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Characterization of Coxiella burnetii outbreak strains
Kuley, Runa - \ 2017
Wageningen University. Promotor(en): Mari Smits; Jerry Wells, co-promotor(en): Alex Bossers. - Wageningen : Wageningen University - ISBN 9789463431514 - 226
coxiella burnetii - q fever - outbreaks - strains - characterization - pathogenesis - zoonoses - virulence - dna sequencing - polymerase chain reaction - livestock farming - netherlands - q-koorts - uitbraken (ziekten) - stammen (biologisch) - karakterisering - pathogenese - zoönosen - virulentie - dna-sequencing - polymerase-kettingreactie - veehouderij - nederland
Q fever is a worldwide zoonotic infectious disease caused by the bacterium Coxiella burnetii. During 2007-2010, the largest Q fever outbreak was reported in The Netherlands, where more than 4000 human cases were registered showing a serious burden of the disease. During this outbreak, goats harboring predominantly the CbNL01 genotype strain were identified as the major source of disease in humans and drastic measures such as mass culling of infected goats were implemented to reduce the spread of the pathogen and control the disease. In order to minimize such complications in the future, it is crucial to have a thorough understanding of the disease causing pathogen and to develop effective Q fever vaccines. The causes of the large Dutch outbreak are not well-understood and one of the main reasons speculated were the hyper-virulent behavior of the circulating C. burnetii isolates. The research described in this thesis focuses on the characterization of C. burnetii outbreak strains isolated from infected goats, cattle, sheep and human clinical materials. Our studies were initiated to better understand the bacterial pathogenesis, virulence, evolution, adaptations in various environments, host immune responses and to identify pathogen related factors that have modulated the disease outbreak. We specifically aimed to identify the virulence factors and mechanisms that contributed to the increased zoonotic potential of the strain associated with the Dutch Q fever outbreak.
The studies presented in this thesis majorly applied Pathogenomic approaches at the genome and transcriptome level to decipher host-pathogen interactions and to develop new tools to study C. burnetii infections. A transcriptome analysis of the outbreak C. burnetii strain of the CbNL01 genotype grown under in vivo and in vitro conditions resulted in the identification of distinct metabolic adaptations and virulence mechanisms of the bacterium. Detailed comparative analysis of complete genome sequences of C. burnetii strains showed a high similarity between strains of the same genotype. Genome sequences of the Dutch outbreak CbNL01 genotype strains were more divergent than the genome sequences of the less prevalent CbNL12 genotype strains and the NM reference strain. The analysis also showed that the high virulence of the outbreak strains was not associated with acquiring novel virulence-related genes arguing against the idea that the Dutch outbreak was due to emergence of hyper-virulent strains though horizontal gene transfer. Among the prominent genetic differences in the CbNL01 outbreak strains compared to CbNL12 and NM, were the presence of several point mutations and increased transposon mediated genome plasticity, which might have contributed to its epidemic potential. Point mutations, especially in a large number of membrane proteins, could also have contributed to the increased zoonotic potential of CbNL01 strains allowing this clone to escape the host immune responses in goats and humans. In addition, mutations in critical genes involved in virulence and evasion of the host immune system could be potentially involved in the increased virulence of the CbNL01 outbreak strains. On the contrary, studies on host immune responses in an in vivo (experimental infections in mice) and an in vitro (human PBMC’s stimulation) model did not show any difference associated with the strain genotype. However, differences in immune responses were found to be associated with the host-origin of the C. burnetii strains. Among different host-origin strains, strains derived from goats and humans generated significantly lower innate and adaptive immune responses than strains derived from cattle, whereas no differences in immune responses were observed when strains were grouped based upon their genotype. These observations support immune evasions as a major virulence strategy of goat and human strains in hosts and further suggest that bacteria originating from goats have a greater potential to cause outbreaks in humans. This indicates that for Q fever prevention purposes goats should be efficiently monitored for the presence of C. burnetii. Taken together, the results described in this thesis suggest that the virulence potential of C. burnetii strains is not only based on genetic differences, but also on other host-adaptation mechanisms such as transposition of genomic elements and/or differential regulation of gene expression. Finally, the results from this thesis provide a framework for future studies in the development of vaccines and diagnostic tools for Q fever.
Structure and fermentation of natural and manufactured lactose-based oligosaccharides
Difilippo, E. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576155 - 128 p.
milks - lactose - oligosaccharides - ingestion - bioactive compounds - isolation - characterization - fermentation - colostrum - food analysis - melksoorten - oligosacchariden - inname - bioactieve verbindingen - isolatie - karakterisering - fermentatie - voedselanalyse
At early stages of life, infant immature intestine is not fully developed, exposing the new-born to potential diseases. Compounds that can exert beneficial actions on the infant intestine are bioactive lactose-based oligosaccharides (LBOs). The natural source of LBOs is mother milk. When human milk is lacking, dietary supplementation with infant formula fortified with manufactured LBOs, such as galacto-oligosaccharides (GOS), is pursued. GOS have been shown to have several properties in common with HMOs. LBOs composition and intestinal fate is extensively described for humans, whereas they are hardly investigated for domestic animal. In this PhD thesis, composition of LBOs in equine and porcine colostrum were described and new structures were elucidated. The analysis were performed mainly using liquid chromatography and capillary electrophoresis techniques. High inter- and intra-individual variation were found for oligosaccharides present in equine and porcine milk. In vivo fermentation fate of porcine milk oligosaccharides (PMOs) was also described analysing PMOs as found in fecal samples of piglets. The results were correlated to existing literature on HMOs. Dietary oligosaccharides are partially present systemically, as suggested from HMO studies. GOS and PMOs in blood, urine and fecal samples from an in vivo feeding trial on piglet were described. Intact dietary oligosaccharides including GOS and milk oligosaccharides from the piglet diet were found in piglet blood and urine samples. All dietary oligosaccharides were fermented/absorbed in vivo, not being detectable in the piglet fecal samples. On the other hand, GOS in vitro fermentation by piglet inoculum delineate a unique fermentation profile regarding GOS size consumption compared to GOS in vitro fermentation by human fecal inoculum. Similar degradation profile regarding GOS linkage types was observed for GOS fermentation by piglet and human inocula.
Isolation, characterization and engineering of Bacillus smithii : a novel thermophilic platform organism for green chemical production
Bosma, E.F. - \ 2015
Wageningen University. Promotor(en): Willem de Vos; John van der Oost, co-promotor(en): Richard van Kranenburg. - Wageningen : Wageningen University - ISBN 9789462575073 - 220
bacillus smithii - bacillus (bacteria) - biobrandstoffen - chemicaliën uit biologische grondstoffen - thermofielen - metabolische profilering - genoomanalyse - mutaties - isolatie - bioengineering - karakterisering - biofuels - biobased chemicals - thermophiles - metabolic profiling - genome analysis - mutations - isolation - characterization
Due to the globally increasing demand for chemicals and fuels and the high environmental impact and limited amount of fossil resources, there is a growing interest in green chemicals and fuels derived from renewable resources. As described in Chapter 1, one of the most feasible alternatives on the short term is microbial conversion of the sugars in biomass to fuels and chemicals in a biorefinery. To be economically and ethically feasible, non-food biomass should be used as a resource, which is often difficult with currently used production organisms. Also, to be economically feasible, the costs of green chemicals and fuels need to be further reduced to be below the costs of products based on fossil resources. To do so, other organisms than the currently most-used platform organisms such as Escherichia coli and Saccharomyces cerevisiae should be used. Ideally, this alternative organism is genetically accessible, has high productivity, titre and yield, is flexible in carbon source, robust, moderately thermophilic, acidophilic, facultatively anaerobic and has little nutritional requirements. The organisms that come closest to these criteria are thermophilic bacilli, which form a diverse class of organisms in the family of Bacillaceae. This thesis describes the isolation, characterization and metabolic engineering of Bacillus smithii, a novel potential thermophilic platform organism.
Chapter 2 provides more detail on the use of thermophilic microorganisms as platform organisms for green chemical production in a biorefinery concept. As commercially available enzyme mixtures used in the simultaneous saccharification and fermentation (SSF) of biomass have their optimum temperature around 50-60°C, using a moderately thermophilic organism would reduce the costs of the SSF process compared to when using mesophiles by reducing the amount of required enzyme. Also, thermophilic processes are less prone to contaminations, and substrate and product solubility are increased. Several successful examples of the application of facultatively anaerobic thermophiles for green chemical production from lignocellulose in an SSF setting are for example Bacillus coagulans for lactic acid production and Bacillus licheniformis for 2,3-butanediol production. However, whereas strongly developed genetic toolboxes are available for current mesophilic production organisms, these tools are still in their infancy for thermophilic organisms. Such tools are required to optimize production and to study metabolism. Thermophilic organisms show a wide variety in metabolism and in many cases the metabolism of these organisms is still poorly understood, hampering full optimization. Chapter 2 furthermore provides an overview of transformation, integration and counter-selection methods currently used for thermophiles. Although several deletion mutants have been constructed using these methods, not all of them are entirely markerless and most are not suited as high-throughput engineering tools, stressing the need for further research in this area.
Despite several facultatively anaerobic thermophiles being described as genetically accessible, this feature is still one the major bottlenecks in developing these organisms into platform organisms. Therefore, in Chapter 3, we set out to isolate a facultatively anaerobic, moderately thermophilic bacterium that was genetically accessible and produced high titers of organic acids. A total of 267 strains of different thermophilic bacilli species were isolated from compost and screened for C5 and C6 sugar utilization and acid production. The 44 best strains were screened for genetic accessibility via electroporation. Only 3 strains tested positive for this, namely Geobacillus thermodenitrificans strains ET 144-2 and ET 251 and B. smithii strain ET 138. In subsequent evaluations in lab-scale bioreactors at 55°C and pH 6.5 on glucose, the two G. thermodenitrificans strains performed poorly whereas B. smithii performed well with high titers, yields and productivity of mainly lactate. In similar lab-scale reactors, this strain also performed well on xylose and at pH 5.5 and was still able to perform for 48 at pH 4.5. The electroporation protocol for this strain was optimized, resulting in a maximum efficiency of 5x103 colonies per µg plasmid pNW33n. Two other B. smithii strains, among which the type strain DSM 4216T, were also shown to be transformable with pNW33n. This is the first time that genetic accessibility is described for B. smithii and it is the first step towards developing it into a platform organism, for which it appears to be suitable based on its efficient C5 and C6 sugar utilization and acid production profile.
In order to become a platform organism and to study its atypical metabolism, a genetic toolbox needs to be established for B. smithii. Chapter 5 describes the development of a markerless gene deletion method for B. smithii. For strains ET 138 and DSM 4216T, the ldhL gene was markerlessly removed via double homologous recombination using plasmid pNW33n. Despite the replicative nature of this plasmid at 55°C, mixtures of single and double crossovers were readily obtained. A pure double crossover deletion mutant was obtained after several transfers on a more defined medium containing acetate or lactate and PCR-based screenings. To eliminate the possibility of mixed genotypes, we subsequently developed a lacZ-counter-selection system, which is based on the toxicity of high X-gal concentrations in the presence of the plasmid-encoded lacZ gene. Using this method, the sporulation-specific sigma factor sigF and pyruvate dehydrogenase complex E1-α pdhA were consecutively removed from the B. smithii ET 138 genome in a markerless way. An initial evaluation of the growth and production profiles of the mutant strains in tubes showed that removal of the ldhL gene eliminates l-lactate production and causes a severe decrease in anaerobic growth and production capacities. B. smithii mutants lacking the sigF gene were unable to sporulate and removal of the pdhA gene eliminated acetate production and rendered the strains auxotrophic for acetate.
Microbubble stability and applications in food
Rovers, T.A.M. - \ 2015
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): Marcel Meinders; Guido Sala. - Wageningen : Wageningen University - ISBN 9789462574755 - 138
microbubbles - eiwit - stabiliteit - karakterisering - voedsel - voedseladditieven - oppervlaktespanningsverlagende stoffen - zuurbehandeling - reologische eigenschappen - sensorische evaluatie - tribologie - druk - verwarming - koelen - protein - stability - characterization - food - food additives - surfactants - acid treatment - rheological properties - sensory evaluation - tribology - pressure - heating - cooling
Aeration of food is considered to be a good method to create a texture and mouthfeel of food products that is liked by the consumer. However, traditional foams are not stable for a prolonged time. Microbubbles are air bubbles covered with a shell that slows down disproportionation significantly and arrests coalescence. Protein stabilized microbubbles are seen as a promising new food ingredient for encapsulation, to replace fat, to create new textures, and to improve sensorial properties of foods. In order to explore the possible functionalities of microbubbles in food systems, a good understanding is required regarding the formation of protein stabilized microbubbles as well as their stability in environments and at conditions encountered in food products. The aim of this research was to investigate the key parameters for applications of microbubbles in food systems. In Chapter 1 an introduction to this topic is given.
In Chapter 2, the effect of the microbubble preparation parameters on the microbubble characteristics, like the microbubble yield, size and stability, was investigated. The protein Bovine Serum Albumin (BSA) and the method sonication was used to manufacture the microbubbles. The manufactured number and stability of microbubbles was highest when they were prepared at a pH around 5 to 6, just above the isoelectric point, and at an ionic strength of 1.0 M. This can be related to the protein coverage at the air/water interface of air bubbles formed during sonication. At a pH close to the isoelectric point the BSA molecules is in its native configuration. Also the repulsion between the proteins is minimized at these pH values and ionic strength. Both the native configuration and the limited repulsion between the proteins result in an optimal protein coverage during the first part of sonication. Also a high protein concentration contributes to a higher surface coverage. The surface coverage is proportional to the protein concentration up to a concentration of 7.5% after which an increase in protein concentration did not lead to a substantial increase in the number of microbubble . In the second part of sonication the protein layer around the air bubble becomes thicker and stronger by heat induced protein-protein interactions. We found that and at a preheating temperature of 55-60°C, about 5 °C below the BSA denaturation temperature, and a final solution temperature of 60-65°C most microbubbles were obtained, while at higher temperatures mainly protein aggregates and (almost) no microbubbles are formed. This suggests that at temperature of around 60°C to 65°C protein aggregated mostly at the air-water interface creating a multi-layered shell, while at higher temperature, they also aggregated in bulk. These aggregates cannot form microbubbles. We found that optimal preparation parameters strongly depend on the protein batch. We hypothesize that the differences in microbubble formation between the protein batches is due to (small) differences in the protein molecular and denaturation properties that determine the temperature at which the molecules start to interact at the air-water interface. Microbubbles made with different protein concentration and preheating temperatures shrunk in time to a radius between 300 nm and 350 nm, after which the size remained constant during further storage. We argue that the driving force for the shrinkage was the Laplace pressure, resulting in an air flux from the bubbles to the solution. We argue that the constant final size can be explained by a thickening of the microbubble shell as a result of the microbubble shrinkage, thereby withstanding the Laplace pressure.
In Chapter 3 and Chapter 4, microbubble stability at environments and conditions representative for food products were studies. In Chapter 3 we investigated the stability upon addition of surfactants and acid, When surfactants or acid were added, the microbubbles disappeared in three subsequent steps. The release of air from the microbubble can be well described with the two-parameter Weibull process. This suggests two processes are responsible for the release of air: 1) a shell-weakening process and 2) a random fracture of the weakened shell. After the air has been released from the microbubble the third process is identified in the microbubble disintegration: 3) the shell disintegrated completely into nanometer-sized particles. The probability of fracture was exponentially proportional to the concentration of acid and surfactant, meaning that a lower average breaking time and a higher decay rate were observed at higher surfactant or acid concentrations. For different surfactants, different decay rates were found. The disintegration of the shell into monomeric proteins upon addition of acid or surfactants shows that the interactions in the shell are non-covalent and most probably hydrophobic. After surfactant addition, there was a significant time gap between complete microbubble decay (release of air) and complete shell disintegration, while after acid addition the time at which the complete disintegration of the shell was observed coincided with the time of complete microbubble decay.
In Chapter 4 the stability of the microbubbles upon pressure treatment, upon fast cooling after heating and at different storage temperatures was studied. The microbubble stability significantly decreased when microbubbles were pressurized above 1 bar overpressure for 15 seconds or heated above 50°C for 2 minutes. Above those pressures the microbubbles became unstable by buckling. Buckling occurred above a critical pressure. This critical pressure is determined by the shell elastic modulus, the thickness of the shell, and the size of the microbubble. Addition of crosslinkers like glutaraldehyde and tannic acid increased the shell elastic modulus. It was shown that microbubbles were stable against all tested temperatures (up to 120°C) and overpressures (4.7 bar) after they were reinforced by crosslinkers. From the average breaking time at different storage temperatures, we deduced that the activation energy to rupture molecular bonds in the microbubbles shell is 27 kT.
In Chapter 5, we investigated the effect of microbubbles on the rheological, tribological sensorial properties of model food systems and we compared this effect to the effect on food systems with emulsion droplets and without an added colloid. We investigated the effect in three model food systems, namely fluids with and without added thickener and a mixed gelatine-agar gel. In a sensory test panellists were asked whether they could discriminate between samples containing microbubbles, emulsion droplets or no added colloid. Emulsions could be sensorially well distinguished from the other two samples, while the microbubble dispersion could not be discriminated from the protein solution. Thus, we concluded that at a volume fraction of 5% of these BSA covered microbubbles were not comparable to oil-in-water emulsions. The good discrimination of emulsion might be ascribed to the fact that emulsion had a lower friction force (measured at shear rates form 10 mm/s to 80 mm/s) than that microbubbles dispersions and protein solutions. Upon mixing emulsions and microbubble dispersions the friction value approximated that of emulsions. This effect was already noticed at only 1.25% (v/v) oil, indicating that microbubbles had not a significant contributions to the friction of these samples. Also microbubble dispersions with and without protein aggregates were compared. The microbubble dispersions with and without thickener containing protein aggregates had a higher viscosity than the those samples without protein aggregates. Protein aggregates in the gelled microbubble sample yielded a higher Young’s modulus and fracture stress. The differences between the gelled samples could be well perceived by the panellists. We attribute this mainly to the fracture properties of the gel. In general we concluded that microbubbles, given their size of ~ 1 mm and volume fraction of 5%, did not contribute to a specific mouthfeel.
Finally in Chapter 6, the results presented in the previous chapters are discussed and put in perspective of the general knowledge on microbubbles production, stability, and applications in food. We described the main mechanisms leading to microbubble formation and stability. We showed that the production parameters significantly influence the interactions in the microbubble shell, and the those interactions highly determine the stability of the microbubbles under several conditions. We reported about limitations of sonication as a method to produce microbubbles suitable for food applications and we provided some ways to overcome these limitations. The use of microbubbles in food systems has been explored and we clearly see possible applications for microbubbles in food. We reported about directions for possible further research.
In this work we made significant progress in understanding the interactions in the microbubble shell and their relation to microbubble stability. We also advanced in comprehension towards possible applications of microbubbles in food.
Enzymatic fingerprinting and modification of acetylated pectins
Remoroza, C.A. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789461739025 - 162
pectinen - acetylering - pectine lyase - polygalacturonase - karakterisering - methodologie - pectins - acetylation - pectin lyase - characterization - methodology
To reveal the ester distribution patterns in acetylated pectins, an enzymatic fingerprinting method usinga combined endo-polygalacturonase (PG) and pectin lyase (PL) treatment followed by hydrophilicinteraction liquid chromatography coupled to electrospray ionization ion trap mass spectrometry with evaporative light scattering detection was developed. This methodpaved the way for the development of the new quantitative parameters degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL). These parameters distinguished the methylester and acetyl group distribution patterns within different sugar beet pectins (SBPs). In the case of pectin having a degree of methylesterification (DM) of >50 and acetylation of ~20, the above approach was insufficient. Hence, a seconddigestion was introduced using a fungal pectin methylesterase and a PG.More than 60% of the total GalA residues present in three SBPs were recovered as monomer and oligomers after the two digestions. The first digestion of the acid extracted commercial SBP revealed the presence of small blocks of nonesterified GalA residues and segments containing large blocks of PL degradable methylesterified and /or acetylated GalA residues. Blocks of partly methylesterified, non-acetylated GalA residues were recognized after the second digestion. These results show that the acetylation pattern is non-random.
A pectin acetylesterase (BliPAE) and a pectin methylesterase (BliPME) from Bacillus licheniformis DSM13 were produced, purified and biochemically characterized. The mode of action of BliPAE and BliPME towards acetylated pectins was revealed using the newly developed enzymatic fingerprinting method. BliPAE specifically deacetylates the O-3 linked acetyl groups of nonmethylesterified galacturonic acid residues in the homogalacturan of pectin. BliPME efficiently de-methylesterifies lemon pectins (DM34-76 ®DM 0) and SBPs (DM 30-73 ®DM 14) in a blockwise manner. BliPME is quite tolerant towards the acetyl groups present within the SBPs. For the first time, a comprehensive experimental characterization was directed to enzymes from B. licheniformis having a PAE and a PME activity.
Enzymatic modification and characterization of xanthan
Kool, M.M. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Henk Schols. - Wageningen : Wageningen University - ISBN 9789461738653 - 136
xanthan - modificatie - enzymen - karakterisering - modification - enzymes - characterization
In this thesis an enzymatic approach for the modification and characterization of xanthans was introduced. Complete backbone degradation of xanthan by cellulases was obtained independent on the molar composition of a xanthan sample. It was shown that only xanthan segments that occurred in a disordered xanthan conformation were susceptible to enzymatic backbone degradation. HILIC-ELSD-MS analysis revealed the presence of six different xanthan repeating units (RUs). All RUs consisted of the same pentasaccharide structure, with different acetyl and pyruvate substitution patterns. Interestingly the presence of an acetyl group at the O-6 position of the outer mannose unit was shown. Analysis of 5 xanthan samples showed that 5–19% of all acetyl groups present are positioned on the outer mannose. Furthermore, the relative abundance of the RUs present in xanthan samples can vary, even when their molar compositions are the same.
Analysis of the transitional behavior of xanthan based on the enzymatic release of the six types of RUs showed that the acetyl groups on the outer mannose, and not on the inner mannose, as was previously reported, are responsible for the stabilization of xanthans conformation. It was proposed that acetylation of the outer mannose also determines the functional properties of a xanthan solution. Furthermore, it was postulated that 1) The RUs that are either acetylated on the outer mannose units or solely acetylated on the inner mannose units are block wise distributed over the xanthan molecule. 2) Pyruvylated RUs and unsubstituted RUs are randomly distributed.
Screening for xanthan modifying enzymes resulted in the discovery of the first two acetyl esterases being active towards xanthan. AXE3, a xylan acetyl esterase produced by Myceliophthora thermophila C1, showed to be specific for the removal of the acetyl groups at the inner mannose unit and was only active towards the disordered xanthan conformation. YesY, a pectin acetyl esterase produced by Bacillus subtilis strain 168, specifically removed the acetyl groups at the outer mannose units and its activity is not influenced by xanthans conformation.
Bruises in Chilean cattle: their characterization, occurrence and relation with pre-slaughter conditions
Strappini, A.C. - \ 2012
Wageningen University. Promotor(en): Bas Kemp; Jos Metz, co-promotor(en): Klaas Frankena; C.B. Gallo. - S.l. : s.n. - ISBN 9789461732255 - 140
rundvee - kneuzingen - kneuzen - karakterisering - risicofactoren - slacht - slachthuizen - karkassen - bewerking - veevervoer - bedwelmen - chili - cattle - bruises - bruising - characterization - risk factors - slaughter - abattoirs - carcasses - handling - transport of animals - stunning - chile
Bruises on cattle carcass affect the quality of the meat and are indicators of poor welfare conditions. According to the literature the occurrence of bruises is related to pre- slaughter conditions, however their contribution is not clear for Chilean cattle. The aim of this thesis was to provide a better understanding of the relationship between pre-slaughter factors and the occurrence of bruises -from loading until slaughter- under Chilean conditions. Therefore in the first study slaughter records of two Chilean slaughterhouses were analysed.It showed that cows and oxen had higher risk to present bruises compared to steers and heifers. Moreover, animals that passed through a livestock market were more prone to present bruises than animals that came directly from the farm. A large difference in carcass bruise prevalence was found between slaughterhouses and this discrepancy was attributed to differences in the use of the Chilean scoring system and to several constraints of the system itself. Thus a new scoring system was developed and its reliability was assessed showing a highagreement when only one observer performs the scoring. An inventory of the gross characteristics of bruises, based on the refined bruising protocol, was carried out. Animals passing through a livestock market have more bruises than animals transported directly from the farm to the slaughterhouse. This thesis presents evidences of rough handling and animals beaten by sticks at markets.In the last study the causal event of bruises during the pre-slaughter periodwas assessed. It showed that rough handling due to inappropriate use of aids to drive animals during loading and unloading, and inadequate stunning facilities at the slaughterhouse were the areas of most risk for bruising. It was concluded that improvements in the design and maintenance of appropriate structures and training of stock people will reduce the occurrence of bruises and in consequence will lead to better welfare conditions of cattle for slaughter.
Passie voor mossen op de proef gesteld: ontdekking, herkenning en ecologie van kwelderknikmos (Bryum warneum)
Weeda, E.J. - \ 2011
Buxbaumiella 90 (2011). - ISSN 0166-5405 - p. 1 - 17.
mossen - vegetatiemonitoring - groeiplaatsen - bedreigde soorten - karakterisering - mosses - vegetation monitoring - sites - endangered species - characterization
Bij vegetatieonderzoek in natte basenrijke pioniermilieus, zoals verzoetende strandvlakten en uitgegraven duinvalleien, vormt het geslacht Bryum een welbekend struikelblok. Heel wat monsters uit dergelijke terreinen heb ik aan Rienk-Jan Bijlsma voorgelegd, en bij herhaling leverde dat als determinatie kwelderknikmos (Bryum warneum) op. Zo raakte ik geïntrigeerd door deze zeldzame maar soms plotseling talrijk optredende mossoort. Het resultaat is het volgende relaas over haar ontdekkingsgeschiedenis en ecologie. Als motto kan de volgende waarschuwing van onze westerburen dienen: alle zeldzame Bryum-soorten van de kust hebben de neiging moeilijk herkenbaar te zijn, en geduld en volharding zijn nuttige deugden als je er greep op wilt krijgen (Porley & Hodgetts 2005, p. 304). Of in het taaleigen van Ger Harmsen (1998): ze kunnen de ‘passie voor mossen’ behoorlijk op de proef stellen.
Breeding programs for indigenous chicken in Ethiopia : analysis of diversity in production systems and chicken populations
Dana, N.M. - \ 2011
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Liesbeth van der Waaij; T. Dessie. - [S.l. : S.n. - ISBN 9789085858720 - 149
kippen - veredelingsprogramma's - genetische verbetering - prestatiekenmerken - dorpen - ecotypen - karakterisering - moleculaire genetica - genetische diversiteit - genetische parameters - agrarische productiesystemen - ethiopië - fowls - breeding programmes - genetic improvement - performance traits - villages - ecotypes - characterization - molecular genetics - genetic diversity - genetic parameters - agricultural production systems - ethiopia
The aim of this research was to generate information required to establish a sustainable breeding program for improving the productivity of locally adapted chickens to enhance the livelihood of rural farmers in Ethiopia. The first step was to characterize village poultry production environments and farmers’ objectives for keeping chickens, and to identify factors affecting the choice of genetic stock used in villages. This was achieved by carrying out a questionnaire survey and a participatory group discussion with village farmers in different geographic regions of Ethiopia. The low input nature of village environments, the prevalence of disease and predators, and other factors such as the use of chickens both as sources of eggs and meat, and income determined the choice of chicken breed used by farmers, and thus, should be considered carefully before initiating new breeding programs. The highest importance attached to adaptation traits and the existence of particular preferences for chickens of certain plumage colours and comb shapes were also found to have effects on developing new breeds for village systems.
The next part of the thesis focused on identifying important and unique gene pools in local populations. This was achieved by characterizing the local chicken ecotypes both morphologically and molecular genetically. This way the genetic difference between the local populations and the level of genetic diversity within the populations was determined. Attributes important in breeding for tropical conditions such as the pea comb gene, and the naked neck gene have been identified. It was also revealed that the variability found within a single population could explain most of the genetic diversity (97%) in Ethiopian chicken populations. The result of this work is important both from conservation and utilization perspective and assists in maintaining indigenous genetic diversity for current and future generations.
Finally, the pedigreed Horro population that was kept on station was used for estimating genetic parameters for the production traits, monthly and cumulative part period egg numbers and growth to 16 weeks of age. Because the pedigreed population was established only recently, data of only 2 generations were available for estimating these genetic parameters. The results are promising but inaccurate due to insufficient amount of data. They would need to be re-estimated when more generations have been produced and thus more data has been generated.
Mapping, isolation and characterization of genes responsible for late blight resistance in potato
Pel, M. - \ 2010
Wageningen University. Promotor(en): Richard Visser; Evert Jacobsen, co-promotor(en): Herman van Eck. - [S.l. : S.n. - ISBN 9789085856368 - 210
solanum tuberosum - aardappelen - phytophthora infestans - ziekteresistentie - genen - genetische kartering - karakterisering - genisolatie - potatoes - disease resistance - genes - genetic mapping - characterization - gene isolation
Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most
devastating diseases on potato. Resistance (R) genes from the wild species Solanum demissum
have been used by breeders to generate late blight resistant cultivars, but resistance was soon
overcome by the pathogen. A more recent screening of a large number of wild species has led
to the identification of novel sources of resistance, many of which are currently being
characterized. R-gene based resistance to any plant pathogen has been conceptualized
according to a model known as gene-for-gene interaction. When matching avirulence (Avr)
and resistance (R) proteins are produced by the pathogen and the plant respectively, a
resistance response is triggered resulting in a hyper-sensitive response (HR) causing necrosis
and cell death at the infection site. If one of these components is missing the plant-pathogen
interaction is compatible and will result in completion of the life cycle of the pathogen. This
thesis describes the cloning and the characterization of the resistant alleles Rpi-vnt1.1, Rpivnt1.2
and Rpi-vnt1.3 from Solanum venturii and their counterpart Avr-vnt1 from
Phytophthora infestans. The Rpi-/Avr- genes pair Rpi-vnt1/Avr-vnt1 along with R3a/Avr3a
and Rpi-blb3/Avr2 have been used to study the genetic and molecular mechanisms behind
tuber blight resistance.
The cloning of Rpi-vnt1 alleles (Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3) was achieved by the
combination of long range PCR (Chapter 2) and a classical map based cloning strategy
(Chapter 3). The long range PCR made use of Tm2 homologous PCR primers, upon
identification of Tm2 sequence homology in associated markers generated with an NBStargeted
fingerprinting technique. Rpi-vnt1 alleles belong to the CC-NBS-LRR class of plant
R genes and encode predicted peptides of 891 and 905 amino acids, respectively, which share
75% amino acid (a.a.) identity with the ToMV resistance protein Tm-22 from tomato.
Compared to Rpi-vnt1.1, the allele Rpi-vnt1.3 harbors a 14 amino acid insertion in the Nterminal
region of the protein and two different amino acids in the LRR domain. Despite these
differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum.
An allele mining study of Rpi-vnt1 alleles across Solanum section Petota showed that the
three functional alleles were confined within S. venturii as only two accessions from the
closely related species S. weberbaueri and S. mochiquense carried Rpi-vnt1.1 (Chapter 4).
Subsequent alignment of Rpi-vnt1-like homologs with Rpi-vnt1 alleles revealed the presence
of illegitimate recombination (IR) signatures suggesting that two successive deletion events
might have occurred in the CC domain. Meanwhile, the construction of a Neighbor Joining tree, based on AFLP data from all the accessions carrying Rpi-vnt1 alleles or Rpi-vnt1-like
homologs showed that Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 alleles belong to a monophyletic
clade. Signatures of illegitimate recombination and the monophyletic grouping of Rpi-vnt1
alleles suggested how Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 could have evolved. Extensive
phenotyping with various Phytophthora isolates identified another Rpi gene in S. venturii
named Rpi-vnt2, complementing the Rpi-vnt1 allelic resistance spectrum. The genetic position
of this second independent locus is not yet identified.
The identification of the matching avirulence factor from the pathogen, Avr-vnt1, was
achieved by using an efficient and high throughput effector screen of resistant wild potato
species (Chapter 5). Avr-vnt1 encodes a typical RXLR-EER effector which expression is
induced 2 days post inoculation. Avr-vnt1 is located on a single locus in the reference strain
T30-4. Among nine isolates, four alleles were identified. The virulent strain EC1 carries a
functional coding sequence of Avr-vnt1 but fails to express the gene.
In Chapter 6, the genetic and molecular mechanisms of tuber late blight have been
investigated. Using transgenic cv. Desiree plants transformed with Rpi-vnt1.1, R3a or Rpiblb3
tuber blight resistance could be studied in an identical genetic background. First, we
demonstrated that transient co-expression of the matching Avr- genes in these transgenic tuber
slices trigger a hypersensitive responses (HR), showing that the presence and interaction of
both proteins is sufficient to establish tuber blight resistance. Second, phenotypic and
molecular analysis of a panel of transformants for Rpi-vnt1.1, R3a and Rpi-blb3, and
transcriptional analysis of the corresponding effectors (Avr-vnt1, Avr3a and Avr2
respectively) during leaf and tuber infection showed that the expression level of a given Rgene
should equal or exceed the expression level of the matching effector in order to trigger
an efficient resistance response in the tuber. Therefore, foliar and tuber late blight resistance
are controlled by similar genetic mechanisms. The perceived lack of phenotypic correlation
between foliage and tuber blight resistance is thus solely due to the tissue specific expression
level of the Rpi-gene.
In the general discussion (Chapter 7), results from the experimental chapters are discussed in
a broader perspective.
Chemical, physical and biological features of Okra pectin
Sengkhamparn, N. - \ 2009
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Henk Schols; T. Sajjaanantakul. - [S.L. : S.n. - ISBN 9789085855293 - 176
pectinen - okra's - karakterisering - fysicochemische eigenschappen - pectins - okras - characterization - physicochemical properties
In Thailand, many plants have been used as vegetables as well as for traditional
medicine. Okra, Abelmoschus esculentus (L.) Moench, is an example of such a plant.
Examples for the medical use are treatment of gastric irritation, treatment of dental
diseases, lowering cholesterol level and preventing cancer. These biological activities are
ascribed to polysaccharide structures of okra in particular pectin structures. However, the
precise structure of okra pectins and also of other polysaccharides in okra pods have been
lacking so far.
In order to obtain detailed information of the different polysaccharides present in
okra, okra cell wall material was prepared from the pulp of okra pods and was then
sequentially extracted with hot buffer, chelating agent, diluted alkali and concentrated
alkali. The sugar (linkage) composition indicated that okra cell wall contained, next to
cellulose, different populations of pectins and hemicelluloses.
The pectic polysaccharides were mainly obtained in the first three extracts having
slightly different chemical structures. The okra pectin extracted by hot buffer was almost a
pure rhamnogalacturonan (RG) I with a high degree of acetylation (DA), covalently linked
to a minor amount of homogalacturonan (HG) having a high degree of methyl esterification
(DM). The chelating agent extractable pectin and the diluted alkali extractable pectin
predominantly contained HG with only minor amounts of RG I. Okra pectins extracted by
hot buffer and with chelating agent had in common that both contained highly branched RG
I with very short side chains containing not more than 3 galactosyl units attached to the
rhamnosyl residues in RG I backbone. Chelating agent extracted okra pectins also carried
arabinan and arabinogalactan type II as neutral side chains and these side chains were even
more abundantly present in the diluted alkali extracted okra pectin.
The hemicellulosic polysaccharides ended up in concentrated alkali extract. From
the sugar (linkage) composition and enzymatic degradation studies using pure and well
defined enzymes, it was concluded that this fraction contained a XXXG–type xyloglucan
and 4-methylglucuronoxylan. The cellulosic polysaccharides were retained in the residue.
The okra hot buffer extractable RG I having a high level of acetyl substitution
appeared to be very well degradable by rhamnogalacturonan hydrolase which was known to
be hindered completely by acetylated substrates. In contrast, an acetylated galacturonic
acid-specific rhamnogalacturonan acetyl esterase was unable to remove acetyl groups from
the RG I molecule of hot buffer extracted okra pectin. For these reasons, the precise
position of the acetyl groups present on enzymatically released oligomers were determined
by Electron Spray Ionization Ion Trap Mass Spectrometry (ESI-IT-MS) and Nuclear
Magnetic Resonance (NMR) spectroscopy. The acetyl groups were found to be
predominantly located at position O-3 of the rhamnosyl moiety, while the methyl esters
seemed to be present only on the HG part of the hot buffer extracted okra pectin. Another
novelty of okra RG-I was the presence of terminal alpha-linked galactosyl substitution at
position O-4 of the rhamnosyl residues within the RG I backbone. These specific features
(acetylated rhamnosyl- and alpha-galactosyl-substitutions) were almost absent in the
chelating agent extracted okra pectin where more commonly known substitutions were
present, including acetylated galacturonosyl residues in the RG I backbone. The unique
structure features of hot buffer extracted okra pectin led to the assumption that these
features may contribute to the rather typical physical properties as well as to the biological
properties found for okra pectin.
In order to understand the effect of the specific structural features of RG I on its
physical properties, the rheological properties of hot buffer extracted okra pectin were
determined and compared to those found for chelating agent extracted okra pectin and for
pectins from other plant materials as reported in the literature. The solutions of hot buffer
extracted okra pectin showed a high viscosity and predominant elastic behaviour which
most probably is caused by strong hydrophobic associations through its acetylated
rhamnosyl residues rather than by methyl esterified galacturonosyl residues as is commonly
the case for pectins. The removal of acetyl groups and methyl esters decreased the
association of the pectin molecules as observed by the light scattering experiment, meaning
that not only viscosity and rheological properties but also association of pectin molecules
were as result of both hydrophobic interactions and charge effects.
The effect of the position of acetyl groups on the bioactivity of okra pectin was
also determined. The complement-fixing activity of okra pectins was found to be affected
by many factors like e.g. the presence of acetyl groups, the size of RG segments and the presence of terminal alpha galactosyl groups and even the three dimensional conformation
of the molecules. The hot buffer extracted okra pectin was also examined for its potential to
modify surfaces of medical devices and implants. The results showed that okra pectin can
be used in coating medical device since it promotes cell apoptosis and shows no
The knowledge described in this thesis provided us with novel information on the
unique structures of okra pectins and may lead to a better understanding of the functional
properties of okra polysaccharides in general and okra pectin in particular and to optimize
the use of okra pectins within the food industry and in medical applications. However,
despite our efforts, at the moment the dependency of the (bio) functionality of okra pectins
on the precise chemical structure are not yet completely understood.
Geochemische schematisering van de ondergrond in het STONE model : organisch stofgehalte in de ondergrond
Boekel, E.M.P.M. - \ 2009
Wageningen : Alterra (Alterra-rapport 1830) - 61
bodemchemie - geochemie - organische stof - bodemtypen - karakterisering - uitspoelen - stikstof - fosfor - modellen - soil chemistry - geochemistry - organic matter - soil types - characterization - leaching - nitrogen - phosphorus - models
De geochemische parametrisering en schematisering van de ondergrond in het STONE-model speelt een belangrijke rol bij het inzichtelijk maken van haalbare nutriëntenconcentraties in het oppervlaktewater op de lange termijn. Op basis van geochemische analyses is het organisch stofgehalte voor de ondiepe ondergrond (> 1m-mv) opnieuw bepaald waarbij twee varianten onderscheiden worden. Het organisch stofgehalte voor beide varianten zijn significant verschillend t.o.v. het organisch stofgehalte in de huidige schematisatie. Het organisch stofgehalte in klei- en veengronden zijn over het algemeen lager, voor zandgronden worden hogere gehalten bepaald. Door veranderingen in organisch stofgehalten kunnen processen in de bodem zodanig worden beïnvloed dat er grote verschillen kunnen ontstaan in de nutriëntenvoorraad in de bodem en de uiteindelijke nutriëntenbelasting naar grond- en oppervlaktewater.
Karakterisering van de virulentie van Nederlandse Meloidogyne chitwoodi populaties
Korthals, G.W. ; Runia, W.T. - \ 2008
meloidogyne chitwoodi - virulentie - nematoda - proeven - landbouwkundig onderzoek - waardplanten - karakterisering - virulence - trials - agricultural research - host plants - characterization
Drie tweejarige proeven op locaties in zuid-oost Nederland en meer dan 20 verschillende M. chitwoodi populaties zijn opgezuiverd, gekarakteriseerd en in kweek gebracht. Deze zijn met potproeven op zeven verschillende gewassen vergeleken
Structural characterization of native pectins
Coenen, G.J. - \ 2007
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Henk Schols. - [S.l.] : S.n. - ISBN 9789085047797 - 152
pectinen - karakterisering - chemische structuur - massaspectrometrie - degradatie - capillaire electroforese - pectins - characterization - chemical structure - mass spectrometry - degradation - capillary electrophoresis
Pectine wordt in levensmiddelen vooral gebruikt als geleermiddel, stabilisator of verdikkingsmiddel in producten zoals jam, yoghurtdranken, vruchten-zuiveldranken en ijs. Daarnaast is er in toenemende mate interesse in het mogelijk gezondheidbevorderend effect van dit polysaccharide. Kennis van de exacte structuur zal bijdragen aan het begrip van de fysiologische functie van pectine in de plant en aan een verdere optimalisering van industriële en medische toepassingen van dit polymeer. Om meer inzicht te krijgen in de structuur werden nieuwe LC-MS en CE-MS methoden ontwikkeld, waarmee verbindingen tussen verschillende structuurelementen konden worden aangetoond. Als gevolg van de verkregen inzichten over de opbouw van pectine, wordt een aanpassing aan het structuurmodel van pectine voorgesteld, waarbij homogalacturonan ketens zowel lineair als vertakt aan rhamnogalacturonan I worden gepositioneerd. De ontwikkelde methoden, kunnen worden toegepast in onderzoek gericht op de opheldering van techno- en biofunctionele eigenschappen van complexe polysaccharidestructuren.
Identificatie, karakterisering en populatieopbouw van nieuwe resistentiebronnen uit wilde Solanum-soorten
Vleeshouwers, V.G.A.A. - \ 2007
aardappelen - solanum - plaagresistentie - phytophthora infestans - identificatie - karakterisering - plantenveredelingsmethoden - potatoes - pest resistance - identification - characterization - plant breeding methods
Identificatie van nieuwe resistentiebronnen tegen Phytophthora infestans is essentieel voor de aardappelveredeling. Onderzoek naar karakterisering en populatieopbouw van nieuwe resistentiebronnen uit wilde Solanum-soorten
Characterisation of cell wall polysaccharides in bilberries and black currants
Hilz, H. - \ 2007
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Henk Schols. - [S.l.] : S.n. - ISBN 9789085046240 - 158
blauwe bosbessen (vaccinium myrtilus) - zwarte bessen - celwandstoffen - pectinen - xyloglucanen - karakterisering - enzymen - drukbehandeling - bessen - bilberries - black currants - cell wall components - pectins - xyloglucans - characterization - enzymes - pressure treatment - berries
During berry juice production, polysaccharides are released from the cell walls and cause thickening and high viscosity when the berries are mashed. Consequences are a low juice yield and a poor colour. This can be prevented by the use of enzymes that degrade these polysaccharides. To use these enzymes most efficiently, the structure and composition of the cell walls had to be known. This thesis describes a detailed composition of the cell walls of bilberries and black currants. The obtained results were used to monitor changes in the cell walls during different treatments such as enzyme treatment and high pressure processing. A higher viscosity and lower juice yield after high pressure processing were assigned to changes in pectin structure and extractability. Based on this knowledge, a synergistic effect was obtained by combining high pressure processing with treatment with commercial enzyme preparations.
Het plantenvirologisch onderzoek bij Plant Research International
Vlugt, R.A.A. van der; Verbeek, M. ; Bouwen, I. ; Kasteel, D.T.J. ; Dullemans, A.M. ; Vink, J. ; Cuperus, C. ; Piron, P.G.M. - \ 2006
Gewasbescherming 37 (2006)5. - ISSN 0166-6495 - p. 227 - 231.
plantenvirussen - plantenziekteverwekkers - plantenziekten - virologie - onderzoeksinstituten - proefstations - universitair onderzoek - identificatie - epidemiologie - karakterisering - plant viruses - plant pathogens - plant diseases - virology - research institutes - experimental stations - university research - identification - epidemiology - characterization
Onze belangrijkste expertises: - Identificeren en karakteriseren van plantenvirussen, hun stammen en isolaten - virusspecifieke symptomen op zowel waardplanten als toetplanten - ontwikkelen van specifieke en gevoelige detectiemethodieken, zowel serologisch (antisera) als moleculair –biologisch - virus-overdracht en verspreiding door vectoren - de epidemiologie en ecologie van virusziekten
Function-structure relationships of acetylated pea starches
Huang, J. - \ 2006
Wageningen University. Promotor(en): Fons Voragen, co-promotor(en): Henk Schols. - [S.l.] : S.n. - ISBN 908504538X - 140
erwten - zetmeel - acetylering - fysicochemische eigenschappen - karakterisering - structuuractiviteitsrelaties - peas - starch - acetylation - physicochemical properties - characterization - structure activity relationships
Cowpea, chickpea and yellow pea starches were studied and the results showed that their properties were strongly related to the chemical fine structures of the starches. Furthermore, granular starches were modified using two types of chemical acetylation reagents and then separated into different size fractions. The amount of introduced acetyl groups was found to depend on the size of the granules for the reaction with rapidly reacting reagent acetic acid anhydride, whereas the amount of introduced acetyl groups was independent from the granule size for reaction with the slowly reacting reagent vinyl acetate. Modification with the two types of reagents resulted in significant difference in physical properties of the starches. The investigation on the chemical fine structure of modified starches suggested that the distribution of acetyl groups over both the amylose and amylopectin populations of the starches was different, not only at molecular level but also at granular level.
Literature review of available techniques to characterize marine and estuarine food webs; with emphasis for application in the model OMEGA
Lange, H.J. de; Brink, N.W. van den - \ 2006
Wageningen : Alterra (Alterra-rapport 1372) - 55
voedselwebben - ecologie - marien milieu - estuaria - karakterisering - modellen - merkers - food webs - ecology - marine environment - estuaries - characterization - models - markers
Food webs can be characterized by use of markers. These are increasingly used in food web studies since they are the only tool that can be used to infer relations through multiple trophic levels. Such a marker is a characteristic of an organism that can be objectively measured and evaluated as an indicator of normal biologic processes. These include fatty acids, stable isotopes, and molecular markers such as immunological markers and DNA markers. This report presents the results of a literature survey on these marker techniques, their use in describing marine and estuarine food webs, and an evaluation of their usefulness for the model OMEGA. Stable isotope analysis is advised as the best method to characterize food webs by means of trophic position and carbon source. Fatty acids can be used to differentiate within trophic groups, especially within the group of primary producers.
Ontwikkelingen rond resistente onkruiden: gevaren en beheersmogelijkheden: themanummer onkruidbeheersing in Nederland, nut en noodzaak
Weide, R.Y. van der; Zeeland, M.G. van; Timmer, R.D. ; Koster, A.T.J. ; Mol, E.S.N. ; Rotteveel, A.J.W. ; Bulcke, R. - \ 2005
Gewasbescherming 36 (2005)2. - ISSN 0166-6495 - p. 98 - 101.
gewasbescherming - onkruidbestrijding - chemische bestrijding - geïntegreerde bestrijding - herbiciden - resistentie tegen herbiciden - controle - onkruiden - tegen herbiciden resistente onkruiden - bedrijfsvoering - karakterisering - plant protection - weed control - chemical control - integrated control - herbicides - herbicide resistance - control - weeds - herbicide resistant weeds - management - characterization
Het optreden van resistentie voor herbiciden bij onkruiden is een gevaarlijke ontwikkeling bnnen de onkruidbestrijding. Door onder andere fusies binnen de gewasbeschermingsindustrie en de kosten voor toelating van middelen, zijn minder middelen met een verschillend werkingsspectrum en een nieuwe werkingswijze beschikbaar. Als de beschikbare herbiciden eenzijdig en grootschalig worden ingezet, ook bij herbicideresistente GM gewassen, kan het verschijnen van resistentie bij onkruiden worden versneld