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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Undernutrition management and the role of protein-enriched meals for older adults
Ziylan, Canan - \ 2016
Wageningen University. Promotor(en): Lisette de Groot; Stefanie Kremer; Annemien Haveman-Nies. - Wageningen : Wageningen University - ISBN 9789462579323 - 148
elderly - elderly nutrition - undernutrition - enrichment - protein - eating patterns - feeding behaviour - meals - nursing homes - ouderen - ouderenvoeding - ondervoeding - verrijking - eiwit - eetpatronen - voedingsgedrag - maaltijden - verpleeghuizen

Undernutrition is a major health problem in the growing elderly population. It is estimated that one in ten Dutch community-dwelling older adults is suffering from undernutrition, and one in three Dutch older adults who receive home care. Undernutrition may lead to many negative consequences, ranging from fatigue and falls to impaired immune function and death. This makes undernutrition an obvious target for preventive measures.

Undernutrition can be defined as “a state of nutrition in which a deficiency or excess (or imbalance) of energy, protein, and other nutrients causes measurable adverse effects on tissue/body form (body shape, size and composition) and function, and clinical outcome”. In addition, it is often described as protein energy malnutrition. Adequate protein intake may to some extent prevent and reverse this process. However, throughout ageing, it becomes increasingly difficult to reach adequate protein intake due to higher protein needs and lower protein intakes. Finding solutions to assist older adults in reaching their optimal protein intake is necessary.

In our overall research project, we considered 1.2g protein per kg weight per day (g/kg/d) as adequate protein intake. In Dutch community-dwelling older adults, protein intake is around 1.0 g/kg/d, implying room for improvement. However, it is possible that many of these older adults deal with physiological changes, medical conditions, and physical and mental limitations that impair their appetite and food provision. For these older adults with higher protein needs, merely recommending that they eat more would not be realistic. It would be more realistic to explore strategies that increase protein intake without having to increase food intake. This calls for the exploration of instruments that match the needs and preferences of older adults: protein-enriched regular products.

One particular group that can be identified as a target group for such products, are older adults who receive home care. Undernutrition prevalence is high in this group, which may be explained by their health problems that led to this dependence on home care. Likewise, many of these older adults also depend on meals-on-wheels. These meals-on-wheels recipients, regardless of whether they receive home care or not, often risk undernutrition too. In both these (overlapping) care-dependent groups, difficulties in adhering to energy and protein recommendations can be discerned. For this reason, enriching the readymade meals that these older adults receive may contribute to the prevention of protein undernutrition by increasing protein intake while keeping food intake the same. Here, protein enrichment instruments can be used to prevent undernutrition, but only when implemented in a timely manner. Adequate undernutrition management systems are therefore necessary to facilitate timely intervention, ensuring that the developed protein-enriched meals are actually offered and effective. For this reason, the overall aim of our research project was to gain insight into the current state of undernutrition management in community-dwelling older adults in the Netherlands and explore the role of protein-enriched regular products as a supportive instrument in protein undernutrition management.

In Study 1 (chapter 2) we explored the experiences of 22 Dutch nutrition and care professionals and researchers with undernutrition awareness, monitoring, and treatment among community-dwelling older adults. This qualitative study among, for example, dietitians, general practitioners, nurse practitioners, and home care nurses provided insight into the current bottlenecks within the existing undernutrition management guidelines. In these telephone interviews, these experts also discussed the current dietary behaviour problems of older adults and their impact on undernutrition risk. The experts’ experiences implied that undernutrition awareness is limited, among both older adults and care professionals. In addition, the interviewees were unclear about which professionals are responsible for monitoring and which monitoring procedures are preferred. The dietitians feel that they become involved too late, leading to decreased treatment effectiveness. In general, the interviewees desired more collaboration and a coherent and feasible allocation of responsibilities regarding undernutrition monitoring and treatment. This implied that the available guidelines on undernutrition management require more attention and facilitation.

In the following mixed-methods study (chapter 3), with interviews, we qualitatively explored the dietary behaviour and undernutrition risk of 12 Dutch elderly meals-on-wheels clients, one of the largest at-risk groups. We followed up on this information by quantifying the topics that emerged from the qualitative exploration of experienced bottlenecks in performing adequate dietary behaviour. For this, we used a survey among 333 meals-on-wheels clients. The interviews with elderly meals-on-wheels clients made clear that they have fixed and habitual eating patterns, while at the same time their appetite had decreased throughout the years. This was confirmed by the survey finding that regular portion size meals were perceived as too large by the oldest group aged over 75y. In addition, as the professionals suggested earlier, the interviewed elderly clients indeed showed limited awareness of undernutrition risk. Simultaneously, the survey showed that almost one in four elderly meals-on-wheels clients was undernourished. These findings led to the conclusion that staying close to the identified dietary habits may facilitate small yet effective modifications within these habits to prevent inadequate nutritional intake. Still, the limited awareness of undernutrition risk was expected to play a limiting role in whether clients believe they need dietary modifications. Consequently, informing them about this need could facilitate their motivation to implement modifications.

After learning about the general dietary behaviour of these older adults, we used this information for Study 3 (chapter 4). We developed two kinds of protein-enriched readymade meals that are in line with the needs and preferences of older adults: one of regular size (450g) and one of reduced size (400g). We tested these meals in a lab setting in 120 community-dwelling older adults in a single-blind randomised crossover trial. One day a week at lunchtime, for four weeks, participants had to consume and evaluate a readymade meal. Overall, regardless of portion size, the protein-enriched meals led to higher protein intakes in vital older adults in a lab setting during lunch. In this crossover study, the participants liked the protein-enriched meals and the regular meals equally. However, we did not find the expected lower ratings of satiety after the reduced-size meals, while one reduced-size enriched meal and another regular-size enriched meal led to higher ratings of subsequent satiety. This higher satiety in the enriched meals could lead to compensational behaviour on the remainder of the day.

After establishing that the protein-enriched meals were effective and acceptable in the lab setting, we moved to the homes of older adults to test the meals in a longer-term study in Study 4 (chapter 5). In this double-blind randomised controlled trial of two weeks, we also included protein-enriched bread to assess whether both this bread and the meals could increase daily protein intake to 1.2g/kg/d in 42 community-dwelling older adults to reach optimal protein intake. We found that the enriched products again led to higher protein intakes and a high liking. The mean protein intake per day was 14.6g higher in the intervention group, which amounted to a protein intake of 1.25g/kg/d, compared with 0.99g/kg/d in the control group. In addition, the meals scored 7.7 out of 10, while the bread scored 7.8 out of 10, which both were comparable with their regular counterparts. Lastly, we found no negative effect of compensational behaviour throughout the day. These promising findings indicated that we achieved a good match between older adults’ needs and preferences regarding protein intake.

In the general discussion of this thesis (chapter 6), we combined our learnings from the four studies to reflect on protein undernutrition management in community-dwelling older adults and the possible role of protein-enriched regular products. We have discussed a conceptual framework consisting of three wheels of protein undernutrition management. In the first wheel regarding awareness, we proposed that limited awareness of adequate nutrition and body composition forms the largest bottleneck in undernutrition management. When this awareness is generated among both older adults and professionals, it will benefit the second wheel of monitoring. Here, we argued that a policy and the actual facilitation of that policy are required for this monitoring to succeed. When the monitoring is performed adequately, in the third wheel, the appropriate treatment can be carried out. We discussed that personalisation and evaluation of this treatment are important conditions. All in all, the public health implications that we have discussed on the basis of our findings can be summarised by the three key messages that could help us ace in adequate protein undernutrition management: address awareness in both older adults and professionals, facilitate continuous collaboration between professionals, and offer protein-enriched products expediently.

Older adults, mealtime-related emotions, and functionalities : tailoring protein-enriched meals
Uijl, Louise C. den - \ 2016
Wageningen 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.

Cater with Care : impact of protein-enriched foods and drinks for elderly people
Beelen, J. - \ 2016
Wageningen University. Promotor(en): Lisette de Groot; Frans Kok, co-promotor(en): Nicole de Roos. - Wageningen : Wageningen University - ISBN 9789462578814 - 142 p.
undernutrition - hospital catering - hospitals - protein - elderly - protein intake - food - beverages - diet studies - dietetics - dietitians - randomized controlled trials - ondervoeding - ziekenhuiscatering - ziekenhuizen - eiwit - ouderen - eiwitinname - voedsel - dranken - dieetstudies - diëtetiek - diëtisten - gestuurd experiment met verloting

Protein undernutrition is a major health concern for older adults, especially for those who are ill. There is growing consensus for a protein intake target of 1.2 - 1.5 gram per kg bodyweight per day (g/kg/d) for these older adults. However, this target is not reached by the majority of older adults. Therefore, more effective and novel strategies to increase protein intake are warranted, including the use of protein-enriched foods and drinks. This thesis evaluated the impact of the developed protein-enriched foods and drinks on protein intake and physical performance among older adults. The studies in this thesis were done as part of the Cater with Care® project; a collaboration between the university, care organizations, and partners from the food industry. The industrial partners developed the products, focusing each on different product categories: Carezzo Nutrition developed bread, pastry, and fresh juices and soups; The Kraft Heinz Company focused on long shelf-life and convenience foods; and the Veal Promotion Foundation produced veal meat.

To fit the products to the needs of the target group, interviews with undernourished older adults (at home or hospitalized) and with dietitians were conducted (chapter 2). These interviews showed that undernutrition awareness is low among older adults. To treat undernutrition by changing their eating habits, older adults need to be aware of their health problem, they need to be willing to change, and they need to be able to understand and implement the dietitian’s advices. This process takes time while undernutrition should be treated immediately. For immediate treatment, enriched products could be used, without first creating awareness. According to the interviewees, enriched products should fit within older adults’ eating habits, and have small portion sizes.

To gain insights in food choices of hospitalized older adults (65 years and older) an observational study was conducted. In this study, energy and protein intakes of 80 hospitalized older patients at low and high risk of undernutrition were assessed (chapter 3). Patients who received an energy- and protein-rich menu, because of their risk of undernutrition, were better able to reach the protein and energy targets than patients with a low risk of undernutrition receiving a standard menu. Based on these results we proposed that all hospitalized older adults – both at low and high risk of undernutrition – should receive an energy- and protein-rich menu.

Subsequently, a pilot study was done in a care home and a rehabilitation center with the aim to explore the potential of the developed protein-enriched products to increase protein intake (chapter 4). Participants did not compensate their consumption of regular protein-rich foods (e.g. dairy, cheese) upon the introduction of protein-enriched foods and drinks. The 22 institutionalized elderly (mean age 83 years) consumed 12 gram protein per day more than they did before the intervention. Consequently, more people met the protein target of 1.2 g/kg/d than before the intervention. We concluded that protein-enriched products enabled institutionalized elderly to reach protein intake targets. Furthermore, we gained valuable feedback to improve the assortment of protein-enriched products for the effectiveness study.

In the final study, effects of the protein-enriched products on protein intake and physical performance were studied in a randomized controlled trial during hospitalization and subsequent recovery at home. During the hospital period in which 147 older patients participated, patients that received protein-enriched products increased their protein intake compared to the control group that already received a protein-rich hospital menu (chapter 5). As a result, 79% of the intervention group reached a protein intake of 1.2 g/kg/d, compared to 48% of the control group. Finally, effects of the protein-enriched products were tested at home, for a longer period (chapter 6). Half of the hospital phase participants (n = 75) continued the intervention at home for 12 weeks. The protein-enriched products were successfully implemented in the daily menu of the older adults: the intervention group had a higher average protein intake (1.5 ± 0.6 g/kg/d) than the control group (1.0 ± 0.4 g/kg/d) during the 12-week intervention period. Seventy-two percent of the intervention group reached a protein intake of 1.2 g/kg/d during the 12-week intervention, compared to 31% of the control group. Protein intake of the intervention group was mainly increased by the following protein-enriched products: bread, dairy drinks, dairy desserts, soups, and fruit juices. However, despite the successful improvement of protein intake, we found no added value on physical performance in the first 6 months after hospitalization.

It was concluded that with the protein-enriched familiar foods and drinks, we have a feasible, acceptable, and appetizing long-term strategy to increase protein intake of older adults in various settings. We envisage a beneficial role of these protein-enriched products in combination with physical activity in older adults with lower protein intakes.

Naar 100% regionaal eiwit : kansen en knelpunten voor eiwitrijke veevoergrondstoffen
Zanders, R. ; Cormont, A. ; Krimpen, M.M. van; Prins, Udo ; Ridder, A. de; Kessel, Hans van; Hartog, H. den; Krajenbrink, Wim ; Pluimers, Jacomijn ; Haren, Rob ; Gankema, P. - \ 2016
Raad voor Regionaal Veevoer - 18 p.
veevoeder - eiwit - veevoederindustrie - veevoeding - eiwitwaarde - streekgebonden producten - duurzame veehouderij - fodder - protein - feed industry - livestock feeding - protein value - regional specialty products - sustainable animal husbandry
Ongeveer de helft van het eiwitrijke veevoer in Nederland wordt geïmporteerd van buiten Europa. Het overgrote deel van deze grondstoffen bestaat uit soja- en palmproducten. Op Europees niveau zijn we voor 96% van onze sojabehoefte en voor 70% van onze totale eiwitbehoefte afhankelijk van import van buiten Europa. In de teeltgebieden, met name in Zuid-Amerika, leidt de grootschalige teelt van deze gewassen tot grote ecologische en sociale schade; door ontbossing van natuurgebieden en daarmee gepaard gaande CO2- uitstoot en biodiversiteitsverlies, door uitputting van de bodem, vervuiling van drinkwater, bedreiging van de lokale voedselvoorziening en gedwongen landonteigening. Regionale eiwitteelt biedt een enorme kans voor de Nederlandse landbouw door het sluiten van kringlopen en het verminderen van milieuschade en sociaal onrechtvaardige omstandigheden. Om knelpunten en kennisvragen te identificeren die een (snelle) transitie naar het gebruik van meer regionaal eiwitrijk veevoer in de weg staan, richtte Milieudefensie in 2015 de Raad voor Regionaal Veevoer op. Dit rapport geeft de bevindingen weer van de Raad voor Regionaal Veevoer, inclusief haar aanbevelingen richting overheid, bedrijfsleven en maatschappelijke organisaties.
Measurement errors in dietary assessment using duplicate portions as reference method
Trijsburg, L.E. - \ 2016
Wageningen University. Promotor(en): Pieter van 't Veer; Anouk Geelen; Jeanne de Vries. - Wageningen : Wageningen University - ISBN 9789462576421 - 128 p.
diet studies - nutritional assessment - questionnaires - reference standards - correction factors - validity - body mass index - regression analysis - food intake - food - protein - potassium - sodium - energy intake - methodology - dieetstudies - voedingstoestandbepaling - vragenlijsten - referentienormen - correctiefactoren - geldigheid - quetelet index - regressieanalyse - voedselopname - voedsel - eiwit - kalium - natrium - energieopname - methodologie

Measurement errors in dietary assessment using duplicate portions as reference method

Laura Trijsburg

Background: As Food Frequency Questionnaires (FFQs) are subject to measurement error, associations between self-reported intake by FFQ and outcome measures should be corrected for measurement error with data from a reference method. Whether the correction is adequate depends on the characteristics of the reference method used in the validation study. The duplicate portion method (DP), compared to the often used 24h recall (24hR), seems a promising reference method as correlated errors between FFQ and DP, such as memory bias, errors in portion size estimations and food composition databases, are not expected.

Aim: This thesis aimed to determine the validity of the DP compared to the 24hR as a reference method for FFQ validation. The second aim was to explore the validity of nutrient densities for DP, 24hR and FFQ. The third aim was to determine the factors associated with misreporting of energy, protein and potassium as estimated by DP, 24hR and FFQ.

Methods: Within the DuPLO-study, a Dutch validation study which is part of the NQplus study, two DPs, two FFQs, two blood and urinary biomarkers and one to fifteen 24hRs (web-based and/or telephone-based) were collected in 198 subjects, within 1.5 years. Also, one or two doubly labelled water measurements were available for 69 participants. Multivariate measurement error models were used to assess proportional scaling bias, error correlations with the FFQ, validity coefficients and attenuation factors. Furthermore linear regression analysis was used to determine the association between misreporting and various factors.

Results: The DP was less influenced by proportional scaling bias, had lower correlated errors with the FFQ and showed higher attenuation factors than the 24hR for potassium, sodium and protein. Also, the DP seemed a better reference method than the 24hR for the assessment of validity coefficients for the FFQ for various fatty acids. The attenuation factors for the FFQ, using either the DP or 24hR as reference method, agreed reasonably well. Furthermore, the DP showed, when using plasma fatty acids as reference, slightly better ranking of participants according to their intake of n-3 fatty acids (0.33) and the n‑3/LA ratio (0.34) than the 24hR (0.22 and 0.24, respectively). Less group level bias was observed for protein and sodium densities compared to their absolute intakes for FFQ, 24hR and DP, but not for potassium. Overall the validity coefficients and attenuation factors for DP, 24hR and FFQ did not improve for nutrient densities compared to absolute intakes, except for the attenuation factor for sodium density. Lastly, BMI proved to be the most consistent determinant associated with misreporting (group level bias) of energy, protein and potassium for DP, 24hR and FFQ. Men tended to underreport protein by the DP, FFQ and 24hR and persons of older age underreported potassium but only by the 24hR and FFQ. Other explorative determinants did not show a consistent association with misreporting of energy or nutrients by the different dietary assessment methods.

Conclusion: With respect to error correlations and attenuation factors the DP performed slightly better than the 24hR as a reference method for validating FFQs in epidemiological research. Furthermore, the use of nutrient densities does not necessarily improve the validity of the dietary intake estimates from DP, 24hR and FFQ. Moreover, it was shown that BMI is an important determinant of misreporting of energy, protein and potassium for these three assessment methods.

Het effect van aminozuuraanbod en -samenstelling van het voer op zoötechnische prestaties van beren gehuisvest onder verschillende sanitaire condities
Meer, Y. van der; Gerrits, W.J.J. ; Jansman, A.J.M. - \ 2016
Wageningen UR Livestock Research (Livestock Research rapport 938) - 32 p.
beren (varkens) - aminozuren - eiwit - voedselsamenstelling - varkensvoeding - mestresultaten - hygiëne - varkenshouderij - zoötechniek - dierlijke productie - boars - amino acids - protein - food composition - pig feeding - fattening performance - hygiene - pig farming - zootechny - animal production
Dit experiment was opgezet om het effect van eiwitniveau (normaal versus verlaagd) en aminozuursamenstelling in het rantsoen te evalueren op de technische prestaties van beren gehuisvest onder een tweetal sanitaire condities.
Grasraffinage en gebruik van grasvezel in de rundveevoeding
Klop, A. ; Durksz, D.L. ; Zonderland, A. ; Koopmans, B. - \ 2015
Wageningen : Wageningen UR, Livestock Research (Livestock Research rapport 790) - 26 p.
veevoeding - melkveevoeding - kalvervoeding - bioraffinage - grasmaaisel - vezels - eiwit - proeven - melkveehouderij - livestock feeding - dairy cattle nutrition - calf feeding - biorefinery - grass clippings - fibres - protein - trials - dairy farming
In 2012 is een proef met melkkoeien uitgevoerd met als doel de waarde van grasvezel te onderzoeken. In het rantsoen van de koeien werd een deel van de graskuil vervangen door grasvezel. De grasvezel kwam beschikbaar na de raffinage van gras. De resultaten van de proef vielen tegen. De voeropname van de koeien die grasvezel kregen was namelijk lager dan van de (controle)koeien die het gangbare rantsoen kregen. De melkgift was eveneens lager op het rantsoen met grasvezel. De oorzaak van de lagere voeropname heeft waarschijnlijk te maken met de versheid en daarmee de smakelijkheid van grasvezel. Daarom is in 2013 besloten om eerst te kijken naar de mogelijkheid om grasvezel te conserveren (in te kuilen), waardoor de kwaliteit en de houdbaarheid mogelijk werd verbeterd. In 2012 is eveneens een oriënterend onderzoek gedaan met graseiwit verstrekt aan kalveren. Graseiwit is het eiwit dat gewonnen wordt uit het grassap en in de proef werd het in gelvorm verstrekt. De resultaten van de proef met kalveren waren uitermate positief. De dieren namen het graseiwit graag op. De groei van de kalveren was vergelijkbaar met de controlegroep.
Drying and hydration of proteins at high concentration
Bouman, J. - \ 2015
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): Renko de Vries. - Wageningen : Wageningen University - ISBN 9789462575509 - 161
eiwit - wei-eiwit - zeïne - drogen - droogmethoden - geneesmiddeltoedieningssystemen - hydratatie - hydrofobiciteit - ph - vacuolen - protein - whey protein - zein - drying - drying methods - drug delivery systems - hydration - hydrophobicity - vacuoles

Proteins are the building blocks of life and serve a wide range of essential functions in organisms. Many metabolic reactions in organisms are catalysed by enzymes, DNA is replicated by proteins and in cells proteins often facilitate active transport of e.g. glucose or ions. Proteins also serve an essential functionality in foods, pharmaceutics, bioplastics and even clothing. Recently, the use of proteins towards higher concentrations is of interest for food, pharmaceutical and medical applications. Nevertheless, the preparation of products with desired product properties can be challenging, when approaching higher protein concentrations. Therefore, in this thesis we investigate proteins at higher concentrations, especially focussing on their drying and hydration behaviour.

In part one of the thesis, the focus is on the dynamics of drying of proteins towards higher concentrations. Dense proteins systems have been scarcely studied compared to proteins at lower concentrations. We address drying behaviour where we focus on the use of whey protein isolate as a model system. In part two of the thesis we focus on the hydration properties of the corn protein zein, where we apply it as a drug excipient. In this part we also investigate the influence of hydration on the release behaviour of drugs into the hydration media.

The drying part (part one) contains two studies. The first study is more fundamental in nature, focussing on the drying of a protein coating. In previous studies mainly the macroscopic properties of protein coatings after drying are investigated, leaving the drying dynamics virtually unexplored. Here we investigate the drying behaviour of the model protein β-lactoglobulin on multiple length scales with an unique combination of in-line techniques. On the microscopic length scale we use dynamic vapour sorption and magnetic resonance imaging while on a smaller length scales, we apply diffusing wave spectroscopy and IR-spectroscopy to monitor the drying process. For all used techniques, the changes in the measured physical properties of the coating as a function of water weight fraction Xw from Xw = 0.8 down to Xw = 0.2 are gradual. However, using dynamic vapour sorption and IR-spectroscopy we measure a sharp change below water weight fractions of Xw = 0.2. We hypothesise that changes in the molecular interactions caused by dehydration of the protein results in a change in the drying kinetics of the film.

In the second study of part one, protein drying is approached on a more applied level, where we study the drying of a spherical droplet. We use single droplet drying as a methods that can model the spray drying process in a simplified and well-controlled way. Sessile droplets are subjected to varying drying conditions such as temperature, initial protein concentration, presence of airflow and droplet rotation. During these experiments the morphological development is monitored by a camera. After drying, scanning electron microscopy and X-ray tomography are used to examine the particles that are formed after complete drying. Irrespective of the conditions used, we observe an initial droplet shrinkage, followed by the nucleation of a hole in the droplet skin, which is followed by the formation of a vacuole. The drying conditions used, strongly influenced the location of the hole and the locking point prior to hole formation. We hypothesise that the location of the hole is caused by local inhomogeneities in protein concentration causing a the nucleation of the hole where the local skin modulus is lowest. Also the locking point of the droplet is found to be due to a inhomogeneity over the whole droplet caused by rapid evaporation. These results can be of importance to understand powder structure and functionality as obtained in spray drying.

In the hydration part (part 2), we investigate the potential of zein as a sole excipient in macroscale caplets obtained by hot melt extrusion (HME) and injection moulding (IM). Zein is good candidate as a sustained release agent, because it is insoluble in two studies. In the first study zein matrices were loaded with the drug paracetamol. Physical mixtures of zein, water and crystalline paracetamol are extruded and injection moulded into caplets. Characterisation of these caplets is performed using differential scanning calorimetry, IR- spectroscopy, scanning electron microscopy and powder X-ray diffraction. The hydration and drug release kinetics from the caplet slices is measured. We find that the drug release kinetics is broadly independent of the dissolution medium and drug loading. The release kinetics is diffusion limited and could be well described by a 2D diffusion model. The results demonstrate that the drug release rate from zein caplet slices can be tuned by its dimensions.

In the second study, a wider range of drugs differing in hydrophobicity is studied. Next to paracetamol, we have used two other model drugs: the hydrophobic indomethacin and the more hydrophilic ranitidine. The zein matrix is capable to stabilize the different dugs in a non-crystalline state, which is promising especially for increasing the bioavailability of poorly water-soluble drugs. Overall crystallinity of the drugs in the caplets increases with its degree of hydrophobicity. For the poorly soluble indomethacin, dissolution rates at low pH were higher from caplet slices, compared to the dissolution rates of indomethacin crystals by themselves. In addition, we found that the electrostatic interactions between zein and drugs can also be used to influence the release kinetics.

Various aspects were found to be of importance both for drying and hydration of concentrated protein systems. The homogeneity during both processes deserves attention as its manipulation can strongly influence final properties if the system. Also the plasticising effect of water on dense proteins is often found essential, when understanding the dynamics of both drying and hydration processes. Finally protein hydrophobicity and its manipulation can provide a window of opportunities in many applications which are involve by drying or hydration.

Microbubble stability and applications in food
Rovers, T.A.M. - \ 2015
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): Marcel Meinders; Guido Sala. - Wageningen : Wageningen University - ISBN 9789462574755 - 138
microbubbles - eiwit - stabiliteit - karakterisering - voedsel - voedseladditieven - oppervlaktespanningsverlagende stoffen - zuurbehandeling - reologische eigenschappen - sensorische evaluatie - tribologie - druk - verwarming - koelen - protein - stability - characterization - food - food additives - surfactants - acid treatment - rheological properties - sensory evaluation - tribology - pressure - heating - cooling

Aeration of food is considered to be a good method to create a texture and mouthfeel of food products that is liked by the consumer. However, traditional foams are not stable for a prolonged time. Microbubbles are air bubbles covered with a shell that slows down disproportionation significantly and arrests coalescence. Protein stabilized microbubbles are seen as a promising new food ingredient for encapsulation, to replace fat, to create new textures, and to improve sensorial properties of foods. In order to explore the possible functionalities of microbubbles in food systems, a good understanding is required regarding the formation of protein stabilized microbubbles as well as their stability in environments and at conditions encountered in food products. The aim of this research was to investigate the key parameters for applications of microbubbles in food systems. In Chapter 1 an introduction to this topic is given.

In Chapter 2, the effect of the microbubble preparation parameters on the microbubble characteristics, like the microbubble yield, size and stability, was investigated. The protein Bovine Serum Albumin (BSA) and the method sonication was used to manufacture the microbubbles. The manufactured number and stability of microbubbles was highest when they were prepared at a pH around 5 to 6, just above the isoelectric point, and at an ionic strength of 1.0 M. This can be related to the protein coverage at the air/water interface of air bubbles formed during sonication. At a pH close to the isoelectric point the BSA molecules is in its native configuration. Also the repulsion between the proteins is minimized at these pH values and ionic strength. Both the native configuration and the limited repulsion between the proteins result in an optimal protein coverage during the first part of sonication. Also a high protein concentration contributes to a higher surface coverage. The surface coverage is proportional to the protein concentration up to a concentration of 7.5% after which an increase in protein concentration did not lead to a substantial increase in the number of microbubble . In the second part of sonication the protein layer around the air bubble becomes thicker and stronger by heat induced protein-protein interactions. We found that and at a preheating temperature of 55-60°C, about 5 °C below the BSA denaturation temperature, and a final solution temperature of 60-65°C most microbubbles were obtained, while at higher temperatures mainly protein aggregates and (almost) no microbubbles are formed. This suggests that at temperature of around 60°C to 65°C protein aggregated mostly at the air-water interface creating a multi-layered shell, while at higher temperature, they also aggregated in bulk. These aggregates cannot form microbubbles. We found that optimal preparation parameters strongly depend on the protein batch. We hypothesize that the differences in microbubble formation between the protein batches is due to (small) differences in the protein molecular and denaturation properties that determine the temperature at which the molecules start to interact at the air-water interface. Microbubbles made with different protein concentration and preheating temperatures shrunk in time to a radius between 300 nm and 350 nm, after which the size remained constant during further storage. We argue that the driving force for the shrinkage was the Laplace pressure, resulting in an air flux from the bubbles to the solution. We argue that the constant final size can be explained by a thickening of the microbubble shell as a result of the microbubble shrinkage, thereby withstanding the Laplace pressure.

In Chapter 3 and Chapter 4, microbubble stability at environments and conditions representative for food products were studies. In Chapter 3 we investigated the stability upon addition of surfactants and acid, When surfactants or acid were added, the microbubbles disappeared in three subsequent steps. The release of air from the microbubble can be well described with the two-parameter Weibull process. This suggests two processes are responsible for the release of air: 1) a shell-weakening process and 2) a random fracture of the weakened shell. After the air has been released from the microbubble the third process is identified in the microbubble disintegration: 3) the shell disintegrated completely into nanometer-sized particles. The probability of fracture was exponentially proportional to the concentration of acid and surfactant, meaning that a lower average breaking time and a higher decay rate were observed at higher surfactant or acid concentrations. For different surfactants, different decay rates were found. The disintegration of the shell into monomeric proteins upon addition of acid or surfactants shows that the interactions in the shell are non-covalent and most probably hydrophobic. After surfactant addition, there was a significant time gap between complete microbubble decay (release of air) and complete shell disintegration, while after acid addition the time at which the complete disintegration of the shell was observed coincided with the time of complete microbubble decay.

In Chapter 4 the stability of the microbubbles upon pressure treatment, upon fast cooling after heating and at different storage temperatures was studied. The microbubble stability significantly decreased when microbubbles were pressurized above 1 bar overpressure for 15 seconds or heated above 50°C for 2 minutes. Above those pressures the microbubbles became unstable by buckling. Buckling occurred above a critical pressure. This critical pressure is determined by the shell elastic modulus, the thickness of the shell, and the size of the microbubble. Addition of crosslinkers like glutaraldehyde and tannic acid increased the shell elastic modulus. It was shown that microbubbles were stable against all tested temperatures (up to 120°C) and overpressures (4.7 bar) after they were reinforced by crosslinkers. From the average breaking time at different storage temperatures, we deduced that the activation energy to rupture molecular bonds in the microbubbles shell is 27 kT.

In Chapter 5, we investigated the effect of microbubbles on the rheological, tribological sensorial properties of model food systems and we compared this effect to the effect on food systems with emulsion droplets and without an added colloid. We investigated the effect in three model food systems, namely fluids with and without added thickener and a mixed gelatine-agar gel. In a sensory test panellists were asked whether they could discriminate between samples containing microbubbles, emulsion droplets or no added colloid. Emulsions could be sensorially well distinguished from the other two samples, while the microbubble dispersion could not be discriminated from the protein solution. Thus, we concluded that at a volume fraction of 5% of these BSA covered microbubbles were not comparable to oil-in-water emulsions. The good discrimination of emulsion might be ascribed to the fact that emulsion had a lower friction force (measured at shear rates form 10 mm/s to 80 mm/s) than that microbubbles dispersions and protein solutions. Upon mixing emulsions and microbubble dispersions the friction value approximated that of emulsions. This effect was already noticed at only 1.25% (v/v) oil, indicating that microbubbles had not a significant contributions to the friction of these samples. Also microbubble dispersions with and without protein aggregates were compared. The microbubble dispersions with and without thickener containing protein aggregates had a higher viscosity than the those samples without protein aggregates. Protein aggregates in the gelled microbubble sample yielded a higher Young’s modulus and fracture stress. The differences between the gelled samples could be well perceived by the panellists. We attribute this mainly to the fracture properties of the gel. In general we concluded that microbubbles, given their size of ~ 1 mm and volume fraction of 5%, did not contribute to a specific mouthfeel.

Finally in Chapter 6, the results presented in the previous chapters are discussed and put in perspective of the general knowledge on microbubbles production, stability, and applications in food. We described the main mechanisms leading to microbubble formation and stability. We showed that the production parameters significantly influence the interactions in the microbubble shell, and the those interactions highly determine the stability of the microbubbles under several conditions. We reported about limitations of sonication as a method to produce microbubbles suitable for food applications and we provided some ways to overcome these limitations. The use of microbubbles in food systems has been explored and we clearly see possible applications for microbubbles in food. We reported about directions for possible further research.

In this work we made significant progress in understanding the interactions in the microbubble shell and their relation to microbubble stability. We also advanced in comprehension towards possible applications of microbubbles in food.

Zeewier voor de toekomst
Ramaker, R. ; Brandenburg, W.A. ; Wald, J. - \ 2015
Resource: weekblad voor Wageningen UR 10 (2015)1. - ISSN 1874-3625 - p. 12 - 15.
mariene gebieden - zeewierenteelt - zeewieren - voedselproducten - oosterschelde - noordzee - toegepast onderzoek - aquatische biomassa - eiwit - financieren - marine areas - seaweed culture - seaweeds - food products - eastern scheldt - north sea - applied research - aquatic biomass - protein - financing
In 2050 moeten grote zeewierplantages op zee voorzien in onze behoefte aan voedsel en grondstoffen. In de Oosterschelde doen Wageningse onderzoekers nu experimenten met duurzame zeewierteelt.
Protein mixtures: interactions and gelation
Ersch, C. - \ 2015
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): A.H. Martin; Paul Venema. - Wageningen : Wageningen University - ISBN 9789462574212 - 199
eiwit - wei-eiwit - sojaeiwit - gelering - gelatine - gels - reologie - structuur - moleculaire interacties - protein - whey protein - soya protein - gelation - gelatin - rheology - structure - molecular interactions

Gelation is a ubiquitous process in the preparation of foods. As most foods are multi constituent mixtures, understanding gelation in mixtures is an important goal in food science. Here we presented a systematic investigation on the influence of molecular interactions on the gelation in protein mixtures. Gelatin gels with added globular protein and globular protein gels with added gelatin were analyzed for their gel microstructure and rheological properties. Mixed gels with altered microstructure (compared to single gels) also differed in modulus from single gels. Mixed gels with microstructures similar to single gels were rheologically similar to single gels. Alterations in microstructure were attributed to segregative phase separation between proteins which occurred during gelation. Gelation was treated as a growth process from macromolecule to space spanning network. At conditions where electrostatic interactions were screened the occurrence of phase separation was attributed to the molecular size ratio between gelling and non-gelling proteins before gelation and changes of this size ratio during gelation. Here only mixtures that during gelation passed a region of high compatibility (similar molecular sizes) before entering a region of decreasing solubility phase separated. For applications this implies that whenever the gelling molecule is larger than the non-gelling molecule phase separation during gelation is unlikely while reversely, if the gelling molecules is smaller than the non-gelling molecule phase separation during gelation typically does occur

Activation and evasion of the type I Interferon response by infectious bronchitis virus : roles of the accessory proteins
Kint, J. - \ 2015
Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789462573376 - 138
interferon - coronavirus - infectieus bronchitisvirus - coronaviridae - immuniteitsreactie - kippen - kippenziekten - pluimveeziekten - vaccinontwikkeling - kwantitatieve methoden - eiwit - virale inmenging - infectious bronchitis virus - immune response - fowls - fowl diseases - poultry diseases - vaccine development - quantitative methods - protein - viral interference

SUMMARY

Viruses are intracellular parasites that exploit the machinery of the host cell to replicate. To defend themselves against invading viruses, animal cells have evolved an anti-viral mechanism, known as the type I interferon response. Through natural selection viruses have in turn evolved mechanisms to counteract or evade the type I IFN response. Coronaviruses are a large group of positive-stranded RNA viruses that cause a range of human and veterinary diseases. Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus and it is the causative agent of a highly contagious respiratory disease of poultry. To date, only few studies have investigated the interaction between IBV and the type I IFN response.

In this thesis, we describe for the first time the activation of the type I interferon response (IFN response) by the Gammacoronavirus IBV, and the repressive role of accessory proteins therein. In Chapter 1 I provide a general introduction into coronaviruses in general and the Gammacoronavirus IBV in particular. I also introduce the IFN response, and highlight differences between the mammalian and chicken IFN response. Finally, I review current knowledge on the roles of coronavirus accessory proteins in counteraction of the IFN response. In Chapter 2 we describe our studies which demonstrated that activation of the IFN response by IBV is dependent on the intracellular double-stranded RNA sensor MDA5. We show that detection of IBV-infection by MDA5 is delayed with respect to the peak of viral replication, and demonstrate that this delay is not due to inhibition of dsRNA detection by IBV. Using mutant viruses that cannot express accessory proteins (null viruses), we found that accessory proteins 3a and 3b of IBV mediate transcription and translation of Ifnβ mRNA.

The observation that IBV delays the activation of the IFN response, prompted us to investigate the sensitivity of IBV to IFN treatment in Chapter 3. Here we show that IBV is relatively resistant to treatment with type I IFN, as relatively high doses of type I IFN are required to decrease propagation of the virus. Next, we studied which viral protein(s) contribute to resistance of IBV to type I IFN and found that absence of accessory proteins 3a and 3b increased sensitivity of IBV to type I IFN, via a presently unknown mechanism. In addition, we observed that independent of accessory proteins 3a and 3b, IBV blocks signaling of IFN by inhibiting phosphorylation and translocation of the transcription factor STAT1. To explain the delayed kinetics of IFN production observed in Chapter 2, we investigated whether delayed protein production was restricted to IFN, or whether IBV, like Alpha- and Betacoronaviruses, inhibits general translation of host proteins (i.e. induces host shutoff). In Chapter 4 we demonstrate that IBV-induced transcription of Ifnβ mRNA leads to the production of relatively little IFN protein. We discovered that limited production of IFN protein by IBV-infected cells is the result of general inhibition of host translation, confirming that IBV induces a shutoff of host-protein production. This finding indicates that evasion of the innate immune system by Gammacoronaviruses may be more similar to that of Alpha- and Betacoronaviruses than previously thought. Using accessory protein null viruses we discovered that accessory protein 5b of IBV is essential for the inhibition of host-protein synthesis by IBV. In Chapter 5 and Chapter 6 we describe the methods used in this thesis to quantify the number of infectious virus particles of IBV as well as methods used to quantify the activation of the type I IFN response in chicken cells. Although the studies described in this thesis have answered several questions about the interaction of IBV with the type I IFN response of its host, they have also raised new questions to be addressed in future research. In the final Chapter of this thesis (Chapter 7), I discuss a number of remaining questions and future perspectives regarding evasion of the IFN response by IBV. Finally, I explore the possible implications of our findings on the in vivo pathogenicity of IBV and on the rational design of attenuated IBV vaccines.

In conclusion, the work described in this thesis demonstrates for the first time how IBV evades, activates, and antagonises the IFN response. Also, this thesis comprises the first study that describes a function for the accessory proteins of IBV and shows that these poorly understood proteins play an important role in antagonism of the type I IFN response.

Molecular design, self-assembly, and material properties of silk-like protein polymers
Beun, L.H. - \ 2015
Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Renko de Vries. - Wageningen : Wageningen University - ISBN 9789462572331 - 160
eiwittechnologie - eiwit - zelf-assemblage - ontwerp - polymerisatie - collageen - protein engineering - protein - self assembly - design - polymerization - collagen

Summary

In this thesis we present the design and characterization of bio-inspired hybrid protein polymers. All polymers are composed of two distinct types of building blocks. The first type is a silk-inspired block that is pH-responsive and can fold and self-assemble into highly ordered structures. The basic structure of this building block is an octapeptide (GAGAGAGX), denoted SX. We include multiple repeats n of this octapeptide (SXn) in our protein polymers. The amino acid X is always an acidic or basic one. This way, the pH of the solvent determines the charge of this amino acid; in the charged state the silk-like blocks repel each other and the proteins are molecularly dissolved. When charge neutralized, the silk-like blocks become hydrophobic and can fold and stack. The second building block is very hydrophilic and acts as a random coil under a wide range of aqueous solvent conditions. The basic structure of this block is a 99 amino acids long sequence of mostly hydrophilic amino acids, that we included either as a single block or as multimers, denoted Cn. The combination of these two blocks in one molecule leads to a pH-responsive protein polymer that switches from hydrophilic to amphiphilic due to solvent pH. The amphiphilic nature of the neutralized protein polymer leads to microscopic phase separation.

All protein polymers were designed by genetic engineering and produced by genetically modified Pichia pastoris in a batch fermentation. A simple ammonium sulfate precipitation was sufficient for all types of proteins to acquire highly pure samples.

The types of hybrid protein polymers we produced and characterized differ in three aspects. Firstly, we designed different silk-like blocks in which the amino acid X had three varieties. We included the acidic amino acid glutamic acid (E) with a pKa ~ 4, to obtain a block that is charged at high pH values and neutral under acidic conditions. We also designed this silk-like block with basic amino acids lysine (K) or histidine (H). These blocks are positively charged under acidic conditions, while being neutral at higher pH. Lysine with a pKa ~ 10 remains charged under slightly alkaline conditions, and is only neutralized at rather extreme pH values. The pKa of histidine (~ 6) means this is the only pH-responsive amino acid that’s almost completely neutral under physiological conditions (a pH of 7.4). This makes histidine the most interesting residue from a biomedical point of view.

The second variety in design is the relative sizes of the two blocks. For any amphiphile, the relative sizes of the two blocks determine the structure that is formed upon self-assembly. The size of the silk-like block was chosen to be 8, 16, 24 or 48 repeats of the octapeptide SXN. The random coil block was included as monomer C1, dimer C2 or tetramer C4.

Our third variation in molecular design is the order of the two building blocks. We constructed two diblock structured protein polymers: C1SH48 and C2SH48. All other protein polymers had a triblock structure. We constructed C2SXNC2 protein polymers, where the self-assembling silk-like domain was the central block, and SX24C4SX24 protein polymers, with telechelic end blocks. All types of protein polymers that we studied are presented in Table 6.1.

Table 6.1: Overview of different protein polymers studied in this thesis.

Telechelic

(Ch. 2)

Varying Silk-Like Block

(Ch. 3 & 4)

Varying Random Coil Block

(Ch. 5)

SE24C4SE24

C2SH8C2

C1SH48

SH24C4SH24

C2SH16C2

C2SH48

SK24C4SK24

C2SH24C2

C2SH48C2

C2SH48C2

In chapter 2 we report on the self-assembly behavior of triblock structured telechelic protein polymers SX24C4SX24. We analyzed the pH-dependent self-assembly into fibrillar structures of three different protein polymers. These proteins only differ in the amino acid X in the silk-like block. We found that all proteins self-assemble into fibrils under solvent conditions at which the amino acid X is uncharged. This self-assembly is completely reversible; changing the solvent pH to a value at which the amino acid X is fully charged, leads to immediate disassembly of the fibrils. The secondary structures of the fibrils are comparable, and are a combination of a random coil corona and a crystalline folded and self-assembled core. The self-assembly process is a pseudo-first order one. Initial fast (heterogeneous) nucleation is followed by elongation of existing fibrils, without the formation of new fibrils. These kinetics lead to monodisperse samples of fibrils. Existing fibrils have at least one living end: addition of new proteins in solution leads to further growth of these fibrils.

In chapter 3 we use super resolution fluorescence microscopy and atomic force microscopy to analyze the self-assembly mechanism of the protein polymer C2SH48C2. Surprisingly, we found that self-assembly of these fibrils is an asymmetric process. The fibrils grow in only one direction with one living end, although the protein polymer that is the building block of these fibrils is highly symmetric. We therefore conclude that nucleation is a heterogeneous process. We observed that once a protein molecule is part of a fibril, it is kinetically trapped. In a timeframe of 3 days, we don’t observe exchange of protein molecules inside fibrils, with proteins in solution. The interactions of uncharged folded protein polymers inside a fibril are simply too strong to overcome to be released into solution. We also report that self-assembly of these fibrils is a process that involves continuous nucleation; elongation of existing fibrils is accompanied by the genesis of new ones. This leads to samples that contain fibrils with a wide variety of sizes, quite different from the populations found for the protein polymers with the inverted block sequence that we presented in chapter 2.

In chapter 4 and 5 we present our findings on several protein polymers in which we varied the relative sizes of the silk-like block and of the random coil blocks. In chapter 4 we present the characterization of protein polymers that have differently sized silk-like domains. We studied 4 protein polymers with the general structure C2SHnC2; the series consisted of n = 8, 16, 24 or 48. The two smallest protein polymers form micelles when charge neutralized (pH 8). The two largest protein polymers form fibrils under these conditions. At low pH, when the silk-like block is highly charged, this block behaves as (extended) random coil, according to circular dichroism measurements. This behavior is consistent for all block sizes that we studied. In the self-assembled state, there is a distinct difference in secondary structure of the micelles and the fibrils. The silk-like core of the micelles has a secondary structure that differs only slightly from the structure in the charged state. It merely acts as a collapsed coil. The secondary structures of the fibril forming protein polymers are very different in neutralized state. Their structures are mutually nearly identical, similar to that of a betaroll.

We observed that the size of the silk-like block has a strong effect on the kinetics of self-assembly. The largest protein polymer C2SH48C2 self-assembles into fibrils at a rate that is over a decade faster than the protein polymer with the smaller silk-like block C2SH24C2. Both fibril forming protein polymers can form hydrogels. There is however a great difference in rigidity of the gels at similar concentrations. The gel that consists of fibrils of C2SH48C2 is a decade stiffer than the one consisting of C2SH24C2. This stronger fibril-fibril interaction due to the more exposed silk-like core of C2SH48C2 clearly has a strong effect on macroscopic gel properties. We used partial enzymatic degradation of the random coil block to determine the influence of decreasing the hydrophilic block on self-assembly behavior. Both micelle forming protein polymers are able to form fibrils after up to 80% of the random coil blocks has been cleaved off. This shows the intrinsic capacity of the silk-like block to form fibrils even at a size as small as 64 amino acids. The fibrils of C2SH24C2 show an increase in interaction after partial cleavage of the random coil block. Individual fibrils start to associate laterally. This is a strong indication that fibrils with smaller random coil blocks can form more rigid hydrogels, based on their increased core-core interaction.

Based on these findings, we designed two new protein polymers with smaller random coil blocks: C1SH48 and C2SH48. With these protein polymers we systematically probe the role of a more exposed silk-like core in gel properties, presented in chapter 5. Both proteins self-assemble into fibrils when neutralized. Fibrils of C1SH48 differ from those of C2SH48 and C2SH48C2 as they start to laterally associate. Surprisingly, the rates at which self-assembling fibrils are formed, are identical for these protein polymers, and also equal to the rate of C2SH48C2­. Apparently, for these sizes of the blocks, the size of the silk-like block is what determines the rate of self-assembly. These two protein polymers attain secondary structures that are very similar to that of C2SH48C2. When looking at macroscopic properties of hydrogels formed by these protein polymers, we do observe a very clear difference. Both C2SH48C2 and C2SH48 form fibril based hydrogels that act as gels with very few (weak) crosslinks. These two gels show similar scaling behavior of modulus with concentration (exponents of 1.52 and 1.66). The attractive interaction of fibrils of C1SH48 leads to a different type of gel. The modulus of this hydrogel scales strongly with concentration (exponent of 2.8), typical for a (physically) cross-linked gel. The latter gels can have much greater moduli than gels of C2SH48C2 and C2SH48, but are also slightly more brittle. The porosity of the gels (an important parameter for biomedical applications) increases when decreasing the size of the random coil domain. However, for this series of protein polymers, the porosity is in the order of 10-100 nm, which makes these gels not suitable for using them to grow 3D cell cultures.

Hoe efficiënt is de energie- en eiwitconversie door melkvee?
Blom, J. ; Hooijdonk, A.C.M. van - \ 2015
VoedingsMagazine 27 (2015)2. - ISSN 0922-8012
melkproductie - eiwit - voedselzekerheid - milieueffect - voedingsstoffen - eiwitkwaliteit - melkveehouderij - milk production - protein - food security - environmental impact - nutrients - protein quality - dairy farming
Melk speelt wereldwijd een belangrijke rol bij het voorzien in de nutriëntenbehoeften van de mens. De koe zet voor mensen niet-eetbaar eiwit uit voer om in eiwit van hoge kwaliteit. Steeds vaker rijst de vraag of deze eiwitconversie voldoende toegevoegde waarde heeft en hoe dit zich verhoudt tot de milieudruk van de zuivelproductie. Professor Toon van Hooijdonk van Wageningen Universiteit ontwikkelt samen met collega's een model om dit inzichtelijk te maken.
Zwavelvoorziening op biologische veebedrijven
Beeckman, A. ; Eekeren, N.J.M. van; Govaerts, W. ; Smolders, E.A.A. - \ 2014
BioKennis bericht Zuivel & rundvlees (2014)30.
zwavel - mineraaltekorten - biologische landbouw - eiwit - zwavelmeststoffen - melkveehouderij - vleesvee - schapen - geiten - diergezondheid - sulfur - mineral deficiencies - organic farming - protein - sulfur fertilizers - dairy farming - beef cattle - sheep - goats - animal health
Door luchtverontreiniging kwam zwavel jarenlang gratis uit de lucht. Nu dit milieu­-probleem is opgelost krijgt de landbouw steeds meer te maken met zwaveltekorten. Zwavel is een essentieel element voor de vorming van verschillende aminozuren (o.a. methionine en cysteine) en daarmee van eiwit. Eiwitvorming is zowel belangrijk voor gewas als dierproductie dus zwaveltekorten komen bij beide voor.
Vitaal naar de eindstreep
Tieland, C.A.B. ; Rest, O. van de; Groot, C.P.G.M. de - \ 2013
WageningenWorld 2013 (2013)4. - ISSN 2210-7908 - p. 26 - 29.
voeding en gezondheid - ouderen - verouderen - vitaminetekorten - vetzuren - eiwit - gezondheidsbevordering - nutrition and health - elderly - aging - vitamin deficiencies - fatty acids - protein - health promotion
Hoe worden we gezond oud? Trainen en extra eiwitten werken, blijkt uit onderzoek van de afdeling Humane voeding. Over het effect van vitamines en omega-3 vetzuren is het laatste woord nog niet gezegd.
Selection and characterization of DNA aptamers
Ruigrok, V.J.B. - \ 2013
Wageningen University. Promotor(en): John van der Oost; Hauke Smidt. - S.l. : s.n. - ISBN 9789461735645 - 152
aptameren - dna - selectie - nucleotiden - technieken - eiwit - rna - aptamers - selection - nucleotides - techniques - protein

This thesis focusses on the selection and characterisation of DNA aptamers and the various aspects related to their selection from large pools of randomized oligonucleotides. Aptamers are affinity tools that can specifically recognize and bind predefined target molecules; this ability, however, is not exclusively associated with aptamers. Antibodies are the most successful affinity tools used today, but alternative affinity tools such as aptamers, engineered binding proteins and molecular imprinted polymers are emerging as sound alternatives. A comparison of their properties is described in Chapter 1. The strength and specificity of the interaction between an affinity tool and its target molecule is an important feature. Generally, an affinity tool should have a high affinity for its target and should be highly specific in order to be useful for research or commercial purposes. One highly advanced method to characterise the interaction between an affinity tool and its target molecule makes use of a Surface Plasmon Resonance (SPR)-based biosensor. Although SPR is an optical phenomenon, in depth knowledge of the physics behind this phenomenon is not required to operate an SPR-based biosensor. Experiments should be performed in a correct way, and therefore it is important to understand how experimental parameters, such as flow rate, ligand density, surface preparation, and reagent quality either improve or adversely affect data quality. Experimental considerations, as well as methods for proper data analysis are discussed in Chapter 2. Data generated within the framework of the 2011 Global Label-free Interaction Benchmark study serves as a typical example.

The ability of aptamers to bind a specific target originates from an intricate interplay between the oligonucleotide sequence and the three dimensional structure that this sequence allows to form. In Chapter 3 this is illustrated by the selection and characterisation of streptavidin-binding aptamers. Five aptamer families were identified, sharing a similar secondary structure. Although slight variations at the actual sequence level are present, two guanines are completely conserved. Using site-specific mutagenesis it was demonstrated that these guanines are essential for streptavidin binding. Binding kinetics and the dissociation constant of each aptamer was determined by SPR and were all within the range of 35-375 nM. Two aptamers can bind one streptavidin tetramer at the same time, as was shown by native mass spectrometry analysis. In addition, the three dimensional structure of the most abundant aptamer was modelled and manually docked to the streptavidin structure, in order to gain more insight in the molecular basis of the interaction. To extend this knowledge even further, crystallisation trails, aiming to obtain a co-crystal structure for the streptavidin-aptamer complex, were performed, and are described in Chapter 4. Unfortunately, these trials did only yield protein crystals, instead of the desired streptavidin-aptamer complex. Therefore, alternative experimental and computational approaches were investigated that could be used to study aptamer-protein interactions. Combining techniques as SPR, small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), and Dynamic light scattering (DLS) could be considered as an alternative to X-ray crystallography. In addition, some of these techniques may provide information on the dynamics of complex formation, whereas crystallography gives a time- and position-averaged image.

Besides streptavidin, another protein, SpaC, was subjected to aptamer selection in this thesis. SpaC is a subunit of pili present on the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG and contains a binding domain for human-mucus. Presence of this binding domain is considered an advantage, because it is already designed to interact with other molecules. Successful production and purification of recombinant SpaC protein is described in Chapter 5, as well as the characterisation of DNA oligonucleotides enriched during subsequent selection rounds. Sequence analysis revealed that specific oligonucleotides are indeed enriched. Furthermore, results of pilot SPR experiments indicated that they bind specifically to SpaC, but more detailed experiments are required to unambiguously demonstrate this.

The dynamics of aptamer enrichment are poorly understood. To address this issue and to gain a more fundamental insight in the aptamer selection process, a multiplexed high throughput sequencing effort was started, which is described in Chapter 6. In this approach samples of 70 selection rounds, derived from 8 distinct aptamer selection experiments, were barcoded, pooled together and sequenced; over 84 million paired-end reads were obtained and analysed. Samples enriched to bind streptavidin show a decrease in α-diversity across subsequent selection rounds. Interestingly, large differences were found between the composition of fractions enriched by affinity elution and thermal elution. Moreover, a small scale comparison of two clone libraries showed that affinity elution, which is expected to enrich more specific binders, also specifically enriches rapid binders.

Supportive SPR experiments have made an important contribution throughout this thesis. The main focus in Chapter 7, however, is on a new application of SPR. The development of a capture approach for supercoiled plasmid DNA, using a triple helix forming oligonucleotide, is described. It could be demonstrated that plasmid DNA can indeed be captured and that SPR can subsequently be used to derive kinetic parameters of a specific interaction with a plasmid. In this particular case the interaction between Lac repressor and its plasmid-based operator was characterised, showing that the association and dissociation rates are ~18 times lower, but that the affinity is the same, when compared to binding to linear operator DNA. This difference underscores the importance of using a DNA substrate with a physiologically relevant topology for studying DNA-protein interactions.

Insects as a sustainable feed ingredient in pig and poultry diets : a feasibility study = Insecten als duurzame diervoedergrondstof in varkens- en pluimveevoeders : een haalbaarheidsstudie
Veldkamp, T. ; Duinkerken, G. van; Huis, A. van; Lakemond, C.M.M. ; Ottevanger, E. ; Bosch, G. ; Boekel, T. van - \ 2012
Lelystad : Wageningen UR Livestock Research (Report / Wageningen UR Livestock Research 638) - 48
insecten - insecten als voedsel - dierlijke producten - veevoeding - varkensvoeding - pluimveevoeding - eiwit - insects - insects as food - animal products - livestock feeding - pig feeding - poultry feeding - protein
A feasibility study to explore application of insects as a sustainable high protein feed ingredient for pig and poultry diets was conducted on request of the Dutch Ministry of Economic Affairs, Agriculture and Innovation. This feasibility study comprised a desk study and a workshop with participants representing the various links in the “insect chain”. The objective of the study was to list, in a joint initiative of industrial stakeholders and scientists, how insects can be used on a large scale as an alternative protein source in feed for pigs and poultry.
Raapzaadeiwitconcentraat en erwteneiwitconcentraat in biologisch biggenvoer = Canola protein concentrate and pea protein concentratrate in diets for organically housed piglets
Peet-Schwering, C.M.C. van der; Binnendijk, G.P. ; Diepen, J.T.M. van - \ 2011
Lelystad : Wageningen UR Livestock Research (Rapport / Wageningen UR Livestock Research 527) - 11
biggenvoeding - biggen - voersamenstelling - eiwit - raapzaadeiwit - erwten - raapzaad - eiwitconcentraten - biologische landbouw - varkenshouderij - voeropname - voederconversie - piglet feeding - piglets - feed formulation - protein - rapeseed protein - peas - rapeseed - protein concentrates - organic farming - pig farming - feed intake - feed conversion
At the Experimental Farm Raalte it was investigated whether canola protein concentrate and pea protein concentrate are suitable protein-rich feedstuffs for organically housed piglets. It is concluded that both protein concentrates are suitable protein-rich feedstuffs for piglets. Feed intake and daily gain were similar and feed conversion was better in weaned piglets that were fed diets with 8.5% pea protein concentrate or 10.0% canola protein concentrate compared to the control group.
Lupine : een gezond alternatief voor boer en burger
Prins, U. ; Vijver, L.P.L. van de - \ 2011
Louis Bolk Instituut
lupinus - teelthandleidingen - akkerbouw - eiwit - vleesvervangers - lupinen - peulvruchten - cultivation manuals - arable farming - protein - meat alternates - lupins - grain legumes
Korte teelthandleiding voor lupine als consumptiegewas en maatschappelijke redenen om lupine te telen.
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