8. Health, Nutrition and Food Science

123

8. Health, Nutrition and Food Science

Health, Nutrition and Food Science

125

Influence of molecular weight of ethylcellulose in aqueous based coatings

Jurgita Kazlauskeab, Sara Almera and Anette Larssonab

aPharmaceutical Technology, Applied Chemistry, Department of Chemistry and Chemical Engineering, Chalmers University of Technology, 412 96 Goteborg,

Sweden bSuMo Biomaterials, a VINN Excellent Center at Chalmers University of Technology,

412 96 Goteborg, Sweden

[email protected] and [email protected]

One of the most commonly used polymers for controlled release from aqueous-

based coatings is ethylcellulose, a water-insoluble cellulose derivative. Previous

investigations on aqueous-based ethylcellulose dispersions are done by using an

ethylcellulose viscosity grade of 20 cps (1-3). Since different molecular weights

of ethylcellulose correlates with different properties for the pure material, it is

natural to think that the properties of aqueous-based coatings produced with

different molecular weights of ethylcellulose will also differ from each other.

Previous work in our group revealed that the molecular weight of ethylcellulose

was important for organic solvent based coatings, there for example mixtures of

hydroxypropyl cellulose and ethylcellulose of different molecular weight gave

different microstructural features and permeabilities (4).

The study shows that the coalescence is decreasing with increasing molecular

weight and the permeability of water is increasing. These findings are important

features in the development of new pharmaceutics with drug release rates

controlled by aqueous-based latex coatings.

References.

1. Frohoff-Hülsmann MA et al., International journal of pharmaceutics. 1999;177(1):69–82. 2. Parikh NH et al., Pharmaceutical research. 1993;10(4):525–34. 3. Muschert S et al., International journal of pharmaceutics. 2009;368(1):138–45. 4. Andersson H et al. European journal of pharmaceutical sciences 2013;48(1–2):240–8.

mailto:[email protected]



126

Temporal transcriptional response to insulin stimulation of myocytes: Impact of type 2 diabetes and obesity

Leif Väremoa, Camilla Scheeleb, Christa Broholmb, Adil Mardinoglua, Intawat Nookaewa, Mathias Uhlénc, Bente Klarlund Pedersenb, Jens Nielsena,c

aBiology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

bThe Centre of Inflammation and Metabolism and the Centre for Physical Activity Research, Department of Infectious Diseases, Rigshospitalet, University of

Copenhagen, Denmark cScience for Life Laboratory, Royal Institute of Technology (KTH), Stockholm,

Sweden

[email protected]

Skeletal muscle is the major target tissue for insulin resistance preceding the

development of type 2 diabetes (T2D). To unravel how this development can be

prevented it is important to map the interaction between insulin and insulin

stimulated responses in human skeletal muscle cells. Therefore, the aim of this

study is to elucidate time-dependent transcriptional responses invoked by insulin

stimulation and in particular to investigate how these temporal profiles manifest

in molecular mechanisms at the level of cellular metabolism and hereby confer

insulin resistance and possible T2D. This is achieved by deep RNA-sequencing

of 96 samples from primary human muscle cells from a clinically well-defined

cohort (covering healthy and diabetic, obese and non-obese, males and females)

and performing thorough data analysis and integration with genome-scale

metabolic modeling. Preliminary results point at a major activity 2 hours after

insulin stimulation and a distinction of implicated processes in the different sub-

groups.

Figure 1. An outline of the experimental design. From a larger cohort, 24 subjects were selected and

divided into four groups (NGT/OB, NGT/non-OB, T2D/OB, T2D/non-OB; NGT=normal glucose tolerant,

OB=obese, T2D=type 2 diabetic) with three females and three males in each group. A vastus lateralis

biopsy was taken from each subject (1), and myoblasts were isolated and differentiated in vitro (2). Fully

differentiated myocytes were serum starved for 2h and stimulated with insulin (3). RNA samples for

sequencing were taken at four time points (4).


127

Taurine bioaccessibility but not bioavailability in Caco-2 cells is increased by the omega-3 fatty acids eicosapentaenoic acid and

docosahexaenoic acid

Andrew Vincent, Nathalie Scheers and Ann-Sofie Sandberg

Food Science, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

Email: [email protected]

BACKGROUND: A diet rich in fish reduces risk factors for cardiovascular

disease. This has been mainly attributed to ω-3 fatty acids but fish contain several

other functional compounds that have proved to be important for cardiovascular

health. Fish also contains high amounts of taurine, which has been shown to

improve vascular health by regulating calcium transport and acting to maintain

membrane stability and redox homeostasis. As these nutrients are seldom

consumed in isolation, it is important to study combinations of nutrients to

understand how they may work synergistically with each other. Omega-3 fatty

acids may influence membrane fluidity and transporters, with a potential impact

on nutrient bioavailability, which could affect the uptake of other nutrients from

fish, including taurine.

OBJECTIVES: To determine if taurine bioaccessibility (uptake) and

bioavailability (basal efflux) is influenced by the ω-3 fatty acids, eicosapentaenoic

acid (EPA) and docosahexaenoic acid (DHA).

METHODS: The human intestinal Caco-2 cell model was used to estimate the

bioaccessibility and bioavailability of taurine. Transport of taurine (20 mM), as

[3H]-taurine (18.5 kBq mL-1), in the presence of DHA (100 μM) and EPA (100

μM) was measured by liquid scintillation counting.

RESULTS: The results indicate that taurine uptake was significantly increased in

the presence of DHA (28 %) and EPA (22 %). Taurine cellular efflux was reduced

by EPA (14 %) and DHA (8 %).

CONCLUSIONS: EPA and DHA increase the bioaccesibility of taurine but do

not affect the efflux, therefore even though local taurine concentrations may be

increased, there does not appear to be an effect on taurine bioavailability.


128

Effect of solvent system and biomass pretreatment method on the yield and quality of fatty acids from Saccharina latissima

Giorgia Tibalderoa,b,c, Joshua Mayersb, Eva Albersb, Ingrid Undelanda

Dept. Biology and Biological Engineering, aDiv. Food and Nutrition Science & bIndustrial Biotechnology, Chalmers University of Technology, Göteborg, Sweden

cUniversità degli studi di Torino, Dept. Scienze della Vita e Biologia dei sistemi, Unit Biochemistry, Turin Italy

Email: [email protected], [email protected]

Although levels of fatty acids in seaweeds are generally low (<10 mg g DW-1),

this fraction is enriched in long chain n-3 polyunsaturated fatty acids. In addition,

this fraction can contain considerable quantities of hydrophobic compounds such

as vitamins, sterols and polyphenols, which still mark seaweed lipids as an

interesting nutritional resource. In the SEAFARM project, Saccharina latissima

harvested from Sweden’s West coast will be subjected to biorefining, with the

aim of maximizing its value as a raw material for food/feed ingredients, chemicals

and energy.

A possible limiting step in this field is the compositional characterization of the

structurally complex seaweed biomass, not least its lipid fraction. The present

study aims to develop extraction methods, which maximize accurate measurement

of this pool. Furthermore, the applicability of different extraction methods to

large-scale biorefinery scenarios is assessed, taking into account that ‘wet-

biomass’ will mostly likely be utilised in such cases.

Firstly, chloroform & methanol systems were tested for fatty acid extraction

(Folch vs. Bligh & Dyer methods) on wet and freeze-dried material, in

conjunction with several pre-treatment methods (mechanical disruption, ultra-

sonication and microwave-assisted extraction). The best pretreatment was then

tested using ethanol and 1-butanol. Direct transesterification was conducted as a

reference method and other techniques appraised with respect to the yield and

profile of extracted fatty acids.

Overall, the modified Bligh & Dyer chloroform:methanol method was the most

effective solvent system in unassisted extractions (5.3 mg g fatty acids DW-1);

microwave and sonication pre-treatments significantly improved fatty acid yields

(7.3 mg g DW-1). Ultra-sonication assisted ethanol extraction performed equally

as well as Bligh & Dyer with pre-treatment (7.6 mg g DW-1). Yields from wet

biomass were generally comparable to those from freeze-dried material. Most

extracts were rich in polyunsaturated fatty acids (40-50%), with up to 0.82 and

0.72 mg g DW-1 being stearidonic and eicosapentaenoic acid, respectively.

Further results and insights into lipid extraction will be presented.




129

Genome Scale Metabolic Modeling of Child Growth, 0-6 months

Avlant Nilssona, Adil Mardinoglua and Jens Nielsen 3a

a Department for Biology and Biological Engineering

[email protected]

Malnutrition of children is a global challenge. Improving our understanding of

child growth may translate in to faster detection and to cost efficient diet

supplements. Genome Scale Metabolic Models (GEMs) have been successfully

applied to many biological systems. Here we make use of a GEM, HMR 2.0 [1],

to model the effect of co-factor deficiency in infants. The concentrations of

metabolites in breast milk are given as input, and the growth of biomass is the

output. The model also includes, weight dependent, maintenance energy

expenditure. Co-factor deficiency is modelled by reducing the metabolic flux

through reactions that require a given cofactor (Figure 1).

Figure 1. Growth trajectories for deficiency of 3 different co factors. The flux through their associated

reactions was limited to 90% of the optimal flux. The 5th, 50th and 95th percentile of WHO growth data

for boys as reference.

References.

[1] Adil Mardinoglu, Rasmus Agren, Caroline Kampf, Anna Asplund, Mathias Uhlen, and Jens

Nielsen. Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in

patients

with non-alcoholic fatty liver disease. Nature communications, 5(May 2013):3083, 2014.


130

In vitro study of gastrointestinal oxidation of marine long chain n-3 polyunsaturated fatty acids – comparison between models based

on human fluids or porcine extracts

Tullberg, C.a, Vegarud, G.b and Undeland, Ia

aDivision of Food and Nutrition Science, Department of Biology and Biological Engineering, Chalmers University of Technology, Sweden

bDivision of Food proteins; Structure and biological function, Department of Chemistry, Biotechnology and Food Science, Norwegian University of

Life Sciences, Norway

[email protected]

Marine long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) have attracted

public awareness due to the positive health effects they have been associated with.

LC n-3 PUFA intake has been connected with reduced risk for many diseases, e.g.

cardiovascular and inflammatory diseases. In vitro studies have shown that fish

lipids can oxidize under gastrointestinal tract (GIT) conditions when using

digestive enzymes of porcine origin (1). The lipid oxidation reaction is known to

generate highly reactive oxidation products that may interact with DNA and

proteins, leading e.g. to various effects on cell function. Some of these oxidation

products are malondialdehyde (MDA) and the α,β-unsaturated aldehydes 4-

hydroxy-trans-2-nonenal (HNE) and 4-hydroxy-trans-2-hexenal (HHE), but little

is known about formation of these compounds in the GIT.

In this study, two GIT in vitro models were set up to study the effect of digestion

on lipid oxidation in cod liver oil. In the first model, digestive enzymes and bile

of porcine origin were used, and in the second model, human digestive juices were

used. The standardised Infogest protocol (2) was applied to the models and

enzymatic activities were matched between them. The digesta was analysed for

reactive oxidation products (MDA, HNE and HHE) by Liquid Chromatography–

Mass Spectrometry (LC-MS) and for free fatty acids by Gas Chromatography-

Mass Spectrometry (GC-MS). Results show that lipid oxidation occurs in both

models under gastric as well as intestinal conditions. However, both time

dependence and maximum levels of oxidation products differed, something which

will be discussed.

References.

1. Larsson, K. et al., J. Agric. Food Chem. (2012) 60:7556-7564

2. Minekus, M. et al., Food & Function (2014) 5:1113-24


131

Eicosapentaenoic and docosahexaenoic acid-enriched high fat diet delays the development of fatty liver in mice

Nikul Soni1, Intawat Nookaew2, 3, Ann-Sofie Sandberg1*, Britt G Gabrielsson1

1Divisions of Food Science and Nutrition and 2Systems Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-41296

Gothenburg, Sweden. Present address: 3Comparative Genomics Group, Biosciences division, Oak Ridge National Library, Oak Ridge, TN 37831, USA.

Scope: To investigate the effects of quality of fat in a high fat diet HFD)

over time on hepatic lipid storage and transcriptome in mice.

Methods and results: Male C57BL/6J mice were fed control, HFD-

EPA/DHA or HFD-corn oil diet for 8 or 12 weeks. Body weights, body

composition, plasma and hepatic triglyceride content were measured. Hepatic

transcriptomes were analysed by microarray followed by gene-set enrichment

analyses. At 8 weeks, the HFD-corn oil mice had higher body weight and

adipose depot mass than the HFD-EPA/DHA but there were no differences

at 12 weeks. Hepatic triglyceride content was low in HFD-EPA/DHA fed

mice at both time- points. Enrichment analyses showed that lipid/fatty acid

biosynthesis; transport and homeostasis were lower in the HFD-corn oil fed

compared with the HFD-EPA/DHA fed mice. Genes encoding proteins

associated to cytoplasmic lipid droplets were expressed at higher levels in

livers from the HFD-corn oil compared to HFD-EPA/DHA mice.

Conclusions: EPA and DHA maintained lower hepatic triglyceride content

despite being fed HFD. The liver transcriptome data implicate that the quality

of dietary fat could modulate Ppar-related gene expression that in turn affects

hepatic lipid storage and maintenance of metabolic health.


132

Serum proportions of eicosapentaenoic acid (EPA) in infants may decrease allergy risk: results from the FARMFLORA birth-cohort

Karin Jonsson1, Malin Barman1, Sara Moberg1, Hilde K Brekke2, Agnes Wold3, and Ann-Sofie Sandberg4

1Food Science, Dept. of Biology and Biological Engineering, Chalmers 2Dept. of Internal Medicine and Clinical Nutrition, University of Gothenburg

3Dept. of Infectious medicine, Clinical Bacteriology section, University of Gothenburg


Background and aim: The allergy prevalence has increased drastically in

countries with a Western lifestyle, and the cause is largely unknown. Some

protective factors have been identified, such as growing up on farms. Data also

suggest that serum composition of polyunsaturated fatty acids (PUFAs) may

affect allergy development. The aims of this study were to: 1) relate fatty acid

composition in cord and infant serum to allergy development; 2) correlate infant

serum fatty acids to maternal diet during pregnancy as well as to diet and breast

milk fatty acids during lactation; 3) relate differences in serum fatty acid

composition to farm residence.

Methods: Twenty-eight farm and 37 non-farm mother-infant pairs were followed

in the FARMFLORA birth-cohort. Fatty acid proportions were analyzed in cord

serum and in child serum and breast milk four months postpartum. Maternal diet

during pregnancy was assessed by food-frequency questionnaires; diet during the

fourth month of lactation was recorded by 24-hour dietary recalls followed by 24-

hour food diaries. Serum fatty acid composition was related to doctors’ diagnosis

of allergy at three years of age as well as to farm residence, and correlated to diet

and breast milk fatty acids.

Results: Proportions in serum at four months of eicosapentaenoic acid (EPA, a

long-chain omega-3 PUFA) were higher in healthy compared to allergic children

(0.48% and 0.16%, respectively, P = .001). The Odds Ratio was 0.47 (P = .01,

95% CI: 0.27-0.83) for every 0.1% increase in EPA, and did not change

substantially after being adjusted for potential confounders. Serum EPA

proportions at four months correlated to maternal fatty fish intake during

pregnancy (rho = 0.4, P = .01) as well as to breast milk proportions and maternal

intake of long-chain omega-3 PUFAs during lactation (rho = 0.4 and 0.5, P = .05

and .01, resp.). When farm children were compared to non-farm children, higher

proportions of arachidonic acid were found in cord serum (14% and 12%,

respectively, P = .001).

Conclusion: Our preliminary data suggest that low proportions of EPA in infants’

serum may increase the risk of developing allergy. The protective effects could

be mediated by EPA per se or by a higher fish intake, which might offer protection

by other means than solely its EPA content.


133

Supercritical CO2 extraction of bilberry (Vaccinium myrtillus L.) seeds: composition and antioxidant activity

Graziele Gustinelli Arantes de Carvalhoab, Marie Almingerb and Lilia Ahrnéab

aSP Technical Research Institute of Sweden bDept. of Biology and Biologial Engineering

Chalmers University of Technology

[email protected]

Bilberries (Vaccinium myrtillus L.) are commonly used as raw material for food

and drinks in Europe, generating seeds as by-products, which contain valuable bio

compounds. The objective of this study was to evaluate the properties of bilberry

seed oil extracted by supercritical fluid extraction.

Extractions were carried out using 50 ±0.1 g of bilberry seeds (milled for 30 s and

moisture content of 4.49%), using a flow rate of 40g CO2/min, for 120 min (to

determine yield) and 80 min (to use for analysis). Pressure at 200, 350 and 500

bars and temperature at 40, 50 and 60°C were selected. Analysis of fatty acid

profile, vitamin E, free radical scavenging activity (DPPH) and peroxide content

were carried out. The mean values of duplicates were compared by one-way

ANOVA and when different, T-test was applied (p<0.05 for both tests).

The yields obtained at 80 min were 8.0-21.4%. Bilberry seed oil showed high

amount of fatty acids (75.1-76.2 g/100 g of oil) and had similar composition

between the different conditions. All extracts had high amount values of 18:2 n-6

(ω6) and 18:3 n-3 (ω3) and the ratio ω6/ω3 < 1. The samples contained 47.1-105.3

mg vitamin E, gamma-tocotrienol/100g of oil. The values for Antioxidant

Activity Index (AAI) obtained were 2.1-3.57. Peroxide values were 1.1-2.0

mekv/kg of oil. The different extraction conditions resulted in significant different

values of vitamin E, DPPH and peroxides. Oils extracted from different conditions

obtained statistical differences in all analysis, except in total fatty acids.

The high amount of ω6 and ω3 and low ω6/ω3 ratio present in all extracts, indicate

high quality. The results for antioxidant capacity, amount of vitamin E show

antioxidative properties. The obtained peroxide values were lower than those

recommended for commercial vegetable oils (≤10) suggesting the extracts are

stable.


134

Sourdough fermentation of wheat flour does not prevent the interaction of transglutaminase 2 with α2-gliadin or gluten

Niklas Engströma, Ann-Sofie Sandberga, and Nathalie Scheersa

aDepartment of Biology and Biological Engineering, Food and Nutrition Science, Chalmers University of Technology


Tissue transglutaminase 2 (TG2) plays a critical role in the initiation of celiac

disease by catalyzing the deamidation of gluten peptides. These deamidated

peptides can initiate an immune response, which leads to intestinal inflammation

and symptoms characteristic of celiac disease. .

Several studies have focused on lactic acid fermentation, often in combination

with enzymes, as a way to degrade gluten in order to alleviate its toxicity to

celiacs. However, incomplete degradation of gluten may lead to an increase in

TG2 binding sites. Therefore, we have investigated how lactic acid fermentation,

similar to that of a normal sourdough, affects the potential binding of TG2 to

gluten protein in wheat flour.

We observed that lactic fermentation of wheat flour, as slurry or as part of

sourdough bread, did not decrease the TG2-mediated transamidation or the

available TG2 binding motif QLP in α2-gliadin to a sufficient extent to be useful

for celiac safe food.


135

Did you eat sourdough bread for supper? A pilot validation study on plasma

Katharina Dihma,b, Henrik Sundéna, Alastair Rossb and Otto Savolainenb

aDivision of Food and Nutrition Science, Department of Biology and Biological Engineering, Chalmers University of Technology

bDivision of Organic Chemistry, Department of Chemistry and Chemical Engineering, Chalmers University of Technology

[email protected]

Food intake biomarkers are a valuable tool in clinical trials and observational

studies aiming to assess the effects of different diets on human health. Recent

work employing metabolomics has identified hydroxy-N-(2-hydroxyphenyl)

acetamide (HHPAA) and N-(2-hydroxyphenyl) acetamide (HPAA) as potential

specific biomarkers for sourdough rye bread intake. However, before being

accepted as validated biomarkers these markers have to be tested across many

studies to assess their performance in different study designs and intake levels.

Our present study aims to validate HPAA and HHPAA as biomarkers and

includes development of a bioanalytical UPLC-QqQ-MS method for measuring

concentration of the proposed biomarkers from plasma and food matrices. The

clinical trial will measure both postprandial kinetics and fasting plasma response

to daily sourdough bread intake over one week. Validation of the proposed

biomarkers can add to the tools available for unravelling the interactions of health

and diet and form the basis for understanding whether they are sufficiently

bioavailable to be of interest as bioactive compounds for human health.



136

Synthesis of novel bioactive compounds produced during sourdough fermentation

Katharina Dihma, b, Henrik Sundéna, Otto Savolainenb and Alastair Rossb

aDivision of Organic Chemistry, Department of Chemistry and Chemical Engineering, Chalmers University of Technology

bDivision of Food and Nutrition Science, Department of Biology and Biological Engineering, Chalmers University of Technology

[email protected]

Benzoxazinoids, a group of bioactive compounds found in wholegrain cereals, are

thought to be partially responsible for a variety of protective health effects that a

wholegrain diet is known for.1,2,3 In order to understand more about these

compounds some relevant Benzoxazinoids and their glycosides were synthesised

(see Fig. 1).

A synthesis using HBOA as a building block in order to form HBOA-glucoside

and HHPAA was found. HBOA was made in a two-step synthesis from 2-

aminophenol and dichloroacetyl chloride. Reduction of HBOA with NaBH4 in

methanol gave HHPAA which was purified by HPLC. Selectively conjugating

HBOA with glucose proved to be difficult but could be accomplished with a

Hg(CN)2 mediated coupling between 2-bromo-2H-1,4-benzoxazin-3(4H)-one

and 2,3,4,6-tetra-O-acetyl-beta-D-glucose. The synthesis of 1, 2 and 3 was

successful and incorporation of deuterium will be attempted using deuterated

nitrophenol as a starting material. This will enable the quantification of 1, 2 and 3

in biological samples.

Fig. 1.

References.

1. A. Esmaillzadeh, P. Mirmiran and F. Azizi, European J. of Clinical Nutrition, 2005, 59, 353-362.

2. K. B. Adhikari et. Al., Mol. Nutr. Food Res. 2013, 57, 1847-1858. 3. K. B. Adhikari et. Al., J. Agric. Food Chem., 2012, 60, 2497-2906.



137

In vitro digestion of rye products: impact of microstructure on glucose and maltose release and digesta viscosity

Daniel Johanssona, José Vázquez Gutiérreza, Marie Almingerb, Rikard Landberga,c

and Maud Langtona

a Dept. of Food Science, Swedish University of Agricultural Sciences, Uppsala b Dept. of Biology and Biological engineering, Chalmers University of Technology

cKarolinska Institutet, Stockholm

[email protected]

Development of tailored foods, which increase fullness and reduce hunger, could

be one way to address the issue of overconsumption of foods which is a strong

contributor to obesity and related disorders. Processing of whole grain rye crisp

breads have been shown to be of importance for postprandial responses in

humans, possibly due to changes in microstructure and physiochemical

composition influencing absorption kinetics of nutrients. The aim of the current

work was to investigate to what extent microstructure and digesta viscosity affect

in vitro starch digestibility.

The digestion of a range of rye products, including porridge, fermented and

unfermented crisp bread, sourdough bread and an extruded product, based on the

same raw material, and a refined wheat bread was studied using two in vitro

methods. A dynamic in vitro model, TIM-1, was used to simulate digestion in the

gastric compartment and the small intestine. Samples were collected during 180

min for characterization of microstructure and to measure release of glucose and

maltose, representing total glucose available for absorption. A static in vitro

method was used for characterization of digesta viscosity after gastric digestion.

There were clear differences in microstructure between the products. The

extruded product had a continuous starch matrix while the other products had a

higher degree of swollen but intact starch granules. In the sourdough bread and to

some extent in the crisp breads, amylose formed a film around the starch granules.

The wheat bread had a continuous protein network and less swollen starch

granules compared to all rye products. In vitro starch digestibility differed

significantly between products (P<0.05) and may be influenced by the presence

of an amylose film as well as starch retrogradation. Further, digesta viscosity was

found to be lower for fermented products, likely due to the degradation of viscous

fibers during the fermentation process.


138

3D Bioprinting of Human Skin

Daphne Hingerta, Sandra Ferreyra Vegaa, Daniel Hägga, Vladimir Kirejevb, Marica Ericsonb,c, and Paul Gatenholma

a Biopolymer Technology, Dept. of Chemistry and Chemical Engineering, Chalmers University of Technology, Göteborg, Sweden

bBiomedical photonics group, Dept. of Chemistry and Molecular Biology, University of Gothenburg, Sweden

cSkinResQU, Centre for Skin Research, University of Gothenburg & Chalmers University of Technology, Göteborg, Sweden

[email protected]

Annually, 11 million people worldwide suffer from burns so severe that they required medical attention1. Out of these 265,000 cases are fatal, this leaves millions of surviving burn victims each year suffering from the disfigurement, disabilities and associated stigma and trauma that follows with the condition. In many countries, burns are among the top causes of disability-adjusted life-years, possibly because young children are at an increased risk of burns. In the year 2000, $211 million were spent on the care of children who had suffered from burns1. Ultimately, by the development of 3D bioprinting technology, patient specific living tissue and organs can be biofabricated with high accuracy using the patients’ own cells. In this project, we explore the feasibility of obtaining 3D bioprinted human skin. Proof-of-principle skin constructs are being developed by alternating one-by-one layer of supporting material and human cells. Firstly, bioink based on nanocellulose and alginate is combined with well-defined density of fibroblasts. Then, the several layers of keratinocytes premixed with melanocytes are deposited by 3D bioprinter. Image data acquired from two photon fluorescence microscopy images of human skin will in the future be used as input for creating a computer-aided design (CAD) file for 3D bioprinting. The biofabricated skin will be stratified in the tailor-made bioreactor. We believe that this emerging technology offers unique opportunity for biofabrication of constructs with the ability to exactly mimic the microarchitecture and thus properties of human skin. The approach can potentially be used in clinics to increase quality of life of burn patients or applied for pharmaceutical and toxicology testing.

Figure 1. Schematic illustration of human skin and 3D bioprinting process.

Acknowledgment Per Börjesson, Jessica Ryler, Jakob Obermüller

References.1. World Health Organisation,

Burns, accessed: March 3, 2015

http://www.who.int/mediacentre/factsheets/

fs365/en/.



139

Postprandial plasma differences after beef and herring based meals in 2-aminoaidipic acid, β-alanine, 4-hydroxyproline, cetoleic acid

and docosahexaenoic acid: a metabolomics study

Ross, A.B.a,b Svelander, C.a,b, Pinto, R.c, Sandberg, A.S.a

a Food and Nutrition Science, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

bChalmers Metabolomics Centre, Chalmers University of Technology, Gothenburg, Sweden

c Bioinformatics Infrastructure for Life Sciences (BILS), Linköpings Universitet, Linköping, Sweden

[email protected]

Dietary guidelines generally recommend increased intake of fish, and reduced

intake of red meat for better long-term health. However, few studies have

compared the metabolic differences between eating meat and fish. We studied the

metabolic response of humans to fish or red meat in 17 overweight males in a

randomised cross-over intervention study. Subjects ate baked herring, pickled

herring or baked beef based meals and post-prandial blood plasma samples were

taken over 7 h. Metabolic profile was measured using gas chromatography-mass

spectrometry. A total of 50 metabolite differences were found for the

comparisons between baked herring and baked beef, or baked herring with pickled

herring. 2-aminoadipic acid, a suggested marker of diabetes risk, was elevated

after the beef meal compared to the herring meals, preceded by a similar rise in

the branch chain amino acid leucine, also suggested to be an early marker of

diabetes risk. Furthermore we found marked rises in β-alanine and 4-

hydroxyproline after beef intake. Herring intake led to greater plasma

concentrations of docosahexaenoic acid (DHA) and cetoleic acid (fatty acid C22:1

n-11), while hippuric acid and benzoic acid differentiated between baked and

pickled herring intake. These results confirm that DHA and cetoleic acid are

biomarkers of herring intake, while β–alanine and 4-hydroxyproline are potential

biomarkers for beef intake. The greater postprandial rise in 2-aminoadipic acid

and leucine suggests a potential role for beef in stimulating insulin secretion,

which may have importance in the context of red meat intake and increased

diabetes risk.



140

A simultaneous metabolic profiling and quantitative multimetabolite metabolomic method for human plasma using gas-chromatography

tandem mass spectrometry

Savolainen, O.-I. Sandberg, A.-S. Ross, A B.

Division of Food and Nutrition Science, Department of Biology and Biological Engineering, Chalmers University of Technology

[email protected]

Metabolomics is an increasingly common tool in the biosciences, yet balancing

the need for broad metabolome coverage and quantification remains a challenge.

To address this challenge, we developed a gas chromatography tandem mass

spectrometry (GC-MS/MS) metabolomics method using a high scanning speed

(20 000 Da/second) GC-MS/MS system that enables simultaneous data

acquisition of both non-targeted full scan and targeted quantitative tandem mass

spectrometry data. Although metabolomics analytics has previously been divided

into either targeted or non-targeted, the method presented herein suggests that

both of these approaches can be brought together into one effective method

allowing reproducible quantification of at least 27 metabolites using multiple

reaction monitoring (MRM) and full mass spectral scan-based detection of 601

metabolic features from human plasma. The method showed good linearity over

normal concentrations in plasma (0.18 - 720 to 4 - 14 400 μM depending on the

metabolite) and good intra- and inter-batch precision (1,8 – 16,7 and 2,6 – 25,1

RSD%). Based on the parameters determined for this method, targeted

quantification using MRM can be expanded to cover at least 508 metabolites. The

new simultaneous targeted and non-targeted metabolomics method will enable

more sensitive and accurate detection of metabolites and biomarkers of interest,

while still allowing detection and identification of unknown metabolites. This

GC-MS/MS method can result in time and cost savings for metabolomics analyses

and demonstrates the utility of GC-MS/MS with high scanning rates for complex

analyses.



141

The origin of symmetry in the metabolism of cancer

Francesco Gattoa, Almut Schulzeb, and Jens Nielsena

aDepartment of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden

bTheodor-Boveri-Institute, Biocenter, Würzburg, Germany

[email protected]

With the advent of next-generation sequencing technologies, it has been

increasingly appreciated the extent of the heterogeneity of cancer genome. The

complexity of cancer genome is daunting for a rational treatment of the disease.

Nevertheless, all cancers converge to some emerging properties, e.g. invasion (1).

Mutations are central in the evolution of most cancers and they are responsible to

select clones that reprogrammed the cellular gene expression to trigger an

outstanding regulation of these emerging properties (2). In this study, we derived

genome-wide associations between cancer-associated mutations and gene

expression in 1082 human primary tumors with the aim to verifying whether

mutations converge in the transcriptional regulation of known or novel molecular

processes, which are hence likely to constitute a universal criterion for selection

in cancer.

We show that 12 cancer mutations converge independently and primarily on the

regulation of metabolic processes, regardless of the cancer type. In particular, we

derived a network of reactions, termed AraX, that involve the glutathione- and

oxygen-mediated metabolism of arachidonic acid and xenobiotics, whose

deregulation is overrepresented by all 12 mutations, an overrepresentation unseen

in any other biological pathway. Finally, we observed that, among all metabolic

pathways, AraX deregulation represents the strongest predictor for survival in

cancer. These findings suggest that mutations independently select for clones with

highly deregulated AraX to gain a selective advantage in cancer evolution.

References.

1. D. Hanahan, R. A. Weinberg, Hallmarks of cancer: the next generation. Cell 144, 646-674 (2011); published online EpubMar 4 (10.1016/j.cell.2011.02.013).

2. B. Vogelstein, K. W. Kinzler, Cancer genes and the pathways they control. Nature medicine 10,

789-799 (2004); published online EpubAug (Doi 10.1038/Nm1087).


142

Effects of pH-shift processing on the bioaccessibility of lipids in the microalgae Nannochloropsis – an in vitro model approach

Lillie Cavoniusa,b, Eva Albersb and Ingrid Undelanda

aChalmers University of Technology, Department of Biology and Biological Engineering, Division of Food and Nutrition Science

bChalmers University of Technology, Department of Biology and Biological Engineering, Division of Industrial Biotechnology


Microalgae are a potential source of human nutrition and animal feed due to the

high content of long-chained n-3 fatty acids (LC n-3 PUFA) and favorable amino

acid profile of some species. Furthermore, microalgae can be cultivated with

minimal nutrient requirements on otherwise non-arable land. We have previously

shown that application of the pH-shift process on Nannochloropsis yields a

product enriched in essential amino acids and with the original amount of LC n-3

PUFA retained (120 mg/g dry weight). Therefore, the product has potential as a

functional food and/or feed ingredient. However, it is not known to which extent

the nutrients are bio-accessible; earlier work has suggests that algal cell walls limit

digestibility in the gastrointestinal tract. Based on the physical changes that take

place during pH-shift processing, e.g. cell lysis and protein

solubilization/precipitation, we hypothesize that the pH-shift process renders the

algal nutrients more available for hydrolysis. The aim of the present study is to

test this hypothesis using a static in vitro digestion model.

Nannochloropsis is subjected to the pH-shift process and nutrient content (fatty

acids, protein, amino acids) is determined before/after processing. To determine

the bio-accessibility of Nannochloropsis processed in various ways, the

microalgae is digested using a static in vitro model of human digestion, based on

the recent international consensus method emerging from the COST FA

1005 Infogest. Intestinal digesta will then be analyzed in respect to degree of

lipolysis using solid phase extraction (SPE) combined with gas chromatography-

mass spectrometry (GC-MS).


143

Antioxidant potential of chia seeds (Salvia hispanica L.)

Renata Adriana LABANCAa,b; Cecilia SVELANDERb; Marie ALMINGERb

aDepartment of Food Science, Federal University of Minas Gerais, Brazil bDepartment of Biology and Biological Engineering, Food and Nutrition Science,

Chalmers University of Technology, Gothenburg, Sweden

E-mail: [email protected]

Human intervention studies have provided evidence for protective effects of

various polyphenol rich foods against chronic diseases. Phenolic compounds are

found in a wide variety of cereals and also in chia seeds, an oilseed native of

Mesoamerica, that are considered pseudocereals. The objective of this study was

to characterize chia (Salvia Hispanica L.) samples (seeds and stabilized flour) by

evaluation of their antioxidant properties using DPPH and ABTS radical

scavenging capacity assay, oxygen radical absorbance capacity (ORAC) assay

and ferric reducing antioxidant potential (FRAP) assays. Total phenolic content

was determined by the Folin-Ciocalteu method. Fatty acid composition and

vitamin E were also analyzed by gas chromatography and mass spectrometry

(GC-MS) and reverse phase high-performance liquid chromatography (HPLC)

respectively. The experiments were carried out using chia samples obtained in

Brazil, Sweden and the Netherlands. Antioxidant properties and total phenolic

content was found to differ significantly between samples. Chia seeds from Brazil

showed higher antioxidant capacity and higher phenolic content (3.56±0.20 mg

GAE/g) compared with the chia seed sample from Sweden (2.00±0.09 mg GAE/g)

and the Netherlands (1.65±0.09 mg GAE/g). The flour from Brazil had the lowest

total content of phenolic compounds (1.20±0.16 mg GAE/g), as well as the lowest

antioxidant capacities in most of the applied assays. It could thus be inferred that

the processing for production of stabilized flour affects these parameters. Within

the samples, the order of antioxidant capacity depended on the assay used. A

significant relationship between antioxidant capacity and total phenolic content

was found, indicating that phenolic compounds are the major contributors to the

antioxidant properties of these cereals. To assess the antioxidant activities of

single compounds or the antioxidant capacity of food extracts, a variety of

methods based on different mechanistic principles must be used in parallel,

because different methods often give different results.


144

Genome-wide identification of mononuclear cell DNA methylation sites potentially affected by infant fish oil supplementation from 9

to 18 months

Mads V. Linda,b, David Martinoc, Laurine BS. Harsløfb, Zdenka O. Kyjovskad, Mette Kristensenb, Lotte Lauritzenb

a Food and Nutritional Science, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

bDepartment of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark

cMurdoch Childrens Research Institute, Royal Children's Hospital, University of Melbourne, Australia

dNational Research Centre for the Working Environment, Copenhagen, Denmark

[email protected]

Long-chain n-3 fatty acid (n-3LCPUFA) intake affects markers of metabolic

syndrome, immune function and brain development. Recent evidence suggests

that some of the effects of n-3LCPUFA might be mediated through epigenetic

mechanisms, especially DNA-methylation. Epigenetic alterations are of particular

interest during pregnancy and early life, since these have been suggested as a

mechanism for programming of later diseases. A parallel randomized control trial

was conducted in 133 9-mo-old, Danish infants who received a teaspoon of fish

oil (FO) or sunflower oil (SO) for 9 mo (median intake=3.8 g/day). DNA was

extracted from buffy coat before and after intervention. Illumina Human

Methylation 450K-arrays were used to explore possible differentiation between

the FO and SO supplemented groups on genome-wide DNA-methylation in a

subset of 12 children. The FO intervention gave rise to a 3-fold increase in

erythrocyte n-3LCPUFA, and reduced plasma triacylglycerol, blood pressure and

ex vivo stimulated interleukin-6 production. Analysis of genome-wide

methylation levels by regression did not find significant differences between

groups after adjustment for multiple testing. Analysis of the top-ranked CpG sites

found 43 CpG’s that appeared to be modified by the intervention with an absolute

difference in methylation of at least 10% (unadjusted P-value <0.01). Methylation

levels at these sites were associated with phenotypic changes mainly in blood

pressure. Our analyses suggest potential epigenome effects associated with

functional outcomes, yet the effect sizes were small and did not reach genome-

wide significance. Thus, while the hypothesis that supplementing infants with n-

3LCPUFA leads to changes in DNA-methylation appears to be well founded,

additional investigation is required as to the overall importance of these changes.


8. Health, Nutrition and Food Science

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