Information on BSc and MSc thesis projects at the ...€¦ · BSc and MSc thesis projects at the Laboratory of Food ... A THESIS RESEARCH PROJECT AT THE LABORATORY OF FOOD CHEMISTRY

L A B O R A T O R Y o f F O O D C H E M I S T R Y

Information on BSc and MSc thesis projects

at the Laboratory of Food Chemistry

November 2014 Go to www.fch.wur.nl ( education)

BSc and MSc thesis projects – Laboratory of Food Chemistry – November 2014

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Table of Contents

CHOOSING AND PERFORMING A THESIS RESEARCH PROJECT AT THE LABORATORY OF FOOD CHEMISTRY ..... 2

PART 1: QUALITY AND/OR PROCESSING OF FOOD (RAW) MATERIALS ............................................................. 4

EFFECT OF THE MAILLARD REACTION ON PROTEIN DIGESTIBILITY (DAIRY SUBJECT) ................................................................. 4 THE ROLE OF MAILLARD REACTIONS IN PROTEIN FOAM STABILITY (DAIRY SUBJECT) ............................................................... 4 SUGAR REACTIVITY ON MAILLARD REACTION OF MILK PROTEINS (DAIRY SUBJECT) ................................................................. 5 INGREDIENTS, INTERFACES AND ICE CREAM (DAIRY SUBJECT) ............................................................................................ 6 FINGERPRINTING OF COMMERCIAL ENZYME PREPARATIONS USING CAPILLARY ELECTROPHORESIS (CE). ..................................... 7 NEW ANALYTICAL TECHNIQUES FOR RESEARCH ON FOOD .................................................................................................. 7 PROTEIN DETERIORATION IN LEAVES AND SEAWEEDS DUE TO ACTIVATION OF THE DEFENCE MECHANISM ................................... 8

PART 2: ENZYMATIC CONVERSION OF FOOD BY-PRODUCTS ............................................................................ 9

C₃-ACIDS PRODUCTION FROM BIOMASS RESIDUES .......................................................................................................... 9 CHARACTERISATION OF RECALCITRANT CARBOHYDRATES IN BIOETHANOL FERMENTATION ..................................................... 10 LPMO ENZYMES – A NEW APPROACH IN BIOMASS DEGRADATION ................................................................................... 11 MORE MUSHROOMS VIA OPTIMIZATION OF THE COMPOSTING PROCESS ............................................................................ 12 HOW MUSHROOMS FEED ON SUGARS ........................................................................................................................ 13

PART 3: HEALTH ASPECTS OF FOOD ............................................................................................................... 14

NOVEL MECHANISMS UNDERLYING THE ANTI-INFLAMMATORY ACTIVITY OF OMEGA-3 FATTY ACIDS ........................................ 14 PHYTOALEXINS, PLANT DEFENCE COMPOUNDS WITH ADDED FUNCTIONALITY ...................................................................... 15 STRUCTURE ACTIVITY RELATIONSHIPS OF ISOFLAVONOIDS AS ANTIMICROBIAL AGENTS .......................................................... 16 EFFECTS OF PROCESSING OF PROTEINS ON THEIR IMMUNOGENICITY: ALLERGY AS A MODEL (DAIRY SUBJECT) ............................ 17 THE FATE OF HUMAN MILK OLIGOSACCHARIDES (HMO) IN INFANT GUT (DAIRY SUBJECT) .................................................. 17 IN VITRO DIGESTION OF PROTEINS FOR FISH FEED .......................................................................................................... 18 BEHAVIOUR AND DIGESTIBILITY OF STARCH IN PIGS ....................................................................................................... 18 THE EFFECT OF PROTEIN STRUCTURE ON DIGESTION DYNAMICS (DAIRY SUBJECT) ................................................................. 19 CHARACTERISATION OF DIETARY FIBER AND INVESTIGATION OF THEIR METABOLIC FATE IN THE HUMAN LARGE INTESTINE ............ 20 EFFECT OF SPECIFIC CARBOHYDRATES ON THE INTESTINAL TRACT OF BROILER CHICKENS AND PIGS ........................................... 21

PART 4: NEW AND/OR IMPROVED INGREDIENTS FOR FOOD .......................................................................... 22

DESIGNING THE POLYPHENOL PROFILE OF TEA FOR IMPROVED TASTE ................................................................................ 22 SUGAR BEET LEAVES: A NEW SOURCE OF PROTEINS? ..................................................................................................... 23 PROGRESS: PROTEINS FROM GREEN SOURCES FOR USE IN BOTH FOOD AND FISH FEED ....................................................... 23 PRODUCTION AND VALORISATION OF HIGH QUALITY PROTEINS FROM INSECTS .................................................................... 24 CHANGES IN PECTIN STRUCTURE DURING THE PROCESSING OF FRUITS AND VEGETABLES ....................................................... 25 USE OF ENZYMATIC CROSSLINKING TO PRODUCE PROTEIN NANOPARTICLES (DAIRY SUBJECT) ................................................. 26 PREPARATION AND CHARACTERISATION OF (ANISOTROPIC) PROTEIN PARTICLES ADDED TO DAIRY PRODUCT AS FAT REPLACERS/FOR

STRUCTURE FORMATION (DAIRY SUBJECT) ................................................................................................................... 26 PREPARATION AND CHARACTERISATION OF MILK PROTEIN BEADS BY GELATION OF THE PROTEIN AT RELATIVELY LOW TEMPERATURE

(COLD GELATION) (DAIRY SUBJECT) ............................................................................................................................ 26

PART 5: EDUCATIONAL DEVELOPMENT .......................................................................................................... 27

DESIGN AND/OR EVALUATION OF DIGITAL LEARNING MATERIAL FOR FOOD CHEMISTRY ........................................................ 27 DEVELOPING NEW LEARNING ACTIVITIES OR IMPROVING EXISTING LEARNING ACTIVITIES ....................................................... 27

BSc and MSc thesis projects – Laboratory of Food Chemistry

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Choosing and performing a thesis research project at the

Laboratory of Food Chemistry

Purpose of a thesis research project

Your thesis research project is an important part of your

study. In the past years you have gained knowledge in

food science with the aid of teachers and supervisors

during lectures, laboratory classes, computer classes,

etc. You learned obtaining information by literature

searches, internet searches, performing experiments,

evaluating your results and writing reports and/or giving

presentations. Also, you have started to a certain extent

to develop a scientific way of reasoning on how to solve

problems. The purpose of a thesis project is to further

develop this skill to analyse and solve a problem in a

scientific way. In addition, a thesis project also perfectly

serves the objective of obtaining knowledge/insight in a

specific area of research.

A thesis research project at the Laboratory of Food Chemistry

After you have chosen a research project, you will start-up this project together with

your supervisor. The supervisor is usually a researcher from our Laboratory. In short,

you will make a research plan, perform experiments by using our equipment and give

some presentations to your fellow-students. The project is finalised with a report and a

final presentation (colloquium).

A thesis research project in Food Chemistry is related to a specific (bio)chemical food

product or biomass conversion issue, which on its turn is related to an industrial

application. In this way we do not solve an industrial problem, as this is the task of our

graduates once they work in industry. Instead, we aim at understanding the mechanisms

behind the problems and we aim to approach this in an academic way. With this

knowledge/insight students are enabled to address (yet unknown) future problems, once

they are graduated.

Within the Laboratory of Food Chemistry we focus our research on a limited number of

topics. By doing so we are able to maintain our internationally well recognized position.

This is also of benefit for students, as on the one hand their research project is well

embedded and on the other hand it is a good reference regarding future employment. As

a consequence the number of areas of research possible for thesis projects is somewhat

restricted. In addition, to guarantee an optimal supervision, it is not always possible to

choose the subject of your interest. This can be due to the fact that other students have

already chosen the subject and for optimal supervision we want to restrict the number of

students per supervisor.

The main research topics of our Laboratory are (see also Table of Contents):

Part 1: Quality and/or processing of food (raw) materials

Part 2: Enzymatic conversion of food by-products

Part 3: Health aspects of food

Part 4: New and/or improved ingredients for food

Part 5: Educational development

Within these topics we deal with several foods and food raw materials such as dairy

products, cereals, fruits, vegetables, legumes, beverages (coffee, tea, beer), etc.


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Choosing a thesis research project

First, formulate for yourself what specific knowledge/skills you want to learn during the

thesis project. If you are e.g. interested in enzymology use this aspect for making your

choice.

Second, is the background of the subject motivating you? If you do not like the

background of the subject it will decrease the chance of success. Be aware that although

it might be interesting to work on an industrial problem or product, it is the underlying

way of performing research that is most important. By doing this you are, as stated

above, enabled to address any industrial future problem. In order to acquire a solid

background in food chemistry this can usually be trained better if one works with specific

components of food rather than with the complete food product itself.

On the following pages you can find the description of the research topics followed by

information on specific thesis projects. I suggest selecting 3 to 4 projects. Make contact

with the supervisors of these projects. You can find the e-mail addresses of the

supervisors at every project description. Have a talk with the supervisors and afterwards

make your choice.

I am sure you will be able to find a subject that fits you. Many students have preceded

you and enjoyed their thesis projects. If there are still some questions or remarks please

do not hesitate to contact me ([email protected], room 1-10, Axis-X).

prof.dr.ir. Harry Gruppen.

Chair of Food Chemistry


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Part 1: Quality and/or processing of food (raw) materials

Effect of the Maillard reaction on protein digestibility (dairy subject)

The Maillard reaction occurs during thermal processing of food such as baking, roasting,

brewing and etc. In the early stage of the reaction, it is between the amino group of a

protein and the reducing end of a carbohydrate (glycation). In later stage, the Maillard

reaction can lead to protein cross-linking. Both of them can change the (techno)-

functional properties of the proteins (e.g. digestibility and foam stability). Therefore, food

industries have a strong interest to control the Maillard reaction and investigate how

much it alters the properties of their product. In the dairy industries, they are especially

curious to know how much the heat treated milk powder affects the protein digestibility.

To study the protein digestibility, we are

using in vitro enzymatic hydrolysis to

mimic gastrointestinal digestion.

Analytical tools such as pH-stat and LC-

MS are used to identify and quantify the

peptide formation. Our aim is to see the

effect of the Maillard reaction (glycation

& cross-linking) on protein digestibility.

Thesis projects:

• Effect of Maillard glycated dairy proteins on enzymatic hydrolysis using Bacillus

licheniformis protease (BLP) and Trypsin.

• Effect of Maillard induced protein cross-linking on protein digestibility.

Information: [email protected]; [email protected]; [email protected]

The role of Maillard reactions in Protein Foam stability (dairy subject)

The foam stability of proteins is of relevance to many food products. While many studies

have been performed to find the link between the properties of the proteins and the

resulting foam such a link has not yet been established. In recent research evidence was

found for the effect of Maillard induced aggregation of proteins in the formation and

stabilization of foam. In this way the functional properties of the proteins could be

boosted significantly in a relatively simple process.

The Maillard reaction starts by linking one saccharide to a free amino-group of a

protein. We observed that as a result of this reaction the proteins started to form

oligomers and small aggregates. Exactly these oligomers and aggregates will be the

subject of study. By selective treatment, or modification of proteins well-defined ‘model’

systems will be produced. These systems should be characterized to the type of

aggregation that was obtained (size, size-distribution, charge or hydrophobicity).

Thesis projects:

Firstly the effect of conditions (protein type, sugar type, temperature / time) will

be studied. Also other reactions than the Maillard reaction could be used to induce

oligomer formation. After formation and characterization of the chemical

properties, some assays can be used to test the changes in foam properties of the

modified variants.

Information: [email protected]


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Sugar reactivity on Maillard reaction of milk proteins (dairy subject)

The Maillard reaction occurs in food products whenever a protein/saccharide mixture is in

the presence of some form of heat. The food industry is not only interested in controlling

the Maillard reaction for its positive effects on food quality (e.g. in bread baking, roasting

meat, brewing of beer), but also in order to control and to minimalize heat damage

during food production (e.g. decrease in available lysine during milk drying, off-flavour

and off-colours). In addition, Maillard glycated proteins may form a new generation of

food ingredients with novel properties.

In the initial step of this reaction, saccharides are attached to existing free amino-groups

on the protein sequence, which in turn will alter the proteins properties like foaming and

emulsifying. The rate and level of reaction will depend on the type of protein present as

well on the reducing sugar present. However, depending on the reaction conditions (pH,

temperature, relative humidity) and reactive components present, several side reactions

may take place influencing the reaction product formation as well as their properties.

It has already been established that not only saccharide type and size, but also the type

of protein plays a role in the Maillard reaction rate. Our intention is to study how different

proteins influences the reactions rate, level of glycation and the structure of the final

product, by focusing on how these proteins react with different sugars, under different

parameters. Important characteristics of the glycated protein will include information on

the amount and location of lysines which have been reacted, and on its solubility (level of

crosslinking during the Maillard reaction).

Thesis projects:

Characterization and quantification of saccharide attached to the protein

sequence, on simple and complex protein/sugar mixtures, at different settings

(pH, temperature, relative humidity) using LC and LC-MS approaches.

Enzymatic fingerprinting of a model glycated protein, to establish which amino

acids have been involved in the glycation process in time (Enzymatic cleavage,

UPLC-MS, ESI-MS/MS)

Information: [email protected], [email protected], [email protected]


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Ingredients, Interfaces and Ice cream (dairy subject)

Ice cream consists for 50 % of air. The air bubbles are stabilised by mixtures of dairy

proteins and low molecular weight (LMW) surfactants. In the production, first the LMW

surfactants adsorb to the interface. Later in the process, the LMW surfactants are

displaced from the interface by the adsorption of proteins. If novel ingredients are used,

this subtle balance between adsorption and desorption is easily disturbed, which can lead

to undesired product properties.

Ingredients Interfaces Ice cream

T0

T1

Objectives:

To control the stability of foam in mixed protein – surfactant systems, the interactions of

proteins and surfactants in the interface needs to be understood. In this project, you will

investigate how the chemical properties of proteins and LMW surfactants alter the

adsorption and desorption of these compounds in mixed systems and relate it to the

stability of foam made from mixed solutions.

Thesis projects:

Investigation of interactions of mixed systems of proteins and LMW surfactants in

bulk solution

o Structure and physico-chemical properties of proteins (e.g. CD, DSC and

protein charge)

o Foam and interfacial properties of mixed systems

You will determine these properties in a well-defined system of dairy proteins and a non-

ionic surfactant in order to investigate the formation of hydrophobic complexes of protein

and surfactants.

The project will involve techniques to determine foam properties (FoamScan), interfacial

properties (automated drop tensiometer) and bulk characteristics (CD, ESI-MS,

Nanosizer).

Information: [email protected]; [email protected]


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Fingerprinting of commercial enzyme preparations using capillary

electrophoresis (CE).

In commercial enzyme preparations, e.g. cellulase preparations, usually a mixture of

enzymes is present. The detailed characterization of such mixtures could be useful in

understanding the differences in their activities. Till now, mainly chromatographic

techniques were used for identification of proteins/ enzymes. In this thesis-subject the

usefulness of capillary electrophoresis to identify several proteins/ enzymes in

commercial enzyme preparations will be studied.

Thesis projects:

Identifying several enzymes present in commercial enzyme preparations by using

CE.

Comparison of CE with chromatographic techniques.

Purification and characterization of their activities of enzymes from commercial

enzymes preparations prior to CE.


New analytical techniques for research on food

During the last years a lot of new techniques and technologies were introduced which

allows the food chemist to monitor reactions which take place in agricultural products

during storage and processing. Nowadays it is possible to monitor chemical reactions in

food products which was impossible only a few years ago. The discipline of proteomics

would not have evolved so quickly without the development of advanced mass

spectrometry techniques. Therefore, it is now possible to determine the structure of

complex mixtures of bio molecules.

The techniques which are available and used at the Laboratory of Food Chemistry are

described well. But for the introduction of these new techniques for the monitoring of

different components it is sometimes necessary to carry out methodology research. For

example: the quantification of components when new HPLC detectors are used, the

better understanding of the fragmentation reactions which take place during the

ionization of oligosaccharides in the LC-MS or the improvement of HPLC separation of

complex mixtures of polysaccharides. The student should have a good perception of

analytical techniques.

Thesis projects:

Application of LC-MS, MALDI-TOF MS, HPLC.

Information: [email protected], [email protected]


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Protein deterioration in leaves and seaweeds due to activation of the defence

mechanism

Plant leaves and seaweeds have been identified as novel sustainable, green, protein

sources. The available protein (roughly 15-35% w/w DM) has potential to be used in both

food and feed applications. Upon protein isolation, cells are disruption and secondary

metabolites are released; the organisms defence mechanism is activated. Some of these

secondary metabolites, e.g. Phenolic monomers in plants and phenolic polymers, (Table

1), are already known to interact with protein and change its properties. Interactions are

based on covalent and non-covalent binding. There is less information on the molecular

composition and possible interactions with protein of seaweed secondary metabolites. Table 1 secondary metabolites present in plants and seaweed and their potential interactions with proteins

Plants Seaweeds

Class Secondary

metabolites Interaction with

protein Secondary

metabolites Interaction with

protein

Phenolic monomers

Phenolic acids, flavonoids

Enzymatic quinone formation, covalent

Phenolic acids, flavonoids

Presence oxidative enzymes unknown

Phenolic polymers

Procyanidins Non-covalent Phlorotannins Non-covalent

Terpenoids Saponins

(triterpene) Unknown

(foaming agent) Mono- di-

triterpenes Unknown

Organo-halogens Chloride, Bromide

derivatives Unknown

Aim

To be able to use green protein sources, a detailed investigation on the possible reactions

occurring during processing has to be done. This involves characterisation of the

compounds present in the sources used, i.e. sugar beet leaf (Beta vulgaris) and brown

seaweed (Laminaria digitata). The main compounds identified will be used for further

studies regarding interactions with proteins and the changes in protein digestibility in

animals.

Thesis project:

Characterisation and quantification of saponins in sugar beet leaf using UHPLC-MS

Development of extraction methods to separate and characterise phenolics,

phlorotannins and terpenoids in brown seaweed by preparative chromatography

combined with UHPLC-MS

Characterisation and quantification of phlorotannins in brown seaweed using

UHPLC-MS and MALDI-TOF-MS techniques



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Part 2: Enzymatic conversion of food by-products

C₃-Acids production from biomass residues

The global awareness of the finiteness of oil reserves make bio-plastics a suitable

alternative for fossil-oil based plastics. Nonetheless, manufacturing of renewable

alternatives for conventional plastics should not be in confrontation with food availability.

The selection of lignocellulosic plant by-products do not affect food products for human

consumption.

Plant cell wall is typically composed of cellulose, lignin and hemicellulose. These three

components are conformed in a complex and recalcitrant structure which is needed to be

degraded before the fermentation step. This is the main bottleneck of the process.

Pretreatment is needed in order to make the lignocellulose structure accessible for

hydrolytic enzymes. Nonetheless, the environmental friendliness of the process has to be

taken into account. This is why mild chemical pretreatments and a strong focus on

enzymes, are preferred. They are being studied aiming the highest degradation of plant

cell wall materials.

Thesis projects:

Isolation and characterization of carbohydrates present in lignocellulosic biomass

(Biomass extractions, Gas Chromatography, Size Exclusion Chromatography,

Anion Exchange Chromatography, Mass Spectrometry).

Study the modification of carbohydrates in biomass structure after the application

of different pretreatments. (Gas Chromatography, Size Exclusion

Chromatography, Anion Exchange Chromatography, MALDI-TOF Mass

Spectrometry).

Mode of action of various enzymes in biomass carbohydrates (enzymatic

degradation). (Size Exclusion Chromatography, Anion Exchange Chromatography,

MALDI-TOF Mass Spectrometry).

Influence of modified lignin structures after pretreatment on the performance of

enzymatic saccharification of plant cell wall materials. (Pyrolysis GC-MS).



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Characterisation of recalcitrant carbohydrates in bioethanol fermentation

The massive use of fossil fuel has negative impacts on the environment. In addition, the

supply of fossil fuel is dwindling. Bioethanol, therefore, is a promising alternative fuel.

The ‘first generation’ bioethanol uses starch from grains as corn and wheat as raw

material. To avoid competition with food supply, the ‘second generation’ bioethanol is

being developed using non-food agricultural waste, such as corn stover (corn plant after

harvesting the corn). The molecular structure of carbohydrates in corn stover is more

complex than that of starch. Despite the attempts that have been done to optimise the

degradation of the various carbohydrates to fermentable monosaccharides, some

carbohydrates remain recalcitrant. If the recalcitrant carbohydrates can be degraded, the

ethanol yield from corn stover can be increased. Characterisation on the recalcitrant

carbohydrates is necessary in further optimisation of the process, including enzyme

selection for the degradation of the corn stover.

Due to the complex structure of the carbohydrates in corn stover, a combination of

enzymes (enzyme cocktail) is required to be able to degrade the various poly-

/oligosaccharides in the sample into monosaccharides. To be able to fine-tune the

composition of the enzyme cocktail, individual enzymes need to be purified and

characterised for its activity and specificity.

Thesis projects:

The thesis project is focused on the degradation of lignocellulosic material from corn

stover, including structural characterisation of the recalcitrant carbohydrates. Purification

and characterisation of enzymes that are needed to degrade the lignocellulosic material

into fermentable monosaccharides will also be performed. Various separation and

analytical techniques including HPAEC, MALDI-TOF/TOF MS and UHPLC-MSn will be used.

Other techniques such as Capillary Electrophoresis (CE) with or without MS can also be

explored.



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LPMO enzymes – a new approach in biomass degradation

Enzymatic degradation of lignocellulosic biomass to obtain fermentable monosaccharides

for secondary generation biofuels and chemicals is a desirable approach for a more

sustainable supply.

Lytic polysaccharide monooxygenases (LPMOs) have shown to increase the breakdown of

lignocellulosic biomass by their oxidoreductive system. Catalysing oxidative cleavage of

polysaccharides, LPMOs are likely to have an important synergistic effect on

lignocellulosic biomass degradation by enhancing the accessibility of the crystalline

material when added to cellulase cocktails.

This work will focus on the biochemical characterisation of various fungal LPMOs.

Furthermore, the application of this enzyme class on different substrates to extend the

possible initiation of biomass degradation will be investigated.

Thesis projects:

Purification of LPMOs and their mode-of-action on hemi(cellulose), (SDS-Page,

HPAEC, MALDI-TOF-MS)

Synergistic effect of LPMOs added to cellulase cocktail on degradation of pre-

treated biomass (HPSEC, HPAEC, MALDI-TOF-MS)

Substrate binding specificity of LPMOs (HPAEC, Fluorescence imaging)


Figure 1: Purification of an LPMO by SEC (Size Exclusion

Chromatography).

Figure 2: HPAEC chromatogram of products obtained upon incubation of cellulose (PASC)

with LPMO (Westereng et al 2011).


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More mushrooms via optimization of the composting process

The mushroom Agaricus Bisporus or “champignon” is industrially grown on wheat straw

compost. Compost is made in such a way that the mushrooms can feed themselves with

the carbohydrates from wheat straw. Understanding and optimizing the composting

process is a key to get good compost quality and hereby good mushroom yields.

The composting process is based on the aerobic fermentation of lignocellulosic biomass

(wheat straw) with chicken and horse manure. The conditions (pH and temperature)

during the composting process must be monitored and controlled. To control and lower

pH values, gypsum (calcium sulfate dihydrate) is added to the initial compost mixture.

The effect of gypsum within the process is still not well understood.

In this project, the effect of gypsum on the fermentation of wheat straw will be

investigated. The main components of wheat straw (cellulose, hemicellulose and lignin)

will be studied and their structure will be analysed after different composting times. The

modification of the wheat straw during composting is the factor which allows mushroom

growth.

Thesis project:

Effect of gypsum on the polysaccharides (cellulose and hemicellose) and polyphenols

(lignin) of wheat straw during fermentation for mushroom growth.

(Gas Chromatography, Anion Exchange Chromatography, Pyrolysis GC/MS)



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How mushrooms feed on sugars

The white button mushroom Agaricus bisporus (“witte champignon”) is one of the most

popular Dutch “vegetables”. A typical person eats one box of champignons per week. So

production of this mushroom is of high importance for Netherlands and reducing the

amount of waste after mushroom production is interesting both from the ecological point

of view as well as financial.

Mushroom itself is low in fat and rich in protein and fibre which makes it also interesting

as a nutrient source. Moreover, it contains vitamins, minerals and bioactive compounds

such as anti-cancer polysaccharides.

The white button mushroom is produced on compost of straw and horse manure. At the

moment we know which components need to be mixed and in which way to make a good

compost but why this ratio of components and certain temperatures are needed is not

known. It is a black box.

For instance, it is not known which sugar components are converted from the compost

(and which are not) and when or how this is of importance for mushroom growth later

on. The need for this information is exemplified by the fact that up to 25% of the

compost is not converted into fungal biomass which means more waste.

The aim of the project is to understanding the conversion of polysaccharides in the

substrate during the process of composting and growth of Agaricus bisporus. You would

investigate which sugars are converted or altered and which are consumed by the

microbes and by the mushroom with a specific focus on characterisation of cell wall

polysaccharides on a molecular level.

Thesis projects:

Fractionation and characterization of the compost for mushroom growth.

Enzymatic degradation studies (Anion Exchange Chromatography, Size Exclusion

Chromatography, several Mass Spectrometry techniques).

Isolation or quantification of bacterial and/or fungal biomass from the compost by

identifying specific components of fungal and bacterial cell wall.

Testing Agaricus transformants for enzyme activities (Anion Exchange

Chromatography, Size Exclusion Chromatography, several Mass Spectrometry

techniques).

Own idea or wishes are welcome (both techniques or topic orientated).

Contact: [email protected], [email protected]

Mushroom compost Full grown mushrooms Mushroom spawn Ready for customers


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Part 3: Health aspects of food

Novel mechanisms underlying the anti-inflammatory activity of omega-3 fatty

acids

Dietary consumption of long chain

omega-3 poly-unsaturated fatty acids

(n-3 LCPUFAs), in particular DHA and

EPA, has been associated with several

positive health effects, including a

reduction of risk factors for

cardiovascular diseases, diabetes,

metabolic syndrome and inflammatory

bowel disease. Evidence is accumulating

that n-3 LCPUFAs contribute to a

decreased inflammatory status via

different mechanisms, which are still

not fully understood. Over last decade,

two mechanisms have been

established: 1) n-6 vs n-3 balance in

membranes was shifted which causes

an anti-inflammatory profile; 2) anti-

inflammatory resolvins were

synthesized from n-3 LCPUFAs in the

body. Recently, a novel explanation was put forward, in which the anti-inflammatory

action was mediated via specific fatty acid amides (FAAs). FAAs are a group of

conjugates formed from diverse fatty acids with e.g. amino acids or ethanolamine.

From a nutritional point of view, FAAs are interesting as some of them might be

important signalling molecules in anti-inflammatory pathways. Therefore, the projects

will investigate the formation and biological activity of FAAs, specifically formed from the

n-3 LCPUFA DHA. First, DHA-derived FAAs will be chemically synthesized and the

structure of the compounds will be confirmed by LC/MS and NMR. Furthermore, their

anti-inflammatory effects will be screened on macrophage cell lines. Key inflammatory

mediators (or markers), such as TNFα, IL-6, will be measured in response to exposure to

the FAAs.

Thesis projects will include practical work at both Food Chemistry and Nutrition and

Pharmacology group.

Thesis projects:

Identification and characterization of N-Docosahexaenol-serine and its immuno-

modulating properties in mouse macrophages

Purification of bioactive fatty acid amides from animal tissue/neuronal cells, especially

DHA derived conjugates by chromatographic techniques

Elucidate the anti-inflammatory activity of DHA derived conjugates using different

biological assays



15

Phytoalexins, plant defence compounds with added functionality

Using prenylation as a tool to diversify and enhance functional properties

Plants of various taxa are known to produce defence compounds, so-called phytoalexins,

when they are exposed to stress. Phytoalexins can possess beneficial functionality such

as (anti-)estrogenic and antimicrobial activity. The process of prenylation, substitution

with a prenyl-group (C5), can be used to enhance or diversify these functionalities. Soy,

a member of the plant family Leguminosae, produces prenylated isoflavonoids during

micro-malting combined with fungal challenging. Prenylation plays a key role in the

functionality of phytoalexins and has been shown to modulate (anti-)estrogenic as well as

antimicrobial activity of phenolics.

In this project we will employ two methods to produce prenylated phenolic compounds:

Micro-malting: germination of plant seeds combined with exposure of seedlings to fungal

growth to stimulate production of (prenylated) phenolics.

Prenyltransferases (PT): prenyltransferase enzymes can be produced by genetically

modified E. coli. PTs can then be used to attach prenyl-groups to various phenolic

compounds.

Thesis projects:

Thesis projects will include a combination of practical work in the fields of chemistry and

microbiology.

Micro-malting seeds with the germinator combined with fungal challenging. This

will be followed by extraction, fractionation, purification and characterisation of

phenolic compounds by (preparative) liquid chromatography (LC) and mass

spectrometry (MS).

Targeted production of prenylated phenolic compounds will be performed by

microbial prenyltransferases (PT). This will involve practical work in genetic

modification of bacteria and purification of the produced PTs. Prenylated phenolics

will be analysed by UPLC-MS.


Germinator used in micro-malting, normally used in the beer brewing industry for pilot-scale malting

Example of a prenylation reaction, the isoflavonoid daidzein is substituted with a prenyl-group


16

Structure activity relationships of isoflavonoids as antimicrobial agents

At present there is a great concern about

the emergence of multidrug resistant

bacterial strains, and continuous research

efforts are being devoted for the

development of new and effective

antimicrobial agents. Many plants from the

Leguminosae family have been used for

medicinal purposes since ancient times in

many parts of the world. Therefore,

substantial attention has been focused on

the exploration and utilization of legume

phytochemicals as alternatives of traditional

antimicrobial agents.

Previous studies have found these plants to be rich in isoflavonoids. Isoflavonoids are

phenolic plant secondary metabolites, and are produced as part of the plant defence

system. Isoflavonoids have been shown to exhibit interesting biological and

pharmacological properties, such as antioxidant, antibacterial, antiviral and anti-

inflammatory activity. These properties seem to be highly structure-dependent.

Prenylation is a type of substitution commonly found in bioactive isoflavonoids. Some

prenylated isoflavonoids have shown strong inhibitory activity towards antimicrobial

resistant strains, as well as synergy with traditional antimicrobials. However, the exact

relation between chemical structure (e.g. number, type and position of substituents in

the isoflavonoid skeleton) and bioactivity is not yet well understood.

In order to develop novel and effective antimicrobials it is necessary to fully understand

these structure-activity relationships (SAR), and elucidate the main chemical features

necessary to have good antimicrobial activity. Different strategies might be employed to

enhance the antimicrobial potential of these compounds (e.g. fermentation, enzymatic

prenylation).

Thesis projects

In general, thesis projects will include practical work from both chemistry and

microbiology.

Study of the effect of germination and fungi-fermentation on the antimicrobial

potential of different Leguminosae seeds.

Characterization and purification of antimicrobial isoflavonoids present in seed

extracts by chromatographic techniques, such as UPLC-MS and preparative HPLC,

in order to determine the main structure-activity relationships.

Elucidation of the mode of action of the main antimicrobial compounds using

different biochemical assays.

Production of microbial prenyltransferases for the in-vitro prenylation of

isoflavonoids, as an alternative for the production of novel antimicrobial

compounds.



17

Effects of processing of proteins on their immunogenicity: allergy as a model (dairy subject)

Food allergy develops in two phases: a sensitisation phase and an elicitation phase, the

latter resulting in clinical symptoms of allergic disease. Sensitisation is the phase during

which immunological priming to the inducing allergen occurs, leading to the evolution of

aTh2-biased immune response and the production of IgE-antibody.

Skewing of adaptive immune response to a Th2-type phenotype and IgE-antibody

production necessarily are preceded by an innate immune response to the allergen.

Activation of receptors on cells of the innate arm of the immune system is crucial for the

initiation of adaptive immune responses.

Processing changes the structural and chemical properties of proteins, and hence of

allergens. Proteins denature, aggregate, bind to lipid structures, and undergo

glycosylation and or glycation (Maillard reaction). These processing-related structural and

chemical changes will influence how the immune system will respond, possibly leading to

eventual allergenicity.

The aim of this project is to study the effect of processing-induced alterations in proteins

(unfolding, fibril formation, aggregation, Maillardation) on the response that they provoke

in (innate) immune cells, such as macrophages and dendritic cells.

Thesis projects:

Characterisation of processing-induced structural alterations in (model) food

proteins (fluorescence, circular dichroïsm, size, electrophoretic behaviour, etc.)

Analysis of immune response to such alterations by exposing model cell cultures

to the proteins by immunologic methods such as phagocytosis and cytokine

production


The Fate of Human Milk

Oligosaccharides (HMO) in Infant Gut (dairy subject)

Milk is the first food to fulfil the need for

the neonates. It provides some potentially

significant physiological and biological

functions such as prebiotic, antipathogenic

and immunomodulatory activities that are

partly resulted from oligosaccharides.

Thesis project:

Oligosaccharides present in human milk

and infant faeces will be identified and quantified by CE-LIF, MALDI-TOF-MS, HILIC-MS,

etc. Mechanisms of metabolisation, conversion and utilisation of HMO

in infant gut will be proposed.

Information: [email protected] and [email protected]

mailto:[email protected]



18

In vitro digestion of proteins for fish feed

Fish for human consumption is increasingly produced through

aquaculture. A lot of the currently farmed fish species have a

high protein requirement in their diet. In order to make

aquaculture more sustainable, novel protein sources from

animal and plant by- products or waste products are

investigated for their suitability as protein sources. These

novel ingredients are often heavily processed which can

negatively impact their digestibility when fed to fish.

To reduce the amount of animal (in vivo) testing, laboratory

(in vitro) techniques are used to screen and assess novel

protein sources. One of the assessment techniques is to

determine the degree of hydrolysis through enzymatic

digestion. Studying the mechanisms behind digestion will

improve our understanding of the digestion processes and

help to develop better predictions for what is happening in

the fish.

Thesis project:

• Comparison of different in vitro digestibility methods

• Identification of controlling factors in in vitro digestion: is it temperature,

substrate concentration, enzyme to substrate ratio or the identity of the

enzyme(s)?

• (Bio)chemical characteristics of the substrate that influence digestibility

• Effects of feed matrix and processing on in vitro protein digestibility


Behaviour and digestibility of starch in pigs

Starch is the main storage carbohydrate in plants and

forms an important source of energy in the diet of

humans and animals. Many different sources of

starch are used in food and feed. This project

focusses on studying the chemical and physical

properties of starch from different sources, and their

physical behaviour and digestibility in a pig model. By

gaining a better insight in the types of starch and their relations to pig digestibility, more

precise models can be developed to predict the feeding value of the starch.

The main questions to be answered in this project, is how and where different starches

present in (processed) feed are digested within a pigs digestive tract, and how this

effects the pig's performance. Firstly, in vitro (lab) digestion trials will be done to adapt

human digestible and resistant starch assays to the animal situation. Later on, these

models will be tested in vivo (in pigs), where digesta of differently fed animals will be

analysed.

Thesis topics:

BSc: Analysis of various (processed) feeds for different starch types, using newly

developed methods.

MSc: Understanding the resistance of starch in the pigs digestive tract, using in

vitro and/or in vivo methods.

Contact: [email protected]; [email protected]


19

The effect of protein structure on digestion dynamics (dairy subject)

Enzymatic digestion of proteins is important in the human digestive tract as well as in the

industrial production of protein hydrolysates (for instance for infant formula). Despite the

many applications of hydrolysates, there are still many questions about the exact

mechanism by which enzymes breakdown the proteins into different peptides. Enzymes

responsible for protein hydrolysis (proteases) are known to have different specificities

(the set of amino acids after which a protease can cleave). The specificity alone however

does not explain the kinetics behind protein hydrolysis, because the different cleavage

sites in a given protein are not digested evenly. Instead, some parts of the protein are

digested more rapidly than others. This phenomenon is yet to be explained.

It is hypothesised that the primary,

secondary and tertiary structure of

the protein and the way these

interact with the enzyme are

responsible for the differences

found. Therefore, the aim of this

project is to clarify the digestion

dynamics in proteolysis by

investigating the relation between

enzyme and protein structure in

vitro, using commercial enzymes and protein isolates. The methods and techniques

developed will help the understanding of in vivo experiments, as well as increase the

understanding and control over hydrolysis in commercial processes.

Thesis projects:

How important is specificity? In this project you will compare the hydrolysis of a

protein by different enzymes that have a similar specificity. This involves

comparison of kinetics, and analysis and comparison of protein hydrolysates using

LC-MS.

Effect of processing on digestibility. Typically, proteins will be heated at some

point during food production. In this project, we study the effect of protein

denaturation on the enzymatic protein digestion dynamics. For this, we combine

the characterization of the denatured proteins, effects of heating conditions, with

analysis and comparison of protein hydrolysates using LC-MS and fluorescence

spectroscopy.



20

Characterisation of dietary fiber and investigation of their metabolic fate in the

human large intestine

Dietary fiber encompass a wide range of molecules with highly diverse chemical and

physical properties. Due to the fact that dietary fibers cannot be degraded in the small

intestine, they will reach the terminal ileum and colon where they are degraded and

fermented by endogenous microbiota. The diversity of the class of dietary fiber and their

degradation products modulate health different. Either directly or via modulation of the

endogenous microbiota. This remains largely unexplored to date.

The objective of this project is to characterise selected dietary fiber components and

metabolites, and to investigate their degradation pattern in complex in vitro models

which simulate the microbiota system in human large intestine. The fate of dietary fiber

components will be monitored and quantified using HPLC and CE methods and unknown

metabolites will be identified using MALDI Tof MS and on-line HPLC-MS and CE-MS.

Figure: Many unknown oligosaccharides fermented by human faecal inocula over 48

hours.

Thesis projects:

Modification, fractionation and characterisation of (novel) dietary fibre products

Investigation of time resolved degradation products during in vitro fermentation of

selected dietary fiber.



21

Effect of specific carbohydrates on the intestinal tract of broiler chickens and

pigs

There is an immense and fast growing pressure

on global availability of nutrients as countries

become more affluent and world’s population

continues to grow. This cannot be solved anymore

by only increasing the number of production

animals. Improvement of feed and food efficiency

in domestic animals is mandatory to ensure the

worldwide demand for food. To achieve that goal

the world is depending upon new technologies in

intensive animal food production systems that ensure a more efficient way for meat

production.

In recent years, researchers have made improvements of feed efficiency which is

attributed to a higher genetic potential, better estimation of nutrient requirements and

more knowledge of feedstuffs to more accurately meet the requirement and prevent

unnecessary nutrient losses. Minor attention has been paid to the improvement of

efficiency by directly influencing the nutrient digestion within the intestinal tract and

influencing the physiology of uptake in the gastrointestinal track of livestock.

It is known from humane studies that carbohydrates have a large influence on

quantitative food intake, nutrient absorption, immunomodulatory activity and health

promotions. In this project, we try to monitor the degradation of polysaccharides (e.g.

PECTIN) in the intestinal tract of pigs. The degradation characteristics will be further

explained by analysing microbiota composition and enzyme activities in pig digesta.

Thesis projects:

Characterizing the pectin degrading enzymes in pig (or rat) colon. Enzymes will

be extracted from pig (or rat) digesta. Pure and well characterized pectins will be

used for enzyme incubation.

Monitoring the degradation of pectins (soy pectin, lemon pectins) and characterize

the oligosaccharides produced during in-vitro fermentation with analytical

techniques



22

Part 4: New and/or improved ingredients for food

Designing the polyphenol profile of tea for improved taste

Tea is, after water, the most consumed beverage worldwide. The typical bitter /

astringent taste and the colour is caused by polyphenols, which are abundantly present

in tea leaves. During tea leaf processing the polyphenols are polymerized due to a

fermentation step. This fermentation is actually the oxidation and condensation of the

polyphenols catalysed by the endogenous tea polyphenol oxidase (PPO). Different levels

of this fermentation step will lead to different kinds of tea. The least (hardly at all)

fermented tea is green tea, followed by oolong and finally black tea. The polyphenol

profile of these different kinds of tea changes with their colour and taste.

The polyphenols present in fresh tea leaves are mainly catechins (belonging to the

flavan-3-ols class), and their gallates. When these catechins are oxidized they can

combine into dimeric, oligomeric and polymeric compounds. The reaction products are

generally categorized as reddish-orange theaflavins (soluble in ethyl acetate), and the

brownish, water-soluble thearubigins. The exact composition of black tea is still poorly

characterized. It has been estimated that one cup of black tea contains around 10,000

different phenolic compounds, which emphasizes the complexity of the mixtures.

In this project it will be investigated whether tea can be processed in a different way, in

order to control and/or design the polyphenol profile of tea with respect to desirable

colour and taste characteristics. For this it is important to isolate the endogenous tea PPO

and characterize its enzymatic activity. The effect of the PPO on the tea polyphenol

profile will be analysed using techniques as liquid chromatography and mass

spectrometry.

Thesis projects:

Isolation and characterization of PPO from tea leaves and comparison of the

enzymatic activity with commercially available PPO (mushroom)

Controlled oxidation of green tea into darker tea using PPO


PPO

O2




23

Sugar beet leaves: A new source of proteins?

There is a lot of interest to find novel sources of proteins

that can be used in food applications. Sugar beet leaves

are currently not used, but may contain up to 20%

protein. These proteins need to be extracted mildly from

the leaves, so that they retain their non-modified- ‘native’

structure needed for optimal techno-functional properties.

At the same time, negative side-reactions, such as the

cross-linking between proteins and phenolic compounds

due to polyphenol-oxidase activity, need to be avoided. In

this project, you will make your own sugar beet leaf

isolate, and study the chemical and techno-functional

properties.

Thesis projects:

Study the effect of the age of sugar beet leaves on composition and protein yield.

Study the effect of different isolation techniques on protein yield and quality.

Characterize the foaming and emulsifying properties of the sugar beet leaves

protein isolate.


PROGRESS: Proteins from green sources for use in both food and fish feed

There is a lot of interest in proteins from algal and

leafy sources, but relatively little is known about

these proteins and their possible functions in food

or feed applications. In this study, colourless water-

soluble protein fractions will be isolated from

microalgae (Nannochloropsis gaditana and

Tetraselmis sp.), cyanobacteria (Spirulina sp.) and

grasses (Tall Fescue) using mild isolation

techniques. These fractions will be analysed and

compared on their composition with respect to

proteins and other compounds. The major protein in

leafy and algal sources, Rubisco, will be studied in

more detail in order to evaluate the source

dependant variations of proteins from different

green sources.

The obtained protein fractions will be further

characterized with respect to their techno-functionalities in food and tested in fish feed

formulations and in food model systems. The impact on gut functioning and structure is

determined in fish trials in order to assess the digestibility, bio-availably and presence of

potential anti-nutritional factors in the protein fractions. Eventually, the results will

provide understanding concerning the functionalities of proteins from green sources in

food and fish feed.

Thesis project:

Techno-functional properties of protein isolates obtained from microalgae or

cyanobacteria

Implications of polyphenol oxidase activity on fish feed processability

In vitro stability and digestibility of microalgal based fish feed pellets



24

Production and valorisation of high quality proteins from insects

There is a growing demand for proteins to feed the increasing world population. It is

estimated that the world population in 2050 will be around 9 billion and that 70% more

food is necessary. An alternative protein source can be insects, the nutritional value of

which is comparable to conventional meat, and the production of which is more

sustainable. Although 80% of the world population consumes insects already

occasionally, consumers in most Western countries appear to dislike such protein

sources. This might change when the protein is added as an ingredient, so that the insect

is not recognisable as such.

To use the insect proteins as a food ingredient, it

is necessary to maintain their techno-functional

properties. For this, protein extraction from the

whole insect under mild extraction conditions is

essential to preserve the characteristics of the

native protein.

Preliminary experiments have shown that

substantial enzymatic browning occurred during

grinding of insects. Enzymatic browning is caused by oxidation of phenolic compounds

and subsequent condensation. This enzymatic reaction is catalysed by polyphenol

oxidase (PPO), which can be activated in insects as a defence reaction during wounding.

The objective of this research is to prevent the enzymatic browning during extraction

water-soluble proteins.

Thesis project:

This project is in collaboration with FQD.

Characterization of precursors and products of enzymatic browning present in the

mealworms with LC-MS

Investigating the enzymatic browning mechanism in mealworms and the

mechanism of inhibiting components

Isolation and characterization of PPO present in insects



25

Changes in pectin structure during the processing of fruits and vegetables

Pectin is one of the main polysaccharides present in fruit and vegetable cell walls being

responsible for firmness. When fruit and vegetable based products are processed, the

structure of pectins may change. Such changes may have a big impact on the texture of

these products. In order to control and to be able to tailor the texture and other physical

properties of the final product, this research is aiming to understand changes in pectin

structures as a result of processing.

The impact of different technologies on pectin structure will be studied (e.g. high

temperature, high pressure, microwave, autoclave, Ohmic heating) by techniques such

as HPAEC, UPLC-MS, MALDI-TOF.

6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 Figure 1: The effect of thermal treatment on pectin size, showing depolymerisation as an

effect of processing

Thesis projects:

Study how processing affects pectin structure, by analysing pectin isolated from

carrots and apples.

Study the interaction between pectin and hemicellulose



26

Use of enzymatic crosslinking to produce protein nanoparticles (dairy subject)

To obtain good product properties, one can choose different types of proteins, coming

from different sources. An alternative is to ‘pre-process’ the proteins to produce protein

isolates with altered functionality. In addition to traditional steps (heating, Maillard),

enzymatic crosslinking can be used in this way.

Currently, peroxidase enzymes have been used to crosslink different

types of proteins. Initial experiments have shown that protein nano-

particles of different sizes (10-80 nm) can be obtained. For these

nanoparticles a significant increase in foam stability was observed. One

of the possible reasons for this is that the particles get stuck between

air-bubbles in a foam, similar to fat globules as in for instance ice-cream.

The aim of this project is to obtain insight in how the size and structure

of crosslinked protein nanoparticles (1) can be controlled by the reaction conditions, and

(2) affect the foam stabilizing properties of the protein.

Thesis projects:

Comparison of heat-induced aggregates to enzymatically cross-linked proteins.

Understanding the relation between reaction conditions and the structure / size of

the formed products (protein nanoparticles)

Study the use of alternative enzymes / substrates in the formation of protein

nanoparticles with different properties.


Preparation and characterisation of (anisotropic) protein particles added to

dairy product as fat replacers/for structure formation (dairy subject)

The theories used to prepare these particles are (partial) enzymatic degradation of

protein, phase separation between milk protein and polysaccharides, and complex

coacervation. Besides chemical characterisation of the particles, microscopy and rheology

will also be important in this line of research. This project will be carried out at

FrieslandCampina, Wageningen.

Thesis project:

Preparation of (anisotropic) protein particles


Preparation and characterisation of milk protein beads by gelation of the

protein at relatively low temperature (cold gelation) (dairy subject)

The influence of amongst other protein composition, salt concentration, and pH on the

bead characteristics will be determined. Bead properties that are important in this line of

research are morphology (microscopy), volume swelling, molecular changes of protein

during gelation, firmness and relaxation of the beads, and (enzymatic) degradation. This

project will be carried out in cooperation with FrieslandCampina, Wageningen.

Thesis project:

Preparation and characterisation of milk protein beads by gelation.



27

Part 5: Educational development

Design and/or evaluation of digital learning material for Food Chemistry

Already for a number of years the Laboratory of Food Chemistry has been designing and

developing digital learning materials. These materials are used in different courses in the

field of food chemistry. With computer applications, learning material can be designed

that can reach certain learning goals in a more efficient way compared to traditional

teaching methods. The possibilities of digital techniques are amongst others easy

visualization, incorporation of movies or animations, the possibility to personalize the

computer program, giving immediate feedback or hints, and interaction, motivation and

activation of students.

This project deals with three different fields: Food Chemistry, Didactics and Information

and Communication Technology (ICT). It is the combination of these three fields that

makes this project interesting. The goal is to design and/or evaluate useful digital

learning materials. You will learn about educational theories, about using computer

technologies and about a certain subject within food chemistry.

Thesis project:

Designing digital learning material for a course in the field of food chemistry.

Depending on the interest of the student and the needs of the teachers we can

describe the topic for the thesis project together.


Developing new learning activities or improving existing learning activities

The Laboratory of Food Chemistry is continuously working to improve our education. One

important development is to develop activating learning activities. Learning activities are

for example laboratory activities, group work, class room activities during lectures,

training questions within readers, etc.

This thesis subject deals with two different fields: Food Chemistry and Didactics. You will

learn how to design and evaluate useful learning activities, by incorporating education

theories, for a certain subject within the field of food chemistry. This means that you will

also gain knowledge about that specific food chemistry subject

Thesis project:

Designing learning material for activating learning activities for a course in the

field of food chemistry. Depending on the interest of the student and the needs of

the teachers we can describe the topic for the thesis project together.


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