Elevated viscosities in a simulated moving bed for γ-aminobutyric acid recovery
Schultze-Jena, A. ; Boon, M.A. ; Vroon, R.C. ; Bussmann, P.J.Th. ; Janssen, A.E.M. ; Padt, A. van der - \ 2020
Journal of Separation Science 43 (2020)7. - ISSN 1615-9306 - p. 1256 - 1264.
chromatography - concentration profile - productivity - simulated moving bed - viscosity
Process streams of agro-food industries are often large and viscous. In order to fractionate such a stream the viscosity can be reduced by either a high temperature or dilution, the former is not an option in case of temperature sensitive components. Such streams are diluted prior to chromatographic fractionation, resulting in even larger volumes and high energy costs for sub-sequential water removal. The influence of feed viscosity on the performance of simulated moving bed chromatography has been investigated in a case study of the recovery of a γ-aminobutyric acid rich fraction from tomato serum. This work addresses the chromatographic system design, evaluates results from a pilot scale operation, and uses these to calculate the productivity and water use at elevated feed concentration. At the two higher feed viscosities (2.5 and 4 mPa·s) water use is lower and productivity higher, compared to the lowest feed viscosity (1 mPa·s). The behavior of the sugars for different feed viscosities can be described well by the model using the ratio of feed to eluent as dilution factor. The behavior of γ-aminobutyric acid is highly concentration dependent and the recovery could not be accurately predicted.
Controlling the self-assembly of protein polymers via heterodimer-forming modules
Domeradzka, Natalia Eliza - \ 2016
Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - pichia pastoris - modules - massaspectrometrie - microscopie - sds-page - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie
Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.
The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.
In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.
Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.
A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.
Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.
In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.
Milk Oligosaccharide Variation in Sow Milk and Milk Oligosaccharide Fermentation in Piglet Intestine
Difilippo, Elisabetta ; Pan, Feipeng ; Logtenberg, Madelon ; Willems, Rianne ; Braber, Saskia ; Fink-Gremmels, Johanna ; Schols, Henk Arie ; Gruppen, Harry - \ 2016
Journal of Agricultural and Food Chemistry 64 (2016)10. - ISSN 0021-8561 - p. 2087 - 2093.
abundance - chromatography - cow - mass analysis - pigs - sugars - variation
Porcine milk oligosaccharides (PMOs) were analyzed in six colostrum and two mature milk samples from Dutch Landrace sows. In total, 35 PMOs were recognized of which 13 were new for the PMO literature: Neutral HexNAc-Hex, β4′-galactosyllactose, putative GalNAc(α/β1-3)Gal(β1-4)Glc, lacto-N-fucopentaose-II, lacto-N-tetraose, galactose substituted lacto-N-neohexaose, lacto-N-hexaose and difucosyl-lacto-N-hexaose, and acidic Neu5Ac(α2-6)GlcNAc(β1-3)Gal(β1-4)Glc, sialyllacto-N-tetraose-a and -b, Neu5Ac2-Hex3, and sialyllacto-N-fucopentaose-II. PMOs were analyzed using capillary electrophoresis with laser-induced florescence detection or mass spectrometry and using liquid chromatography with mass spectrometry. Interindividual variation regarding PMO presence and concentration was observed between porcine milks. Within a limited sample set, a 43% decrease of the major PMOs was found during a 1 w lactation period. Interestingly, while some PMOs decreased, some other PMOs increased in concentration. PMOs were also monitored in fecal samples of suckling piglets. In feces of 1-2 d old piglets, few intact PMOs were found, indicating considerable PMO fermentation at early stage of life.
Identifying and naming plant-pathogenic fungi: past, present, and future
Crous, P.W. ; Hawksworth, D.L. ; Wingfield, M.J. - \ 2015
Annual Review of Phytopathology 53 (2015). - ISSN 0066-4286 - p. 247 - 267.
molecular systematics - polyphyletic nature - polyphasic approach - mycorrhizal fungi - species concepts - taxonomy - genus - classification - identification - chromatography
Scientific names are crucial in communicating knowledge about fungi. In plant pathology, they link information regarding the biology, host range, distribution, and potential risk. Our understanding of fungal biodiversity and fungal systematics has undergone an exponential leap, incorporating genomics, web-based systems, and DNA data for rapid identification to link species to metadata. The impact of our ability to recognize hitherto unknown organisms on plant pathology and trade is enormous and continues to grow. Major challenges for phytomycology are intertwined with the Genera of Fungi project, which adds DNA barcodes to known biodiversity and corrects the application of old, established names via epi- or neotypification. Implementing the one fungus–one name system and linking names to validated type specimens, cultures, and reference sequences will provide the foundation on which the future of plant pathology and the communication of names of plant pathogens will rest.
The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase—A means of separating amino acids for synthesizing biobased chemicals
Teng, Y. ; Scott, E.L. ; Sanders, J.P.M. - \ 2014
Biotechnology Progress 30 (2014)3. - ISSN 8756-7938 - p. 681 - 688.
nitrogen-containing chemicals - bulk chemicals - electrodialysis - biorefinery - biomass - immobilization - chromatography - alginate
Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate a-decarboxylase (GAD, Type I, EC 22.214.171.124) was applied to specifically convert Glu into c-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme’s initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu.
Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides
Dallas, D.C. ; Weinborn, V. ; Moura Bell, J.M.L.N. de; Wang, M. ; Parker, E.A. ; Guerrero, A. ; Hettinga, K.A. ; Lebrilla, C.B. ; German, J.B. ; Barile, D. - \ 2014
Food Research International 63 (2014)part B. - ISSN 0963-9969 - p. 203 - 209.
holstein-friesian colostrum - globule-membrane proteome - bovine-milk - mass-spectrometry - peptone fraction - beta-casein - chromatography - proteolysis - components - system
Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane yielding whey permeate. Research shows that bovine milk from healthy cows contains hundreds of naturally occurring peptides – many of which are homologous with known antimicrobial and immunomodulatory peptides – and nearly 50 oligosaccharide compositions (not including structural isomers). As these endogenous peptides and oligosaccharides have low-molecular weight and whey permeate is currently an under-utilized product stream of the dairy industry, we hypothesized that whey permeate may serve as an inexpensive source of naturally occurring functional peptides and oligosaccharides. Laboratory fractionation of endogenous peptides and oligosaccharides from bovine colostrum sweet whey was expanded to pilot-scale. The membrane fractionation methodology used was similar to the methods commonly used industrially to produce whey protein concentrate and whey permeate. Pilot-scale fractionation was compared to laboratory-scale fractionation with regard to the identified peptides and oligosaccharide compositions. Results were interpreted on the basis of whether industrial whey permeate could eventually serve as a source of functional peptides and oligosaccharides. The majority (96%) of peptide sequences and the majority (96%) of oligosaccharide compositions found in the laboratory-scale process were mirrored in the pilot-scale process. Moreover, the pilot-scale process recovered an additional 33 peptides and 1 oligosaccharide not identified from the laboratory-scale extraction. Both laboratory- and pilot-scale processes yielded peptides deriving primarily from the protein ß-casein. The similarity of the laboratory- and pilot-scale's resulting peptide and oligosaccharide profiles demonstrates that whey permeate can serve as an industrial-scale source of bovine milk peptides and oligosaccharides.
Cross-platform comparative analyses of genetic variation in amino acid content in potato tubers
Carreno-Quintero, N. ; Undas, A.K. ; Bachem, C.W.B. ; Mumm, R. ; Visser, R.G.F. ; Bouwmeester, H.J. ; Keurentjes, J.J.B. - \ 2014
Metabolomics 10 (2014)6. - ISSN 1573-3882 - p. 1239 - 1257.
mass-spectrometry - metabolic networks - mixed-model - late blight - qtl - arabidopsis - methionine - biosynthesis - diversity - chromatography
Many of the biochemical pathways for plant amino acid metabolism are known and, at least in model species, most of the genes encoding the biosynthetic enzymes have been identified. How the accumulation of amino acids is regulated is much less well understood and for this genetic analysis can be instrumental. In potato, the nutritional value of the tubers is often determined by the content of essential amino acids such as lysine, tyrosine, methionine and cysteine. Better insight into the genetic determinants underlying the variation in amino acid accumulation in potato could support efforts to improve tuber nutritional quality by breeding. In this study, we used a diploid potato mapping population to explore the genetic basis of amino acid content. Hereto, we compared the use of one non-targeted and two targeted analytical approaches for amino acid analysis, allowing the evaluation of the robustness of amino acid quantification and the number and strength of detected quantitative trait locis (QTLs) across the different analytical platforms. Assessment of the three methodologies revealed a comparable detection of amino acids using non-targeted and targeted approaches. QTL detection across the different analytical platforms was similar, although slight differences in strength and explained variance were observed. The QTL regions were subsequently studied to provide candidate genes for the genetic regulation of amino acid accumulation in potato. Our results are discussed in the context of the detection of amino acid variation and its implications for the identification of QTLs.
A systematic approach to obtain validated partial least square models for predicting lipoprotein subclasses from serum NMR spectra
Mihaleva, V.V. ; Schalkwijk, D.B. van; Graaf, A.A. de; Duynhoven, J.P.M. van; Dorsten, F.A. van; Vervoort, J.J.M. ; Smilde, A.K. ; Westerhuis, J.A. ; Jacobs, D.M. - \ 2014
Analytical Chemistry 86 (2014)1. - ISSN 0003-2700 - p. 543 - 550.
nuclear-magnetic-resonance - low-density lipoprotein - plasma-lipoproteins - insulin-resistance - regression-models - spectroscopy - quantification - chromatography - abnormalities - chemometrics
A systematic approach is described for building validated PLS models that predict cholesterol and triglyceride concentrations in lipoprotein subclasses in fasting serum from a normolipidemic, healthy population. The PLS models were built on diffusion-edited (1)H NMR spectra and calibrated on HPLC-derived lipoprotein subclasses. The PLS models were validated using an independent test set. In addition to total VLDL, LDL, and HDL lipoproteins, statistically significant PLS models were obtained for 13 subclasses, including 5 VLDLs (particle size 64-31.3 nm), 4 LDLs (particle size 28.6-20.7 nm) and 4 HDLs (particle size 13.5-9.8 nm). The best models were obtained for triglycerides in VLDL (0.82
Multivariate PAT solutions for biopharmaceutical cultivation: current progress and limitations
Mercier, S.M. ; Diepenbroek, B. ; Wijffels, R.H. ; Streefland, M. - \ 2014
Trends in Biotechnology 32 (2014)6. - ISSN 0167-7799 - p. 329 - 336.
process analytical technology - principal component analysis - monitoring batch processes - cell-culture - biotechnology - spectroscopy - quality - chromatography - fermentation - chemometrics
Increasingly elaborate and voluminous datasets are generated by the (bio)pharmaceutical industry and are a major challenge for application of PAT and QbD principles. Multivariate data analysis (MVDA) is required to delineate relevant process information from large multi-factorial and multi-collinear datasets. Here the key role of MVDA for industrial (bio)process data is discussed, with a focus on progress and limitations of MVDA as a PAT solution for biopharmaceutical cultivation processes. MVDA based models were proven useful and should be routinely implemented for bioprocesses. It is concluded that although the highest level of PAT with process control within its design space in real-time during manufacturing is not reached yet, MVDA will be central to reach this ultimate objective for cell cultivations.
The potential impact of membrane cascading on downstream processing of oligosaccharides
Patil, N.V. ; Janssen, A.E.M. ; Boom, R.M. - \ 2014
Chemical Engineering Science 106 (2014)2014. - ISSN 0009-2509 - p. 86 - 98.
nanofiltration - separation - ultrafiltration - chromatography - fractionation - purification - saccharides
To assess the potential use of ideal nanofiltration cascades for the industrial fractionation of oligosac- charides, simulations of single, three and five stage NF cascades were carried out.Three and five stage ideal cascades show significant improvement in separation with diafiltration compared to single stage systems.The calculations do imply different membrane areas in each stage of the cascade.The ratio of the sieving coefficients of abinary mixture over the membrane plays an important role in determining the relation between yield and purity in a cascade system.A thigh sieving coefficient ratios ,both yield and purity increase concurrently in a three stage system,whereas at a low ratio of the sieving coefficients, the yield and purity become inversely proportional on the retentate side. In a five stage systems,both yield and purity become inversely proportional at high and low sieving coefficient ratios. A five stage cascade system installation would be optima lfor most applications since at very low local separation factors sufficient separation and yield could be achieved.
Characterization of Romboutsia ilealis gen. nov., sp. nov., isolated from the gastro-intestinal tract of a rat and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen. nov., Intestinibacter gen. nov., Terrisporobacter gen. nov. and Asaccharospora gen. nov.
Gerritsen, J. ; Fuentes Enriquez de Salamanca, S. ; Grievink, W. ; Niftrik, L. van; Tindall, B.J. ; Timmerman, H.M. ; Rijkers, G.T. ; Smidt, H. - \ 2014
International Journal of Systematic and Evolutionary Microbiology 64 (2014)Pt. 5. - ISSN 1466-5026 - p. 1600 - 1616.
ribosomal-rna genes - acid methyl-esters - lipid-composition - deoxyribonucleic-acid - electron microscopy - renaturation rates - dna hybridization - polar lipids - bacteria - chromatography
A Gram-positive staining, rod-shaped, non-motile, spore-forming obligately anaerobic bacterium, designated CRIBT, was isolated from the gastro-intestinal tract of a rat and characterized. The major cellular fatty acids of strain CRIBT were saturated and unsaturated straight chain C12-C19 fatty acids, with C16:0 being the predominant fatty acid. The polar lipid profile comprised six glycolipids, four phospholipids and one lipid that did not stain with any of the specific spray reagents used. The only quinone was MK-6. The predominating cell wall sugars were glucose and galactose. The peptidoglycan type of strain CRIBT was A1d lanthionine-direct. The genomic DNA G+C content of strain CRIBT was 28.1 mol %. On the basis of 16S rRNA gene sequence similarity, strain CRIBT was most closely related to a number of Clostridium species, including C. lituseburense (97.2 %), C. glycolicum (96.2 %), C. mayombei (96.2 %), C. bartlettii (96.0 %) and C. irregulare (95.5 %). All these species show very low 16S rRNA gene sequence similarity (
Tropane alkaloids in food: poisoning incidents
Adamse, P. ; Egmond, H.P. van; Noordam, M.Y. ; Mulder, P.P.J. ; Nijs, W.C.M. de - \ 2014
Quality Assurance and Safety of Crops & Foods 6 (2014)1. - ISSN 1757-8361 - p. 15 - 24.
datura-stramonium seeds - capillary-electrophoresis - analytical toxicology - mass-spectrometry - atropine - ingestion - leaves - enantioseparation - identification - chromatography
A large number of wild and cultured plants produce secondary metabolites that can be toxic to humans and animals. The present study aims to provide insight into the routes of (un)intentional poisonings of humans by tropane alkaloids. Poisonings of humans by tropane alkaloids occur as unintended ingestions (contamination, mislabelling: thirteen reports; mistaken identity: eleven reports) or intended ingestions (overdoses: nine reports). Contamination of food occurs when toxic plant (parts) are accidentally mixed with edible plants during harvest or processing. Concentrations are usually highest in roots and seeds. Intended ingestions can be the result of consumption for recreational purposes (hallucinogenic effects) or for medical properties (e.g. treatment of arthritis, use as anaesthetic), or homicides and suicides. Carry-over of plant toxins in feed into food products of animal origin does not appear to be a relevant source of exposure. There are several analytical methods available for monitoring tropane alkaloids in food and feed but no regulatory limits have been set. The toxic doses are often not clear due to the lack of analytical data in the cases reported. Human foods that potentially contain tropane alkaloids are herbal teas, herbal preparations, blue-or black berries and edible flowers. Contamination has also been found in beans, buckwheat, soybean and linseed.
Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters
Faassen, E.J. ; Beekman-Lukassen, W.D. ; Lurling, M. - \ 2013
PLoS ONE 8 (2013)6. - ISSN 1932-6203 - 7 p.
methylamino-l-alanine - amino-acid - mass-spectrometry - cyanobacteria - chromatography - seeds - guam
The neurotoxin ß-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA.
Characterisation of 3-aminoquinoline-derivatised isomeric oligogalacturonic acid by travelling-wave ion mobility mass spectrometry
Huang, J.H. ; Bakx, E.J. ; Gruppen, H. ; Schols, H.A. - \ 2013
Rapid Communications in Mass Spectrometry 27 (2013)20. - ISSN 0951-4198 - p. 2279 - 2285.
cell walls - oligosaccharides - matrix - chromatography - separation - pectin - quantification - resolution
RATIONALE Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. METHODS A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. RESULTS TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. CONCLUSIONS Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.
Comparison of xanthans by the relative abundance of its six constituent repeating units
Kool, M.M. ; Gruppen, H. ; Sworn, G. ; Schols, H.A. - \ 2013
Carbohydrate Polymers 98 (2013)1. - ISSN 0144-8617 - p. 914 - 921.
xanthomonas-campestris - rheological properties - pyruvate content - polysaccharide - gum - acetyl - chromatography - hydrolysis - transition
Five xanthans were hydrolyzed to their repeating units using cellulases. Hydrophilic interaction chromatography with online electrospray ionization ion trap mass spectrometry and evaporative light scattering detection was used to analyze the oligomers released. It was concluded that six different pentamer repeating units (RUs) exists within a xanthan sample. The most abundant RU shows acetylation on the inner mannose and pyruvylation on the outer mannose. The second most abundant RU shows acetylation on both the inner and the outer mannose. It becomes clear that more variations in the xanthan structure exist than generally recognized. Comparison of five different xanthan samples revealed that, although the molecular composition of xanthan samples can be exactly the same, the ratio in which the RUs occur can differ significantly. It is, therefore, concluded that xanthan samples should be characterized for both, their molecular composition and the relative abundance of the RUs present
Separation and identification of individual alginate oligosaccharides in the feces of alginate-fed pigs
Jonathan, M.C. ; Bosch, G. ; Schols, H.A. ; Gruppen, H. - \ 2013
Journal of Agricultural and Food Chemistry 61 (2013)3. - ISSN 0021-8561 - p. 553 - 560.
mass-spectrometry - capillary-electrophoresis - molecular-weight - dietary fiber - growth - acid - cells - chromatography - fermentation - hydrolysis
This research aimed to develop a method for analyzing specific alginate oligosaccharides (AOS) in a complex matrix such as pig feces. The data obtained were used to study alginate degradation by the microbiota in the large intestine during adaptation, including the individual variation between pigs. A method using an UHPLC system with an ethylene bridged hybrid (BEH) amide column coupled with MSn detection was able to distinguish saturated and unsaturated AOS with DP 2–10. Isomers of unsaturated trimer and tetramer could be separated and annotated. In the feces, saturated and unsaturated AOS were present. The presence of unsaturated AOS indicates that the microbiota produced alginate lyase. The microbiota utilized unsaturated AOS more than saturated AOS. The results also suggested that guluronic acid at the reducing end of AOS inhibits the utilization by microbiota during the first weeks of adaptation. After adaptation, the microbiota was able to utilize a broader range of AOS.
Online Preconcentration-IC-ICP-MS for Selenium Quantification and Speciation at Ultratraces
Lenz, M. ; Floor, G.H. ; Winkel, L.H.E. ; Román-Ross, G. ; Corvini, P.F.X. - \ 2012
Environmental Science and Technology 46 (2012)21. - ISSN 0013-936X - p. 11988 - 11994.
plasma-mass spectrometry - water samples - environmental-samples - chromatography - food - soil - interferences - extraction - behavior - coal
Selenium (Se) is of key importance to human health with a very narrow concentration range of optimal dietary intake. Due to the inherent analytical challenge linked with the low natural abundance, information on precise and accurate Se speciation in deficient environments is hardly existent. This study presents a novel approach to determine Se species-specifically at ultratraces, by online coupling of a preconcentration (trap) column to an ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS) system. It is demonstrated that with this robust and work/time efficient method, the predominant selenium oxyanions, selenite (SeIV) and selenate (SeVI), can be quantified down to 7.3 and 8.3 picogram total Se, respectively, in an overall analytical time of 420 s, only. The applicability for environmental samples was proven on pristine volcanic ashes collected from seven different volcanoes. The high sensitivity of the novel approach allowed to determine speciation in samples that were strongly depleted in total selenium (
The composition of carcass volatile profiles in relation to storage time and climate conditions
Kasper, J. ; Mumm, R. ; Ruther, J. - \ 2012
Forensic Science International 223 (2012)1-3. - ISSN 0379-0738 - p. 64 - 71.
organic-compounds - human decomposition - human cadavers - human remains - bacteria - identification - chromatography - preferences - succession - headspace
After death organisms are decomposed by a variety of enzymes and microorganisms. The decay is typically accompanied by the emission of a plethora of volatile organic compounds responsible for the unpleasant odour of a carcass and thus, for the attraction of necrophagous insects. The composition of carcass-related odour profiles strongly depends on the composition of macro-nutrients like fat, carbohydrates, and particularly protein, as well as on the presence of oxygen which influences the community of microorganisms colonising the corpse. The impact of abiotic factors like temperature and humidity on carcass-related volatile emission is less well understood although these parameters are known to have a strong impact on the growth of microorganisms. In the present study we investigated the volatile succession released from dead mice stored for one, ten and 30 days under warm/hot (wh, 22 degrees C/80-90% RH) or cold/dry (cd, 12 degrees C/40-60% RH) climate conditions. We identified 51 typical carcass volatiles by coupled gas chromatography-mass spectrometry and analysed the volatile profiles by multivariate statistical methods to find compounds characterising the different stages. Dead mice stored under wh conditions released volatiles much faster, in higher amounts, and in a greater diversity than those stored under cd conditions. The relatively low amount of sulphur chemicals released under cd conditions were most striking. The results are discussed with respect to their possible applicability in forensic science and insect ecology studies. (C) 2012 Elsevier Ireland Ltd. All rights reserved.
Pesticide residues in individual versus composite samples of apples after fine or coarse spray quality application
Poulsen, M. ; Wenneker, M. ; Withagen, J.C.M. ; Christensen, H.B. - \ 2012
Crop Protection 35 (2012). - ISSN 0261-2194 - p. 5 - 14.
captan residues - multiresidue method - variability - fruit - chromatography - orchard - weather - deposit - trees - time
In this study, field trials on fine and coarse spray quality application of pesticides on apples were performed. The main objectives were to study the variation of pesticide residue levels in individual fruits versus composite samples, and the effect of standard fine spray quality application versus coarse spray quality application on residue levels. The applications included boscalid, bupirimate, captan, fenoxycarb, indoxacarb, pirimicarb, pyraclostrobin and thiophanate-methyl. Apples were collected from four zones in the tree and pesticide residues were detected in the individual apples. None of the results for the pesticides residues measured in individual apples exceeded the EU Maximum Residue Levels (MRLs). However, there was a large variation in the residues levels in the apples, with levels from 0.01 to 1.4 mg kg-1 for captan, the pesticide with the highest variation, and from 0.01 to 0.2 mg kg-1 for pyraclostrobin, the pesticide with the lowest variation. Residues of fenoxycarb and indoxacarb were only found in a few apples, probably due to the early application time of these two compounds. The evaluation of the effect of spray quality did not show any major difference between fine and coarse spray quality, except for carbendazim, the degradation product of thiophanate-methyl, where fine spray quality resulted in higher carbendazim residue levels than coarse spray quality. To examine the relationship between individual results and average results from ten apples, 20 composite samples were statistically constructed from sets of ten of the individual results. The variability factors for the individual samples (n = 80) at the 97.5 percentile were calculated for both standard and air induction nozzle application and were in the range of 0.9–9.4. The variability factor of seven used when EU member states calculate possible exceeding of Acute Reference Dose (ARfD) was adequate to encompass almost all the average results from the analyses of ten individual apples. However, for captan up to 9% of the results were not covered depending on which of the mathematically constructed composite concentrations was chosen. The variability factor of three, recommend by Codex, seems to be too low, because up to 30% of the apple samples for captan were not covered if the worst case scenario was chosen. The factor of three seems was also too low for thiophanate-methyl
In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans
Jonathan, M.C. ; Borne, J.J.G.C. van den; Wiechen, P. van; Souza Da Silva, C. ; Schols, H.A. ; Gruppen, H. - \ 2012
Food Chemistry 133 (2012)3. - ISSN 0308-8146 - p. 889 - 897.
gastrointestinal-tract - colonic function - fatty-acids - polysaccharides - kinetics - starch - gas - fermentability - chromatography - degradation
In vitro fermentation of twelve dietary fibers by fecal inocula from pigs and humans were performed. The fibers included homoglucans, mannans, fructans, polyuronides, and complex heteroglycans. Gas production, short chain fatty acid production and fiber degradation products were monitored during fermentation. Human inoculum has more ability to ferment resistant starch and fibers containing uronic acids. In contrast, pig inoculum is able to ferment cellulose, which is hardly fermented by human inoculum. The sugar and linkage composition of the fibers has an important influence on fiber fermentation patterns. Fibers containing uronic acids induced the production of acetate, whereas fibers containing neutral sugars induced the production of propionate or butyrate. Fermentation of the fructans showed that molecular size could be an influential factor, and fermentation of complex heteroglycans showed that the arrangement of sugars in the molecules may also affect the fermentation patterns. This experiment also shows that monitoring of fiber degradation products is important for understanding how fibers are degraded during fermentation.