolized. Without mannitol in the medium trehalose synthesis was slower and parallelled the breakdown of the reserve materials glycogen and PHB. 13C-NMR experiments with 25-fold concentrated cell-suspensions using 13C 1 -mannitol as substrate revealed that 20% of the trehalose synthesized was derived from the substrate, but 80% from other sources. Therefore, trehalose synthesis occurred from the internal pool of glycogen and/or PHB, whether mannitol was present or not. Cells of strains TA- 1 and SU-47 that had accumulated trehalose metabolized this compound again in a low osmolarity environment. As trehalose is a general occurring compound found in cells of Rhizobium at much lower concentrations the higher concentrations which were measured during the conditions of osmotic stress could be explained by the role of trehalose as osmo-protectant. The cellular phosphoglycerol-substituted and neutral cyclic (1,2)-β-glucans of NaCl-free grown SU-47 cells were not degraded nor excreted after the NaCl-shock. With in vivo31P-NMR the glycerol-1-phosphate substituted cyclic glucans in cell suspensions of strain SU-47 could well be observed.
InCHAPTER7 the synthesis of cyclic (1,2)-β-glucans from UDP-[ 14C]-glucose by a crude membrane preparation and whole cells of Rhizobium leguminosarum by trifolii TA-1 was investigated. The observed enhanced excretion of cyclic (1,2)-β-glucans by strain TA-1 was due to an increased permeability of the outer membrane for cyclic glucans. This was concluded from the following observations: (i) Incubation of repeatedly frozen and thawed TA-1 cells with UDP- 14C-glucose resulted in the appearance of a labelled glucan in the medium. Cells grown in the presence of NaCl and cells cultured at 33°C showed the same effect, while cells cultured at 25°C in the absence of NaCl did excrete only little cyclic glucans; (ii) the presence of 0.1 mM EDTA, a complexing agent of divalent cations which results in a weakened LPS layer, also induced glucan excretion in growing cultures of strain TA- 1; (iii) R. leguminosarum bv viciae RBL5515, exoB8 ::Tn5 mutant cells excreted a labelled glucan fraction, while the parent strain RBL-5515 did not. The mutant was affected in the production of EPS, CPS, and LPS (CHAPTER9), which all might explain the greater permeability of the mutant cells towards cyclic (1,2)-β-glucans. Hardly any difference in biosynthetic activity was observed between membrane fractions of TA-1 cells grown in the presence (0.2 M) or absence of NaCl. Glucan formation in vitro and glucan excretion by whole cells was strongly inhibited in the presence of 50 mg/ml cyclic glucan, indicating biosynthesis of cyclic (1,2)-β-glucans in strain TA-1 to be controlled by end-product inhibition. Therefore, the constant loss of glucans from osmotically-stressed TA-1 cells prevented end- product inhibition and resulted in glucan accumulation in the medium of up to 1600 mg/l. Cyclic (1,2)-β-glucans from Rhizobium meliloti and Agrobacterium tumefaciens are thought to be involved in both infection behaviour and osmo- regulation at low osmotic pressure of the medium. Combining the results obtained on the synthesis of cyclic glucans, it is doubtful that cyclic glucans of R . leguminosarum play an important role in osmo-adaptation at low osmolarity, for the following reasons: (i) cellular glucan concentrations are much lower than in R. meliloti; (ii) the great majority of the glucans in R. leguminosarum are neutral molecules, and (iii) no repression of glucan synthesis at enhanced osmolarity of the medium occurs.
InCHAPTER8 Rhizobium leguminosarum bv trifolii TA-1 was used a model organism to study the influence of growth rate and medium, composition on exopolymer production. Circumstances leading to a high CPS-yield also lead often to concomitantly high EPS-production, rendering the medium viscous, and making the process of recovery of CPS more difficult. In continuous cultures, EPS was at every dilution rate (between D=0.02-0.12 h -1) the most abundant polysaccharide present in production medium, while CPS synthesis occurred only at low specific growth rates. Only low amounts of cyclic glucans were excreted (10-30 mg/l). In production medium EPS was synthetized in the active phase of growth and continued in stationary phase (up to 1.6-2.1 g/l CPS-synthesis which takes place only in stationary phase and in the presence of excess mannitol, was produced up to 1.8 g/l during batch incubation for 14 days at 25°C. Maximal CPS production was 2.9 g CPS/l medium, with 1 g protein as biomass in a medium containing 20 g/l mannitol and 2 g/l glutamic acid. The maximum specific growth rate was μ max =0.133 h -1. To washed cells at which 10 g/l mannitol was added CPS synthesis reached 2.1 g/l, but EPS- synthesis was lower (0.8 g/l).
InCHAPTER9 the polysaccharide production by Rhizobium leguminosarum RBL5515 and some Tn::5 generated mutants affected in polysaccharide synthesis was studied in production medium. The EPS of the wildtype strain was composed of a K-36 type octasaccharide repeating unit with sugar composition of glucose: glucuronic acid: galactose in the ratios of 5:2:1. The CPS from the R. leguminosarum strains investigated had all a constant sugar composition of glucose: galactose: mannose in the ratios of 1:4:1. The presence or absence of either the pRL1JI or pSym5, the Sym plasmids of R. leguminosarum bv viciae and trifolii, respectively, did not influence the quantities of these polysaccharides synthesized, being comparable to strain TA-1. The production patterns of exopolysaccharides of their Tn::5 generated mutants were clearly different from most wild-type production patterns by the synthesis of altered EPS-structures, low production of CPS and/or EPS, and enhanced excretion of cyclic (1,2)-β-glucans (all mutants; 400-1000 mg/l) as compared to strain RBL5515 (50 mg/l). The mutants RBL5515, exo4 ::Tn5, RBL5515, exoB8 ::Tn5 and RBL5515, exo344 ::Tn5 formed 5-20% of wildtype EPS-level. Both mutants RBL5515, exoB8 Tn::5 and RBL5515, exo344 ::Tn5 synthesized a truncated EPS with a heptasaccharide-repeating unit missing the terminal galactose in the side chain. While the RBL5515, exoB8 ::Tn5 mutant was shown to be devoid of UDP-glucose 4' epimerase activity, the activity of this enzyme and of other enzymes involved in the synthesis of UDP-galactose were comparable in RBL5515, exo344 ::Tn5 and the wildtype. It was concluded that RBL5515, exo344 ::Tn5 was affected in a galactose transferase activity. Since this mutant failed to nodulate plants belonging to the pea inoculation group, and the presence of the terminal galactose in the side chain of the EPS of R. leguminosarum bv viciae was not required for succesful nodulation, it was postulated that the amount of EPS produced by RBIL5515 exo344 ::Tn5 and the herewith related viscosity is insufficient for nodulation.
Finally, CPS of R. leguminosarum is a polymer which forms gels already at 0.2 % w/ v which is even lower than for agar and therefore has considerable potential applications. Cyclic (1,2)-β-glucans are potentially useful molecules because of their relatively hydrophobic internal space, which make them suitable as an inclusion agent. By choosing the adequate cultural conditions and/or strains, R.leguminosarum was able to produce CPS (CHAPTERS8, 9) or cyclic glucans (CHAPTERS3,5) as the main polysaccharides. The aim of this thesis was to extend the knowledge on the production of Rhizobium polysaccharides as influenced by cultural conditions. The results described in this thesis work can help to stimulate the application-oriented research on hydrocolloïds (CPS, EPS) and inclusion agents (cyclic glucans), and may lead to a better understanding of the biological role of these compounds.
Structural characteristics of wheat flour arabinoxylans = Structuurkenmerken van arabinoxylanen uit tarwebloem
Gruppen, H. - \ 1992
Agricultural University. Promotor(en): A.G.J. Voragen; R.J. Hamer. - S.l. : Gruppen - ISBN 9789054850489 - 157
tarwebloem - polysacchariden - wheat flour - polysaccharides - cum laude
A procedure has been developed for the isolation of highly purified water-unextractable cell wall material (WUS) from wheat flour. From this material arabinoxylans were extracted via sequential extraction with Ba(OH) 2 , KOH and NaOH solutions. The first extract comprised arabinoxylans only, which represented 80% of all arabinoxylans present in the WUS. The arabinoxylans in this extract were fractionated using anion exchange chromatography and graded- ethanol precipitation. Neutral sugar and glycosidic linkage analysis revealed great similarities between these alkali-extractable arabinoxylans and similarly fractionated water-extractable arabinoxylans, isolated from the same wheat flour. Enzymic analysis using different endo-(1-4)- β-D-xylanases from Aspergillus awamori showed, however, that the water-extractable arabinoxylans were degraded both faster and to a larger extent than the alkali-extractable arabinoxylans.
Based on the structure and abundance of enzyme-resistant polymeric material and different oligosaccharides, the latter being identified with 1H-NMR analysis, present in the enzyme digests, a structural model is proposed for the arabinoxylans. This model shows that the arabinoxylan contains highly branched regions, interlinked with less branched regions which include subregions of unsubstituted xylose up to 7 contiguous residues. Variation in arabinose/xylose ratio between different arabinoxylan is due to variation in the relative proportion as well as the composition of the less branched regions. With increasing arabinose/xylose ratio of the arabinoxylans a decreasing extent of enzymic degradation observed. The enzymes used had different effects on the degradation of WUS and extracted arabinoxylans as well as on the baking performance of wheat flour.
Extracellular polysaccharides as target compounds for the immunological detection of Aspergillus and Penicillium in food
Kamphuis, H.J. - \ 1992
Agricultural University. Promotor(en): F.M. Rombouts; S.H.W. Notermans. - S.l. : Kamphuis - ISBN 9789054850199 - 157
voedselbesmetting - voedselmicrobiologie - aspergillus - penicillium - polysacchariden - food contamination - food microbiology - aspergillus - penicillium - polysaccharides
This thesis is devoted to the immunological detection of Aspergillus and Penicillium in food products. More specifically, the immunogenicity, antigenicity, production and structure of the water-soluble extracellular polysaccharides (EPS) of these moulds have been studied, and a latex-agglutination assay, based on the detection of EPS has been developed.
For the detection of moulds many methods are available, each of them with specific advantages and disadvantages, mostly related to reliability and applicability ( Chapter 2 ).
An overview of the immunogenicity and antigenicity of EPS produced by moulds is presented in Chapter 3 . The role of β(1,5)-galactofuranoside sequences as epitopes of galactomannans from Aspergillus and Penicillium is documented. Antigenically specific polyclonal antibodies raised against P.digitatum EPS are directed towards β(1,5)-linked galactofuranosyl residues. These antibodies react specifically with EPS from Aspergillus and Penicillium.
Synthetic tetramers and heptamers of β(1,5)-linked galactofuranosides are conjugated to tetanus toxoid and polyclonal antibodies are raised in rabbits against these synthetic immunogens ( Chapter 4 ). Antibodies obtained after immunisation with the heptamer conjugate possess the same genus specific antigenicity as the antibodies raised against P.digitatum EPS. No reactions are observed with the Penicillium subgenus biverticillium species and species belonging to genera other than Aspergillus and Penicillium . In contrast, antibodies raised against the tetramer conjugate reacted only with six out of 24 tested Aspergillus and Penicillium strains.
From Glucanex, a Trichoderma harzianum enzyme preparation, an exo-β-D- galactofuranosidase is purified. This enzyme is used to hydrolyse specifically the immunodominant β(1,5)-linked galactofuranosyl residues from Aspergillus and Penicillium EPS. This enzyme alleviates the antigenicity of the EPS completely ( Chapter 5 ).
Additionally, the reductive cleavage method for determination of the glycosidic bonds revealed that the β(1,5)-linked galactofuranosyl side chains in P.digitatum EPS carry side-chains of β(1,6)-1inked galactofuranosyl residues. These results allowed to propose a new structural model for the antigenically active galactofuranoside side chains of Penicillium galactomannans.
In Chapter 6 , the production of antigenic EPS by P. aurantiogriseum and P. digitatum has been described under various growth conditions. Antigenic EPS was produced under almost all conditions investigated. However, both P.aurantiogriseum and P.digitatum do not produce antigenic EPS on lactate as the carbon source. Also, P.camemberti isolated from a mould fermented cheese (Camembert) does not produce antigenic EPS on lactate, althought, P.camemberti produces antigenic EPS on other substrates. The monosaccharide composition of the EPS produced by P.aurantiogriseum and P.digitatum under various conditions varies considerably.
Immunopotent acid-labile β(1,5)-linked galactofuranosyl residues of Aspergillus fumigatus, Aspergillus niger and Penicillium digitatum EPS were acid-hydrolysed ( Chapter 7 ). Antibodies are raised against these acid-treated extracellular polysaccharides. It was supposed that these acid-treated EPS preparations would elicit antibodies with a broader specificity, making them useful in the detection of nearly all or all moulds occurring in food products. However, the antibodies obtained are more species specific and are generally directed to glucosyl and/or mannosyl residues of the EPS.
Antibodies raised against P.digitatum EPS are used for the development of a rapid and reliable latex-agglutination assay for the detection of Aspergillus and Penicillium in food and feed ( Chapter 8 ). The reliability of the assay is enhanced by using a synthetic epitope, a tetramer of β(1,5)-Iinked galactofuranosides. With this tetramer false-positive results can easily be recognised.
Finally, in Chapter 9 the applicability of the developed latex-agglutination assay is tested in both comparative and collaborative studies. The significance of extracellular polysaccharides produced by moulds for the detection of moulds in food and feed is discussed.
Literatuuronderzoek over toepassing en analyse van polysacchariden in levensmiddelen
Essers, M.L. ; Muuse, B.G. - \ 1982
Wageningen : RIKILT (Verslag / RIKILT 82.32) - 18
polysacchariden - koolhydraten - verdikkingsmiddelen - voedseladditieven - voedselanalyse - analytische methoden - polysaccharides - carbohydrates - thickeners - food additives - food analysis - analytical methods
Doel van dit onderzoek is: Verkrijgen van inzicht in de toepassingen en de bepalingsmetboden van polysacchariden in levensmiddelen, teneinde de verschillende verdikkingsmiddelen te kunnen bepalen. Bestudeerd werden de wettelijke regelingen in Nederland, toepassingen in levensmiddelen en de analytiek van polysacchariden in levensmiddelen.
Function, structure and metabolism of the intracellular polysaccharide of Arthrobacter
Zevenhuizen, L.P.T.M. - \ 1966
Wageningen : Veenman (Mededelingen / Landbouwhogeschool 66-10) - 80
arthrobacter - polysacchariden - corynebacteriaceae - arthrobacter - polysaccharides - corynebacteriaceae
|Arabanases in phytopathogenic fungi
Fuchs, A. ; Jobson, J.A. ; Wouts, W.M. - \ 1965
Wageningen : [s.n.] (Mededeling van het Laboratorium voor phytopathologie no. 216) - 4
plantenziekteverwekkende schimmels - polysacchariden - plant pathogenic fungi - polysaccharides
|Chromatografisch onderzoek naar de samenstelling van de polysacchariden uit de celwand in verband met de analyse van ruwvoeders
Gaillard, B.D.E. - \ 1954
Wageningen : [s.n.] - 65
polysacchariden - voer - chemische samenstelling - voedergewassen - zoötechniek - fysiologie - celwanden - polysaccharides - feeds - chemical composition - fodder crops - zootechny - physiology - cell walls
De verandering van de rotatie en van het geleidend vermogen van enkele suikers in waterige oplossing met en zonder boorzuur
Verschuur, R. - \ 1927
Wageningen : Veenman (Mededelingen van de Landbouwhogeschool te Wageningen dl. 31, verh. 1) - 118
monosacchariden - polysacchariden - elektrochemie - monosaccharides - polysaccharides - electrochemistry