Staff Publications

Staff Publications

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

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

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Selective and mild fractionation of microalgal proteins and pigments using aqueous two-phase systems
Suarez Ruiz, Catalina A. ; Emmery, Daniel P. ; Wijffels, Rene H. ; Eppink, Michel H.M. ; Berg, Corjan van den - \ 2018
Journal of Chemical Technology and Biotechnology 93 (2018)9. - ISSN 0268-2575 - p. 2774 - 2783.
aqueous two-phase systems - cholinium-based ionic liquids - microalgae biorefinery - pigments - proteins

BACKGROUND: Microalgal biomass is generally used to produce a single product instead of valorizing all of the cellular components. The biomass production and downstream processes are too expensive if only one product is valorized. A new approach was proposed for the simultaneous and selective partitioning of pigments and proteins from disrupted Neochloris oleoabundans cultivated under saline and freshwater conditions. RESULTS: An aqueous two-phase system composed of polyethylene glycol and cholinium dihydrogen phosphate selectively separated microalgal pigments from microalgal proteins. 97.3 ± 1.0% of lutein and 51.6 ± 2.3% of chlorophyll were recovered in the polymer-rich phase. Simultaneously, up to 92.2 ± 2.0% of proteins were recovered in a third phase (interface) in between the aqueous phases (interface). The recovered proteins, including Rubisco with a molecular weight of ∼560 kDa, seem to be intact and pigments did not suffer degradation, demonstrating the mildness of this system for fractionating microalgal biomolecules. CONCLUSION: The ability of aqueous two-phase systems (ATPSs) to simultaneously and efficiently fractionate different biomolecules in a mild manner from disrupted microalgae is demonstrated. This is an important step towards the development of a multiproduct microalgae biorefinery.

Dossier Veranderend eetpatroon
Mols, H. ; Kortstee, H.J.M. ; Warnaar, M. ; Methorst, B. ; Sijtsema, S.J. ; Dagevos, H. ; Onwezen, M.C. ; Ingenbleek, P.T.M. ; Genderen, R.A. van - \ 2017
Wageningen : Groen Kennisnet
nutrition - feeding habits - consumers - consumption - proteins - health
De trends in voedselland volgen elkaar in snel tempo op. Gezond, duurzaam, natuurlijk, gemak, out of home, fairtrade zijn trenditems die we in kranten, vakbladen en actualiteitenrubrieken, vlogs en blogs dagelijks kunnen ervaren.
Maar leidt het ook tot gewenste veranderingen in de voedselkeuze van de consument naar een gezonder en duurzamer eetpatroon.
Sugar beet leaves: from biorefinery to techno-functionality
Kiskini, Alexandra - \ 2017
University. Promotor(en): Harry Gruppen, co-promotor(en): Peter Wierenga. - Wageningen : Wageningen University - ISBN 9789463436793 - 141
sugarbeet - sugarbeet tops - biorefinery - bioprocess engineering - proteins - isolation techniques - physiological age - suikerbieten - suikerbietenloof - bioraffinage - bioproceskunde - eiwitten - isolatietechnieken - fysiologische leeftijd

Sugar beet leaves (SBL), which are a side stream of the sugar beets cultivation, are currently left unexploited after sugar beets have been harvested. The general aim of this thesis was to study the biorefinery of SBL, with a special focus on the isolation of proteins. To reach this aim the research was divided into three sub-aims: 1) to determine whether there is variability in the chemical composition of the leaves due to pre-harvest conditions (plant age), 2) to evaluate the variability of the techno-functionality of leaf soluble protein concentrate (LSPC) due to system conditions and 3) to extend current product and process synthesis approaches to enable the design of biorefining process. To address the first aim, SBL collected at different time points were used. Despite a small variation in the chemical composition of the leaves of different plant ages, a large effect of the plant age on the quality of LSPC was observed. In particular, LSPC from old plants was brown (indicative of polyphenol oxidase - PPO - activity), whereas LSPC from young plants was yellow. Based on these data, samples extracted with sodium disulfite (to inhibit PPO-mediated browning) were used for further experiments. The obtained LSPC consisted mainly of protein (69.3% w/w db (N∙5.23)) and carbohydrates (5.1% w/w db; half of which was charged carbohydrates). The main protein present in LSPC was Rubisco. The emulsion and foam properties of LSPC were studied as a function of protein concentration (Cp), pH and ionic strength (I). The minimal Cp of LSPC needed to form a stable emulsion (Ccr) was comparable to that of other widely used plant proteins, such as soy protein isolate. A critical ζ-potential (ζcr ~ 11 mV) was identified, below which flocculation occurs. At pH 8.0 and high I (0.5 M) the Ccr was higher than at low I (0.01 M), which relates to a higher protein adsorbed amount at the interface (Γmax). The foam ability (FA) of LSPC increased with Cp at all conditions tested. The FA was related to the soluble and not to the total Cp in the bulk. Interestingly, the minimal Cp; i.e.CcrFA needed to reach highest FA was constant as a function of pH. At high I (0.5 M) LSPC had higher FA than at low I (0.01 M), which was related to the faster adsorption of proteins at the interface. A minimum Cp was required to form stable foams. At pH 3.0 and 5.0 the foam stability of LSPC was higher than at pH 8.0. This was postulated to be due to formation of aggregates (between proteins or between proteins and charged carbohydrates). From these data it was shown that the techno-functional properties of LSPC could be linked to the molecular and interfacial properties of the dominant proteins in the concentrates. Thus, predictions for the techno-functional properties of impure systems, such as LSPC, can be made using only the known molecular properties of the dominant proteins and a small set of experiments. The knowledge acquired through the previous studies was used to adapt an existing methodology; namely the product-driven-process synthesis (PDPS) methodology, to extend its use in biorefinery. The adapted PDPS contained 4 novel steps, which facilitated its use in biorefinery. To illustrate how this new approach can be used in practice, a case study of a sugar beet leaves biorefinery was presented.

Production of protein‐based polymers in Pichia pastoris
Werten, Marc W.T. - \ 2017
University. Promotor(en): Martien Cohen Stuart; Gerrit Eggink, co-promotor(en): Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789463436069 - 241
proteins - polymers - pichia pastoris - gelatin - proteolysis - biosynthesis - eiwitten - polymeren - gelatine - proteolyse - biosynthese

From a chemistry perspective, proteins can be thought of as polymers of amino acids, linked by amide bonds. They feature unsurpassed control over monomer sequence and molecular size. The amino acid sequence of proteins determines their three-dimensional folded structure, resulting in unique properties. Proteins such as collagen, elastin, and silk play a crucial structure-forming role in various tissues and animal architecture such as spider webs. These proteins are characterized by highly repetitive amino acid sequences, and can reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. These unique properties have sparked the interest of material scientists, and mimics of these proteins have been designed and produced as heterologous proteins in suitable expression systems.

The most commonly employed host for these so-called protein-based polymers, or protein polymers for short, is the bacterium Escherichia coli. In this thesis, we explored the use of an alternative platform, namely the methylotrophic yeast Pichia pastoris (Komagataella phafii). This organism is well-known for its often relatively high yields, and offers a choice between intracellular and secretory production. Secretion of the polymer into the medium provides a highly effective first purification step, and precludes the need for cell disruption procedures that are cost-prohibitive at an industrial scale.

We evaluated the secretory production in P. pastoris of various protein polymers: murine collagen fragments (gelatins), a de novo-designed highly hydrophilic gelatin, silk-like proteins, hydrogel-forming triblock copolymers with collagen-inspired end blocks, block copolymers with heterodimer-forming modules, and silk-inspired triblock copolymers that feature integrin-binding or proteoglycan-binding cell-adhesive motifs. All of these protein polymers were produced at g/L levels, and various bioprocessing and strain engineering strategies were employed to address problems such as proteolytic degradation and other undesired posttranslational modifications. The basic physicochemical properties of the polymers produced were studied, which revealed interesting characteristics. Some of these polymers show promise for further development towards biomedical applications such as tissue engineering and controlled drug release.

Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding
Vissers, Anne M. - \ 2017
University. Promotor(en): Harry Gruppen; Wouter Hendriks, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789463432023 - 154
phenols - leaves - seaweeds - tannins - beta vulgaris - laminaria - proteins - catechol oxidase - nuclear magnetic resonance spectroscopy - in vitro - mass spectrometry - browning - fermentation - animal feeding - fenolen - bladeren - zeewieren - tanninen - eiwitten - kernmagnetische resonantiespectroscopie - massaspectrometrie - bruinkleuring - fermentatie - diervoedering

Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation.

Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties.
High molecular weight PhT from L. digitata could potentially modify protein properties by non‑covalent interactions. L. digitata contained PhT with subunits mainly connected via C‑O-C linkages, as determined using NMR spectroscopy. Further mass spectrometric analysis revealed the presence of a wide range of oligomers with degrees of polymerisation between 3 and 27. The interaction between PhT and proteins (b-casein and bovine serum albumin) was studied using model systems with different pH values, representing the various environments throughout the ruminants digestive tract. Phlorotannins bound to protein independent of pH, and broadened the pH range of protein precipitation from 0.5 to ~1.5 pH unit around the protein’s pI. At the pH of the abomasum of 2-3, the proteins re-solubilised again, presumably by increase in their net charge. Due to their ability to form water insoluble complexes, PhT could improve ruminal fermentation in vitro in a dose dependent manner, resulting in lower methane production and ammonia (NH3) concentration. The decreased NH3 concentration reflected decreased dietary protein breakdown in the rumen, which is considered a nutritional and environmental benefit.

FeedOmics, an approach to evaluate the functional properties of protein containing feed ingredients
Kar, Soumya K. - \ 2017
University. Promotor(en): Mari Smits; Jerry Wells, co-promotor(en): Alfons Jansman; Dirkjan Schokker. - Wageningen : Wageningen University - ISBN 9789463434461 - 254
compound feeds - ingredients - protein sources - proteins - functional properties - metabolism - feed formulation - protein digestion - proteomics - digestive tract - nutrition physiology - animal nutrition - livestock feeding - mengvoer - ingrediënten - eiwitbronnen - eiwitten - functionele eigenschappen - metabolisme - voersamenstelling - eiwitvertering - eiwitexpressieanalyse - spijsverteringskanaal - voedingsfysiologie - diervoeding - veevoeding

This thesis presents FeedOmics approach as a toolkit, to evaluate (novel) protein containing feed ingredients of different origin considering both their nutritional and functional value in terms of their capacity to support or modify nutrient supply, the animal’s physiology, tissue development and functioning. Such knowledge may contribute to introduce novel and/or alternative protein containing feed ingredients in the diet of livestock, thus creating a sustainable food supply for growing human population.

In vivo 1H NMR methods to study dynamics of chloroplast water and thylakoid membrane lipids in leaves and in photosynthetic microorganisms
Pagadala, Shanthi - \ 2017
University. Promotor(en): Herbert van Amerongen, co-promotor(en): Henk van As. - Wageningen : Wageningen University - ISBN 9789463431569 - 130
cell membranes - membranes - chloroplasts - thylakoids - photosynthesis - in vivo experimentation - stress conditions - stress - proteins - lipids - mobility - dynamics - celmembranen - membranen - chloroplasten - thylakoïden - fotosynthese - in vivo experimenten - stress omstandigheden - eiwitten - lipiden - mobiliteit - dynamica

Dynamics of thylakoid membranes and mobility of pigment-protein complexes therein are essential for survival of photosynthetic organisms under changing environmental conditions. The published approaches to probe mobility of the thylakoid membrane lipids and protein complexes are either dependent on the use of external labels or are used only for in vitro studies. Here, we present non-invasive 1H NMR methods (DOSY and DRCOSY) to study dynamics of water in chloroplasts, lipids in oil bodies and in thylakoid membranes and pigment-protein complexes under complete in vivo conditions in leaf disks of F. benjamina and A. platanoides and in suspensions of the green alga Chlamydomonas reinhardtii and blue-green alga Synechocystissp.PCC 6803.

In leaf disks of Ficus benjamina and Acer platanoides, water in chloroplasts could be clearly discriminated from other pools. Both water in chloroplasts, and water in vacuoles of palisade and spongy cells showed resonances in the high field part of the spectra (with respect to pure water), in contrast to what has been reported in literature. Subepidermal cells (present only in F. benjamina but not in A. platanoides) may act as a water storage, buffer pool during drought. This pool prevented the fast loss of water from the chloroplasts. Nutrient stress and excess salt stress resulted in accumulated lipid bodies and in striking differences in the dynamics and spectra/composition of the different components. T2 values of the different components are compared with those observed in suspensions of Synechocystissp.PCC 6803. The differences in membrane composition (ratio of the different membrane lipids) were clearly observed in the DANS of the oil bodies and the (thylakoid) membranes, but the diffusion coefficients were quite comparable. Also the DANS of the component that is assigned to the pigment-protein complexes are quite different, reflecting the differed composition. The diffusion coefficients of this component in isolated spinach thylakoids and in C. reinhardtii are very comparable, but about a factor of 10 lower with respect to that of Synechocystis at short diffusion times. The dynamics of these complexes in these systems are thus quite different.

The ribosome regulates flavodoxin folding
Houwman, Joseline A. - \ 2017
University. Promotor(en): Willem van Berkel, co-promotor(en): Carlo van Mierlo. - Wageningen : Wageningen University - ISBN 9789463431453 - 176
proteins - flavoproteins - cofactors - ribosomes - biosynthesis - eiwitten - flavoproteinen - co-factoren - ribosomen - biosynthese

During and after their translation by the ribosome, folding of polypeptides to biologically active proteins is of vital importance for all living organisms. Gaining knowledge about nascent chain folding is required to enhance our understanding of protein folding in the cell. This in turn allows us to obtain insights into factors responsible for protein misfolding, aggregation, and, potentially, for numerous devastating pathologies.

In Chapter 1 the model protein flavodoxin is introduced. Also theories about protein folding are presented, which led to the concept of the “folding energy landscape”. Flavodoxin folds via a misfolded off-pathway intermediate, which is molten globular and forms extensively during its refolding in vitro.

In Chapter 2 we show that flavodoxin also populates an on-pathway molten globule during its folding. In the F44Y variant of apoflavodoxin, lowering the ionic strength induces the off-pathway molten globule state. By adding the cofactor FMN, we could follow aspects of the folding of this protein, as off-pathway molten globular flavodoxin first has to unfold and subsequently refold before FMN can bind. Thus, presence of the off-pathway molten globule retards FMN binding. We determined the presence of the off-pathway molten globule at decreasing ionic strengths with cofactor binding kinetics and polarized time-resolved tryptophan fluorescence spectroscopy. Comparison of both data sets revealed the presence of another, concurrently present molten globule. This species is most likely on-pathway to native protein. To our knowledge this is the first time that two concurrent molten globules have been discovered that reside on folding routes of decidedly different nature (i.e., on- and off-pathway ones).

While much work has been done on the folding of flavodoxin in vitro, the next step is to elucidate how this protein folds in vivo. In Chapter 3 the first insights into cotranslational flavodoxin folding are presented. By using ribosomal nascent chains (RNCs) we could determine that when flavodoxin is fully exposed outside the ribosome it can bind its cofactor. However, while its five C-terminal amino acids are still sequestered in the ribosomal exit tunnel, the protein is in a non-native state and cannot bind FMN. Thus the last step in production of this flavoprotein in vivo is the binding of cofactor.

Chapter 4 reveals the influence of the ribosome on formation of the off-pathway molten globule of flavodoxin. By using RNCs of the F44Y variant of apoflavodoxin, we proved that the ribosome restrains formation of this molten globule. This discovery was possible by exploiting the findings of Chapter 2 and Chapter 3, namely that cofactor binding kinetics slow down when off-pathway molten globule is present and that a fully exposed, natively folded flavodoxin nascent chain binds FMN. For F44Y RNCs no retardation in FMN binding occurs, whereas cofactor binding slows down in case of isolated, full-length F44Y in the molten globule state. Thus the ribosome restrains formation of molten globules in stalled nascent flavodoxin. This is possibly due to electrostatic repulsion of the nascent chain by the ribosomal surface, as both are negatively charged, leading to entropic stabilization of native protein at physiological ionic strength.

In Chapter 5 we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These proteins form intermediates that are off-pathway to native protein and several of these species are molten globules. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. The experimentally characterized off-pathway intermediates seem to be of α-helical nature. We discuss the probing of the cotranslational folding of flavodoxin as a first step towards a molecular description of how flavodoxin-like proteins fold in vivo.

Finally, Chapter 6 touches upon the implications of our findings and possible applications in biotechnology, health and disease. A finding that has potential application is the role FMN has as a chemical chaperone. This chemical chaperone can already work at the cotranslational level, as binding of FMN stabilizes a nascent chain and thereby protects the nascent chain against degradation.

The transcriptome as early marker of diet-related health : evidence in energy restriction studies in humans
Bussel, Inge P.G. van - \ 2017
University. Promotor(en): Sander Kersten, co-promotor(en): Lydia Afman. - Wageningen : Wageningen University - ISBN 9789463430678 - 194
energy restricted diets - energy intake - gene expression - genomes - proteins - endurance - food composition - human nutrition research - energiearme diëten - energieopname - genexpressie - genomen - eiwitten - uithoudingsvermogen - voedselsamenstelling - voedingsonderzoek bij de mens

Background: Nutrition research is facing several challenges with respect to finding diet related health effects. The effects of nutrition on health are subtle, show high interindividual variations in response, and can take long before they become visual. Recently, the definition of health has been redefined as an organism’s ability to adapt to challenges and ‘this definition’ can be extended to metabolic health. In the metabolic context the ability to adapt has been named ‘phenotypic flexibility’. A potential new tool to magnify the effects of diet on health is the application of challenge tests. Combined with a comprehensive tool such as transcriptomics, the study of challenge tests before and after an intervention might be able to test a change in phenotypic flexibility. A dietary intervention well-known to improve health through weight loss is energy restriction (ER). ER can be used as a model to examine the potential of challenge tests in combination with transcriptomics to magnify diet-induced effects on health. As opposed to ER, caloric restriction (CR) is a reduction in energy intake aimed at improving health and life span in non-obese subjects and not directly aimed at weight loss. In this thesis, we aimed to investigate the use of the transcriptome as an early and sensitive marker of diet-related health.

Methods: First we studied the consequences of age on the effects of CR on the peripheral blood mononuclear cells (PBMCs) transcriptome. For that purpose, we compared the changes in gene expression in PBMCs from old men with the changes in gene expression in PBMCs from young men upon three weeks of 30% CR. To study the effect of a change in dietary composition during ER, we compared the changes in gene expression upon a 12 weeks high protein 25% ER diet with the changes in gene expression upon a 12 weeks normal protein 25% ER diet in white adipose tissue (WAT). Next, we investigated the added value of measuring the PBMC transcriptome during challenge tests compared to measuring the PBMC transcriptome in the fasted state to magnify the effects of ER on health. This was investigated by measuring the changes in gene expression upon an oral glucose tolerance test (OGTT) and upon a mixed meal test (MMT), both before and after 12 weeks of 20% ER. Finally, we determined the differences between a challenge test consisting of glucose alone, the OGTT, or consisting of glucose plus other macronutrients, the MMT, on the PBMC transcriptome in diet-related health.

Results: We observed that the transcriptome of PBMCs of healthy young men had a higher responsiveness in immune response pathways compared to the transcriptome of PBMCs of aged men upon CR (chapter 2). Also, we showed that upon a normal protein-ER diet the transcriptome of WAT showed a decrease in pathways involved in immune response and inflammasome, whereas no such effect was found upon a high protein-ER diet. These effect were observed while parameters such as weight loss, glucose, and waist circumference did not change due to the different protein quantities (chapter 3). 12 weeks of 20% ER was shown to increase phenotypic flexibility as reflected by a faster and more pronounced downregulation of OXPHOS, cell adhesion, and DNA replication during the OGTT compared to the control diet (chapter 4). Finally, two challenge tests consisting of either glucose (OGTT) or glucose plus fat and protein (MMT), were shown to result in a larger overlap than difference in the changes in gene expression of PBMCs (chapter 5).

Conclusions: Based on the differential changes in gene expression upon CR at different ages, we concluded that age is an important modulator in the response to CR. As a high protein ER diet induced transcriptional changes seemed to reflect less beneficial health effects than a normal protein ER diet we concluded that the diet composition is important in the health-effect of ER as measured by the transcriptome. Based on the faster PBMCs changes in gene expression during an OGTT upon 12 weeks of 20% ER, we concluded that the PBMC transcriptome combined with a challenge test can reflect changes in phenotypic flexibility. This makes challenge tests a suitable tool to study diet-related health effects. Finally, based on the changes in gene expression of the MMT and OGTT, we conclude that glucose in a challenge test is the main denominator of the postprandial changes in gene expression in the first two hours. Overall, these results lead to the conclusion that the transcriptome, especially in combination with challenges test, can be used as an early marker of diet-related health. The direct relation to health still needs to be investigated, but the possibility to use the transcriptome as an early marker of diet-related health gives rise to a better understanding of the effects of nutrition on health.

Tuning for light and more : engineering phototrophy and membrane proteins in Escherichia coli
Claassens, Nicolaas J.H.P. - \ 2017
University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Vitor Martins dos Santos. - Wageningen : Wageningen University - ISBN 9789463430920 - 328
escherichia coli - phototropism - membranes - proteins - light - photosystem i - gene expression - fototropie - membranen - eiwitten - licht - fotosysteem i - genexpressie

The application of microbial and plant photosynthesis for biobased production on the one hand has a huge potential but on the other hand photosynthesis has serious limitations regarding its efficiency. Hence, studying both fundamental features of photosynthetic processes and engineering of photosystems is of paramount interest, exploring the engineering of photosystems is the overarching aim of this thesis. As described in Chapter 1, natural photosystems may be modified or transplanted to allow for more efficient conversion of solar light energy into biochemical energy. Hereto ambitious proposals to engineer photosystems have been made, and to realize those endeavors the disciplines of synthetic and systems biology are required. To explore how to apply and improve those disciplines hereto, the work described in this thesis has focused on the transplantation of simple photosystems (proton-pumping rhodopsins; PPRs) into the cell membrane of the heterotrophic model bacterium Escherichia coli. Both in silico analyses, including metabolic and thermodynamic modeling (Chapter 3) and a series of experimental studies on transplanting PPR photosystems (Chapters 4,6 and 7) were performed, which identified several challenges, limitations and most importantly opportunities. This thesis also describes the application of novel tools to substantially improve the functional production of PPRs and a variety of other membrane proteins in E. coli.

Chapter 2 provides more details on previously reported examples of heterologous expression of PPRs in several hosts, and on the physiological impact of these transplanted photosystems. Based on this evaluation, some suggestions are made to improve and further exploit the transplantation of these photosystems.

In Chapter 3 a systematic, integrated in silico analysis is made of anaerobic, photo-electro-autotrophic synthetic metabolism in E. coli, consisting of (i) a PPR photosystem for ATP regeneration, (ii) an electron uptake pathway, and (iii) a natural or synthetic carbon fixation pathway. Constraint-based metabolic modelling of E. coli central metabolism is used, in combination with kinetic and thermodynamic pathway analyses. The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the PPR photosystems, and the relatively high ATP consumption due to maintenance. In general these analyses illustrate the potential of in silico analyses to identify potential bottlenecks and solutions in complex designs for autotrophic and photosynthetic metabolism, as a basis for subsequent experimental implementation.

To tackle a main bottleneck of PPR systems: their functional membrane-embedded production level, the heterologous production in E. coli of the proton-pumping rhodopsins from Gloeobacter violaceus (GR) and from Thermus thermophilus JL18 (TR) is quantified and experimentally optimized in Chapter 4. High constitutive production of both rhodopsin proteins is achieved by fine-tuning transcription and translation. Besides the canonical retinal pigment, the GR system has the ability to bind a light-harvesting antennae pigment, echinenone. After optimization of the heterologous pigment biosynthesis pathways for either retinal or echinenone production, appropriate quantities of retinal or echinenone for PPR reconstitution were detected in E. coli. Association of echinenone with GR broadens its absorption spectrum in E. coli, broadening the potential for light-harvesting also to blue light. Optimization of the branched pathway for simultaneous biosynthesis of both retinal and echinenone has been attempted by using a smart library of variable Ribosome Binding Sites (RBSs) with varying strengths (RedLibs). In general, the here described approaches towards improved functional production of rhodopsin photosystems in E. coli and their pigments may prove more widely applicable for heterologous production of more complex photosystems and other systems.

In Chapter 5 an up-to-date overview is provided on how codon usage can influence functional protein production. The fact that all known organisms have an incomplete set of tRNAs, indicates that biased codon usage could act as a general mechanism that allows for fine-tuning the translation speed. Although translation initiation is the key control step in protein production, it is broadly accepted that codon bias, especially in regions further downstream of the start codon, can contribute to the translation efficiency by tuning the translation elongation rate. Modulation of the translation speed depends on a combination of factors, including the secondary structure of the transcript (more or less RNA hairpins), the codon usage landscape (frequent and more rare codons) and for bacteria also RBS-like sequences at which ribosomes can pause. The complex combination of interdependent factors related to codon usage that can influence translation initiation and elongation. This complexity makes that the design of synthetic genes for heterologous expression is still in its infancy, and despite the availability of some codon usage algorithms, it is often as well a matter of trial and error.

In Chapter 6 the effect of different codon usage algorithms (optimization and harmonization) has been experimentally tested for heterologous production of membrane proteins. Apart from the codon usage algorithms also the combined effect of transcriptional fine-tuning in E. coli LEMO21(DE3) was assessed. The overproduction of 6 different membrane-embedded proteins, including 4 PPR variants (from bacteria, archaea and eukaryotes), was tested. For production of tested PPR variants, the different codon usage algorithms hardly influenced production, while transcriptional tuning had a large impact on production levels. Interestingly, for the other two tested non-PPR membrane proteins, some codon usage variants significantly improved production on top of transcriptional tuning. For both these proteins the codon-optimization algorithm reduced functional production below that of the wild-type codon variant, while the harmonization algorithm gave significantly higher production, equal or even higher than for the wild-type variant.

In Chapter 7 it is demonstrated that a translational-tuning system can be used to successfully optimize the expression of several membrane proteins, based on initial findings presented in Chapter 4. The employed, recently developed Bicistronic Design (BCD) system is based on translational coupling of a gene encoding a short leader peptide and the gene of interest that is under control of a variable ribosome binding site. A standardized library of 22 RBSs allows for precise, gene context-independent, fine-tuning of expression of this second gene, here encoding a membrane protein. For all four membrane proteins tested in this study the BCD approach resulted in 3 to 7-fold higher protein levels than those obtained by two other recently developed methods for optimizing membrane protein production. The presented approach allows for inducer-free, constitutive, high-level production of membrane proteins in E. coli, which can be widely applicable for both membrane protein purification studies as well as for synthetic biology projects involving membrane proteins.

In Chapter 8 a broad review and perspectives are provided on the potential of microbial autotrophs for the production of value-added compounds from CO2. Both photoautotrophic and chemolithoautotrophic production platforms are discussed, and recent progress in improving their efficiency and production potential is highlighted. Transplantation efforts for photosystems, but also for CO2 fixation pathways and electron uptake systems are discussed. An overview is provided on novel in silico and experimental approaches to engineer components related to autotrophy in heterotrophic and autotrophic model hosts, including approaches applied in this thesis. Future avenues are discussed for realizing more efficient autotrophic production platforms.

Finally, in Chapter 9 and 10 the work in this thesis is summarized and a general discussion is provided on future avenues for engineering of PPR photosystems, photosystems in general and on the optimization of membrane protein production.

Studying fast dynamics in biological complexes : from photosynthesis in vivo to single DNA molecules in vitro
Farooq, Shazia - \ 2017
University. Promotor(en): Herbert van Amerongen, co-promotor(en): Johannes Hohlbein. - Wageningen : Wageningen University - ISBN 9789463431002 - 149
biology - dna - proteins - interactions - probability analysis - förster resonance energy transfer - fluorescence - spectroscopy - photosynthesis - biologie - eiwitten - interacties - waarschijnlijkheidsanalyse - förster resonantie-energieoverdracht - fluorescentie - spectroscopie - fotosynthese

During the last decades, fluorescence spectroscopy has emerged as a powerful tool in the fields of biophysics, biotechnology, biochemistry, cellular biology and the medical sciences. These techniques are highly sensitive, and allow us to study the structure and dynamics of (bio)molecular systems (Valeur 2001). A significant advantage of fluorescence techniques is that they can often be non-invasive and measurements can be performed in real time. In this thesis different advanced fluorescence methods will be used to study two important biological processes: (1) DNA dynamics and (2) plant photosynthesis. The first part aims at improving the smFRET technique for the analysis of DNA dynamics and other fast conformational changes. This improvement is made by combining and developing instrumentation and data evaluation tools. The second part is the continuous development of time-resolved fluorescence spectroscopy methods, as well their application in the field of photosynthesis to study ultrafast processes in thylakoid membranes and leaves. The two fluorescence techniques are technically and conceptually very different, but they are both designed for analysis of biomolecular systems. In this thesis, the techniques are applied to study energy transfer and dynamical changes in DNAs, thylakoid membranes and leaves.

REFERENCE: VALEUR B 2001. Molecular Fluorescence: Principles and Applications. 1 ed: Wiley-VCH.

Structuring oil by protein building blocks
Vries, Auke de - \ 2017
University. Promotor(en): Erik van der Linden, co-promotor(en): Elke Scholten. - Wageningen : Wageningen University - ISBN 9789463430760 - 167
gelation - gels - proteins - mechanical properties - oils - solidification - gelering - eiwitten - mechanische eigenschappen - oliën - hardwording

Over the recent years, structuring of oil into ‘organogels’ or ‘oleogels’ has gained much attention amongst colloid-, material,- and food scientists. Potentially, these oleogels could be used as an alternative for saturated- and trans fats in food products. To develop oleogels as a suitable replacement for saturated fats, the requirements go beyond merely providing a solid appearance to an otherwise liquid oil. For food applications, the gelator should be a well-known ingredient for food manufacturers, have a good nutritional value, and contribute to ‘clean labelling’. Proteins meet all these requirements and could therefore be of high potential. The general concept of protein-based oil gelation fits well into the growing general interest to reduce solid fats from food products along with increase in flexibility in terms of choice of ingredients. In this thesis, the suitability of proteins as a structuring agent for liquid oil was investigated and the rheological behaviour was described. To create protein oleogels, heat-set whey protein gels and protein aggregates, or ‘building blocks’, are created in an aqueous environment. Then, the aqueous phase is exchanged for a liquid oil phase via an intermediate solvent. It was show that by using this procedure, the created protein building blocks are highly efficient in creating oleogels. It is encouraging to see that the interactions between proteins can be altered by simple changes to the system, such as changing the oil type, water addition, or applying a heat treatment. This leads to the possibility to effectively and substantially tune the rheological properties of the final oleogel, such as its gel strength or yielding behaviour.

Of proteins and processing: mechanisms of protein damage upon rapeseed processing and their effects on nutritional value
Salazar Villanea, Sergio - \ 2017
University. Promotor(en): Wouter Hendriks; Harry Gruppen, co-promotor(en): Thomas van der Poel; E.M.A.M. Bruininx. - Wageningen : Wageningen University - ISBN 9789462579873 - 182
animal nutrition - rapeseed - rapeseed protein - feed processing - proteins - digestion - pigs - diervoeding - raapzaad - raapzaadeiwit - voedermiddelbewerking - eiwitten - spijsvertering - varkens

Hydrothermal processing is a common practice during the manufacture of protein-rich feed ingredients, such as rapeseed meal (RSM), and feeds. This processing step can induce physical and chemical changes to the proteins, thereby reducing the digestibility and utilization of crude protein (CP) and amino acids (AA). Whilst most literature has linked the chemical changes to the proteins with negative effects on protein digestibility, the effects of the physical changes of the protein conformation have not been considered simultaneously. Hence, the aim of this thesis was to provide further insight into the mechanisms of protein damage during ingredient/feed processing and their effects on protein hydrolysis/digestibility. In Chapter 2, the available literature on the physical changes that occur to vegetable proteins used in swine diets after processing was reviewed. Overall, hydrothermal processing increases the contents of intermolecular/intramolecular β-sheets and disulfide bonds, which were negatively correlated to protein digestibility. The correlations, however, were dependent on the type of protein analysed. When the physico-chemical changes in the proteins occur during processing of the ingredients, proteins usually become less responsive to further processing treatments. Rapeseed proteins were used as model, as this oilseed is hydrothermally processed during the oil extraction process and is further processed when incorporated in animal diets.

Protein damage during production of rapeseed meal

There is high variability in the nutritional value of commercial RSM. The variation is mainly due to the conditions used during the desolventization/toasting step. Therefore, the aim of the experiment in Chapter 3 was to characterize the secondary structure and chemical changes that occur during toasting of RSM and their effects on in vitro protein digestibility. A cold defatted RSM was toasted for 120 min with samples obtained every 20 min. Increasing the toasting time from 0 to 120 min increased protein denaturation by 3-fold and decreased protein solubility by 4-fold, lysine content by 23% and the reactive lysine content by 37%. The proportion of intermolecular β-sheets increased after the initial 20 min of toasting, but steadily decreased thereafter. The contrary was observed for the proportion of α-helices. The changes in the secondary structure of proteins were not correlated to the rest of the physical and chemical changes. Therefore, changes in the secondary structure of proteins cannot be considered good indicators of damage to proteins due to hydrothermal processing. The rate of protein hydrolysis decreased by 2-fold when toasting time was increased from 0 min to 120 min. The changes in protein solubility and lysine/reactive lysine contents were positively correlated to the rate of protein hydrolysis. Changes to the physical conformation of rapeseed proteins occur at faster rates during toasting compared to chemical changes.

In Chapter 4, it was hypothesized that the decrease in the rate of protein hydrolysis with increasing toasting time was due to the reduction in protein solubility. In order to test this hypothesis, the soluble and insoluble protein fractions from each of the RSM studied in Chapter 3 were separated and hydrolysed. Hydrolysis kinetics and the molecular size distribution of the peptides resulting after hydrolysis were analysed. The extent of hydrolysis of the insoluble protein fraction was 44% higher than that of the soluble protein fraction. The rate of hydrolysis of the soluble protein fraction separated from the hydrothermally treated RSM was 3-9 fold higher than that of the insoluble protein fraction. In the insoluble fraction, formation of both disulfide bonds and Maillard reaction products (MRP) (fructosyl-lysine [FL], carboxymethyl-lysine [CML] and carboxyethyl-lysine [CEL]) was noticed, which explains the decrease in the rate of protein hydrolysis with longer toasting times. Overall, increasing the toasting time of the whole RSM and the insoluble protein fraction increased the size of the peptides resulting after enzymatic hydrolysis. A shift in the mechanism of protein hydrolysis from a more one-by-one type to a more zipper-type likely explained the correlations between the rate of hydrolysis and the molecular size distribution after hydrolysis. Protein solubility seems to be a key parameter for understanding the decrease in the rate of protein hydrolysis with increasing toasting time.

The correlations between two in vitro protein digestibility methods and the standardised ileal digestibility in growing pigs of severe thermally-treated soybean and rapeseed meals were studied in Chapter 5. Soybean meal and RSM were toasted in the presence of lignosulfonate in order to induce severe thermal damage to the proteins. In vitro protein digestibility was analysed using the two-step enzymatic method (pepsin at pH 2.0 and pancreatin at pH 6.8) and the pH-STAT method. The standardised ileal digestibility values were obtained from a previous experiment, in which ileal-cannulated growing pigs were used. The degree of hydrolysis after 10 min was positively correlated (r = 0.95, P = 0.046) to the standardised ileal CP digestibility. The in vitro rate of protein hydrolysis using the pH-STAT method and CP digestibility using the two-step enzymatic method tended to be positively correlated to the standardised ileal digestibility of CP (r = 0.91, P = 0.09, for both in vitro methods). In conclusion, both in vitro methods might be used for the in vivo digestibility of severe thermally-treated ingredients.

Effects of diet processing on protein digestibility of RSM with different extents of damage

The processed ingredients (e.g. rapeseed meal) are mixed with other ingredients and processed further during the compound feed manufacturing process. The effects of the diet processing methods (e.g. pelleting and extrusion) on protein digestibility could depend on the extent of the damage of the ingredients used. The aim of Chapter 6 was to test the effects of toasting time of rapeseed meal, diet processing method and the interaction between both on protein digestion along the gastrointestinal tract and apparent/standardised ileal digestibility of CP and AA. Mash, pelleted and extruded diets were manufactured using either 0, 60 or 120 min toasted RSM as the only protein source, for a total of 9 different experimental diets. Whilst increasing the toasting time decreased the contents of lysine and reactive lysine in the diets, no effects were noticed after pelleting or extrusion of the diets compared to the mash. The mean particle size of the diets was reduced from 479 μm in the mash diets to 309 and 211 μm after pelleting and extrusion, respectively. A total of 81 growing boars were individually fed with one of the experimental diets. Following euthanasia, the small intestine was divided in 3 sections of equal length and the contents of the final 1.5 m of each small intestine section were sampled. The apparent CP digestibility for each section of the gastrointestinal tract was used to calculate the rate of CP digestion based on a second order equation. The rate of digestion was higher in the diets containing 0 min toasted RSM compared to the diets that contained 60 or 120 min toasted RSM. The diet processing method tended to affect the rate of protein digestion, with higher rates for the extruded > pelleted > mash diets. Significant effects of the interaction between toasting time and diet processing method were found on the apparent ileal CP digestibility. Whilst a lower apparent ileal CP digestibility was found in the 120 min toasted RSM mash diet compared to the 0 and 60 min toasted RSM mash diets, no differences were observed between the different toasting times in the pelleted and extruded diets. Similar significant interactions were noticed for the apparent ileal digestibility of some dispensable and indispensable AA (e.g. arginine, isoleucine, leucine, methionine, threonine, alanine, glycine, proline, serine). Pelleting of the 60 and 120 min toasted RSM diets increased the standardized ileal digestible CP content by 6 and 15%, respectively, compared to the 60 and 120 min toasted mash diets. Extrusion of the 0, 60 and 120 min toasted RSM diets increased the standardized ileal digestible CP content by 5, 9 and 12%, respectively, compared to the 0, 60 and 120 min toasted mash diets. Similar positive effects of pelleting and extrusion were obtained for the apparent ileal digestible contents of lysine and reactive lysine, especially for the diets that contained RSM toasted for longer times. In conclusion, the severe effects of protein damage during the production of RSM on protein digestibility can be (partially) ameliorated by processing of the diets.

Processing of ingredients and diets can lead to the formation of early (e.g. FL) and advanced (e.g. CML and CEL) MRP. These MRP have been associated with common metabolic disorders, for example atherosclerosis. Absorption of dietary MRP has been previously estimated based on indirect measurements, such as concentrations in blood, urine and faeces, which could be biased by endogenous formation of MRP and deposition in tissues. Hence, the aim of Chapter 7 was to measure the apparent ileal digestibility of early (FL, determined as furosine after acid hydrolysis) and advanced (CML and CEL) MRP. The same diets and ileal digesta samples as in Chapter 6 were used in this study. The 0 min toasted RSM diets (mash, pelleted and extruded) were excluded from this study. Sucrose was added to these diets immediately before feeding in order to increase their feed intake, which could confound the determination of the apparent ileal digestibility of the MRP for these treatments. The content of FL was higher in the 120 min compared to the 60 min toasted RSM diets, whereas it was lower in the extruded diets compared to the pelleted and mash diets. The decrease in FL content after extrusion of the diets can probably be related to conversion of the early into advanced MRP, as the content of CML was higher in the extruded diets compared to the pelleted and mash diets. The contents of FL, CML and CEL were positively correlated to the contents of these compounds in the ileal chyme. The apparent ileal digestibility of FL, CML and CEL for the different diets ranged from -8.5 to 19.1%, -0.2 to 18.3% and 3.6 to 30%, respectively. In conclusion, the apparent ileal digestibility of the early and advanced MRP from thermally-treated RSM diets in growing pigs were overall low and did not seem to be related to the contents of these compounds in the diets.

The results of this thesis indicate that the changes to the physical conformation of proteins during toasting of RSM occur at a faster rate than chemical changes. Both types of changes affect protein solubility, which is important in determining the rate of protein hydrolysis. It is suggested in this thesis that the rate of protein hydrolysis of hydrothermally-processed ingredients is probably the main contributing factor for the in vivo protein digestibility. This was demonstrated, as increasing the toasting time of RSM decreased the rate of CP digestion in growing pigs. The negative effects of longer toasting times on CP and AA digestibility, however, could be ameliorated by pelleting and extrusion of the diets. The positive effects of diet processing methods (pelleting and extrusion) on the digestibility of damaged proteins from thermally-treated ingredients should be taken into account in feed evaluation studies and formulation of practical diets.

Energie en eiwitwaardeschatting gras-klaverkuilen
Schooten, H.A. van; Spek, J.W. - \ 2016
Wageningen : Wageningen Livestock Research (Wageningen Livestock Research rapport 994) - 33
grasklaver - eiwitten - energie - voedingswaarde - biochemie - diervoedering - grass-clover swards - proteins - energy - nutritive value - biochemistry - animal feeding
Mild disintegration of green microalgae and macroalgae
Postma, Richard - \ 2016
University. Promotor(en): Michel Eppink; Rene Wijffels, co-promotor(en): Giuseppe Olivieri. - Wageningen : Wageningen University - ISBN 9789462579477 - 181
algae - chlorella vulgaris - bioprocess engineering - biorefinery - proteins - milling - carbohydrates - biobased economy - disintegrators - technology - extraction - algen - bioproceskunde - bioraffinage - eiwitten - maling - koolhydraten - desintegrators - technologie - extractie

An increased worldwide protein demand for food and feed and the necessity to release the water soluble proteins in the first stage of the cascade biorefinery require the development of mild protein extraction technologies. Cell disintegration is the first hurdle and is considered as one of the most energy consuming steps. Therefore, this thesis focused on the development of a mild, scalable and energy efficient disintegration technology for green microalgae and macroalgae (seaweed) aimed on extraction of water soluble components (like proteins and carbohydrates).

For microalgae disintegration, two main technologies were investigated. First of all the conventional technology bead milling and second a novel approach using Pulsed Electric Field (PEF). In Chapter 2 a benchmark was set by means of bead milling for the release of water soluble protein from the green microalgae Chlorella vulgaris. Overall, protein yields between 32 and 42% were achieved, while the energy consumption was reduced with 85% by selective protein extraction to values as low as 0.81 kWh kgDW-1. Remarkably, the benchmark was much better than expected.

In Chapter 3 the bead mill was further optimized by decreasing the applied bead size, furthermore the applicability of bead milling on two additional microalgae species (Neochloris oleoabundans, Tetraselmis suecica) was shown. In addition, to be able to better understand the disintegration mechanism, the so-called stress model was applied. This model describes the comminution process in a bead mill as function of the amount of bead contacts and the force of each impact. The release kinetics could be improved and thereby the specific energy consumption could be reduced to 0.45‒0.47 kWh kgDW-1 by using 0.3 mm beads for all algae.

Chapter 4 describes a screening on the applicability of PEF, over a broad range of operating conditions, for the extraction of water soluble proteins from the microalgae C. vulgaris and N. oleoabundans. No substantial protein yields were observed under the investigated conditions. This led to the conclusion that PEF is not suitable to release water soluble proteins, not even at specific energy consumptions much higher than those for the benchmark, bead milling.

In Chapter 5 it was attempted to improve the performance of PEF by investigating the synergistic effect with the processing temperature. The PEF experiments were performed using a pilot scale continuous flow electroporation unit in which the processing temperature was controlled between 25 – 65 °C. The results showed that under the tested conditions, the combined PEF-Temperature treatment did not cause substantial disintegration of the algal cells to effectively release water soluble proteins.

In addition to the microalgae, macroalgae were subject of investigation in the search for new protein sources in Chapter 6. Four batch technologies were used to disintegrate the green macroalgae Ulva lactuca, being; osmotic shock, enzyme incubation, PEF and High Shear Homogenization (HSH). In descending order the highest protein yields per treatment; HSH (~40%) > enzyme degradation (~25%) > osmotic shock (~20%) > PEF (~15%).

In the final chapter the main results and remaining bottlenecks are discussed and a future outlook on microalgae disintegration is presented. To date, bead milling is the only technology able to disintegrate fresh microalgae at specific energy consumptions below 10% of the total energy available from the algae and release substantial amounts of water soluble protein. The future outlook was based on a techno-economic evaluation, which showed that the cultivation costs are limiting the economic feasibility of microalgae biorefinery. Future focus should be on the cultivation.

Older adults, mealtime-related emotions, and functionalities : tailoring protein-enriched meals
Uijl, Louise C. den - \ 2016
University. Promotor(en): Kees de Graaf, co-promotor(en): Stefanie Kremer; Gerry Jager. - Wageningen : Wageningen University - ISBN 9789462578920 - 178
meals - emotions - elderly nutrition - elderly - smell - food preferences - protein - proteins - questionnaires - young adults - chocolate - maaltijden - emoties - ouderenvoeding - ouderen - reuk - voedselvoorkeuren - eiwit - eiwitten - vragenlijsten - jongvolwassenen - chocolade

Background and aim

Dietary proteins are of special interest for the heterogeneous group of older adults, since these people do not always have an adequate protein intake. When protein-rich products are better aligned with the requirements of older persons, an adequate nutrient intake is more likely. In this thesis we therefore explored two approaches for tailoring protein-enriched meals to older consumer subgroups; emotion-based and functionality-based. We expected a better ‘product-cluster fit’ (i.e. a more positive meal experience) when the clusters’ meal associations are congruent to their mealtime expectations.

Methods

We conducted an online survey in which vital community-dwelling older adults (n=392) reported their mealtime-related emotions and mealtime functionality. Using a hierarchical clustering analysis we described clusters within our population. Subsequently, we explored the extent to which the expectations of these clusters can be applied for the development of tailored protein-enriched meals. For the emotion-based approach, we conducted two central location tests (CLTs, n=461) to explore older adults’ food-evoked emotions. For the functionality-based approach we conducted in-depth interviews in order to get further insights regarding functional mealtime expectations and attitudes towards proteins and protein-enrichment. Based on the latter insights we tailored PE meal concepts to two functionality-based segments. In a final home-use test, the members of the functionality-based segments (n=91) prepared and evaluated the tailored PE meal concepts.

Results

The emotion-based approach resulted in four clusters; pleasurable averages, adventurous arousals, convivial indulgers, and indifferent restrictives. These emotions that these segments associated with their mealtimes varied along the two dimensions valence and arousal. However, from both CLTs we learned that the variation in valence-arousal as observed for mealtime-related emotions was not observed for emotions related to actual foods. The latter makes it challenging to identify products that evoke emotions congruent to the mealtime expectations of the emotion-based clusters.

With regard to the functionality-based approach, we encountered three clusters; physical nutritioners, cosy socialisers, and thoughtless averages. The cosy socialisers value the social interactions and cosiness during their mealtimes, whereas the physical nutritioners focus more on the health and nutrient aspects of meals. Thoughtless averages have the least distinctive mealtime expectations. We translated these functional mealtime expectations into two PE meal concepts; one tailored to cosy socialisers and one tailored to physical nutritioners. These meal concepts were well-accepted by the participants. However, congruency between mealtime expectations and functional meal associations did not result in a better ‘product-cluster fit’.

Conclusions

Given the challenge to identify congruency between the meal associations and the mealtime expectations of the emotion-based clusters, we consider the emotion-based approach to be not yet actionable enough as a basis for tailoring PE products to older consumers. In contrast, the functionality-based approach appeared to be more promising, since the functional meal expectations could be translated to well-accepted tailored PE meal concepts. However, the effectivity of our functionality-based approach was not yet confirmed in this thesis, since congruency between functional meal associations and functional meal expectations did not necessarily result in a better ‘product-cluster fit’. Future studies, focussing on e.g. other meal types, are recommended to further explore mealtime functionality as a basis for tailoring PE meals to older consumer subgroups.

Water-binding of protein particles
Peters, J.P.C.M. - \ 2016
University. Promotor(en): Atze Jan van der Goot; Remko Boom. - Wageningen : Wageningen University - ISBN 9789462578623 - 198 p.
whey protein - proteins - particles - water binding capacity - water - nuclear magnetic resonance - wei-eiwit - eiwitten - deeltjes - waterbindend vermogen - kernmagnetische resonantie

As overweight and obesity become more prevalent in society, the demand for food products that can help maintain body weight increases. One way to make such products is by decreasing the protein and fat content through increasing the water content. This thesis describes the potential of protein microparticles (MPs) to bind ≥ 90% w/w water tightly without negatively influencing sensory perception. Therefore, two types of MPs were prepared: heat- and cold-gelated MPs. In addition, pea protein isolate, soy protein isolate, lupin protein concentrate and vital wheat gluten particles were used. To determine their water-binding capacity (WBC), pellets were made by centrifuging dispersions of those particles. These measurements showed that some of these pellets can bind ≥ 90% w/w water. However, the WBC of a pellet contains both water bound within and between the particles, which means that the WBC of a pellet is always unequal to the WBC of the protein particles themselves. To gain more insight in these two water domains and the pellet as a whole, a combination of time domain nuclear magnetic resonance and microscopy was found to be useful. From the measurements, it was concluded that the WBC of a pellet is determined by the structure of protein particles (nanostructure) as well as the structure between particles (microstructure). Overall, the WBC of pellets being larger than that of particles themselves suggests that pellets are a good alternative to the use of MPs for the purpose of increasing the water content of a product, provided they remain a network in the product.

Quantitative and ecological aspects of Listeria monocytogenes population heterogeneity
Metselaar, K.I. - \ 2016
University. Promotor(en): Marcel Zwietering; Tjakko Abee, co-promotor(en): Heidy den Besten. - Wageningen : Wageningen University - ISBN 9789462577664 - 173 p.
listeria - listeria monocytogenes - stress - stress tolerance - ribosomes - proteins - lactobacillus plantarum - behaviour - ecological assessment - genome analysis - dna sequencing - resistance - heterogeneity - stresstolerantie - ribosomen - eiwitten - gedrag - ecologische beoordeling - genoomanalyse - dna-sequencing - weerstand - heterogeniteit

Bacterial stress response and heterogeneity therein is one of the biggest challenges posed by minimal processing. Heterogeneity and resulting tailing representing a more resistant fraction of the population, can have several causes and can be transient or stably in nature. Stable increased stress resistance is caused by alterations in the genome and therefore inheritable and is referred to as stable stress resistant variants. Also L. monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress resistant variants. Adverse environments were shown to select for stable stress resistant variants. The objective of the research described in this thesis was to evaluate if L. monocytogenes population diversity and the presence of stable resistant variants is a general phenomenon that is observed upon different types of stress exposure, to get more insight in the mechanisms leading to increased resistance and to evaluate the ecological behaviour and potential impact on food safety of these stable resistant variants. Acid stress was chosen as it is an important hurdle both in food preservation, as well as in stomach survival.

First, the non-linear inactivation kinetics of L. monocytogenes upon acid exposure were quantitatively described. A commonly used biphasic inactivation model was reparameterized, which improved the statistical performance of the model and resulted in more accurate estimation of the resistant fraction within L. monocytogenes WT populations. The observed tailing suggested that stable stress resistant variants might also be found upon acid exposure. Indeed, 23 stable acid resistant variants of L. monocytogenes LO28 were isolated from the tail after exposure of late-exponential phase cells to pH 3.5 for 90 min, with different degrees of acid resistance amongst them. Increased acid resistance showed to be significantly correlated to reduced growth rate. Studying the growth boundaries of the WT and a representative set of variants indicated that the increased resistance of the variants was only related to survival of severe pH stress but did not allow for better growth or survival at mild pH stress.
A set of variants were further characterized phenotypically and cluster analysis was performed. This resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation and virulence indicators. A higher glutamate decarboxylase (GAD) activity correlated with increased acid resistance. Whole genome sequencing of a set of variants was performed and revealed mutations in rpsU, encoding ribosomal protein S21. This rpsU mutation was found in all 11 variants comprising the largest phenotypic cluster, indicating a potential role of this ribosomal protein in stress resistance. Mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and HHP resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants.

Next, the performance in mixed species biofilms with Lactobacillus plantarum was evaluated, as well as their benzalkonium chloride (BAC) resistance in these biofilms. It was hypothesized that the acid resistant variants might also show better survival in biofilms with L. plantarum, which provide an acidic environment by lactose fermentation with pH values below the growth boundary of L. monocytogenes when biofilms mature. L. monocytogenes LO28 WT and eight acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, which could be correlated to increased survival in the mixed biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms.

Lastly, a set of robustness and fitness parameters of WT and variants was obtained and used to model their growth behaviour under combined mild stress conditions and to model their performance in a simulated food chain. This gave more insight in the trade-off between increased stress resistance and growth capacity. Predictions of performance were validated in single and mixed cultures by plate counts and by qPCR in which WT and an rpsU deletion variant were distinguished by specific primers. Growth predictions for WT and rpsU deletion variant were matching the experimental data generally well. Globally, the variants are more robust than the WT but the WT grows faster than most variants. Validation of performance in a simulated food chain consisting of subsequent growth and inactivation steps, confirmed the trend of higher growth fitness and lower stress robustness for the WT compared to the rpsU variant. This quantitative data set provides insights into the conditions which can select for stress resistant variants in industrial settings and their potential persistence in food processing environments.

In conclusion, the work presented in this thesis highlights the population diversity of L. monocytogenes and the impact of environmental conditions on the population composition, which is of great importance for minimal processing. The work of this thesis resulted in more insight in the mechanisms underlying increased resistance of stress resistant variants and quantitative data on the behaviour of stress resistant variants which can be implemented in predictive microbiology and quantitative risk assessments aiming at finding the balance between food safety and food quality.

Co-assembled DNA-protein polymer bottlebrushes : main-chain stiffening & liquid crystallinity
Storm, I.M. - \ 2016
University. Promotor(en): Martien Cohen Stuart; Frans Leermakers, co-promotor(en): Renko de Vries. - Wageningen : Wageningen University - ISBN 9789462577466 - 161 p.
polymers - liquid crystals - dna - proteins - polymeren - vloeibare kristallen - eiwitten

Bottlebrushes are macromolecules consisting of a backbone polymer onto which side chains are either physically or chemically grafted. Early theories suggested that attaching side chains to a (flexible) backbone molecule would induce the so-called main-chain stiffening effect. This newly formed bottlebrush molecule should therefore behave as a semi-flexible polymer rather than a flexible polymer. Due to this semi-flexible behaviour bottlebrushes should also be able to show liquid crystalline behaviour. However, there are very few examples of bottlebrush systems that are able to make liquid crystalline phases. In this thesis, we present a co-assembled bottlebrush system that consist of DNA as the backbone molecule and genetically engineered protein polymers as side chains. This co-assembled system is one of the few bottlebrush systems that actually does show liquid crystalline behaviour. This ability makes this bottlebrush system a perfect system to explain why it is so very difficult to make liquid crystalline phases with bottlebrushes. We have shown that attaching side chains will, at first, result in an effectively more flexible bottlebrush system. Only for systems with very densely packed and long side chains is the stiffness of the bottlebrush molecule increasing. Moreover, with osmotic stress experiments we have shown that the presence of free polymers also has a negative influence on the stiffness of bottlebrush molecules and hence this reduces the tendency for the system to form liquid crystals.

Silky gels for cells : Self-assembling protein-based polymers for use in tissue engineering
Wlodarczyk, M.K. - \ 2016
University. Promotor(en): Martien Cohen Stuart; Marleen Kamperman; S.C.G. Leeuwenburgh. - Wageningen : Wageningen University - ISBN 9789462576230 - 194 p.
polymers - proteins - biomedical engineering - biomaterials - recombinant dna - transplantation - compatibility - encapsulation - heparin - biodegradation - physical properties - polymeren - eiwitten - biomedische techniek - biomaterialen - transplantatie - compatibiliteit - inkapselen - heparine - biodegradatie - fysische eigenschappen

Tissue engineering is a relatively new, but actively developing field of biomedical science. It aims at organ or tissue regeneration by use of scaffolds, which are usually seeded with cells prior to implantation, and stimulated by bioactive cues or growth factors. It is a promising and valuable alternative to the use of transplants, for which the demand is greater than the supply, and for which application is connected with high risk of rejection and infection due to immunosuppressant medication. One of the main challenges of tissue engineering, that we tried to address in this thesis, is the design of biocompatible and functional biomaterials that could serve as cell scaffold. We investigated, if protein-based polymers, more specifically, if the de novo designed, C2SH48C2 copolymer, which self-assembles into fibers upon a pH-trigger, is a suitable material for cell scaffolds.

In Chapter 2 we described the design and production, by means of recombinant DNA technology, of C2SH48C2. The protein was efficiently secreted by Pichia pastoris at high yields of g/l levels and we proposed an effective purification method. We showed that fibers and gels form by self-assembly upon pH adjustment, and that rheological properties of the obtained hydrogels depend on the total protein concentration. In view of potential biomedical applications, erosion studies were performed, which indicated that the gels exhibited long term stability in conditions mimicking those in body fluid. The biocompatibility of the gel scaffolds was demonstrated in a 2D cell culture study. However, despite the cell viability, a low proliferation rate was observed.

To improve cell performance in contact with C2SH48C2 hydrogels (Chapter 3) we incorporated active domains in the C2SH48C2 protein by recombinant functionalization. We described the synthesis of two protein variants: (1) BRGDC2SH48C2, N-terminally enriched in integrin-binding domains (RGD) and (2) BKRSRC2SH48C2, N-terminally enriched in heparin binding domains (KRSR). We showed precise control over the amount of active domains in the final gels, by simply mixing the variants of the proteins in the desired molar ratio before inducing gelation. A 23-day cell culture study, performed using MG-63 cells, revealed that the presence of RGD and KRSR domains positively influenced cell attachment, spreading and activity. A synergistic effect was observed, i.e. scaffolds containing both bioactive domains yielded fully confluent layers of cells at an earlier stage during cell culture than the other gels. We concluded that cell behavior can be controlled by tuning the content of functional domains.

In Chapter 4, we tested the suitability of the C2SH48C2 protein, enriched in RGD domains, for cell encapsulation, as the conditions of 3D cell culturing are more similar to the environment of cells in the body. We independently varied gel stiffness (by means of protein concentration) and functional motif (RGD) density, and analyzed the influence of these parameters on the cellular response. The viability and proliferation of MG-63 cells, encapsulated in the gels at different protein concentrations and RGD densities, was investigated with a cell activity assay, and by quantitative analysis of confocal pictures of nuclei (DAPI stain) and F-actin (phalloidin). We showed that optimal cell behavior is obtained in the presence of RGD domains and at low protein concentrations. The results indicated that RGD functionality is not the sole requirement; the gel matrix needs to exhibit the right mechanical properties and architecture to allow for cell growth, cytoplasmic extension and migration.

Finally, in Chapter 5, we showed that active domains (here KRSR) can serve multiple functions in the material. We demonstrated the cross-linking ability of KRSR domains in the presence of heparin, leading to structural and mechanical changes in the scaffolds. In dilute systems (0.1 % (w/v)), heparin increases the rate of fiber growth, and induces fiber bundling. At higher protein concentrations, leading to the hydrogel formation (2 % (w/v)), the gelation rate and final storage modulus can be tuned by the amount of heparin and KRSR domain density. We concluded that with this approach, the material properties of C2SH48C2 protein gels can be effectively and simply controlled in a straightforward and biocompatible way.

In Chapter 6 we described the main requirements for biomaterials and discussed to what extent they are fulfilled by protein-based polymers, and in particular, by the presented C2SH48C2 protein. The main advantages over alternative materials, and the challenges that need to be addressed before application in tissue engineering becomes a reality, were discussed. We ended with suggestions to improve the properties of C2SH48C2 protein for use as a biomaterial, especially its biodegradability, and its structural and mechanical properties.

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