ABS The Finnish Graduate School on Applied Bioscience: Bioengineering, Food & Nutrition, Environment

SUMMARIES OF THE THESIS RESEARCH

13th ANNUAL SEMINAR

26 August 2008 EE Building, Lecture Hall “Walter”(Viikki) University of Helsinki Agnes Sjöbergin katu 2, Helsinki

ORGANIZERS ABS GRADUATE SCHOOL FINNISH FOOD RESEARCH FOUNDATION

SUMMARIES OF THE THESIS RESEARCH

Issued by the Board of the ABS Graduate School

CONTENTS

ORGANIZATION OF THE ABS GRADUATE SCHOOL ....................................................2 ABSTRACTS (PhD students financed by the ABS Graduate School in 2008) Constantin Camelia (University of Helsinki / Dept of Applied Chemistry and Microbiology) ..5 Hakovirta Janetta (University of Helsinki / Dept of Applied Chemistry and Microbiology)......6 Hannula Minna (University of Helsinki / Dept of Food and Environmental Hygiene)..............7 Hinderink Katja (University of Helsinki / Dept of Food and Environmental Hygiene).............8 Hätönen Katja (National Public Health Institute / Nutrition Unit)...........................................9 Järvinen Riikka (University of Turku / Dept of Biochemistry and Food Chemistry)...............10 Jääskeläinen Elina (University of Helsinki / Dept of Applied Chemistry and Microbiology)......11 Kanerva Päivi (University of Helsinki / Dept of Food Technology) .......................................12 Kantala Tuija (University of Helsinki / Dept of Food and Environmental Hygiene)...............13 Kassinen Anna (University of Helsinki / Dept of Basic Veterinary Sciences).........................14 Kemi Virpi (University of Helsinki / Dept of Applied Chemistry and Microbiology) .............15 Kinnunen Paula (University of Helsinki / Dept of Basic Veterinary Sciences) .......................16 Kokkonen Meri (Evira / Research Department)....................................................................17 Koponen Jani (University of Kuopio / Food and Health Research Centre) ............................18 Krogius-Kurikka Lotta (University of Helsinki / Dept of Basic Veterinary Sciences).............19 Kylli Petri (University of Helsinki / Dept of Applied Chemistry and Microbiology)...............20 Lappalainen Tiina (University of Kuopio / Dept of Clinical Nutrition) ..................................21 Larmo Petra (University of Turku / Dept of Biochemistry and Food Chemistry)....................22 Leskinen Heidi (University of Turku / Dept of Biochemistry and Food Chemistry) ...............23 Markkula Annukka (University of Helsinki / Dept of Food and Environmental Hygiene)......24 Marttinen Maija (University of Helsinki / Dept of Applied Chemistry and Microbiology)......25 Moilanen Ulla (Helsinki Univ. of Technology / Dept of Biotechnol. and Chem. Technol.) .....26 Mykkänen Otto (Univ. of Kuopio/Dept of Clinical Nutrition; Food and Health Res. Centre) 27 Nakari Ulla-Maija (National Public Health Institute / Enteric Bacteria Laboratory).............28 Pakarinen Jaakko (University of Helsinki / Dept of Applied Chemistry and Microbiology)....29 Peltola Minna (University of Helsinki / Dept of Applied Chemistry and Microbiology) .........30 Pohjanheimo Terhi (Univ. of Turku / Dept of Biochemistry and Food Chemistry; FFF)........31 Suokko Aki (University of Helsinki / Dept of Basic Veterinary Sciences)..............................32 Söderholm Henna (University of Helsinki / Dept of Food and Environmental Hygiene)........33 Tolppanen Anna-Maija (University of Kuopio / Dept of Clinical Nutrition) ..........................34 Usvalampi Anne (Helsinki Univ. of Technology / Dept of Biotechnol. and Chem. Technol.) .35 Viljakainen Heli T. (University of Helsinki / Dept of Applied Chemistry and Microbiology) .36 Zheng Jie (University of Turku / Dept of Biochemistry and Food Chemistry) ........................37

ORGANIZATION OF THE ABS GRADUATE SCHOOL

University in charge:

University of Helsinki

Director in Charge:

Prof. Lea Hyvönen Department of Food Technology P.O. Box 66 FI-00014 University of Helsinki Finland Tel: +358 9 191 58215 Fax: +358 9 191 58460 E-mail: [email protected]

Vice Director:

Prof. Mirja Salkinoja-Salonen Department of Applied Chemistry and Microbiology P.O. Box 56, Biocenter 1 (Viikinkaari 9) FI-00014 University of Helsinki Finland Tel: +358 9 191 59300 Fax: +358 9 191 59301 E-mail: [email protected]

Coordinator:

Laila Partanen, M.Sc. Department of Food Technology P.O. Box 66 FI-00014 University of Helsinki Finland Tel: +358 9 191 58213 Fax: +358 9 191 58460 E-mail: [email protected]

Management Board: The highest authority in the ABS Graduate School is held by the Management Board (MB) where each associated university and research institute is represented. The associated universities and the research institutes of the ABS Graduate School are:

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Universities 1. University of Helsinki Faculty of Agriculture and Forestry Department of Applied Chemistry and Microbiology Department of Food Technology MB: Prof. Lea Hyvönen / Prof. Mirja Salkinoja-Salonen Coordinator: Laila Partanen, M.Sc.

Faculty of Veterinary Medicine Department of Basic Veterinary Sciences Division of Microbiology and Epidemiology Division of Pathology and Parasitology Division of Veterinary Physiology Department of Food and Environmental Hygiene MB: Prof. Airi Palva / Prof. Hannu Korkeala

2. University of Kuopio Faculty of Medicine Department of Clinical Nutrition Faculty of Natural and Environmental Sciences Institute of Applied Biotechnology MB: Prof. Atte von Wright / Prof. Hannu Mykkänen

3. University of Turku Faculty of Mathematics and Natural Sciences Department of Biochemistry and Food Chemistry MB: Prof. Heikki Kallio / Prof. Seppo Salminen

4. Helsinki University of Technology Faculty of Chemistry and Materials Sciences Department of Biotechnology and Chemical Technology Biochemistry and Microbiology Bioprocess Technology MB: Prof. Katrina Nordström / Prof. Matti Leisola

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Research institutes 5. MTT Agrifood Research Finland MB: Prof. Hannu Korhonen / Anne Pihlanto, PhD

6. National Public Health Institute (KTL) Department of Bacterial and Inflammatory Diseases Enteric Bacteria Laboratory Department of Health Promotion and Chronic Disease Prevention Nutrition Unit Department of Health and Functional Capacity Biomarker Laboratory MB: Doc. Liisa Valsta / Prof. Anja Siitonen

7. Finnish Food Safety Authority Evira Research Department MB: Prof. Tuula Honkanen-Buzalski / Prof. Vesa Myllys

8. VTT Technical Research Centre of Finland MB: Prof. Juha Ahvenainen / Prof. Kaisa Poutanen

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Non-protein toxin producing Bacillus strains: characterization and significance for food safety PhD student: Camelia Constantin (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: camelia.constantin @helsinki.fi Supervisors: Prof. Mirja Salkinoja-Salonen (University of Helsinki / Department of Applied Chemistry and Microbiology) Funding: ABS Graduate School _____________________________________________________________________________ Introduction B. cereus is recognized as one of the most frequent food poisoning agents and it produces an enterotoxin and a stable nonprotein toxin, cereulide (emetic), that can be even fatal. Cereulide is a heat stable peptide toxin, non-ribosomally produced, whose structure and mechanism of action was just recently elucidated. Species from Bacillus subtilis group, like other Bacillus species comprise bacteria ubiquitary in environment. Despite their ubiquity, the involvement of Bacillus subtillis species in food poisoning has been inconclusive. Aim Design a quantitative assay to measure cereulide, characterize the cereulide producing subset of B. cereus and detect and investigate other Bacillus species producing non-protein toxins. Results and discussion We were the first to develop a quantitative chemical assay for cereulide. This was a breakthrough in the cereulide research and became a very powerful tool of investigation. Results of this study are published in article 1. Using a large number of strains of wide origins and a polyphasic approach, we found that the cereulide producing B. cereus are genetically and biologically more diverse than hitherto assumed in the literature. This study was the first demonstration of diversity within this subset of B. cereus. (published in article 2).

Further studies were carried out on how different media and temperatures influences the cereulide production. The manuscript with this work was submitted (see paper3). We recently extracted a heat stable toxic substance, identified as amylosin, produced by strains of B. subtilis and B. mojavensis from two food poisoning outbreaks. This is the first report on amylosin production in food and by other species than B. amyloliquefaciens. The manuscript with these results is currently in preparation. Articles 1. Häggblom, M., Apetroaie, C., Andersson, M.A., and Salkinoja-Salonen, M.S. 2002. Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under different conditions. Appl. Environ. Microbiol. 68 (5): 2479-2483. 2. Apetroaie, C., Andersson, M.A., Spröer, C., Tsitko, I., Shaheen, R., Jääskeläinen, E.L., Wijnands, L.M., Heikkilä, R., and Salkinoja-Salonen, M.S. 2005. Cereulide producing strains of Bacillus cereus show diversity. Arch. Microbiol. 184 (3): 141-151. 3. Apetroaie-Constantin, C., Shaheen, R., Andrup, L., Smidt, L., Rita, H., and Salkinoja-Salonen, M.S. 2008. Environment driven cereulide production by emetic strains of Bacillus cereus. International Journal of Food Microbiology, manuscript number FOODD-07-00635 (conditionally accepted). 5

Modern techniques in detection, identification and quantification of bacteria and peptides from foods PhD student: Janetta Hakovirta (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: [email protected] Supervisor: Prof. Per Saris (University of Helsinki / Department of Applied Chemistry and Microbiology) Funding: ABS Graduate School, Academy of Finland, Los Alamos National Laboratory _____________________________________________________________________________ Introduction The microbial and preservative contents of foods are strictly regulated to assure their safety to the consumer. During foodrelated disease outbreaks and every day control of food products, it is crucial to be able to detect, identify, and quantify quickly and accurately the presence of microbes and preservatives. Aim This thesis focuses on developing easier and quicker methods for bacterial identification and the development of a sensitive bioassay for quantification of nisin, a food preservative. Results and discussion Ultrasensitive flow cytometry (FCM) was studied as an alternative for the time consuming PFGE, the golden method in epidemiological studies, to obtain a fingerprinting pattern based on enzymatically digested DNA. With this method, DNA fragment sizing was comparable and accurate in identifying different species and strains, such as Staphylococcus aureus and Escherichia coli, and the analysis time was 16 hours shorter when compared to PFGE. Another fingerprinting method is singleenzyme amplified fragment length polymorphism (SE-AFLP), which is based on PCR amplification of enzymatically digested DNA. A method was developed for the identification of different species and

strains of Bacilli, Staphylococci, E. coli and Yersinia using SE-AFLP technique. A Lactococcus lactis strain containing a plasmid with the gfpuv gene under the control of the nisin-inducible nisA promoter and nisRK genes for the two-component signal transduction system, in its chromosome was constructed. This biosensor strain correlates the amount of nisin in the environment by transducing the production of the measurable green fluorescent protein (GFP). This strain can quantify nisin from various food matrices with detection limits ranging from 10 pg/ml in culture supernatant to 3.6 ng/g in cheese. Articles 1. Kim, Y., Jett, J., Larson, E., Penttila, J., Marrone, B., and Keller, R. 1999. Bacterial fingerprinting by flow cytometry: bacterial species discrimination. Cytometry 36: 324-332. 2. Larson, E., Hakovirta, J., Jett, J., Burde, S., Keller, R., and Marrone, B. 2000. Rapid DNA fingerprinting of pathogens by flow cytometry. Cytometry 41: 203-208. 3. Velappan, N., Snodgrass, J., Hakovirta, J., Marrone, B., and Burde, S. 2001. Rapid identification of pathogenic bacteria by single-enzyme amplified fragment length polymorphism analysis. Diag. Microbiol. Infect. Dis. 39: 77-83. 4. Hakovirta, J., Reunanen, J., and Saris, P. 2006. Bioassay for nisin in milk, processed cheese, salad dressings, canned tomatoes, and liquid egg products. Appl. Environ. Microbiol. 72: 1001-1005. 6

Effect of putative efflux pump inhibitors and inducers on the antimicrobial susceptibility of Campylobacter jejuni and C. coli PhD student: Minna Hannula (University of Helsinki / Department of Food and Environmental Hygiene) E-mail: [email protected] Supervisors: Prof. Marja-Liisa Hänninen (University of Helsinki / Department of Food and Environmental Hygiene) Funding: ABS Graduate School _____________________________________________________________________________ Introduction Campylobacteriosis is the leading cause of bacterial gastroenteritis in industrialized countries. In sever cases erythromycin and ciprofloxacin are the drugs of choice for treatment. However, the rapid increase of resistant strains as well as the emergence of multiple resistant strains are of growing concern. Efflux pumps, particularly the resistancenodulation division type CmeABC pump, have an important role in the antimicrobial resistance of Campylobacter jejuni and C. coli. This pump is able to produce a significant decrease in the susceptibility of Campylobacter to a number of antimicrobials. Efforts have been made to find substances able to reverse the action of the efflux pumps or act as tools for further understanding of the mechanisms underlying antimicrobial resistance. Aim The object of this investigation was to study the effect of putative efflux pump inhibitors, phenyl-arginine- -naphthylamide (PA N) and 1-(1-naphthylmethyl)piperazine (NMP), as well as the effect of putative efflux pump inducers, sodium salicylate and sodium deoxycholate, in the MIC-levels of C. jejuni and C. coli to erythromycin, ciprofloxacin, kanamycin, tetracycline and rifampicin. Results and discussion PA N induced an 8–32-fold and 8–64-fold reduction in the MIC-values of the studied strains for erythromycin and rifampicin,

respectively. NMP produced a smaller, 2– 8-fold reduction for the same antimicrobials. In regards to the putative efflux pump inducers, sodium deoxycholate had little or no effect on the MIC-values of the studied antimicrobials, whereas sodium salicylate brought about a small, (2–4-fold) but reproducible, increase in the MIC-values for ciprofloxacin, and in 7/17 of the studied strains a two-fold growth in the MIC-values for rifampicin. These results confirm the importance of efflux in the Campylobacter resistance and highlight the role of efflux pump affecting agents as tools for research aiming at the inhibition of CmeABC and reversal of antimicrobial resistance in Campylobacter. Articles 1. Hänninen, M.-L., and Hannula, M. 2007. Spontaneous mutation frequency and emergence of ciprofloxacin resistance in Campylobacter jejuni and Campylobacter coli. J. Antimicrob. Chemother. 60: 1251-1257. 2. Hannula, M., and Hänninen, M.-L. 2007. Phylogenetic analysis of Helicobacter species based on partial gyrB gene sequences. Int. J. Syst. Evol. Microbiol. 57: 444-449. 3. Laatu, M., Rautelin, H., and Hänninen, M.-L. 2005. Susceptibility of Campylobacter hyointestinalis subsp. hyointestinalis to antimicrobial agents and characterization of quinolone-resistant strains. J. Antimicrob. Chemother. 55: 182-187. 7

Growth of Group I Clostridium botulinum at Extreme Temperature PhD student: Katja Hinderink (University of Helsinki / Department of Food and Environmental Hygiene) E-mail: [email protected] Supervisors: Prof. Hannu Korkeala and Doc. Miia Lindström (University of Helsinki / Department of Food and Environmental Hygiene) Funding: ABS Graduate School ___________________________________________________________________________ Introduction Clostridium botulinum, a Gram-positive, endospore forming, anaerobic bacterium, produces a highly potent neurotoxin causing the paralytic disease botulism. The most well-known form foodborne botulism emerges due to intake of preformed neurotoxin with food. The heat stable spores of C. botulinum may survive food processing and under anaerobic conditions spores can germinate and form a toxic culture. Temperature is a very important factor controlling growth of C. botulinum in foods. There is only little information available on strain variation and temperature tolerance and adaptation to stressful temperature of Group I C. botulinum. Aim The aim of the study is to gain information on the tolerance and adaptation of Group I C. botulinum to extreme temperature and the underlying genetic mechanisms. Furthermore growth of 23 strains at extreme temperatures has been studied. Results and discussion The minimum and maximum growth temperatures and maximum growth rates at 20°C, 37°C and 42°C of 23 Group I C. botulinum strains have been determined. The strains showed an unexpectedly broad variation in growth permitting temperatures. The minimum growth temperatures varied from 12.8°C to 16.5°C, whereas the maximum growth temperatures showed even larger variation from 40.9°C to 48.0°C. Particularly within the toxin type B large variation between

strains was found reflecting the diverse genetic background of the studied strains shown previously in AFLP analysis. Seven type B strains grew faster at 42°C than at 37°C and had a higher maximum growth temperature than the average of all studied strains. In contrast, three type A strains had higher than average maximum growth temperatures in combination with lower growth rates at 42°C than at 37°C. This shows that the optimum growth temperature of some Group I C. botulinum strains is higher than the commonly expected 37°C and that the maximum growth temperature of a strain does not allow drawing conclusions on its growth performance at high temperature. The revealed strain variation between Group I C. botulinum strains has to be taken carefully in consideration when designing experiments to evaluate the safety of food in future. To study heat shock response and adaptation to high temperature one Group I C. botulinum strain has been grown at 39°C in continuous culture in a fermentor. After stabilization of the culture the temperature was shifted to 45°C. Samples were taken before and at different time points after the heat shock and after adaptation of the culture. RNA was isolated, reverse transcribed into cDNA and hybridized onto DNA microarrays. The data analysis is ongoing.

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The Glycemic and Insulinemic Responses of the Finnish Foods: measurement and modification PhD student: Katja Hätönen (National Public Health Institute / Department of Health Promotion and Chronic Disease Prevention / Nutrition Unit) E-mail: [email protected] Supervisors: Doc. Liisa Valsta (National Public Health Institute), Research Prof. Jarmo Virtamo (National Public Health Institute) and Prof. Johan Eriksson (University of Helsinki; National Public Health Institute) Funding: ABS Graduate School, Academy of Finland, Finnish Cultural Foundation, National Public Health Institute ___________________________________________________________________________ Introduction Diet is an important environmental factor regulating body weight, metabolism and the risk of many chronic diseases. A diet of low glycemic response is associated with less insulin resistance and lower prevalence of metabolic syndrome as well as with lower incidence of type 2 diabetes and coronary artery disease. Glycemic index (GI) ranks foods according to their effects on postprandial glucose response. Several factors can modify glycemic and insulinemic responses, for example the fat/protein content, subject characteristics, etc. Aim To study the effect of methodological choices of GI measurement on glycemic responses and to study the modifying effect of carbohydrate quality and load, other nutrients (protein, fat) and subject characteristics (weight, body composition, status of glucose and insulin metabolism) on the glycemic, insulinemic, IGF/IGFBP system and satiety responses.

research is focusing on modifying factors of glycemic and insulinemic responses. The postprandial tests for the PhD study have been completed, except the ongoing study which tests the effect of the quality of carbohydrates on glycemic, insulinemic, IGF/IGFBP system and satiety responses in subjects with different background characteristics. The analyses of the completed postprandial tests are in progress and the results will be published in the next few years. Articles 1. Hätönen, K.A., Similä, M.E., Virtamo, J.R., Eriksson, J.G., Hannila. M.-L., Sinkko, H.K., Sundvall, J.E., Mykkänen, H.M., and Valsta, L.M. 2006. Methodologic considerations in the measurement of glycemic index: glycemic response to rye bread, oatmeal porridge, and mashed potato. Am. J. Clin. Nutr. 84: 1055-1061. 2. Wolever, T.M.S., Brand-Miller, J.C., Abernethy, J. et al. 2008. Measuring the glycemic index of foods. Am. J. Nutr. 87: 247S-257S.

Results and discussion In the first phase of this project the effects of certain methodological choices on postprandial glycemic response were examined. The results showed that capillary blood sampling should be preferred in the measurement of GI, and reference tests with glucose or white bread should be performed at least twice. Future 9

Cutin and suberin in berries; composition and clinical function as fibre PhD student: Riikka Järvinen (University of Kuopio / Department of Biochemistry and Food Chemistry) E-mail: [email protected] Supervisors: Prof. Heikki Kallio (University of Turku) Funding: ABS Graduate School, National Technology Agency of Finland (Tekes) _____________________________________________________________________________ Introduction Northern berries are regarded as natural high value food products and sources of many essential nutritional components. Berry skin contains in addition to cellulose various nutritionally important polymeric compounds e.g. cutin. The composition of aliphatic hydroxyacid monomers in the cutin of berries has not been widely studied. Also the importance of cutin polymer as possible part of fibre in human nutrition has not been shown in clinical studies. Aims There are two main aims of this study: 1. investigate the composition of cutin as polymer and after depolymerization as monomers 2. investigate the effect of cutin containing material in postprandial glycemia and/or lipemia and absorbtion of selected nutrients. Cutin monomer profiles are created of 12 wild or cultivated berries (together 18 varieties) from Finland, of which cutin from different currants (red, white, black, green) will be further studied for the annual changes and influence of the location of cultivation, northern and southern Finland. Polymer investigations will be carried out by using microscopy and Solid-State NMR spectroscopy techniques.

Results and discussion Cutin (i.e., extractive free isolated cuticular membrane) from berry varieties was depolymerized by NaOMe-catalyzed methanolysis. Monomer yield was determined gravimetrically as the CHCl3soluble material after methanolysis. Yields of obtained depolymerized material varied widely between different berries, ranging from 2 % in strawberry (Fragaria x ananassa) to 64 % in Rose-hip (Rosa rugosa). The differences in the amount of ester-linked cutin in berries may have a strong influence on the various functions of cuticle in berries and also as dietary fibre, which will be studied later. Cutin monomers have been identified as trimethylsilyl derivatives with GC-EIMS. MS-library has been created for the cutin monomers. The berry cutin monomer mixtures are mostly composed of longchain (C-16 to C-22) aliphatic -hydroxy acids, with mid-chain functionalities such as epoxy, oxo and hydroxy groups. Also phenolic compounds and suberin-like diacids with mid-chain functionalities were detected. Differences in monomer composition have influence on the polymer structure and its functions, which will be studied later. Article Kallio, H., Nieminen, R., Tuomasjukka, S., and Hakala, M. 2006. Cutin composition of five Finnish berries. J. Agric. Food Chem. 54: 457-462.

Importance of cutin as a part of dietary fibre will be studied in randomized, crossover postprandial clinical trial of 15 healthy persons. 10

Assessment and control of Bacillus cereus emetic toxin in food PhD student: Elina Jääskeläinen (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: [email protected] Supervisors: Prof. Mirja Salkinoja-Salonen (University of Helsinki / Department of Applied Chemistry and Microbiology) Funding: ABS Graduate School, Finnish Centre of Excellence in Integrative Photosynthesis and Bioactive Compound Research grant 2008-2013 _____________________________________________________________________________ Introduction Despite of improving levels of hygiene, the incidence of registered foodborne disease has been in same level many years. Bacillus cereus was the most common bacterial cause of foodborne disesases in year 2006 in Finland. One reason for that can be B. cereus emetic toxin, cereulide. Aim This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing B. cereus. Results and discussion I developed methods for extraction and quantitative analysis of cereulide directly from food. I used organic solvents; methanol, ethanol and pentane in extraction. In bakery products I made extraction by using high temperature (100°C) and pressure (103.4 bar). Alternative way made extraction is flooded plain food with ethanol and allowed equilibrate stationary at room temperature. I used this protocoll for cereulide extraction from potato puree and penne. These extraction methods are important improvement steps for study of B. cereus emetic foodpoisoning. Before cereulide extraction was made by using water, when yield was poor and variable. By using bioassay it is possible to rapidly detect which food is causing the poisoning. The identification of cereulide was made by using mass spectorometry. I used

cereulide specific ions, m/z (± 0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 µg g-1 wet wt ) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, when stored at non refrigeration temperatures. B. cereus is an ubiquitous spore former and is difficult to eliminate from food. It is therefore important to know which conditions will affect the formation of cereulide. My research showed that the cereulide content was strongly affected by the growth environment. Storage under N2 atmosphere prevented the production of cereulide. But when CO2 was presence, the low amount of O2 (< 1%) did not prevent the production of cereulide. Also nutrients affects cereulide production. Adding free aminoacids leucine and valine stimulated cereulide production. These aminoacids are peptide bonded natural consitituent in all proteins. Interestingly, adding peptide bonded leucine and valine had no effect on cereulide production. Reference Jääskeläinen E. Assessment and control of Bacillus cereus emetic toxin in food. Ph.D. thesis, University of Helsinki, Finland. Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki 1/2008. 11

Prolamins in gluten-free foods PhD student: Päivi Kanerva (University of Helsinki / Department of Food Technology) E-mail: [email protected] Supervisors: Prof. Hannu Salovaara (University of Helsinki) and University lecturer Tuula Sontag-Strohm (University of Helsinki) Funding: ABS Graduate School, Tekes and Raisio Research Foundation _____________________________________________________________________________ Introduction Alcohol soluble protein fraction of wheat, barley and rye is responsible for the harmful immune response in people with gluten intolerance. Therefore, they need to avoid products that may contain these proteins. Wheat is the main contaminant in most gluten-free food products. Therefore, wheat proteins and peptides involved in immune response have been studied in many publications. However, in oat products barley and rye are occasionally main contaminants too. Celiac-harmful proteins from gluten-free products are determined by immunological methods. The methods are based on a wheat prolamin standard and the results are calculated based on the characteristics of wheat prolamins. Barley and rye are closely related to wheat and their proteins have many similarities to wheat proteins. However, some important differences exist between the protein fractions of these three cereals. These differences cause errors to the analysis results when determining the total gluten content of the sample. Aim The aim of the research is to develop a method for quantification of prolamins of wheat, rye and barley in gluten-free products. Results and discussion The results of our studies show that differences between barley, rye and wheat prolamins lead to inaccuracies in gluten-free assays and this can be avoided by choosing a standard that better matches to the investigated protein.

The performances of enzyme-linked immunosorbent assays (ELISA) based on monoclonal omega-gliadin and R5 antibodies have been tested in this study. The omega-gliadin antibody based ELISA was used for the analysis of prolamins in beer samples (1). The omega-gliadin antibody cross-reacted weakly with barley prolamins, which lead to underestimations of actual prolamin concentrations. The R5 antibody based ELISA was used to detect barley contaminations in oat flour samples (2). In this study, the oat samples were deliberately contaminated with barley flour. The R5-ELISA cross-reacted strongly with barley proteins, which lead to overestimation of the actual prolamin concentrations. In both assays, the results improved greatly when using a more appropriate standard. Instead of the gliadin standard of the assay, we used a hordein standard that was extracted from either malted barley (1) or from barley four (2). Articles 1. Kanerva, P., Sontag-Strohm, T., and Lehtonen, P. 2005. Determination of Prolamins in Beers by ELISA and SDSPAGE. J. Inst. Brew. 111: 61-64. 2. Kanerva, P.M., Sontag-Strohm, T.S., Ryöppy, P.H., Alho-Lehto, P., and Salovaara, H.O. 2006. Analysis of Barley Contamination in Oats using R5 and Omega-Gliadin Antibodies. J. Cereal Sci. 44: 347-352.

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Endemic hepatitis E virus in Finland PhD student: Tuija Kantala (University of Helsinki / Department of Food and Environmental Hygiene) E-mail: [email protected] Supervisors: Doc. Leena Maunula and Prof. Emer. Carl-Henrik von Bonsdorff (University of Helsinki / Department of Food and Environmental Hygiene) Funding: ABS Graduate School, EU (project FP6-SP22-CT-2004-502571) _____________________________________________________________________________ Introduction Hepatitis E virus (HEV) is an important cause of fecally transmitted viral hepatitis, especially in developing countries. Recently, HEV isolates have been identified in humans and swine in many countries where it is considered non- endemic. HEV appears to be common among pigs worldwide. HEV is also considered to be a potential zoonotic agent. It is suggested that swine could function as a reservoir of the HEV strains that can infect humans. Aim The objectives of the Ph.D. study are to investigate if endemic HEV is present in swine in Finland and if there are non-travel related cases of HEV that could be of zoonotic origin in humans in Finland. The aim is also molecular genetic and epidemiologic characterization of the viruses found in the study. Results and discussion HEV in swine in Finland Overall, HEV RNA was detected in serum and/or fecal samples of 17% of 157 individual pigs from 44% of 23 different swine farms around Finland by real-time RT-PCR. A survey was made in a swine evaluation station to further examine the prevalence of HEV in pigs of different ages and the transmission of the virus from HEV positive pigs to HEV negative pigs living in a same pen. Of pooled fecal samples collected from the floors of 48 pens in the evaluation station, 25% were positive for HEV RNA. Of 44 pigs that were introduced to the station at the age of 2-3 months and divided into pens to be raised in mixed

groups, 34% were positive for HEV RNA. No new positive pigs were detected 4 and 10 weeks later. HEV in humans in Finland IgM and/or IgG antibodies against HEV by both ELISA test and immunoblotting were found in 21 (20%) from 105 samples from human patients diagnosed with acute non-A, B or C hepatitis. HEV RNA was detected in 8 of those 21 samples. Five of the 8 HEV RNA positive patients had a history of recent travel to an area where HEV is endemic. Sequences from the RNA-dependent RNA polymerase gene region of the HEV genome have so far been obtained from 5 of the viruses found from humans and 8 of the viruses found from swine. All human viruses belong to HEV genotype 1, which suggests that they have been imported from areas endemic to HEV. All porcine viruses belong to HEV genotype 3 which is the most common genotype in swine worldwide. According to our results, there is endemic hepatitis E virus among pigs in Finland. It seems that the virus is not easily transmitted from one pig to another. Of the HEV positive pigs, 96% were not older than 4 months of age. The virus seems to appear mostly in pigs between 2-4 months of age. Further investigation is required to tell whether there are non-travel related cases of HEV in humans in Finland, and if those cases are of zoonotic origin.

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Characterization of human faecal microbial community of people with irritable bowel syndrome by molecular biology methods PhD student: Anna Kassinen (University of Helsinki / Dept of Basic Veterinary Sciences) E-mail: [email protected] Supervisors: Prof. Airi Palva (University of Helsinki / Faculty of Veterinary Medicine) Funding: ABS Graduate School, Tekes _____________________________________________________________________________ Introduction Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with unknown aetiology. The possibility of quantitative or qualitative changes in the intestinal microbiota playing a role in developing IBS or as a result of IBS is being studied in our research group.

On the basis of the gathered sequence data, real-time qPCR analyses have been developed for analysis of individual samples. Thus far phylotypes from the genera Coprococcus, Collinsella, and Coprobacillus have shown a significant difference (P < 0.05) between IBS patients and healthy subjects.

Aim Our objective was to evaluate and develop molecular methods for quantifying bacteria from faecal samples and to characterize the microbial population in the faeces of IBS subjects and healthy volunteers.

Our results show that the faecal microbiota is altered in IBS and that further research is justified.

Results and discussion Real-time quantitative PCR (qPCR) proved to be an effective and sensitive method for quantifying bacteria from faecal samples and an extensive set of 16S rDNA-targeted primers for quantification bacteria in faecal samples by real-time qPCR were developed. For qualitative analysis of the faecal microbiota samples of 24 IBS patients were pooled according to their symptom subtype and 23 control individuals were pooled as one. The samples were percent guanine plus cytosine (%GC) profiled and fractioned prior to cloning and sequencing of the partial 16S rRNA gene. A total of 3 753 sequences from 12 clone libraries were phylogenetically analysed. A comparatively high proportion of sequences affiliated with Bacteroides and Actinobacteria in the mixed type IBS sample and control sample, respectively.

Articles 1. Malinen, E., Kassinen, A., Rinttilä, T., and Palva, A. 2003. Comparison of realtime PCR with SYBR Green I or 5'nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology 149: 269-277. 2. Rinttilä, T., Kassinen, A., Malinen, E., Krogius, L., and Palva, A. 2004. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J. Appl. Microbiol. 97: 1166-1177. 3. Kassinen, A., Krogius-Kurikka, L., Mäkivuokko, H., Rinttilä, T., Paulin, L., Corander, J., Malinen, E., Apajalahti, J., and Palva, A. 2007. The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects. Gastroenterology 133: 24-33.

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The effects of dietary phosphorus and calcium-to-phosphorus ratio on bone metabolism in Finnish females PhD student: Virpi Kemi (University of Helsinki / Department Applied Chemistry and Microbiology) E-mail: [email protected] Supervisors: Doc. Christel Lamberg-Allardt (University of Helsinki) Funding: ABS Graduate School, Tekes, Juho Vainio Foundation, Ella and Georg Ehrnrooth Foundation, Finnish Konkordia Fund _____________________________________________________________________________ Introduction In many countries the dietary intake of phosphorus is 2-to 3-fold higher than the recommended dietary reference intake (DRI) for phosphorus. Furthermore, the intake of phosphorus has risen during the last decades, with an increasing intake from processed foods with phosphatecontaining food-additives (AP). Although phosphorus is an essential mineral for bone, it is consumed in amounts that are too high for optimal bone health. An overly ample phosphorus intake has been suggested to be deleterious to bone through increased parathyroid hormone (PTH) secretion, especially when dietary calcium intake is low. Aim The main aim of the studies is to investigate the effects of dietary phosphorus and calcium-to-phosphorus ratio on calcium and bone metabolism. Results and discussion In the first controlled study dietary phosphorus increased dose-dependently SPTH. In addition, bone resorption increased and serum ionized calcium (SiCa) concentration and bone formation decreased. In the second controlled study S-PTH concentration decreased and S-iCa concentration increased dose-responsively with the increasing calcium doses. The bone formation did not differ significantly but bone resorption decreased significantly with both 600 mg and 1200 mg calcium doses. Acutely high dietary phosphorus

intake and low dietary Ca:P ratio have negative effects on bone metabolism. In the cross-sectional study we found that phosphate additives (AP) may have more harmful effects on bone than other phosphorus sources, as indicated by higher mean S-PTH concentration among participants who consumed AP-containing foods. Furthermore, high total habitual dietary phosphorus intake affected S-PTH unfavourably. Articles 1. Kemi, V., Kärkkäinen, M., and Lamberg-Allardt, C. 2006. High phosphorus intakes acutely and negatively affect Ca and bone metabolism in a dosedependent manner in healthy young females. Br. J. Nutr. 96: 545-552. 2. Kemi, V., Karp, H., Kärkkäinen, M., Laitinen, K., and Lamberg-Allardt, C. 2007. Increased calcium intake does not completely counteract the effects of increased phosphorus intake on bone: An acute dose-response study in healthy females. Br. J. Nutr. 2007 Oct 1; 1-8 [Epub ahead of print]. 3. Kemi, V., Rita, H., Kärkkäinen, M., Viljakainen, H., Laaksonen, M., Outila, T., and Lamberg-Allardt, C. Habitual high phosphorus intake and foods with phosphate additives negatively affect serum parathyroid hormone concentration: A cross-sectional study on healthy premenopausal women. Public Health Nutr. (conditionally accepted).

15

Orthopoxviruses in wild rodents PhD student: Paula Kinnunen (University of Helsinki / Department of Basic Veterinary Sciences) E-mail: [email protected] Supervisors: Prof. Olli Vapalahti, Prof. Airi Palva and Prof. Emer. Antti Vaheri (University of Helsinki) Funding: ABS Graduate School, University of Helsinki, EU grant QLR2-CT-2002-01358, the Farmos Foundation for Research and Science, the Foundation for Support of Veterinary Research, the Foundation for Finnish Veterinary Medicine _____________________________________________________________________________ Introduction The genus Orthopoxvirus (OPV) within the family Poxviridae consists of cross-reactive virus species. Some of them are zoonotic, like vaccinia (VACV) and cowpox (CPXV) viruses; others infect most likely one host species only, e.g. variola virus. After the cessation of VACV vaccinations, CPXV and other orthopoxvirus infections have become more important. The reservoir of CPXV is wild rodents. Despite the name, the virus is transmitted from rodents to humans mainly by cats. In Finland, a generalized cowpox eruption was diagnosed in a girl in year 2000 (1). Since then, orthopoxvirus-antibodies have been found, besides in humans, also in Finnish cats, dogs, horses, cattle, lynx and wild rodents (1). Aim Aim of this study was to find out, how common OPV infections are in wild rodents, and which OPV species are involved, enabling us to deduce potential zoonotic risk. Results and discussion Blood and tissue samples were collected from wild rodents in Finland (n = 301) and, for comparison, in the republic of Buryatia in Central Asia (n = 486). Blood samples were studied by immunofluorescence assay using CPXV as antigen. The IgG prevalences varied depending on trapping location up to 69%.

Panels containing high seroprevalence were screened for orthopoxvirus DNA. Nucleic acids were extracted from lung tissue and used as a template for real-time PCR established to amplify a part of hemagglutinin (HA) gene of all OPVs known to exist in Eurasia. Despite the sensitivity of the assay (24 copies of HAgene), only one PCR-positive lung sample was found (n = 316). The amplicon was identified as CPXV by sequencing. The PCR results show that ongoing infections with high virus load are rare as in most of those rodents already infected (ab positive) less than 24 copies of OPV genome exist in 0.2 -0.4 mg (40 – 400 cells) of the studied samples that ends up into the PCR. Most probably, the virus has been cleared by the immune system. However, one ongoing CPXV infection was found proving that the method is functional. In addition, antibody findings indicate that orthopoxviruses are regionally common in Finnish and Buryatian rodents, which might cause a zoonotic risk. Article Pelkonen, P.M., Tarvainen, K., Hynninen, A., Kallio, E.R., Henttonen, K., Palva, A., Vaheri, A., and Vapalahti, O. 2003. Cowpox with severe, generalized eruption, Finland. Emerg. Infect. Dis. 9 (11): 1458-1461.

16

A novel approach to chemical and toxicological analyses of fungal toxins in cereals and feed PhD student: Meri Kokkonen (Finnish Food Safety Authority, Evira / Research Department) E-mail: [email protected] Supervisors: Prof. Kimmo Peltonen (Evira), Dr. Marika Jestoi (Evira) and Prof. Vieno Piironen (University of Helsinki) Funding: ABS Graduate School, Evira, Ministry of Agriculture and Forestry _____________________________________________________________________________ Introduction Mycotoxins are secondary metabolites of filamentous fungi. They are common contaminants in cereals and may evoke a broad range of toxic properties. Therefore, mycotoxins pose a health risk to both human and animals. The analysis of mycotoxins is challenging as they are usually present in minute concentrations in complex sample matrices and may occur in various combinations. To get a comprehensive idea of the quality of a sample, multi-residue methods aiming at simultaneous detection of several mycotoxins by a single method have become popular. A feasible technique in this kind of approach is liquid chromatography coupled to mass spectrometry (LC/MS). In addition to chemical methods, in vitro -cytotoxicity bioassays can be used to provide complementary information and help better understand the biological effects of mycotoxins and their mixtures. Aim The objective of this study is to develop and apply new methods for mycotoxin analyses. A multi-component LC-MS/MS method for determining several mycotoxins in cereals has been developed. In addition, the applicability of in vitro – cytotoxicity tests will be studied to evaluate the toxicity of fungal strains. The effect of environmental conditions on the mycotoxin producing capabilities of Fusarium strains isolated from Finnish fields will be studied with the help of the

developed chemical and in vitro –toxicity methods. Results and discussion A multi-analyte LC-MS/MS method for determining 31 different Fusarium, Aspergillus, Penicillium and Claviceps toxins was developed and validated for cereals. The selected mycotoxins were extracted from cereal matrix by accelerated solvent extraction technique. The crude extract with minimum clean-up was injected to LC-MS/MS system. The analytes were separated in two different chromatographic runs and detected by tandem MS using electrospray ionization. The validation showed that the method performance was satisfactory for (semi-) quantitative work, limits of quantification varying between 1 and 1250 µg/kg, recoveries mostly between 51 and 122 % and repeatability being from 2 to 26 % within-day and 14 to 28 % between-days (as RSD). The developed multi-mycotoxin method allows simultaneous cost- and time-saving determination of several coexisting toxins and screening-type work at sufficiently low concentration levels. The multi-method is currently being applied for analysing the mycotoxin profiles of Finnish Fusarium strains both in cereal grains and on solid culture media.

17

Effect of enzyme-aided processing on flavonoids in berries PhD student: Jani Koponen (University of Kuopio / Food and Health Research Centre) E-mail: [email protected] Supervisors: Doc. Riitta Törrönen and Prof. Kaisa Poutanen (University of Kuopio / Food and Health Research Centre) and Senior Assistant Seppo Auriola (University of Kuopio / Department of Pharmaceutical Chemistry) Funding: ABS Graduate School, EU (project QLk1-CT-2002-02364), Ministry of Agriculture and Forestry, Jenny and Antti Wihuri Foundation _____________________________________________________________________________ Introduction Berries are rich in flavonoids, especially anthocyanins, a large group of polyphenolic compounds. Flavonoids are secondary plant metabolites and can be associated with health-promoting effects, e.g. antioxidative, anticarcinogenic and antimicrobial activities. Enzymes such as pectinases are currently used in industrial berry processing to increase the juice yield. Enzymatic treatment can enhance the extractability of phenolic components from the berry matrix, but the impact of these treatments on the fate of flavonoids is yet unknown. Aim The objectives of this doctoral thesis are to investigate the anthocyanin content among Finnish foods and to evaluate the effecst of enzyme-aided juice processing on flavonoids found in berries. Results and discussion The anthocyanin content in foods consumed in Finland varied in berries from 0 to 564 mg/100 g, in fruits from 0 to 66 mg/100 g, and in vegetables from 0 to 75 mg/100 g of fresh weight. The highest anthocyanin contents were found in bilberry (Vaccinium myrtillus) and chokeberry (Aronia melanocarpa). In fruits and vegetables, the highest levels were detected in cherry (Prunus avium) and red cabbage (Brassica oleracea var. capitata "f. rubra"), respectively. Enzyme-aided processing elevated the extractability of anthocyanins and flavonols into bilberry and black currant juices as compared to non-enzymatic processing. The

increase for total anthocyanidins was up to 83 % (bilberry) and 58 % (black currant), and for total flavonols up to 41 % (bilberry) and 49 % (black currant). Enzyme-aided processing decreased the level of bilberry flavonoid glycosides by hydrolyzing these compounds to aglyconic forms, when high dosages of enzyme preparations were used. These results show that berries, especially dark blue berries are an excellent source of anthocyanins. This data will be included in the Finnish Food Composition Database Fineli and used for calculation of the average dietary intake of anthocyanins from the Finnish diet. In addition, the results indicate that enzyme preparations used in the production of berry juices improve the juice yield and the extraction of anthocyanins and flavonols into the juice. Thus, enzyme-aided processing offers a powerful tool for improving the health value of berry juices. Articles 1. Koponen, J.M., Happonen, A.M., Mattila, P.H., and Törrönen, A.R. 2007. J. Agric. Food Chem. 55: 1612-1619. 2. Buchert, J., Koponen, J., Suutarinen, M., Mustranta, A., Lille, M., Törrönen, R., and Poutanen, K. 2005. J. Sci. Food Agric. 85: 2548-2556. 3. Koponen, J.M., Buchert, J., Poutanen, K.S., and Törrönen, A.R. Eur. Food Res. Tech. (in press). 4. Koponen, J.M., Happonen, A.M., Buchert, J., Kontkanen, H., Auriola, S., Poutanen, K.S., and Törrönen, A.R. J. Agric. Food Chem. (in press).

18

The faecal microbiota of healthy individuals and patients with diarrhoea predominant irritable bowel syndrome PhD student: Lotta Krogius-Kurikka (University of Helsinki / Department of Basic Veterinary Sciences) E-mail: [email protected] Supervisors: Prof. Airi Palva (University of Helsinki / Department of Basic Veterinary Sciences) Funding: ABS Graduate School, Tekes, Academy of Finland _____________________________________________________________________________ Introduction Gastrointestinal (GI) microbiota plays an important role in human health and disease via its essential metabolic, trophic and protective functions on its host. The microbiota constitutes a highly complex ecosystem and the majority of the bacteria in the GI tract are unculturable. Alterations of GI microbiota in the irritable bowel syndrome (IBS) have been discovered, especially in diarrhoea predominant IBS patients (IBS-d). Alleviation of IBS symptoms with probiotic treatment has also been observed, but its effect to the microbiota has not been properly reported. Aim The aim of this study is to reveal the composition of faecal microbiota of healthy humans and subjects with IBS-d using high troughput cloning and sequencing of percent guanine and cytosine profiled samples. Furthermore, effect of multispecies probiotic supplementation on intestinal microbiota of IBS patients is studied using quantitative real time PCR (qPCR) in two placebo controlled intervention studies.

subjects with the IBS-d subjects showed, that the structure of the compared libraries differ. The faecal samples of one intervention study have been analysed (1.). On the basis of the phylogenetic analysis of the constructed clone libraries in this work and in the work by Kassinen et al. (Gastroenterology 2007 133:24-33), qPCR assays have been constructed, and will be utilised to the analysis of the faecal samples of the other intervention study. The analyses of the results are in progress and even more assays will be performed. Article Kajander, K., Krogius-Kurikka, L., Rinttilä, T., Karjalainen, H., Palva, A., and Korpela, R. 2007. Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome. Aliment. Pharmacol. Ther. 26: 463-473.

Results and discussion Massive 16S RNA gene clone libraries from the faecal microbiota of healthy subjects as well as IBS-d subjects have been created. The phylogenetic analysis of the control subjects microbiota revealed a high number of phylotypes. Results from the comparison of the sequence libraries from control 19

Bioactive berry encapsulation

phenolics:

isolation,

analysis,

and

protection

by

PhD student: Petri Kylli (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: [email protected] Supervisors: Docent Marina Heinonen (University of Helsinki / Department of Applied Chemistry and Microbiology) Funding: ABS Graduate School, TEKES _____________________________________________________________________________ Introduction Plants are rich in bioactive phenolic compounds. They have been considered anti-inflammatory, anticarcinogenic, and antimicrobial agents as well as antioxidants. Polymeric fraction of procyanidins are discovered being the most effective antioxidant in the procyanidins. Phenolic compounds are very reactive compounds. Their molecular structure is easily altered during storage and processing leading to decrease in the bioactivity. For examble they are very susceptible to heating and light. Bioactive compounds can be protected by encapsulation into the carbohydrate matrix. Aim The aim of this study is to isolate and characterize phenolic compounds present in berries and to test their bioactivities. Encapsulation to protect these bioactive compounds is also studied. Results and discussion The research has focused on isolation and characterization of rowanberry phenolics and their bioactivities. Rowanberry phenolics were found to be effective free radical scavengers and excellent inhibitors of lipid oxidation in liposomes and emulsions (Table 1.). The effect of the nature of conjugation of phenolic acids, abundant in rowanberries, was also investigated using synthesized hydroxycinnamic acids. The experiment showed the differences between the

esterification positions to the antioxidant activity. Table 1. Inhibition of hexanal formation by rowanberry phenolic extracts. cultivar Rowanberry Burka Titan Granatnaja Zoltaja

2,1 µg/mL 90.3 ± 1.5 85.9 ± 0.9 86.3 ± 2.3 87.9 ± 0.7 90.4 ± 0.6

hexanal 4,2 µg/mL 95.7 ± 2.6 96.9 ± 2.1 96.2 ± 2.8 96.6 ± 2.7 97.0 ± 1.0

8,4 µg/mL 96.8 ± 0.1 92.7 ± 1.2 97.2 ± 0.1 96.5 ± 0.9 97.1 ± 0.1

Polymeric proanthocyanidins from cranberries and lingonberries were extracted and further fractionated with preparative scale LC-MS-FL method. Polymeric proanthocyanidins show excellent antioxidant activity toward lipid oxidation. To investigate means for the protection of bioactive phenolics cloudberry the ellagitannin fraction was encapsulated in maltodextrin. The efficiency and stability of microencapsules were tested showing that the higher the molecular weight of the maltodextrin the higher stability and efficiency was achieved. Further research will focus on bioactivities of the phenolic compounds in berry leaves and cell cultures. Article Kylli, P., Nousiainen, P., Biely, P., Sipilä, J., Tenkanen, M., and Heinonen, M. 2008. Antioxidant Potential of Hydroxycinnamic Acid Glycoside Esters. J. Agric. Food Chem. (conditionally accepted). 20

Effect of lifestyle changes on genetic factors regulating inflammation and lipid metabolism in obesity PhD student: Tiina Lappalainen (University of Kuopio / Department of Clinical Nutrition) E-mail: [email protected] Supervisors: Prof. Helena Gylling, Doc. Ursula Schwab and Marjukka Kolehmainen, PhD (University of Kuopio / Department of Clinical Nutrition; Food and Health Research Centre) Funding: ABS Graduate School, Academy of Finland _____________________________________________________________________________ Introduction Obesity is a health problem that is increasing at an alarming rate. Obesityassociated metabolic abnormalities, e.g. insulin resistance and inflammation, can be improved by weight loss and increased physical activity. However, the knowledge of the molecular basis eliciting beneficial metabolic changes, especially in adipose tissue, is partial. Aim The objective of this thesis is to identify inflammation and lipid metabolism related genes which are differentially expressed in adipose tissue of lean and obese subjects. Serum amyloid A (SAA) is one of the novel links between increased adipose tissue mass and low-grade inflammation in obesity. However, little is known about the effects of lifestyle changes and factors regulating its serum concentration and mRNA levels. Furthermore, in this doctoral thesis the role of different polymorphisms in the obesity associated genes (e.g. SAA and FTO) will be evaluated in the Finnish Diabetes Prevention Study (DPS) population. Results and discussion We investigated the association between SAA and leptin in obese and normal weight subjects and analysed the effect of weight reduction on serum SAA concentration and gene expression in adipose tissue of obese subjects. We found the BMI-independent positive association between SAA and leptin, which suggest the interaction of these

two adipokines. Weight loss of at least 5% increased SAA mRNA expression. These findings may have implications in understanding their role in low-grade inflammation in obesity. In the second subwork, we have examined the mRNA expressions of adipogenic genes (e.g. stearoyl-CoA desaturase 1 and lipin-1). Data showed that obese subjects, especially women, have significantly impaired mRNA expression of adipogenic genes. This support the hypothesis that impaired expression of adipogenic genes may result in the development of larger adipocytes, and thus, metabolic disturbances of adipose tissue metabolism. Moreover, we found the down-regulation of the enzyme regulating lipid oxidation, palmitoyl-CoA oxidase 1, along with weight reduction, which may have a role in weight regain. These findings will provide information on obesity-induced changes in lipid metabolism-related factors in adipose tissue, and, on the effects of lifestyle interventions on these factors. In the third subwork, the association between the common variant of FTO and body mass index was confirmed in the DPS study population, especially in women. Article Lappalainen, T. et al. 2008. Serum concentrations and expressions of serum amyloid A and leptin in adipose tissue are interrelated: the Genobin Study. Eur. J. Endocrinol. 158 (3): 331-339.

21

Effect of sea buckthorn berries on circulating concentrations of cholesterol, triacylgycerols, and flavonols in healthy adults PhD student: Petra Larmo (University of Turku / Department of Biochemistry and Food Chemistry) E-mail: [email protected] Supervisors: Prof. Raija Tahvonen (University of Turku / Functional Foods Forum / EPANET) and Prof. Heikki Kallio (University of Turku / Department of Biochemistry and Food Chemistry) Funding: ABS Graduate School, Turku University Foundation, Finnish Cultural Foundation _____________________________________________________________________________ Introduction Epidemiological studies indicate that consumption of flavonoids has an inverse association with the risk of cardiovascular diseases (CVD). Aim The objective was to study the effect of flavonoid-rich sea buckthorn berry (SBB) on circulating lipid markers associated with CVD risk, and plasma flavonol concentration in healthy volunteers. Methods, results and discussion A double-blind, randomized, placebocontrolled parallel design was used. During the 90 days study period, the participants daily consumed either 28 g of SBB or placebo. Two-hundred fifty-four volunteers were recruited, 229 completed the study period and gave all the necessary samples. For flavonol analyses a random sample of 40 participants was generated. Fasting blood samples for the analysis of CVD risk factors and flavonols were obtained at the beginning and end of the study period. Compared to the placebo, the consumption of SBB increased the plasma concentration of the flavonol isorhamnetin significantly. The concentrations of quercetin and kaemferol increased but the change was not significant. SBB did not affect the serum total, HDL, or LDL cholesterol, or the serum triacylglycerol concentrations.

The participants in this study had healthy diets. In Chinese intervention studies SBB juice and sea buckthorn total flavonoids reduced the plasma triacylglycerol and/or cholesterol levels of hypertriglycemic participants without clear effects on the corresponding parameters of subjects with normal triacylglycerol levels. The mean serum lipid concentrations of the participants in our study were within the limits of the European recommendations. It is also possible that the effects would have required more time to develop, even though the study period was long compared to the majority of earlier intervention studies on flavonols and flavonol containing foods. In conclusion, the consumption of SBB significantly increased the plasma concentration of isorhamnetin, indicating that it is a good dietary source. However, this did not convert to affecting the circulating concentrations of lipid markers in healthy, normolipidemic adults having healthy dietary habits. Article Larmo, P., Alin, J., Salminen, E., Kallio, H., and Tahvonen, R. Effects of sea buckthorn berries on infections and inflammation: a double-blind, randomized, placebo-controlled trial. Eur. J. Clin. Nutr. Advance online publication, 27 June 2007; doi:10.1038/sj.ejcn.160281.

22

Regioisomeric structure determination of triacylglycerols in dietary fats and oils by high-performance liquid chromatography and mass spectrometry PhD student: Heidi Leskinen (University of Turku/ Department of Biochemistry and Food Chemistry) E-mail: [email protected] Supervisors: Prof. Heikki Kallio (University of Turku) and Jukka-Pekka Suomela, PhD (University of Turku) Funding: ABS Graduate School, The Finnish Foundation for Economic and Technology Sciences – KAUTE, the Raisio Group Research Foundation, and the Turku University Foundation _____________________________________________________________________________

Mass spectrometry (MS) is a powerful tool in analyzing TAGs, because information about the regioisomeric structure of individual TAGs can be obtained directly without enzymatic treatment of the sample. During the dissociation of TAGs in MS, FA in sn-2 position is cleaved off less efficiently than the sn-1/3 FAs. Liquid chromatographic (LC) separation of TAGs containing different FA isomers (doublebond positional isomerism, cis/trans isomerism) prior MS is essential, because these cannot be differentiated by MS. Aim Different methods combining MS and LC systems are tested, developed and applied in order to investigate the regioisomeric structure of TAGs in dietary fats and oils. Results and discussion The positional isomers (sn-ABA and snAAB + sn-BAA; A and B denotes different FAs) of different TAGs were quantified in lard, rapeseed oil, sunflower seed oil, and sunflower seed oil by different LC-MS systems. The analyses were made with

reference TAGs (pure regioisomers) and dietary oils and fat. The three ionization methods used were positive atmospheric pressure chemical ionization (APCI), positive electrospray ionization (ESI), and ammonia negative ion chemical ionization (NICI). In ESI either ammonium adducts or silver ion adducts were prepared during ionization. 2.0 [LnL]+ / [LL]+

Introduction Dietary fats are mainly composed of triacylglycerols (TAG). The positional distribution of the three fatty acids (FA) in glycerol moiety of TAG has an effect on the chemical, technological and nutritional properties of fats and oils, and in natural TAGs the distribution is nonrandom.

R2 = 0.979

1.8 1.6 1.4 1.2 0

25 50 75 100

sn -ABA content, %

Figure 1. APCI-MS calibration curve of TAG 18:3(n-3)/18:2/18:2 (Ln/L/L) calculated based on the reference TAG analysis. y-axis: The ratio of DAG ion intensities; x-axis: 100 * sn-LLnL / (snLLnL + sn-LnLL + sn-LLLn). Article Leskinen, H., Suomela, J.-P., and Kallio, H. 2007. Quantification of triacylglycerol regioisomers in oils and fat using different mass spectrometric and liquid chromatographic methods. Rapid Commun. in Mass Spectrom. 21: 2361-2373.

23

Prevalence of Listeria monocytogenes in raw food and the role of inlH in adaptation of L. monocytogenes to different environments PhD student: Annukka Markkula (University of Helsinki / Department of Food and Environmental Hygiene) E-mail: [email protected] Supervisors: Prof. Hannu Korkeala (University of Helsinki / Department of Food and Environmental Hygiene) Funding: ABS Graduate School, Walter Ehrström Foundation, University of Helsinki _____________________________________________________________________________ Introduction Listeria monocytogenes is a hazardous food borne pathogen ubiquitous in nature. It is common finding in raw food and food processing environments. Some internalization associated surface proteins of L. monocytogenes, which belong to the group of leucine-rich repeat containing proteins (LRR proteins), are known to mediate species specific differences in the virulence of L. monocytogenes. The role of virulence associated LRR-protein InlH in differences in strain or niche specificity of L. monocytogenes is not known. Aim The aim of the study is to examine the prevalence and biodiversity of L. monocytogenes in different sources. The role of inlH in niche specificity and in the ability of L. monocytogenes to cause persistent contaminations in food processing environments will be studied. Results and discussion In the first part of the study prevalence and strain variation of L. monocytogenes in raw fish and tonsils of pigs were examined. A total of 4% and 14% of raw fish samples and samples from tonsils of pigs, respectively, were positive for L. monocytogenes. The genetic diversity of L. monocytognes strains was wide in both sources. Different L. monocytogenes strains were found from fish and pig samples.

L. monocytogenes strains isolated from clinical, food, food processing plant and feed samples will be examined by PCR and sequencing. In the third part of the study the role of inlH in the attachment of L. monocytogenes to food processing surfaces will be examined using inlH mutated and wild type L. monocytogenes strains. Also, the expression of inlH in different growth environments will be studied by RT-PCR. Articles 1. Markkula, A., Autio, T., Lundén, J., and Korkeala, H. 2005. Raw and processed fish show identical Listeria monocytogenes genotypes with pulsed-field gel electrophoresis. J. Food Prot. 68: 1228-1231. 2. Autio, T., Markkula, A., Hellström, S., Niskanen, T., Lundén, J., and Korkeala, H. 2004. Prevalence and genetic diversity of Listeria monocytogenes in the tonsils of pigs. J. Food Prot. 67: 805-808.

In the second part of the study strain and niche specificity and the structure of inlH in 24

Effect of Plant Sterols and Stanols on Adenoma Formation in ApcMin/+ mice PhD student: Maija Marttinen (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: [email protected] Supervisors: Prof. Marja Mutanen, PhD Anne-Maria Pajari (University of Helsinki) Funding: ABS Graduate School, Yrjö Jahnsson Foundation _____________________________________________________________________________ Introduction Colorectal cancer (CRC) incidence has been increasing in many Western countries for several decades. Diet rich in plant foods is known to decrease CRC risk, whereas high intake of fat and cholesterol is associated with an increased risk. Plant sterols have been shown to inhibit cell growth in vitro, however, the effect of long-term consumption of plant sterol enriched foods on cancer development needs to be studied further. The ApcMin/+ mice carry a heterozygous germ line mutation in the Apc tumour suppressor gene. The homologous gene is mutated in patients with hereditary colon cancer (FAP, familial adenomatosis polyposis), as well as in 80% of sporadic colorectal cancer cases. In ApcMin/+ mouse, the loss of heterozygosity in Apc leads to the development of multiple adenomas in the small intestine. Aim The aim of this PhD study is to investigate the effect of plant sterol and stanol intake on intestinal adenoma formation and cell signalling in ApcMin/+ mice. Results and discussion In Study I, the mice were fed either a 0,8% (w/w) plant stanol diet or a control diet without plant stanol esters. In Study II, the mice were assigned either to a plant sterol group or a control group. The plant sterol content of the experimental diet in study II was 0,7% (w/w). The plant stanol and sterol esters were added to the experimental diets in the form of freeze-dried products with

plant stanol respectively.

or

sterol

enrichment,

The number of intestinal adenomas was significantly increased by both plant stanol and sterol feeding. The adenoma size was not affected in either study. The levels of signalling proteins were measured by Western blotting method. In Study I, mucosal -catenin and p53 levels were unchanged, however, nuclear cyclin D1 and activated MAPK-protein levels were significantly higher in the plant stanol fed mice indicating of increased cell growth and proliferation. In Study I, the mucosal cholesterol level was the same in both groups. In Study II, the mucosal -catenin and cyclin D1 were unaffected by plant sterol diet. The mucosal cholesterol level was lower after plant sterol feeding. The results suggest that different mechanisms are involved in adenoma formation by plant sterol and stanol feeding. In a recent study (Study III), the mice were fed plant sterols in different doses (0.2%; 0.4% and 0.8%). The analysis of the results are in process. Studies I and II show that plant sterols and stanols induce the initiation of tumourigenesis in ApcMin/+ mouse. The cell proliferation seems to be upregulated already in the ‘healthy’ tissue after plant stanol feeding. Currently, the mechanism behind the initiation of adenoma formation caused by plant sterols and stanols is being studied in more detail. The results of the study have not been published yet.

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Production of ligninolytic enzymes on solid-state cultivations PhD student: Ulla Moilanen (Helsinki University of Technology / Department of Biotechnology and Chemical Technology) E-mail: [email protected] Supervisors: Prof. Matti Leisola, Dr Ossi Pastinen (Helsinki University of Technology) and Prof. Annele Hatakka (University of Helsinki) Funding: ABS Graduate School _____________________________________________________________________________ Introduction Solid-state cultivation means cultivation of microbes on solid medium without liquid phase. The method is especially applicable to fungi because dry and solid medium is a natural growth environment for most fungi. Some enzymes can be produced hundredfold on solid media compared to liquid. In spite of their benefits solid-state cultivation methods and technology are not as well developed as they are in liquid cultivations. Laccase and manganese peroxidase (MnP) are ligninolytic enzymes. Fungi utilise them when degrading wood lignin into smaller molecular weight compounds. Ligninolytic enzymes have low substrate specificity and that is why they can be used in many applications such as biopulping, waste water treatment, soil remediation or baking. At the moment laccase is the only enzyme used commercially. MnP is sold just for analytical purposes. It can only be produced in small concentrations in liquid medium. However, there are promising results with white-rot fungi on solid culture media. Aim The aim of this research is to develop an economically feasible process to produce ligninolytic enzymes on solid-state cultivation media. A central part of the work is developing solid-state cultivation techniques. Results and discussion We screened 18 different fungi and three different industrial waste materials for the production of laccase and MnP. From the

tested industrial waste materials oat husks were the most promising substrate for fungal cultivations. From the screened strains Cerrena unicolor showed the greatest potential for industrial use because of its ability to produce significant amounts of both MnP and laccase at the same time. Next we optimized the culture medium for the production of C. unicolor ligninolytic enzymes. We supplemented oat husks with different inducers like Cu and Mn and with extra nutrients. The experiments were done in 100 g scale. From the tested supplements the addition of oat meal yielded the best enzyme activities, improving laccase activity by 120 % to 300 nkat/g DW and MnP activity by 190 % to 230 nkat/g DW. In order to produce these enzymes industrially new solid-state reactor types have to be available. We have developed two 3-10 kg scale solid-state reactors. With the reactors we have successfully repeated the best cultivations described above. We have also developed on-line measurement systems for measuring viable biomass in solid-state cultivations. To study the industrial potential of the produced enzymes we tested the ability of C. unicolor laccase to decolourise several artificial textile dye baths. We showed that laccase removed colour efficiently and thus it has potential to be used in industrial applications.

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Identifying molecular targets of bilberry consumption PhD student: Otto Mykkänen (University of Kuopio / Department of Clinical Nutrition; Food and Health Research Centre) E-mail: [email protected] Supervisors: Doc. Riitta Törrönen, Doc. Leena Pulkkinen and Dr Thomas Dunlop (University of Kuopio) Funding: ABS Graduate School, Yrjö Jahnsson Foundation, Juho Vainio Foundation _____________________________________________________________________________ Introduction Obesity is an increasing risk factor for the development of chronic diseases such as metabolic syndrome, type 2 diabetes and cardiovascular diseases. Recent studies have shown that anthocyanin-rich extracts ameliorate obesity, insulin resistance and dyslipidemia induced by high-fat diet in mice. Anthocyanins represent a major group of flavonoids in berries and are especially high in Finnish bilberries (lat. Vaccinium myrtillus). A nutrigenomics approach utilizing microarrays offers a novel tool to study the changes in gene expression and demonstrate the wide range of molecular targets of berry consumption. Aim This thesis will investigate the molecular effects of bilberry consumption in mice with normal metabolism and in mice fed with high-fat diet to develop obesity related metabolic disturbances. Research Mice of C57BL/6J strain fed with high-fat diet (45-60 E% fat) has been proven to be a useful model to study diet induced obesity related metabolic disturbances such as hyperinsulinemia, hyperglycemia and hypertension. We feed 4 -week old male mice with one of the following diets for 12 weeks: 1) standard rodent diet, 2) standard diet + bilberries, 3) high-fat diet, 4) high-fat diet + bilberries. The effective dose of freezedried berries (w/w) was verified in a pilotstudy. Body weights are measured weekly

and tissue weights at the end of the experiment. To analyse obesity related metabolic disturbances several physiological parameters are measured: glucose, insulin, lipid and hormone levels; glucose tolerance and insulin sensitivity tests; magnetic resonance imaging (MRI) for measuring body fat in vivo and indirect calorimetric set up with TSE LabMaster System® to measure metabolic and physical activities well as feeding and drinking behaviour. The mice are sacrificed at 12 weeks and tissue samples from liver, adipose tissue, skeletal muscle and brain are collected. Total RNA and proteins are isolated and RNA further purified and processed for hybridizations on whole mouse genome chips using Illumina platform. Microarrays are performed on 4-6 animals per group and verified with quantitative PCR. Also Western Blot analysis and additional immunohisto-chemical methods are used later to determine protein expression. Current Status of the project The pilot study and the first animal experiment have been done during the year 2007 and the second animal experiment will be finished in spring 2008. Data from these animal experiments and especially measurements of physiological parameters are reported in the first manuscript. Tissue samples for gene expression and histological analyses are collected during these experiments and reported in the following manuscripts.

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Microbiological characteristics of Campylobacter strains isolated from Finnish patients PhD student: Ulla-Maija Nakari (National Public Health Institute / Department of Bacterial and Inflammatory Diseases / Enteric Bacteria Laboratory) E-mail: [email protected] Supervisor: Prof. Anja Siitonen (National Public Health Institute) Funding: ABS Graduate School, National Public Health Institute _____________________________________________________________________________ Introduction Campylobacter spp. are the most common bacterial causes of human intestinal infections in Finland, there are 3500-4000 cases annually. There is a seasonal peak in the number of cases in July and August. The Finnish clinical microbiology laboratories report their Campylobacter findings to the National Infectious Disease Register. The strains are identified to the species level by the clinical microbiology laboratories but no epidemiological typing is carried out. Epidemiological typing would enable differentiation of the strains and provide tools for epidemiological investigations. Aim The aim of my PhD study is to obtain new information about the epidemiology of Campylobacter infections in Finland. For the research material, a large set of Campylobacter strains isolated from patients was collected. One of the purposes of the study was to investigate temporal trends, and geographical and demographical distribution of Campylobacter infections in Finland. Campylobacter strains were typed by heat-stable serotyping. Travel history of the patients was asked to determine the impact of travel on the seasonality of the infections. Results and discussion Of the 2364 Campylobacter strains collected, 39% were of domestic origin, 53% were associated with foreign travel and the travel history of the patient was not known in 8%. The proportion of travel –

related cases was 54-68% in patients aged 10 to 59, and lower in younger and older patients. The overall incidence of Campylobacter infection varied between 61/100 000 and 76/ 100 000 in 2002-2005. The incidence was highest (148/100 000) in the age group 25-29 and lowest in children aged 5 to 9 (21/100 000), and in patients older than 74 years (24/100 000). The incidence varied seasonally: 63% of the domestic cases and 25% of the travelassociated cases were detected in July and August, indicating that the seasonal peak is mostly caused by domestic infections. The domestic C. jejuni strains isolated from patients were serotyped. The most common serotypes were HS 2 (12%), HS 12 (12%), HS 4-complex (9%), HS 1,44 (8%) and HS 6,7 (7%). The proportion of non-typeable strains was high, 34%. All of these serotypes contributed to the seasonal peak. Serotype HS 2 was the most common serotype in 0-59 year old patients whereas among patients 60 years or older, HS 12 was more frequent than HS 2.(Nakari, Puhakka et al. 2008). Articles 1. Nakari, UM., Laaksonen, K., Korkeila, M., and Siitonen, A. 2005. Comparative typing of Campylobacter jejuni by heat-stable serotyping and PCR-based restriction fragment length polymorphism analysis. J Clin Microbiol 43 (3): 1166-1170. 2. Nakari, UM., Puhakka, A., and Siitonen, A. 2008. Correct identification and discrimination between Campylobacter jejuni and C. coli by a standardized hippurate test and species-specific polymerase chain reaction. Eur J Clin Microbiol Infect Dis. Mar 4 (Epub). 28

Environmental Reservoirs of Mycobacteria Causing Porcine and Human Infection PhD student: Jaakko Pakarinen (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: [email protected] Supervisors: Prof. Mirja Salkinoja-Salonen (University of Helsinki) and Doc. Terhi AliVehmas (University of Helsinki) Funding: ABS Graduate School, TEKES grant no 2265/31/03, Academy of Finland grant no 53305 ___________________________________________________________________________ Introduction Nearly half of the currently known nontuberculous Mycobacterium (NTM) species (n=112) are opportunistic pathogens that are transmitted from the environment to humans and animals. Pig mycobacteriosis is the most common animal mycobacterial disease in Finland with a long-term average prevalence 0.34%. The high genetic similarity between human and porcine NTM isolates indicates common sources of infection (i. e. soil, water, dust) for pigs and humans. Besides opportunistic environmental pathogens such as NTM, humans are exposed to dustborne immunomodulatory bacteria, which contribute to the development of atopic diseases such as asthma and allergy. Aim The aim of research is to develop and apply DNA based methods for detection of opportunistic and immunomodulatory bacteria in the environment. Results and discussion We found evidence of mycobacterial proliferation in bedding materials on four high-prevalence pig farms by Mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization (RNA analyses done by Timo Nieminen, Univ. of Oulu) (article 1). We used the qPCR and 16S rRNA sandwich hybridization assays also to estimate the density and metabolic activity

of mycobacteria in selected soils (article 2). Using DNA cloning we showed that urban house dust, causing Th2-type immunity in mice and human dendritic cells, contained mainly human commensal bacteria, while Th1 reactive hay barn dust contained a large diversity of bacteria (immunological analyses done by the Finnish Inst. of Occupational Health) (article 3). Articles 1. Pakarinen, J., Nieminen, T., Tirkkonen, T., Tsitko, I., Alivehmas, T, Neubauer, P., and Salkinoja-Salonen, M.S. 2007. Proliferation of mycobacteria in a piggery environment revealed by mycobacteriumspecific real-time quantitative PCR and 16S rRNA sandwich hybridization. Vet. Microbiol. 120: 105-112. 2. Nieminen, T., Pakarinen, J., Tsitko, I., Salkinoja-Salonen, M., Ali-Vehmas, T., Breitenstein, A., and Neubauer, P. 2006. 16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils. J. Microbiol. Methods. 67: 44-55.

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Preventing adhesion of vegetative bacteria and spores by polarization and radical formations on steel surfaces in industrial processes PhD student: Minna Peltola (University of Helsinki / Department of Applied Chemistry and Microbiology) E-mail: [email protected] Supervisors: Prof. Mirja Salkinoja-Salonen (University of Helsinki / Department of Applied Chemistry and Microbiology) Funding: ABS Graduate School _____________________________________________________________________________ Introduction Microbial hygienity of the productive means is necessity for good quality of the food production. The most difficult problem for hygienity is the spore-forming bacteria such as Bacillus cereus. The spores are able to adhere to different surface material and are resistant to almost all permitted detergent and disinfectants used in food industry. Sensitivity have been found mostly to oxygenous radicals producing antimicrobial substances; hydrogen peroxide, peracetic acid and ozone. For exposing the spores to oxygen radicals we use two previously designed instruments DBA (Double Biofilm Analyzer) and RadBox. DBA allows studying polarisation effect on biofilms on metal surfaces. The RadBox is designed for detecting presence and amount of oxygen radicals. Using the DBA, we found out that polarisation under certain conditions prevented biofilm formation and reduced the existing biofilm. As model organisms, we used the pink slime bacteria Deinococcus geothermalis. Antimicrobial effects of polarisation are not understood. Our hypothesis is that oxygen radicals and pH changes near the surface might be involved.

reactive towards oxygen radicals and H+ ion. To study effect of polarisation on biofilm (spores and vegetative cells) on the metal surface. In addition, we study the physiology of adherence mechanism of biofilm bacteria D. geothermalis at protein level and microscopically with field emission scanning electron microscope (FESEM) and confocal laser scanning microscope (CLSM). Results and discussion D. geothermalis adhere to the surfaces with specific adhesion threads. Proteins of the threads are similar to type IV-pilin proteins. PAS (Periodic acid-Shiff) staining and carbohydrate specific lectin staining combined with CLSM showed that threads are coated with sugars. Articles 1. Saarimaa, C., Peltola, M., Raulio, M., Neu, T.R., Salkinoja-Salonen, M.S., and Neubauer, P. 2006. Characterisation of adhesion threads of Deinococcus geothermalis as Type IV pili. J. Bacteriol. 188 (19): 7016-7021. 2. Peltola, M., Neu, T.R., Raulio, M., Kolari, M., and Salkinoja-Salonen, M.S. 2008. Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study. Environ. Microbiol. (accepted).

Aim The aim of research is to find out quality and quantity of the oxygen radicals formed during polarisation using fluorogenic dyes 30

Identifying the factors that make the healthy food to taste good; Interactions between chemical composition, sensory quality and acceptance PhD student: Terhi Pohjanheimo (University of Turku / Department of Biochemistry and Food Chemistry; Functional Foods Forum (FFF)) E-mail: [email protected] Supervisors: Prof. Raija Tahvonen (University of Turku / Functional Foods Forum) and Dr Mari Sandell (University of Turku / Functional Foods Forum) Funding: ABS Graduate School, Tekes, Finnish Cultural Foundation _____________________________________________________________________________ Introduction An increasing interest for healthy and functional food has multiplied the amount of new products on the market. Even if a food meets the strict health and nutritional requirements facilitating a health claim, it is unlikely to be accepted by consumers unless they do like the flavour. Sensory quality of food, which is strongly affected by chemical composition, plays an important role in acceptance of food. However, consumers are individuals in their preferences, and differences between consumers cannot be neglected. Characterization of the interactions between chemical composition, sensory quality and hedonic responses is essential for successful development of new healthy food products. Identifying both qualityand market-related factors that make the healthy food to taste good in different consumer segments is interesting also from the academic point of view.

Results and discussion At present, the data material has been collected in various consumer tests and food products have been analyzed both in sensory quality and chemical key characteristics. We have found that consumers’food motivations were shown to influence on sensory perception of certain food products. For example, highly health-motivated subjects rated the taste of certain breads and health drinks significantly higher than subjects with low health-motivation. On the other hand, gender or age did not have an effect on the liking in model foods studied. Based on our studies, there are substantial and systematic evidence for the ability of brand and package information to heighten or lower consumers’ perceived sensation intensity of food products.

Aim The aim of the work is to characterize the interactions between chemical composition, sensory quality and acceptability in healthy model foods, when the variation of product composition, consumers and product information are taken into account. The emphasis of this study is to reveal the critical sensory and chemical quality characteristics that are explaining the acceptability of model products in different consumer segments.

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The stress responses of probiotic bacteria PhD student: Aki Suokko (University of Helsinki / Dept of Basic Veterinary Sciences) E-mail: [email protected] Supervisors: Doc. Pekka Varmanen (University of Helsinki / Faculty of Veterinary Medicine) Funding: ABS Graduate School, J. and A. Wihuri foundation, TEKES, Suomen Akatemia _____________________________________________________________________________ Introduction Environmental challenges or stresses cause accumulation of nonnative proteins within the cytoplasm. Bacteria respond to stress with elevated expression of stress response proteins, which fold or dispose nonnative proteins. Lactobacilli and bifidobacteria are increasingly studied due to their probiotic benefits. To promote health, probiotic bacteria must resist stresses in industrial processes and selective conditions of GIT. Aim This project aims to develop methods to study stress-induced proteome of probiotic bacteria. The main focus is to resolve the physiological role of Clp-proteins in stress responses of selected probiotic srains. Results and discussion Experimental methods for protein radiolabelling and 2-D gel electrophoresis were optimized for four probiotic strains to study changes in their protein synthesis during stress. In B. longum, several proteins, including classical chaperons, were found to be induced both during heat shock and by bile salts, indicating that heat and bile salts responses are closely related in this organism. For the probiotic Lactobacillus strains, similar proteomic approaches were demonstrated suitable for stress response studies. Using genetic approaches only one L. rhamnosus strain among four strains studied was found to harbour two copies of genes encoding chaperone ClpL (named clpL1 and clpL2). Both clpL genes were found to be increasingly expressed in after heat stress. Moreover, our results strongly indicate that the L. rhamnosus strain has

acquired the clpL2 from L. plantarum by horizontal gene transfer and present the first example of transfer of a stress gene between Lactobacillus species. A L. gasseri clpL mutant strain was shown to be 1000-fold more sensitive to lethal heat than its isogenic control. Furthermore, L. gasseri clpL is a novel member of HrcA (heat regulator for chaperones) regulon. The expression level of ClpL in a probiotic strain might be a good indicator whether the adaptation to technologically relevant conditions has been succesful. Articles 1. Suokko, A., Savijoki, K., Malinen, E., Palva, A., and Varmanen, P. 2005. Characterization of a mobile clpL gene from Lactobacillus rhamnosus. Appl. Environ. Microbiol. 71: 2061-2069. 2. Savijoki, K., Suokko, A., Palva, A., Valmu, L., Kalkkinen, N., and Varmanen, P. 2005. Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by [35S]methionine labelling and two-dimensional gel electrophoresis. FEMS Microbiol. Lett. 248: 207-215. 3. Savijoki, K., Suokko, A., Palva, A., and Varmanen, P. 2006. New convenient defined media for [S]methionine labelling and proteomic analyses of probiotic lactobacilli. Lett. Appl. Microbiol. 42: 202-209. 4. Suokko, A., Poutanen, M., Savijoki, K., Kalkkinen, N., and Varmanen, P. 2008. ClpL is essential for induction of thermotolerance and is potentially part of the HrcA regulon in Lactobacillus gasseri. Proteomics 8: 1029-1041.

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Cold tolerance of Clostridium botulinum PhD student: Henna Söderholm (University of Helsinki / Department of Food and Environmental Hygiene) E-mail: [email protected] Supervisors: Prof. Hannu Korkeala and Doc. Miia Lindström (University of Helsinki / Department of Food and Environmental Hygiene) Funding: ABS Graduate School ___________________________________________________________________________ Introduction Clostridium botulinum is an anaerobic, Gram-positive, spore-forming bacterium. It is widely distributed in the environment and therefore easily ends up contaminating raw material of foods. C. botulinum produces highly potent neurotoxin, which causes a life threatening disease called botulism. Food-related botulism is an intoxication that results from the ingestion of food contaminated with the toxin. The ability of some strains of C. botulinum to grow and produce this potentially lethal toxin in foodstuffs preserved at low temperatures poses a serious risk for food safety. Aim The aim of the study is to obtain new information about the genetics related to cold tolerance in C. botulinum. The results can be used in food safety as well as at a level of basic research. The ability of C. botulinum to grow at low temperatures and the functions of the cold-related genes are studied. Both conventional and molecular biological methods are employed. Results and discussion In the first part of the study genome-wide transcriptional analysis with DNA microarrays (Institute of Food Research, Norwich, UK) was employed to study the effect of low temperature on gene expression of group I Clostridium botulinum type A strain ATCC 3502 and to screen for interesting genes for further research. Based on log odds scores of differential expression, approximately 700 genes were either up- or down-regulated

(p