International Conference Foodomics

U n i ve rs i t y o f B o l o g n a M a rc h e Po l y t e c h n i c U n i ve rsi t y

I nternational C onference on

F ood o mics 2 8 t h a n d 2 9 t h M ay, 2 0 0 9 Cesena, Italy

Abstract Book

U n ive rsit y o f Bo l o gna M a rc he Po l y t ec h n ic U n ive rsi t y

I nternational C onference on

F ood o mics 28

th

and 29

th

M ay, 2 0 0 9

Cesena, Italy

Abstract Book

Cover photographic composition by Gianfranco Picone Abstract book composition by Francesca Danesi & Gianfranco Picone

International Conference on FOOD-OMICS

Preface FOOD-OMICS A science for nutrition, health and wellness in the post-genomic era Since the human genome was completely sequenced in 2003, a new postgenomic era appeared in the “life sciences”. It was soon clear that, with the conclusion of this ambitious project, new paths were traced, leading to improve human health and well-being. From genomics, that studies the structure of the whole genome of an organism through its mapping, three other major -omics sciences were originated: transcriptomics, which studies and defines the entire set of transcripts (m-RNA), in relation to different environmental factors that can have effects on the comprehensive biological pathways; proteomics, which deals with the study of the whole protein complement of an organism, including species resulting from changes occurring after the m-RNA translation; and finally metabolomics, addressing the definition of a “molecular fingerprint” linked to all the small molecules of metabolites, which are the end product of gene expression. Transcriptomics, proteomics and metabolomics are all dynamic realms, as affected by interactions between the body and external stimuli. It is for this reason that a great interest has developed relating to the influences that biotic and abiotic factors exert on gene expression. Among the abiotic factors, of predominant importance is the action of food components in the regulation of genes, which may intervene, in a positive or negative way, on the risk of occurrence of certain human diseases. This approach is fundamental to the science of nutrigenomics, the principal purpose of which is aimed at understanding the mechanisms by which dietary intake modulates the function of genes and, hence, metabolism. In this context, it is clear that nutrition, when studied at the molecular level using a holistic view, acquires dignity of –omics science. Nutritional genomics, or nutrigenomics, is therefore an integrated science that would explain, at genetic and molecular level, how nutrients, commonly taken by the diet, may alter the expression and/or the genetic structure of an individual, thus altering the balance between health and disease. It is with this approach that questions related to a proper nutrition must be addressed, based on the concept that food and drink affect in several ways the health state of each individual consumer. I

A Science for Nutrition, Health and Wellness in the Post-Genomic Era

The ability to identify the relationship between diet and disease is a key element in the precise primary preventive strategies aimed at improving the health conditions of the population and of the individual. In this vein, we must also consider how, during the evolution of food consumption, the introduction of new nutritional factors, associated with a substantial changes in the way of producing foods, has an impact on the health of the human organism. Special attention deserve the effects of (bio)technological changes on the genetic and molecular composition of the food, that ultimately could affect the interactions between nutrients and the human genome. Indeed, the progressive development of molecular biology has made it possible to prepare numerous protocols for genetic manipulation of plants and animals intended for human consumption. This development has provided new opportunities in the food sector in terms of increasing production and improving quality. At the same time, however, the introduction of biotechnology in food has raised considerable doubts, especially among consumers who demand new concepts and new parameters defining (Total Food Quality) and ensure (Food safety) the total quality of a food. A multidisciplinary approach is required, dealing with the study of food and humans, from genes to metabolites, including the analysis of nutritional components, both endogenous or introduced by (bio)technology, of healthy components and allergenic or toxic substances, such as xenobiotics, as well as the evaluation of presence of pathogenic microorganisms and their toxic metabolites. The focus is then to be put to the study of all interactions in order to give better regulations and to ensure security and quality to food productions. Therefore, the application of the -omics approach in food sciences (Foodomics) plays an important role in the understanding of the nutritive potentials of a conventional or modified food; on the other hand, allows us to better understand why not all foodstuffs can be eaten by everyone, so encouraging the development of a "technology" for producing food made to measure, or of a "science" for the preparation of personalized diets. The international conference "Food-omics: a science for nutrition, health and wellness in the post-genomic era" will touch different topics with two sessions that address the definition of molecular quality of conventional food, the effects of (bio)technological processing on the molecular profile of food, the interaction II


between nutrients and human genes, the study of the nutritional and general health state of humans through the definition of their metabolic profile, the beneficial or toxic effects of individual classes of substances isolated from food, and more yet. The conference will be held in Cesena, a city located in the Italian territory marked by a strong eno-gastronomic tradition, location of the Food Sciences Campus of Alma Mater - University of Bologna. The conference will consist of invited lectures organized in thematic sessions covering, as much as possible for a two-days frame, the comprehensive genomics, proteomics and metabo(l/n)omics of food and its interactions with humans. Due to the multidisciplinary coverage of conference, all speakers will offer a general view of their leading research work. Francesco Capozzi Chair of the Scientific Committee

Giuseppe Placucci Chair of the Organizing Committee

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SCIENTIFIC COMMITTEE Francesco Capozzi - chair Cesena, Italy Søren B. Engelsen Copenhagen, Denmark

Harry Kuiper Wageningen, The Netherlands

Jose A. López Jimenez Murcia, Spain

Luisa Mannina Campobasso, Italy

ORGANIZING COMMITTEE Giuseppe Placucci - chair Cesena, Italy Elena Babini Cesena, Italy

Alessandra Bordoni Bologna, Italy

Achille Franchini Bologna. Italy

Bruno Mezzetti Ancona, Italy

SECRETARIAT E-mail: [email protected] Francesca Danesi Cesena, Italy

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Mattia Di Nunzio Cesena, Italy

Gianfranco Picone Cesena, Italy


ORGANIZED BY

University of Bologna

Marche Polytechnic University

Linked with the COST863 International Summer Berry School

Under the sponsorship of

Supported by

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Speakers’ Abstracts

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Improving meat quality by regulating animal feeding and genetics: recent advances in poultry Cécile Berria, Elisabeth Le Bihan-Duvala, Vonick Sibuta,b, Maamer Jlalia, Vérane Gigaudb and Michel J. Duclosa a INRA, UR83 Recherches Avicoles, F-37380 Nouzilly, France b Institut Technique de l’Aviculture, 37380 Nouzilly, France With changes similar to those that occurred in the swine industry, the poultry market is now characterized by an increasing diversity of further processed products. In 2006, the French market share for the whole chicken was 39% against 38% for cut and 23% for processed products. As a consequence, poultry companies are now involved in food technology and product development, and the improvement of meat processing ability has become a more prevalent concern. In chickens, as in pigs post-mortem pH is a key factor controlling meat quality [1]. Variations in meat ultimate pH (pHu) are responsible for variations in several breast meat properties, including water-holding capacity, color and firmness [2]. Low ultimate pH results in “acid meat”, with a pale aspect and reduced water-holding capacity, while high ultimate pH leads to DFD (dark, firm, dry) meat with dark color and poor storage quality. At the genetic level, there is a very strong negative correlation between the ultimate pH of breast meat and the level of muscle glycogen estimated by the Glycolytic Potential (GP) at slaughter time (rg -0.97). The GP was also shown to be highly heritable (h2 0.43) [2]. Understanding the mechanisms and identifying the genes controlling muscle glycogen stores constitutes a promising way to better control and improve chicken breast meat properties. It would allow developing useful breeding tools, such as molecular markers, to select birds with expected meat properties, and help optimizing rearing practices, via the study of gene regulation. [1] C. Berri, E. Le Bihan-Duval, M. Debut, V. Santé-Lhoutellier, E. Baéza, V. Gigaud, Y. Jégo, M. J. Duclos (2007) Consequence of muscle hypertrophy on characteristics of Pectoralis major muscle and breast meat quality of broiler chickens. J. Anim. Sci. 85: 2005-2011. [2] E. Le Bihan-Duval, M. Debut, C. Berri, N. Sellier, V. Santé-Lhoutellier, Y. Jégo, C. Beaumont 2008 Chicken meat quality: genetic variability and relationship with growth and muscle characteristics. BMC Genetics 9: 53.

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Auxin and jasmonic acid interaction in the control of late stamen development in Arabidopsis Valentina Cecchetti1, Giuseppina Falasca2, MariaMaddalena Altamura2, Karin Ljung3, Paolo Costantino1 and Maura Cardarelli4 [email protected] 1 Dipartimento di Genetica e Biologia Molecolare, 2Dipartimento di Biologia Vegetale, 4Istituto Biologia e Patologia Molecolari, CNR, Università La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy, 3Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Umeå, Sweden. In plants, growth and development are controlled by a limited number of hormones whose roles and interplay begin now to be unravelled at the genetic and molecular level. Among plant hormones, auxin plays arguably the most prominent role, and its mechanism of action has been clarified in diverse developmental processes. Based on an extensive genetic and molecular analysis in Arabidopsis, we have proposed a model on the role of auxin in late stamen developmental processes: anther dehiscence, pollen maturation and filament elongation. Expression of auxin-sensitive reporter constructs indicates that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 indicate that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. In tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes by analysing the mdr1 pgp1 mutants affected in auxin transport, and show that while auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, auxin transport contributes to the independent regulation of preanthesis filament elongation. Our model envisages that a peak in auxin concentration at the end of meiosis triggers filament elongation and prevents premature anther dehiscence and pollen maturation; the subsequent decline in auxin concentration releases the block and triggers these processes (1).

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To further test our model we have measured auxin concentration in anthers at different developmental stages and determined the relative role of single tir, afb receptors in the different stamen developmental processes. It has been demonstrated that the phytormone jasmonic acid (JA) is involved in the final stages of anther dehiscence pollen maturation and pre-anthesis filament elongation, whereas we established that auxin acts on the initial stages of these processes. To determine if auxin acts on the levels of JA the expression of JA-biosynthesis genes and the JA content are being measured in floral buds at different stages of development. The role of auxin in late stamen development will be discussed in view of our model and recent data. 1) Cecchetti, V., Altamura, M.M, Falasca, G., Costantino, P. and Cardarelli, M. (2008) Auxin regulates Arabidopsis anther dehiscence, pollen maturation and filament elongation. Plant Cell, 20, 1760-1774.

Metabolomics and other methods for tracing the origin of chicken meat Ian J. Colquhoun, Yvonne M. Gunning and Simon D. Kelly Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK There are a number of driving forces for the development of reliable analytical methods to verify the provenance of the food we eat. The UK Food Standards Agency has consulted the public on a number of key issues relating to food labelling. The FSA’s findings clearly demonstrated that ‘country of origin labelling’ was high on the consumers’ list of demands for change. There is growing enthusiasm among consumers for high quality food with a clear regional identity. As well as heightened consumer awareness there are a number of important legislative driving forces for reliable techniques to verify food provenance, such as Protected Food Names (Protected Designation of Origin and Protected Geographical Indication). Recent food scares have raised public concerns especially with regard to the origins of meat and its means of production. The EU FP6 project TRACE addresses the primary objective of the European Food Quality and Safety Priority to protect the health and well-being of citizens by assuring the quality of their food and improving control of food production. TRACE aims to support this objective by providing a transparent means of assessing the identity of a food product together with a system for monitoring the authenticity of that product. 5


A major theme of TRACE has been exploitation of the natural variation that occurs in the isotopic content of the bio-elements hydrogen, carbon, oxygen and sulphur to determine geographical origin of chicken meat and dietary patterns that can act as indicators of geographical location. We have also explored whether metabolite profiling methods (based on 1H NMR and LC/MS/MS) in combination with univariate and multivariate statistical methods can be used to indicate geographical origin. Chicken meat samples from Europe, S. America, Thailand and China have been collected and analysed using metabolite profiling and isotope ratio methods. For ease of interpretation a Partial Least Squares – Discriminant Analysis method was used (NOPLS) to determine if the samples could be classified using the NMR and LC/MS/MS data. A systematic pair-wise comparison showed that each group could be discriminated from every other group with a good rate of success by either method. Good success rates were also obtained in distinguishing samples from one group versus all other samples combined. A number of amino acids, amino acid derivatives and ammonium compounds were identified as compounds mainly responsible for the discrimination. Features of the metabolite profiling approach will be discussed in comparison with the better established stable isotope methods.

When food meets man: the epigenetic approach Emma De Fabiania, Nico Mitroa, Federica Gilardi a, Andrea Galmozzia, Donatella Carusoa,b, Maurizio Crestania a Department of Pharmacological Sciences, Università degli Studi di Milano, Italy b Center for the Characterization and Safe Use of Natural Products “Giovanni Galli” Post-translational modifications of chromatin, including histones and other DNAassociated proteins, impose dramatic changes in chromatin structure and nucleosomal organization, thus profoundly affecting gene transcription. Collectively, these events contribute to the epigenetic control of gene transcription. In recent years, a wide array of post-translational modifications, e.g. acetylation, methylation, phosphorylation, ubiquitinylation, have been discovered to occur in the regulation of gene expression, and a number of regulators modulating these reactions have been discovered as well. According to this view, the phenotypic response either to food intake, and to individual nutrients should be investigated also considering post-translational modifications 6


at the level of histone proteins and/or transcription factors and other DNAinteracting proteins. Bile acids are required for digestion and absorption of lipids in the small intestine and are produced in the liver from cholesterol, through complex and highly regulated metabolic pathways. Bile acid synthesis is regulated at the transcriptional level through several mechanisms, some of which are linked to environmental conditions, including the nutritional state. At this regard we found that the transcriptional regulation of the genes involved in bile acid synthesis is strictly associated to the fasted-to-fed cycle, at least in animal models, since it is repressed in the fed state and up-regulated during fasting [1]. More interestingly, by applying technologies based on chromatin immunoprecipitation, we could demonstrate that the underlying mechanism consists in the formation of an active (fasting-state signaling) or repressive (fed-state signaling) complex at the promoter of the gene encoding cholesterol 7-hydroxylase, a key enzyme in bile acid synthesis [1]. We lately discovered that bile acids returning to the liver in the post-prandial phase, promote the nuclear import of histone deacetylase 7 (HDAC7) in liver cells, which contributes to the assembly of a repressive complex at the promoter of the cholesterol 7-hydroxylase gene [2]. It can be argued that adaptation to the nutritional state may occur at other DNA regions relevant in gene transcription. Indeed we found that similar changes in chromatin structure occur at the level of key genes in gluconeogenesis, another metabolic pathway strictly linked to the fasted-to-fed cycle [1]. However, it should be underlined that promoter-specific assembly of repressive/activating complexes makes this type of regulation extremely gene-specific. The advances in our understanding of the mechanisms contributing to the epigenetic regulation of gene transcription have also implied the discovery of small molecules modulating enzymes and proteins involved in the posttranslational modifications of histones and other DNA-interacting proteins. In particular, several compounds present in foods and nutrients display the ability to modify chromatin and other DNA-associated factors. For example, isothiocyanates found in cruciferous vegetables and garlic allyl compounds have been shown to inhibit HDACs. This biological activity contributes to the proapoptotic properties of these compounds and ultimately, to the chemopreventive effects of vegetable-rich diets. Another example is represented by resveratrol, a phenolic compound exhibiting the ability to activate sirtuins, a unique class of histone deacetylases, implicated in a wide variety of biological processes, including longevity and energy metabolism. In fact, it was shown that 7


resveratrol administration increases aerobic capacity in mice and protects animals against diet-induced obesity and insulin resistance [3]. These beneficial effects of resveratrol are associated to induction of genes for oxidative phosphorylation and mitochondrial biogenesis in skeletal muscles and largely dependent on its ability to activate sirtuins [3]. Mitochondrial biogenesis is most likely dependent on activation/induction of specific trancription factors and cofactors. In fact, we could demonstrate that resveratrol is able to increase the transcriptional activity of the mitochondrial transcription factor A (mTFA) promoter in myoblasts [4]. In summary, the elucidation of the epigenetic mechanisms governing gene transcription and expression will help understand the complexity of the adaptive response to food intake and to better define the biological properties of some nutrients. [1] De Fabiani E, Mitro N, Gilardi F, Caruso D, Galli G, Crestani M. Coordinated Control of Cholesterol Catabolism to Bile Acids and of Gluconeogenesis via a Novel Mechanism of Transcription Regulation Linked to the Fasted-to-fed Cycle. J Biol Chem 2003; 278:39124-39132. [2] Mitro N, Godio C, De Fabiani E, et al. Insights in the regulation of cholesterol 7alpha-hydroxylase gene reveal a target for modulating bile acid synthesis. Hepatology 2007; 46:885-97. [3] Lagouge M, Argmann C, Gerhart-Hines Z, et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell 2006; 127:1109-22. [4] Galmozzi A, Mitro N, Crestani M, De Fabiani E, unpublished observations

NMR spectroscopy and chemometrics: new interdisciplinary playgrounds for nutritionalists and food scientists Søren Balling Engelsen Department of Food Science, Faculty of Life Sciences, Copenhagen University, DK1958 Frederiksberg, Denmark The number of applications of multivariate data analysis (chemometrics) to series of NMR spectra is rapidly increasing due to an emerging interest for quantitative NMR spectroscopy e.g. in the pharmaceutical and food industries. Nutrimetabolomics is a newly emerging field of "omics" research concerned with the comprehensive characterization of metabolites in human body fluids as a 8


function of nutritional status and/or intervention. In humans, biologically relevant samples can easily be obtained from blood, sweat, urine and faeces. Since the establishment of NMR metabonomics [1] for studying responses to pathophysiological stimuli substantial efforts has been devoted to the application of NMR screening methods for blood and urine. The extension to nutrimetabolomics is straight forward. The prospect of nutri-metabolomics is gigantic as it provides a tool for objective monitoring the human metabolic response to dietary interventions and lifestyle habits and thus has the potential to become the most valuable tool for research in food related life style diseases such as obesity and cardiovascular diseases. Through nutri-metabolomics it will be possible to investigate and monitor the influence of nutritional interventions upon phenotype or genotype via specific biochemical profiles and events. A serious challenge in the use of NMR in nutri-metabolomic studies lies between the technical capacity to generate data and the human capacity to interpret and integrate these data. In a split second 1H NMR can generate 65.000 variables which hold information about all the protons in a biological sample. Effective multivariate evaluation of such large data sets is of key importance in order to obtain meaningful and interpretable results. This paper gives an analysis of advantages and limitations of applying some advanced chemometric procedures [2-4] for selected classification and quantification problems in food and nutritional sciences [5-7]. [1] J.K. Nicholson, J.C. Lindon & E. Holmes: 'Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica (1999) 29:11811189 [2] R. Bro. PARAFAC. Tutorial and applications. Chemom. Intell. Lab. Syst. (1997) 38:149-171 [3] L. Nørgaard, A. Saudland, J. Wagner, J.P. Nielsen, L. Munck & S.B. Engelsen, Interval partial least squares regression (iPLS): A comparative chemometric study with an example from near infrared spectroscopy, Applied Spectroscopy (2000) 54:413419. [4] L. Nørgaard, R. Bro, F. Westad & S.B. Engelsen, A modification of canonical variates analysis to handle highly collinear multivariate data, Journal of Chemometrics (2006) 20:425-435. [5] M. Dyrby, M. Petersen, A.K. Whittaker, L. Lambert, L. Nørgaard, R. Bro & S.B. Engelsen: Analysis of lipoproteins using 2D diffusion-edited NMR spectroscopy and multi-way chemometrics. Analytica Chimica Acta (2005) 531:209-216.

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A Science for Nutrition, Health and Wellness in the Post-Genomic Era [6] H. Winning, E. Roldán-Marín, L.O. Dragsted, N. Viereck, M. Poulsen, C. SánchezMoreno, M.P. Canoc & S.B. Engelsen, An exploratory in vivo metabonomic investigation reveals dimethylsulfone as a robust biomarker for onion intake, submitted (2009). [7] F. Savorani, G. Picone, A. Badiani, P. Fagioli, F. Capozzi & S.B. Engelsen, Metabolic profiling and aquaculture differentiation of gilthead sea bream by 1H NMR metabonomics, submitted (2009).

Could studies on gene-nutrient interaction maintain the promise of good health and/or prevention of chronic diseases Lynnette R. Ferguson Discipline of Nutrition, Faculty of Medical & Health Sciences, The University of Auckland, Auckland, New Zealand There is good reason to believe that what is good dietary advice for one individual may be inadequate or even dangerous for another (1). Many of the differences between individuals relate to variations in single nucleotide polymorphisms (SNPs), which modify the way in which nutrients affect the expression of genes. For example, our studies in Inflammatory Bowel diseases have shown that SNPs in several key genes are associated with increased disease risk of this disease in New Zealand, but that the pattern of genes associated with the disease risk differs from that seen in some studies in large areas such as Europe. One important explanation of these differences is that the risk is not associated with the gene per se, but results from a gene-diet interaction (2). Thus, the maintenance of optimal health or prevention of disease will be most effective if it is based on an understanding of genetics and genomics. Differences between individuals can be compared at a population level using the technologies of genetic epidemiology, and can be applied to optimal developments of functional foods, as well as optimised personalised dietary advice (1). It is increasingly apparent that studies on gene-nutrient interactions are essential, in order to maintain optimal health and prevent chronic diseases. [1]. LR Ferguson (2009) Nutrigenomics Approaches to Functional Foods. JADA 209: 452-458 [2]. I Petermann, CM Triggs, C Huebner, DY Han, RB Gearry, ML Barclay, PS Demmers, A McCulloch, LR Ferguson (2009) Mushroom intolerance: a novel dietgene interaction in Crohn’s disease, Br. J. Nutr., online Mar 31 2009, doi:10.1017/S0007114509276446 10


Genetic resources and breeding for improved nutritional quality in berry crops Chad Finn United States Department of Agriculture -Agricultural Research Service; Horticultural Crops Research Laboratory; Corvallis, Oregon USA Selecting the best, biggest, highest yielding genotypes is as old as humankind. As society shifted from a hunter-gather to a sedentary, agriculturally oriented lifestyle, our ancestors selected their favorite genotypes to plant near their homes. This repeated selecting of the best drove the development of most of our cultivated crops for thousands of years. Only recently, especially in the 19th and 20th Century’s did formal plant breeding begin to play a major role in crop improvement. Berries were among the last crops to be domesticated and to have formal improvement efforts begun on them. This recent domestication means that it is still relatively easy to go back to species material for new traits but this also means that the knowledge base that we draw on as scientists is relatively small. With a better understanding of human nutrition and the fact that many public health issues could be alleviated with an increased consumption of fruits and vegetables, it is important to assess how we might improve berry cultivars to help meet this need. While there are a host of potential new technologies and knowledge associated with genomics/metabolomics/proteomics, for the foreseeable future we are going to primarily rely on traditional breeding techniques to improve cultivars. Also for the foreseeable future, it will be important to approach improvement with the same, tried and true objectives of trying to improve fruit desirability combined with cultivars that reduce production costs making the fruit available to the greatest possible portion of the population. Polyphenolics/anthocyanins, which are a primary component of fruit color in berries, are very good for human nutrition for reasons that are not fully understood. Improving fruit color is a relatively straight forward and good way to improve the nutrition of many berries and a significant amount of research has been conducted in this area. As we are confronted with the latest nutritional fancy of the consuming public (i.e. antioxidants), it is important that we not forget the primary objectives, while at the same time stand ready to incorporate new tools and knowledge into our breeding programs as soon as it makes sense to do so. Some suitable references Clark, J.R., E.T. Stafne, H.K. Hall, and C.E. Finn. 2007. Blackberry breeding and genetics. Plant Breeding Reviews 29:19-144.

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Connor, A.M., C.E. Finn, T.K. McGhie, and P.A. Alspach. 2005. Genetic and environmental variation in anthocyanins and their relationship to antioxidant activity in blackberry and hybridberry cultivars. J. Amer. Soc. Hort. Sci. 130:680687. Connor, A.M., C.E. Finn, and P.A. Alspach. 2005. Genotypic and environmental variation in antioxidant activity and total phenolic content among blackberry and hybridberry cultivars. J. Amer. Soc. Hort. Sci. 130:527-533. Deighton, N. R. Brennan, C. Finn, and H.V. Davies. 2000. Antioxidant properties of domesticated and wild Rubus species. J. Sci. Food Agric. 80:1307-1313. Finn C.E. and J.F. Hancock. 2008. Raspberries. p. 359-392. In: J. F. Hancock (ed), Temperate fruit crop breeding: Germplasm to genomics. Springer Finn C.E. 2008. Blackberries. p 83-114. In: J. F. Hancock (ed), Temperate fruit crop breeding: Germplasm to genomics. Springer Luby, J.J. and D.V. Shaw. 2009. Plant Breeders perspecitves onimproving yeidl and quality traits in horticultural food crops. J. Amer. Soc. Hort Sci. 44:20-22. Moyer, R., K. Hummer, C. Finn, B. Frei, and R. Wrolstad. 2002. Anthocyanins, phenolics and antioxidant capacity in diverse small fruits: Vaccinium, Rubus and Ribes. J. Agric. Food Chem. 50:519-525. Siriwoharn, T, R.E. Wrolstad, C.E. Finn, and C.B. Pereira. 2004. Influence of cultivar, maturity and sampling on blackberry (Rubus L. hybrids) anthocyanins, polyphenolics, and antioxidant properties. J. Agric. Food Chem. 52:8021-8030. Wrolstad, R.E., T. Ngo, C.E. Finn, and Y. Zhao. 2008. Color quality of fresh and processed strawberries. ACS Symp. Series 983:18-42.

Functional Genomics in Strawberry (Fragaria spp.) as Applied to Questions of Fruit Yield and Quality Kevin M. Folta Horticultural Sciences Department and the Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL, USA [email protected] Strawberry (Fragaria spp.) is an excellent system to test gene function, particularly with regard to genes that may affect fruit quality and character. Protocols have been devised for rapid transformation and regeneration of diploid (F. vesca; 5AF7) and octoploid (F. x ananassa; LF9) genotypes, providing a means for functional gene tests and gene discovery in planta. A comprehensive 12


transcriptome has been described for strawberry fruits in both diploid and octoploid strawberry, revealing the identity of the components that potentially shape important horticultural characters. Our laboratory has focused on previously uncharacterized genes and found transcripts that affect fruit shape and size, along with plant stature. Other novel genes contribute to higher yields and robust flowering. Manipulation of blue light photosensors also affects flowering and will potentially modulate nutritional content of fruit. These studies are linked to direct tests of consumer preferences in an effort to develop markers associated with flavor and fruit quality. The findings of these studies may likely translate well to other rosaceaeous crops, as the mechanisms that underlie important traits are probably similar between species.

Animal derived foods in relation to chronic diseases D Ian Givens Nutritional Sciences Research Unit, Animal Science Research Group, School of Agriculture, Policy and Development, University of Reading, United Kingdom. In spite of the recognised benefits of reducing saturated fatty acid (SFA) intake few parts of the EU meet recognised targets. Milk and dairy products represent the single largest source of dietary SFA in most countries yet epidemiological evidence indicates that milk has cardio-protective properties [1] such that simply reducing consumption of dairy foods to meet SFA targets may not be a sound public health approach. This paper explores the options for replacing some of the SFA in milk fat with cis-MUFA through alteration of the diet of the dairy cow and the evidence that such changes can improve the indicators for CVD. In addition, the beneficial effects of long chain (LC) (carbon chain 20) n-3 PUFA are well documented but recent evidence indicates that few people achieve the UK daily recommended intake for adults of 450 mg of EPA + DHA per day. In many parts of Europe the daily intake of EPA + DHA by adults and especially young people is less than 100 mg per day, since many never eat oily fish [2]. There is also concern that intake of n-6 PUFA has increased excessively and is aggravating the effects of low LC n-3 intake. Poultry meat contributes small but worthwhile amounts of EPA + DHA and studies to enrich the EPA + DHA content of animal-derived foods will be described and how this would impact on habitual intake. Research is however required to characterise the benefits associated with lipid-modified foods and to understand how the active compounds in natural foods can be 13


enhanced. In the future, the role of animal nutrition in creating foods closer to the optimum composition for chronic health is likely to be more important particularly in relation to an increasingly aged population. It will also require political will to ensure that financial incentives to create such foods are in place. [1] P.C. Elwood, D.I. Givens, A.D. Beswick, A.M. Fehily, J.E. Pickering, J.Gallacher (2008) The survival advantage of milk and dairy consumption: An overview of evidence from cohort studies of vascular diseases, diabetes and cancer. J. Am. Coll. Nutr. 27: 723S-734S [2] D.I.Givens, R.A.Gibbs (2008) Current intakes of EPA and DHA in European populations and the potential of animal-derived foods to increase them. Proc.Nutr. Soc. 67: 273-280

The use of omics technologies as part of the comparative safety assessment of newly developed (GM) plant varieties Esther J. Kok and Jeroen P. van Dijk RIKILT - Institute of Food Safety - The Netherlands Different international advisory platforms (OECD, FAO/WHO) have advocated the use of omics technologies in the identification and assessment of unintended side effects of plant breeding strategies, primarily for those strategies that comprise genetic modification steps. Nowadays, omics technologies have shown their robustness in different areas of research and several studies have shown that omics technologies can have added value in safety assessment strategies of novel (GM) plant varieties. This may become increasingly relevant now that new and enhanced techniques are being used as part of plant breeding strategies with, as a result, plants with altered traits that may be more complex and change the physiology of the plant more profoundly than was the case so far. Another trend shows that more often traits are modified to the benefit of the consumer, that may influence metabolic routes that have not often been targeted so far and/or that may affect consumers to a larger extent. These developments will have implications for the safety assessment of the resulting plant products. As omics technologies can provide a more holistic profile of the newly bred plant varieties, with emphasis on the edible parts, they may trace alterations in the plant’s physiology that remain undetected on the basis of so-called targeted analyses. In recent years, large European projects, such as the integrated European research project SafeFoods (www.safefoods.nl), have, amongst many 14


other issues, further investigated the applicability of omics technologies to this end. The results of these projects will be discussed, also in the light of the current regulatory division between products of breeding strategies using recombinantDNA techniques and other breeding strategies.

When man ingests polyphenols: what happens to the polyphenols and how do they influence cardiovascular function? Paul A Kroon Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom. Polyphenols are plant secondary metabolites and are present ubiquitously in plant foods. The largest group of plant polyphenols are the flavonoids and these have received particular scientific attention since it was shown that increased consumption of particular flavonoid subclasses was associated with reduced risk of certain age-related diseases such as cardiovascular disease[1] (REF). Subsequently, researchers sought to prove that flavonoids do indeed protect against age-related diseases, and in particular, research effort focussed on identifying potential mechanisms. The very numerous in vitro studies reported subsequently have identified a large number of cellular responses and noncellular actions of flavonoids that appear to be consistent with an ability to protect against disease. However, the majority have been flawed in that they have not considered what happens to dietary flavonoids once ingested. In particular, it has become clear that (i) flavonoids and other polyphenols are only partially absorbed from the gastrointestinal tract, and (ii) that the small portion that is absorbed is efficiently metabolised such that the forms reaching the blood are not the same structures as those in the food, and their physicochemical and biological properties are substantially altered[2]. In the first part of this presentation, the current state of the art concerning flavonoid absorption and metabolism will be briefly reviewed. The key factors influencing the extent of polyphenol absorption will be discussed, the achievable plasma concentrations described, and the changes occurring in polyphenol structure (typically glucuronidation, sulfation and methylation) will be highlighted. In the second part of the presentation, the influence of metabolism on the biological properties of flavonoids will be addressed. For many years, my group 15


have used quercetin, a pentahydroxylated flavonol, as a model flavonoid to address this question. I will present data from studies using cultured cell models showing that in most cases the highly efficient conjugation of quercetin that occurs during absorption effectively eliminates many of the potentially beneficial properties of the un-conjugated parent molecule. I will also present examples where certain of the conjugated derivatives of quercetin found in vivo retain substantial biological activity. Hence, use of an appropriately designed in vitro model can identify mechanisms that are plausible in vivo. To end with, I will present some data where we used transcriptomics to interrogate the changes in vascular endothelial cells in vitro in response to various catechins[3]. This study provided tremendous insights into which structures were bioactive and which were not, and identified the likely pathways and mechanisms by which procyanidin-rich foods affect endothelial function. References: [1] Hertog et al. (1993) Lancet 342, 1007-1011; [2] Kroon et al. (2004) Am J Clin Nutr 80, 15-21; [3] Garcia-Conesa et al. (2009) Mol Nutr Food Res 53, 266-276.

Functional genomics of allergen gene families in fruits Marzban Gorji, Maghuly Fatemeh and Laimer Margit Plant Biotechnology Unit, IAM, Department of Biotechnology, VIBT BOKU, 1190 Vienna, Austria The Plant Biotechnology Unit of the Dept of Biotechnology is devoted to questions concerning the production of healthy food in a safe environment trying to provide a few suggestions on biotechnological approaches to solve allergen related issues from the viewpoint of the breeder, the farmer and the consumer. The presentation will attempt to present an overview over the work on allergens present in fruit crops carried out at the PBU, Vienna, beginning from the first description of Mal d 1 in apple (Vanek-Krebitz et al. 1995). In the attempt to elucidate the biological function of this important apple allergen, promoter function studies and the detection of MdAP were achieved (Pühringer et al. 2000, 2003). The development of essential detection tools was continued in the frame of the EU project SAFE to answer questions concerning the localization of apple allergens (Marzban et al. 2005), the influence of production and storage conditions on the allergen content of apples (Sancho et al. 2006, 2008), the 16


potential role of pollen as additional source of sensitisation (Marzban et al. 2006), the patients response to allergens (Asero et al. 2006) and the genetic structure of apple genome in relation to major allergens (Gao et al. 2005). The creation of valid genetic and proteomic allergen identification tools allow a fast evaluation of individual sensitisation patterns (Herndl et al. 2007, Marzban et al. 2008b, 2009a). Research was continued in the field of small fruits and berries in the frame of COST Action 863 (Euroberry) (Marzban et al. 2005, 2008, 2009b,c) References Asero R., Marzban G., Martinelli A., Zaccarini M. and Laimer da Câmara Machado M. 2006. Search for low allergenic apple cultivars for birch pollen-allergic patients: is there a correlation between in vitro assays and patient response? European Annals of Allergy and Clinical Immunology 38: 94-98. Gao ZS, van de Weg WE, Schaart JG, van der Meer IM, Kodde L, Laimer M, Breiteneder H, Hoffmann-Sommergruber K, Gilissen LJ. 2005. Linkage map positions and allelic diversity of two Mal d 3 (non-specific lipid transfer protein) genes in the cultivated apple (Malus domestica). Theor Appl Genet. 2005 Jan 13: 1432-1442. Herndl A., Marzban G., Kolarich D., Hahn R., Boscia D., Hemmer W., Maghuly F., Stoyanova E., Katinger H. and Laimer M. 2007. Mapping of Malus domestica allergens by two-dimensional electrophoresis and IgE-reactivity. Electrophoresis 28: 437-448. Marzban G., Pühringer H., R. Dey, S. Brynda, Martinelli A., Zaccarini M., Kolarich D., Altmann F., Katinger H. and Laimer M. 2005. Localisation and distribution of major apple allergens in fruit tissue. Plant Science 169:387-394. Marzban G., Mansfeld A., Hemmer W., Stoyanova E., Katinger H. and Laimer da Câmara Machado M. 2005. Fruit cross-reactive allergens: a theme of uprising interest to consumers health. Biofactors. 23: 235 -241. Marzban G., Mansfeld A., Herndl A., Jäger S., Stoyanova M.E.,, Hemmer W., Katinger H. and Laimer M. 2006. Direct evidence for the presence of allergens in Rosaceae fruit-tree pollen. Aerobiologia 22: 237-245. Marzban G., Herndl A., Kolarich D., Maghuly F., Mansfeld A., Hemmer W., Katinger H. and Laimer M. 2008a. Identification of four IgE-reactive proteins in raspberry (Rubus ideaeus L.). Mol. Nutr. Food Res. DOI 10.1002/mnfr.200700518 Marzban G., Herndl A., Maghuly F., Katinger H. and Laimer M. 2008b. Mapping of fruit allergens by two dimensional electrophoresis and immunodetection. Expert Review of Proteomics 5: 61-75. 17


Marzban G., Herndl A., Pietrozotto S., Banerjee S., Obinger C., Maghuly F., Hahn R., Boscia D., Katinger H. and Laimer M. 2009a. Conformational changes of Mal d 2, a thaumatin-like apple allergen, induced by food processing. Food Chem. Doi: 101016/jfoodchem.2008.06051 Marzban G., Maghuly F., Herndl A., Katinger H. and Laimer M. 2009b. Challenges to screening and identification of putative allergens in berry fruits of the Rosaceae family. Biofactors. accepted. Marzban G., Herndl A., Mezzetti B., Tulipani S., Battino M. and Laimer M. 2009c. Strawberry PRPs fluctuate depending on genotype and seasonal conditions. Acta Hort. in press. Pühringer H., Moll D., Hoffmann-Sommergruber K., Watillon B., Katinger H. and Laimer da Câmara Machado M. 2000. The promoter of an apple YPR10 gene, encoding the major apple allergen Mal d 1, is stress and pathogen-inducible. Plant Science 152: 35-50. Pühringer H., Zinöcker I., Marzban G., Katinger H., Laimer M. 2003. MdAP, a novel protein in apple, is associated with the major allergen Mal d 1. Gene 321: 173-183. Sancho A.I., Foxall R., Beowne T., Dey R., Zuidmeer L., Waldron K.W., van Ree R., Hoffmann-Sommergruber K., Laimer M. and Mills E.N.C. 2006. Effect of postharvest storage on the expression of the apple allergen Mal d 1. J. of Agricultural and Food Chemistry 54 (16) 5917-5923 Sancho A.I., van Ree R., van Leeuwen A., Meulenbroek E.J., van de Weg E., Gilissen L., Puehringer H., Laimer M., Martinelli A., Zaccharini M., Vazquez-Cortés S., Fernandez Rivas M., Hoffmann-Sommergruber K., Mills E.N.C. and Zuidmeer L. 2008. Measurement of lipid transfer protein in 88 apple cultivars. Intl. Archives of Allergy and Immunology. 146:19-26. Vanek-Krebitz M., Hoffmann-Sommergruber K., Laimer da Câmara M., Susani M., Ebner C. Kraft D., Scheiner O. and Breiteneder H. 1995. Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen allergen. Biochem. Biophys. Res. Comm. 214(2): 538-551

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Individual human phenotypes in metabolic space and time Claudio Luchinat Magnetic Resonance Center, University of Florence, Via Luigi Sacconi, 6 50019 Sesto Fiorentino, Italy [email protected] We have recently shown that a thorough statistical analysis performed on NMR spectra of human urine samples reveals an invariant part characteristic of each person, which can be extracted from the analysis of multiple samples of each single subject1.This finding (i) provides evidence that individual metabolic phenotypes may exist and (ii) opens new perspectives to metabonomic studies, based on the possibility of eliminating the daily ‘‘noise’’ by multiple sample collection. This study has been extended to more subjects, confirming the original findings and showing that the discriminant phenotype space is far from being “saturated”. Furthermore, a new collection of samples from the same individuals after two-three years shows that the individual phenotype is relatively stable over this period at least in the examined cohort of young healthy adults2. In phenotyping diseases, blood-derived samples often yield better discrimination than urine samples, probably due to the lower day-to-day variability3,4. Our findings suggest that collection of multiple urine samples may alleviate this problem. Separation of the variable and the invariant parts of the urine NMR fingerprints may actually help add significance to disease-related features in the fingerprints themselves. References 1. Assfalg, M., Bertini, I., Colangiuli, D., Luchinat, C., Schaefer, H., Schuetz, B., Spraul, M., Evidence of different metabolic phenotypes in humans, Proc. Natl. Acad. Sci., 2008, 105, 1420-1424. 2. Bernini, P., Bertini, I., Luchinat, C., Nepi, S., Saccenti, E., Schaefer, H., Schuetz, B., Spraul, M., Tenori, L., Individual human phenotypes in metabolic space and time, submitted 3. Bertini, I., Calabrò, A., De Carli, V., Luchinat, C., Nepi, S., Porfirio, B., Renzi, D., Saccenti, E., Tenori, L., The metabonomic signature of celiac disease, J. Proteome Research, 2009, 8, 170-177. 4. Bertini, Luchinat, Di Leo et al., A metabolomic fingerprint for detection of micrometastatic disease in early breast cancer, in preparation.

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Monitoring of healthy metabolic trajectory with nutritional metabolomics Sebastiano Collino, François-Pierre Martin*, Sunil Kochhar, Serge Rezzi Nestlé Research Center - Switzerland Metabolomics is a well established analytical approach for the analysis of physiological regulatory processes via the metabolic profiling of biofluids and tissues in living organisms indicative. Metabolic profiling techniques based on proton nuclear magnetic resonance (1H NMR) spectroscopy and mass spectrometry (MS) techniques generate complex metabolic profiles that result of multiple metabolic pathway interactions under the influence of environment, lifestyle, genetics, and gut microbiota factors. Multivariate statistical analysis assures the recovery of metabolite patterns indicative of the physiological state or metabolic activity in response to well defined parameters. Its potentials are fully exploited in the field of “nutrimetabolomics” which aims at assessing the effects of active ingredients and foods in individuals. However, one of the greatest challenges in nutrition research is to understand the role of the critical interactions between mammalian organisms and environmental factors, including the gut microbiota, in determining diverse physiological processes and the outcome of nutritional interventions. Due to the recent developments made in this area, nutrimetabolomics is now foreseen as a powerful approach for future nutritional programs tailored at health maintenance and disease prevention.

Cellular and Molecular Immunological Events in Food Allergy Claudio Nicoletti Mucosal Immunology, Integrated Program of GI tract Biology, BBSRC-IFR, Norwich, UK Food allergy is an increasing medical problem in western world but unfortunately, due to our lack of knowledge on many basic issues a therapy is not available yet. Indeed, strict avoidance of the causal food is the only safe approach to the problem. In our laboratory we are interested in understanding the cellular and molecular events underlying adverse reaction to food, the ultimate aim being the identification of therapeutic targets. Allergic reaction is considered to be a T helper (TH) type 2 dominated immune reaction to food components; however the events leading to this type of response are still largely unknown. T helper cells originate from naive T cells following cognate interaction 20


with antigen-presenting cells (APC), mainly dendritic cells (DC). Thus, with this in mind we have investigated the regulatory properties of DCs in allergic/sensitized mice and subsequently humans. Our main interest was to dissect in details various aspects of T cell-DC interaction. We observed that several immunoregulatory features of “allergic” DCs are altered compared to DCs from healthy, non-sensitized donors. We found that DCs from allergic mice and humans tend to survive T cell-mediated apoptosis, an important regulatory mechanism used by the immune system to keep under control and otherwise uncontrollable immune reaction. Further experiments have shown that apoptosis-resistant DCs have the ability to induce IgE production when transferred into naïve syngenic recipients. Also, DCs from allergic donors failed to produce the TH 1-polarizing cytokine IL-12 following appropriate stimuli. The consequences of these alterations of critical immunoregulatory pathways are discussed.

Functional genomics of fruit development in horticultural plants Tiziana Pandolfini Faculty of Science, University of Verona The productivity and agronomic value of many horticultural plants depends on the number of fruits per plant, and on the size, weight and quality of the fruit. Fruit development is a complex process that include molecular, biochemical and structural changes temporally and spatially coordinated. Tomato represents the model for fleshy fruits and the most studied species because of its commercial importance. Tomato development can be divided in three phases. The first phase corresponds to ovule fertilization and fruit set which represents the onset of ovary growth and development into fruit. The second and third phases correspond to fruit growth due to cell division and cell enlargement, respectively and they are followed by ripening. Genetic and biochemical aspects of relatively late stages of fruit growth and maturation have been extensively investigated over the last 15 years. Early phases of fruit development, including fruit set and initial ovary growth, have received less attention, despite their importance for both basic understanding and applied purposes. A model system suitable for studying fruit initiation is represented by parthenocarpic plants that develop fruits without pollination/fertilization. In these plants genes controlling fruit growth can be monitored independently from those involving in embryo and 21


seed development. The transcriptome analysis of pre-anthesis parthenocarpic tomato flower buds has been used to identify genes expressed during early phases of fruit development. 212 transcripts were differentially expressed in parthenocarpic flower buds, 65 of them (31%) with unknown function. The study of the expression pattern during flower and fruit development and a genetic approach based on RNA-interference-mediated gene suppression has been undertaken for some of the genes identified. Recently, the screening carried out led to the discovery of two genes, named Aucsia genes, necessarily required for fruit initiation in tomato, in that their functional suppression caused parthenocarpic fruit development.

Soft Fruit - A Nutritional Nirvana Derek Stewart Head of Plant Products and Food Quality, Scottish Crop Research Institute, Dundee, DD2 5DA, Scotland UK The agricultural policies of the developed world have meant that, for these countries at least, food is plentiful but changing eating patterns have seen an increase in the consumption of ready-made meals and food elevated with respect to sugar and fat. The knock-on effect of this is evident in the rapidly increasing level of obesity in the western world with 20% of males and 25% of females now classified as obese in the United States. Associated with this and the related dietary shift are increases in the incidence of degenerative diseases such as atherosclerosis, some cancers etc. Fundamental, clinical and epidemiological research into the basal causes and consequences of these diseases has highlighted the gross and specific benefits of including a significant level of fruit and vegetable within the diet. Polyphenols are ubiquitous in plants and, with respect to dietary plant sources, particularly prevalent in many fruit species and in particular in the common soft fruit such as strawberries, blackcurrant, raspberries etc. As a result of the putative potent health benefits of these compounds, crop (including fruit) breeding has undergone a paradigm shift with the adoption of global metabolite screening rapidly becoming an integral part of the process. Evidence will be presented to show that soft fruit extracts and components exhibit beneficial health effects in model and real systems. In additions a stratagem for integrating these targets into new and ongoing

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breeding programmes will be outlined using a joint molecular genetic and metabolomics approach

Activation of human transcription factors by food components Adrie J.M. Verhoeven Department of Biochemistry, Cardiovascular Research School COEUR, Erasmus Medical Center, Rotterdam, The Netherlands Food components, or their intracellular metabolites, affect gene expression through modulation of the activity of transcription factors (TFs). This occurs directly, either through binding to the TFs, e.g. as is the case for lipids and the nuclear receptor family, or through induction of the post-translation modification of TFs, e.g. in the case of the deacetylation of PGC1, LXR and FoxO by resveratrol. Alternatively, this may occur indirectly through only partly understood mechanisms, eg. the modulation of SREBP by unsaturated fatty acids. We are interested in the regulation by unsaturated fatty acids of the hepatic lipase (HL) gene, which encodes an extracellular liver enzyme involved in plasma lipoprotein remodeling and reverse cholesterol transport. (Patho)physiological data suggest that HL is upregulated by increased fatty acid supply to the liver, and downregulated by statins. These effects were mimicked in human hepatoma HepG2 cells by using oleate and atorvastatin. Simultaneously, SREBP activity was oppositely regulated. We hypothesized that oleate reduces SREBP activity which in turn leads to increased HL expression. Unsaturated fatty acids indeed downregulated SREBP activity, possibly through the increased flux of cholesterol from the plasma membrane to the ER. In transient transfection assays, SREBP2 overexpression reduced HL promoter activity, but this occurred relatively late. The inhibitory effect of SREBP2 required an intact USF binding site in the HL promoter, and SREBP2 overexpression led to reduced USF1 levels. Complete downregulation of SREBP activity by incubation of HepG2 cells with cholesterol + 25OH-cholesterol, however, failed to affect HL promoter activity, suggesting that the oleate-induced activation of HL expression is not mediated by the suppression of SREBP activity. Finally, we show that oleate increases the amount of USF1, thereby explaining the increase in HL expression. Our data show that food components may affect gene expression through modulation of unexpected networks of transcription factors.

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These studies were supported in part by grants from the Programma Marco Polo, University of Bologna (M. di Nunzio) and the Dutch Heart Foundation (grant number 2001B174).

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Posters’ Abstracts

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Influence of storage time in relation to chemical composition and antioxidant values in berry fruit Liaqat Ali, Birgitta Svensson, Beatrix Alsanius And Marie E. Olsson Department of Horticulture, Swedish University of Agricultural Sciences, P.O.Box 103, SE-23053 Alnarp, Sweden Post harvest life of the berry fruits is important from nutrient and health aspects. The metabolism is continuing although the fruit is detached from the plant and the content of nutrients as well as other healthful compounds such as antioxidants might therefore be changed and therefore it is important to preserve the nutritional value of the berry fruits. In this study raspberries (Rubus idaeus, cv. Polka) and blackberries (Rubus fructicosus, cv. Loch Ness) were subjected to different storage periods (0-9 days) at 2°C to analyze the effect of storage on the contents of different nutrients (Vit.C and sugars) and antioxidants (ellagic acid, anthocyanins and total phenolics). High performance liquid chromatography with photodiode array detection was used for these analyses. Ellagic acid was stable in both raspberries and blackberries (103.06 to 113.70 and 172.24 to 181.59 mg/100g f.w. respectively) during storage. Anthocyanins increased during storage (24.05 to 29.01 mg/100g f.w.) in raspberries but decreased in blackberries (67.70 to 57.04 mg/100g f.w.). Vitamin C in raspberries ranged from 21.94 to 27.96 mg/100g f.w. but was comparably low in blackberries (10.78 to 12.11 mg/100g f.w.). Significant changes in total phenolics and sugars during different storage periods (0-9 days) were found. Both total phenolics and sugars decreased and then increased with increasing storage time. The results suggest that storage caused no negative effects on the content of sugars and antioxidants. Keywords: Anthocyanins, antioxidants, ascorbic acid, blackberries, ellagic acid, raspberries, storage, sugars and total phenolics.

The influence of the planting substrate on fruits quality of high bush blueberry cultivars recently released in Romania Irina Ancu, Paulina Mladin, Mihai Iancu, Alina Nuta, Sergiu Ancu and Mladin Gheorghe Research Institute For Fruit Growing Pitesti, Romania The largest area planted with highbush blueberry culture (Vaccinium corymbosum L.) are located in the United States, and recently started to become 27


a commercial crop in Europe (Kader et al. 1996). In Romania blueberry was introduced for the first time in 1968 at the RIFG Pitesti-Romania. This species requires special growing conditions: soils with high organic matter, low pH and good permeability. Such soils is found in Romania in small areas. Currently, in the country are approximately 50 hectares of highbush blueberry culture and to maintain soil acidity at a optimum pH for this species (4,8-5,5) chemical and organic amendments are usually applied. Most blueberries (95%) produced on these plantations are exported to UE countries. Fruit are appreciated for their low caloric content, richess in bioactive compounds with high antioxidative activity and important anticarcinogenic effect (Schmidt et al. 2005). Thus in the period 2005-2008 at RIFG- Pitesti Mărăcineni studies on the quality fruit on 6 cultivars, recently released in Romania were performed. Simultan, Delicia, Lax, Compact, Augusta, Azur cvs. were grown on 3 planting substrates (peat 50%+ manure 50%; coniferous litter 100%, peat 50% + 50% manure +50 g of sulfur in the planting hole). After 3 years of investigations it was found that all cultivars planted on litter coniferous substrate registered significantly lower values of the fruit quality indicators. The highest average fruit weight value was recorded: Delicia (2.15 g), Auguta (1.86 g), and Lax (1.83 g). The highest fruit content of dry soluble matter was recorded at Simultan cv. (16.09% Brix). Simultan shown also the firmnest fruits (0.67 N). [1] Kader F., Rovel B., Girardin M., 1996. Fraction and identification of the phenolic coimpounds of highbush blueberries (Vaccinium corymbosum L.) Food Chem. 55 (1): 35-40. [2] Schmidt B.M., Erdman Jr., J.W., Lila M.A., 2005. Effects of food processing on blueberry antiproliferation and antioxidant activity. J. Food Sci. 70(6): 19-26

Antioxidant capacity in Strawberry fruits: Evaluation in different varieties and field culture methods. María Teresa Ariza-Fernández, Pedro Domínguez , Rafael Sesmero, Luis Miranda , Rosalía Villalba, María Teresa Cid, José Manuel López-Aranda, José Federico Sánchez-Sevilla, Carmen Soria, Juan Jesús Medina. IFAPA, Consejería Agricultura y Pesca, Junta de Andalucía. In the last years, an increasing interest in the presence of certain compounds in food which are beneficial to human health is arising. In plant-derived food, these natural-occurring compounds are part of the secondary metabolism of many 28


kinds of fruits and vegetable products and are known as Phytochemicals. The antioxidant capacity of Phytochemicals as well as its health-promoting and/or disease-preventing property is currently being intensely studied by the scientific community. Food products such as virgin olive oil, wine, beer and spinach have been reported to contain a large amount of antioxidant molecules, which protect the organism against harmful metabolism products such as Reactive Oxygen Species. Moreover, a number of studies have correlated the intake of antioxidant compounds with a less incidence of multiple kinds of diseases, including Alzheimer and Cancer. Some other studies described a possible role of antioxidants as natural antifungal compounds, contributing to lengthen the postharvest life of the crop [1]. Among the studied fruits, berries represent an interesting group due to their high content in antioxidants [2]. In strawberry fruits, Phenolic acids represent a major group of antioxidants [3], which includes the chemical subgroup of Flavonoids. In turn, Anthocyanins corresponds to a kind of pigment within the group of Flavonoids which, apart from giving the typical red colour to the strawberry receptacle, also has been reported to have a high antioxidant activity. Finally, it has been reported that the content in antioxidants in the strawberry fruits depends on the genotype, environmental conditions (such as temperature, relative humidity, radiation...) and the field culture method [4]. Traditionally, in the framework of the Spanish National Program for the obtention of new varieties of Strawberry (Convenio INIA CC05-024-C3-1), the selection process of the emerging varieties only included basic agronomical fruit traits such as production, taste, shape, firmness, colour, etc. Nevertheless, since the increasing interest of antioxidants as beneficial traits for human food consumption, the evaluation of the 2007 crop season included three new antioxidant-related parameters: Phenolic Acids, Flavonoids and Anthocyanins. The aim of the present study is to analyze the content of these three parameters in three advanced selections of the mentioned National Program in different growing conditions, and compare such contents to three registered varieties. The advanced selections (namely, 1601-1, 1802-2 and 1823-1) and the varieties (‘Aguedilla’, ‘Camarosa’ and ‘Ventana’) were planted in ground and soilless conditions and in two different planting dates: early (06/10/2006) and conventional (19/10/2006). The plants position followed a randomly distribution, using a split-plot statistical design with three replicates. Two measures were taken along the growing season: mid of February and mid of April, following spectroscopic- based methods. The results showed no significant differences between crop systems and planting date for any of the analyzed parameters. 29


Likewise, no differences were found between varieties regarding Phenolic Acids content, being the averages over 100 mg / 100 g FW in all cases.. On the other hand, significant differences were found in Flavonoids and Anthocyanins contents in the different varieties. Regarding Flavonoids, the advanced selections showed similar or lower levels than the varieties, showing the selection 1601-1 significantly lower content. In the case of Anthocyanins, the selection 1823-1 showed the lowest level, whereas 1601-1 and 1802-2 had similar values to the variety Ventana, which in turn presented the lowest Anthocyanin content for the registered varieties. For the purpose of a potential breeding program aimed to improve the antioxidant capacity of new strawberry varieties, the results showed that the parameters of interests are Flavonoids and Anthocyanins contents rather than Phenolic acids, since the higher variance of the formers would let the selection of parental plants with acceptable levels of these kind of antioxidants. The analysis of the more than 300 different varieties in the Fragaria Genus Collection located in the IFAPA Churriana (Málaga, Spain) would be the next step for the selection of parental genotypes with optimal levels. This work was supported by the INIA Agreement CC05-024-C3-1 and INIA Project RTA 2007-00012. [1] C. Hébert, M.T. Charles, L. Gauthier, C. Willemot, S. Khanizadeh, J. Cousineau (2002). Strawberry proanthocyanidins: Biochemical markers for Botrytis cinerea resistance and shelf life predictability. Acta Hort. 567. 659-662. [2] S.Y.Wang, W. Zheng (2001). Effect of plant growth temperature on antioxidant capacity in strawberry. J. Agric. Food Chem. 49. 4977–4982. [3] B.R.Cordenunsi, J.R. Oliveira do Nascimento, M.I. Genovese, F.M. Lajolo (2002). Influence of cultivar on quality parameters and chemical composition on strawberry fruits grown in Brazil. J Agric Food Chem. 50. 2581-2586. [4] Hancock, J.F. (1999). Strawberries. CAB Internacional. Wallingford, UK.

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Hot vs Cold water steeping of different teas: does it affect antioxidant activity? Tiziana Bacchetti,a, Elisabetta Venditti a, Luca Tiano,a Patricia Carloni,b Lucedio Greci,b Elisabetta Damiania* a Dipartimento di Biochimica, Biologia e Genetica, bDipartimento di Idraulica, Strade, Ambiente e Chimica, Università Politecnica delle Marche, Ancona I-60131, ITALY Tea, second only to water, is the most highly consumed beverage worldwide and whose medicinal and health properties have been widely explored, in particular the antioxidant properties. It is prepared from the young, tender leaves of the Camellia sinensis (L.) which undergo different manufacture procedures to give various types of teas: black, green, white, oolong. In addition, the growing season, geographic region, processing of the leaves and whether teas are blended or unblended, create the vast selection available commercially and contribute to each tea’s uniqueness. Also the methods of preparing the beverage also varies worldwide and could influence tea properties. The aim of the present study was specifically to investigate whether antioxidant activity of teas may be affected by hot or cold water steeping and how this may be related to their polyphenol content and metal chelating activity. At this purpose, a set of five loose tea samples were analyzed which included four unblended teas: black tea (Chinese Lapsang Souchong), white tea (chinese Pai Mu Tan), green tea (China Special Gunpowder), oolong tea (from Fujian Province - China), and one blended black tea (Lyons Gold Brand) were analyzed following their infusion in either hot water (90 °C, 7 mins) or cold water (room temperature, 2 hrs). White tea shoed higher antioxidant activity measured as hydrogen donating ability using the ABTS and DMPD assays. No significant differences amongst hot or cold teas has been observed, except in the case of white tea where significantly higher values were obtained under cold water steeping (p1.4, and p-value≤0.05, 142 and 14 genes resulted differentially expressed in stages 01 and 02, respectively. An analysis to show the most represented Kegg pathways among the differentially expressed genes was performed (Fig. 1). At stage 01, among the 93 upregulated genes in Sarda (which is by far the most productive dairy breed) we could recognize caseins αS2, β and K. In addition to milk protein genes, we identified genes involved in processes linked to both lactation and mammary involution, as oxidative metabolism, apoptosis, cell cycle control, oncogenes, ubiquitination pathway and cell communication (focal 34


adhesion adherens junction), endocrine system (insulin signalling pathway, adipocytokine signalling pathway). At stage 02 only 8 genes were upregulated in Gentile and 27 in Sarda. We can remark the overexpression in Sarda of some interesting genes as those encoding casein K, and proteins involved in oxidoreductase activity (like TGOLN2 and FTH1) and in ECM-interaction (like COL1A2). Finally, we can observe in Sarda an overexpression of genes implicated in lipolysis, like lipase (DAGLB) and phospholipase (PLD3). Lipolysis is particularly important in sheep cheese, due to the high fat content and lipase activity. Figure 1. KEGG pathway analysis of the differentially expressed genes.

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Fluorescent differential display analysis of genome expression in Lactobacillus sakei strains under stress conditions Maria Grazia Bonomo, Maria Anna Sico and Giovanni Salzano Department of Biology, Defence and Agro-Forestal Biotechnology, University of Basilicata, Italy Lactic acid bacteria are widely used in meat technology and an efficient control of these microbiological processes requires an increase in the knowledge of bacterial behaviour under stress conditions. Lactobacillus (Lb.) sakei is used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors such as temperature, pH and salt concentration. This study was focused on the behaviour of Lb. sakei strains, chosen as potential autochthonous starters, under various growth conditions relative to acid, osmotic and heat stress treatments. Growth and survival were investigated by measuring OD at 600 nm and colony forming units (CFU) on MRS agar plates in order to study cell viability. Survival was observed at each condition tested with a significant decline of surviving cells after exposure especially to 60°C, 55°C and pH 2.5. Bacterial growth showed a drop of the average maximal population to 68.3% (± 2.6%), 60.2% (± 15.1%) and 24.2% (± 7.7%) at 50°C, 55°C and 60°C respectively. The acid stress caused a reduction over 80%, while the osmotic stress a lowering to 27%. Significant average decreases in growth rates were also obtained. Moreover, the differential expression of genome in response to different stress conditions was analyzed by the fluorescent differential display (FDD) technique. FDD was carried out by using a random 6-carboxyfluorescein labeled 5’-anchored M13 primer (M13-FAM) and this methodology provided a useful approach to find differential bands linked to different stress responses. Distinct bands in response to different stresses, such as low pH values, elevated temperatures and high osmolarity, were obtained and the identification of their sequences is currently under way. The resulting fingerprints showed the presence and the absence of specific products between each stressed sample and the control. This study obtained the development and the optimization of a technique that allows the identification of gene expression changes, associated with differential microbial behaviour under different stress conditions with a better stress response definition and a better discrimination of starter cultures. Our work provided an innovative FDD method, with an high level of reproducibility and quality for studying and probing the knowledge of the relation between differential genome expression and different stresses tolerance. 36


Molecular mechanisms related to the reduction of cholesterol biosynthesis by n-3 PUFAs Elisa Boschetti, Francesca Danesi, Veronica Valli, Vitaliano Tugnoli and Alessandra Bordoni Department of Biochemistry “G. Moruzzi” - University of Bologna, Italy High plasma levels of total cholesterol (Chol) and LDL-Chol are important risk factors for cardiovascular diseases (CVDs). Although Chol is introduced with diet, in the human body Chol biosynthesis represents the most important source. Recently, the possibility to reduce Chol concentration through the regulation of genes implied in its biosynthesis has been hypothesized. In this light n-3 polyunsaturated fatty acids (PUFAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), appear particularly interesting. N-3 PUFAs are widely known as triglyceride (TG)-lowering agents, and have been recently demonstrated to prevent age-related modifications of CHOL metabolism [1]. EPA and DHA play a key role in the prevention or progression of human diseases by different mechanisms, i.e. by modulating membrane lipid composition and by affecting metabolic and signal-transduction pathways. Furthermore, a direct control of gene expression by n-3 PUFAs through directed or undirected interaction with nuclear receptors has been recognized. In a recent work, we have demonstrated that n-3 PUFAs modulate gene expression in cultured rat cardiomyocytes [2]. In this work the effect of an n-3 PUFAs enriched diet on the expression of genes related to endogenous cholesterol production has been evaluated in the liver of control and spontaneously hypertensive rats (SHRs). SHRs rats were chosen since they are considered a suitable animal model of the metabolic syndrome (MS) [3]. First, the expression of sterol regulatory element binding proteins (SREBP1 and SREBP2), the transcription factors regulating the expression of genes involved in Chol, fatty acid and TG biosynthesis, was evaluated. Then the expression of gene related to SREBP master regulation (3hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAR), the limiting enzyme in Chol biosynthesis; low-density lipoprotein receptor (LDLR), that mediates the clearance of LDL particles from the blood; and acyl-coenzyme A cholesterol transferase (ACAT2), liable of cholesterol esterification) was verified. Rats fed the n-3 PUFA enriched diet evidenced significant differences in the expression of these genes, all in agreement with a Chol lowering effect. Our results could therefore better explain the molecular mechanism of n-3 PUFAs related to cholesterolemia reduction.

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[1] C. Martini, V. Pallottini, E. De Marinis, M. Marino, G. Cavallini, A. Donati, S. Straniero, A. Trentalance (2008) Omega-3 as well as caloric restriction prevent the age-related modifications of cholesterol metabolism. Mech Ageing Dev. 129: 12. 722-727. [2] A. Bordoni, A. Astolfi, L. Morandi, A. Pession, F. Danesi, M. Di Nunzio, M. Franzoni, P.L. Biagi, A. Pession (2007) N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure. FEBS Lett. 581: 5. 923-929. [3] M. Oron-Herman, Y. Kamari, E. Grossman, G. Yeger, E. Peleg, Z. Shabtay, A. Shamiss, Y. Sharabi (2008) Metabolic syndrome: comparison of the two commonly used animal models. Am J Hypertens. 21: 9. 1018-1022.

Bean genomic polymorphism uncovered by a systematic approach based on βtubulin gene sequence organization Luca Braglia, Elena Ponzoni, Diego Breviario Institute of Agricultural Biology and Biotecnology (IBBA)-C.N.R. Milano, Italy Common bean (Phaseolus vulgaris L.) is the most widely consumed grain legume in the world. Among the five domesticated species, P. vulgaris is the major source of protein for rural and urban populations in Latin America and eastern Africa. Its balance of essential amino acids in seeds is complementary to that in grains of cereals, providing a positive healthy contribute. In Italy, different local ecotypes are known, cultivated in small areas with traditional cropping practices and reported to provide an important yet differential nutritional contribution. However, traditional breeding programs are limited by the under-utilization of the available genetic diversity and the evaluation of phenotypic differences remains the method for evolutionary and pedigree relationship determination. Advances in molecular biology have allowed the development of rapid, sensitive and specific screening methods to study genetic diversity and relatedness between plant species and varieties. One recently developed, useful genotyping method is based on intron length polymorphisms present in members of the plant β-tubulin gene family (cTBP). The genomic organization of these genes, that encode proteins of relevance for growth, is such to allow a multiple approach for detection of genetic diversity. In addition to the classic cTBP method, we introduce a new approach that uncovers gene-sequence variability in the 5’upstream region of the beta-tubulin genes. Applied on different bean ecotypes, this AFLP like technique holds the potential 38


to become a new powerful DNA fingerprinting tool for studying genetic relationships and diversity.

Metagenomics analysis of microbiota from fermented sausages before ripening Matteo Busconia, Dario Rossia, Mariangela Marudellia, Corrado Foghera a Istituto di agronomia, genetica e coltivazioni erbacee, sezione di botanica e genetica vegetale, Facoltà di Agraria, Università Cattolica del Sacro Cuore, Italia. It is well known that several microorganisms cannot be grown readily in pure culture, and that culturing does not capture the full spectrum of microbial diversity of a given environment [1, 2]. For such a reason, several cultureindependent methods were designed to describe the phylogenetic diversity in several environments. Up to now, only few papers describing the application of culture independent methods to identify the microflora in sausages have been published [2]. In this study, the biodiversity of the bacterial population of two naturally fermented sausages, produced in northern Italy, has been evaluated by using a metagenomics approach and the identification of the different species was carried out by direct retrieval and analysis of 16S rDNA sequences free of cultural bias. The analyses were carried out not at the end of the fermentation but before the ripening and for each sausage we considered three different sections of the product: the whole transversal section, only the central region, and only the lateral region, that differ for the water activity (WA) and pH values. The sampling for each of the areas was carried out in order to avoid possible contamination as a consequence of the cut. Total DNA has been extracted, and prokaryotic DNA was further analysed using two universal primers for 16S rRNA genes. PCR products were ligated in plasmid vector and then transformed into chemically competent Escherichia coli cells. For each sausage 120 random clones were selected: 40 from whole section, 40 from central region, and 40 from lateral region. Plasmid DNAs were extracted, directly sequenced and finally the sequence data were used in search for homology by database screening. The genus Lactobacillus was the most represented in both sausages, with one of the samples showing a greater biodiversity with at least 16 different species respect to the 8 species detected in the other sample. Some species were present in all the three sections (Lactobacillus sakei, L. curvatus) while other species were found only in the central (Leuconostoc mesenteroides, uncultured Leuconostoc) or the lateral (Staphylococcus equorum, Staphylococcus saprophyticus) section. 39


At least four species belonging to the genus Enterococcus were detected and among the others also a sequence showing the greatest homologies with Shigella flexneri was found. [1] Handelsman, J., 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev 68, 4: 669-685. [2] Rantsiou, K., Cocolin, L., 2006. New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods: a review. Int. J. Food Microbiol. 108: 255-267.

Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors Eugenio Butelli1, Lucilla Titta2, Marco Giorgio2, Hans-Peter Mock3, Andrea Matros3, Silke Peterek3, Elio G W M Schijlen4, Robert D Hall5, Arnaud G Bovy4, Jie Luo1 & Cathie Martin1 Nat Biotechnol. 2008 Nov;26(11):1301-8. 1 John Innes Centre, Norwich Research Park, Colney, , UK; 2Experimental Oncology Dept., European Institute of Oncology, , Milano, Italy; 3Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany; 4Plant Research International, Wageningen, The Netherlands; 5 Centre for BioSystems Genomics Wageningen, The Netherlands. Dietary consumption of anthocyanins, a class of pigments produced by higher plants, has been associated with protection against a broad range of human diseases. However, anthocyanin levels in the most commonly eaten fruits and vegetables may be inadequate to confer optimal benefits. When we expressed two transcription factors from snapdragon in tomato, the fruit of the plants accumulated anthocyanins at levels substantially higher than previously reported for efforts to engineer anthocyanin accumulation in tomato and at concentrations comparable to the anthocyanin levels found in blackberries and blueberries. Expression of the two transgenes enhanced the hydrophilic antioxidant capacity of tomato fruit three-fold and resulted in fruit with intense purple coloration in both peel 40


and flesh. In a pilot test, cancer-susceptible Trp53–/– mice fed a diet supplemented with the high-anthocyanin tomatoes showed a significant extension of life span. Whole and cross-section of ripe wild-type and Del/Ros1N tomato fruit. Scale bar 2cm.

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H NMR fingerprinting of soybean extracts, with emphasis on isoflavones identification and quantification Augusta Caligiania, Gerardo Pallaa, Annalisa Maiettib, Martina Cirlinia, Vincenzo Brandolinib a Department of Organic and Industrial Chemistry, University of Parma, Italy; b Department of Pharmaceutical Science, University of Ferrara, Italy Soybean (Glycine max L.), traditionally produced and consumed in China, is nowadays one of the most important agricultural commodities in the world, cultivated mainly for its high protein and oil contents. In the past decades, numerous studies have shown the health promoting effects of the soy components; regular consumption of soyfoods can reduce the incidence of breast, colon, and prostate cancers [1], prevent heart disease and osteoporosis, and alleviate menopausal symptoms [2]. Among the many soy components examined, isoflavones are the most promising components responsible for the health benefits of soy [3]. As a consequence of these findings, many functional foods and food supplements based on soy ingredients have been developed and commercialized. Soybean components, in particular isoflavones, can vary greatly, depending on the varieties, cultivation typology etc., so an important issue is to dispose of rapid analytical methods for the characterization of soybean and soy products. NMR spectroscopy has become more and more important in food science, mainly as a fingerprinting technique but also as a quantitative analysis tool. In this experimental work the 1H NMR spectra of hydroalcoholic extracts of different defatted soybean varieties, coming from traditional and organic farming, were registered. 41


In the spectra it is possible to point out signals belonging to the groups of amino acids, carbohydrates, organic acids, aromatic substances. A group of signals were tentatively identified as saponines. In the aromatic zone, the isoflavone signals resulted of particular interest: genistein, daidzein, genistin, daidzin, malonil genistin, acetyl genistin, malonil daidzin signals were assigned, and several isoflavones were quantified in soybean samples with a good agreement with literature data (mean values of total isoflavones were 2.16 g/Kg for organic soybean samples and 1.59 g/Kg for traditional samples). A metabolomic analysis of soybean extracts was also performed, elaborating all the information obtained from the 1H NMR spectra by multivariate statistical analysis. The spectra were divided in zones of 0.04 ppm and automatically integrated, considering also the unidentified signals. The data obtained, representing a fingerprint of the samples, were statistically elaborated with principal component analysis (PCA). The score plot shows that the main separation is between traditional and organic samples; moreover, a significant dispersion can be observed inside the two principal groups, demonstrating an important effect of the varieties on the pattern of the plant metabolites. This study represents a further confirmation that 1H NMR coupled with multivariate statistical analysis is a powerful tool for the metabolomic analysis of food matrices, offering the advantages of the simple sample preparation and the rapidity of data acquisition. In the particular case of soybean, this method might have many interesting applications, for examples to study the different distribution of metabolites in OGM soy, or for the screening of food and food supplements based on soy component (e.g. isoflavones supplements) present on the market. [1] J. Isanga, G.N. Zhang (2008) Soybean bioactive components and their implications to health - A review. Food Rev. Intern. 24: 2. 252-276. [2] M. Messina (2005) Soyfoods and soybean isoflavones for promoting bone health. Agro Food Industry Hi-Tech 16: 3. 30-32. [3] M. Messina, O. Kucuk, J. W. Lampe (2006) An overview of the health effects of isoflavones with an emphasis on prostate cancer risk and prostate-specific antigen levels. J. AOAC Intern. 89: 4. 1121-1134. 42


An LC-EIMS method for a rapid profiling of free fatty acids in biological fluids Achille Cappiello, Veronica Termopoli, Helga Trufelli, Pierangela Palma, Giorgio Famiglini Dipartimento di Scienze Geologiche, Tecnologie Chimiche e Ambientali, Università di Urbino “Carlo Bo” Introduction: We propose an innovative approach for the simultaneous profiling of individual free fatty acids (FFAs) in human plasma using liquid chromatography coupled to a direct-electron ionization mass spectrometer (LC-Direct-EI-MS). The characterization of FFAs in plasma is of major interest in metabolomics because of their prominent role in metabolic pathways. Although these compounds represent a small fraction of total plasma lipids, they are major substrates for energy metabolism and also act as mediators in signal transduction. Alterations of FFAs levels occur under many pathological conditions such as nutritional and metabolic disorders, diabetes, cancer, cardiovascular and gastrointestinal disease. Therefore, the development of fast and sensitive methods for detecting unesterified FFAs in plasma represents an essential step in studying lipid metabolism in vivo. Method: Several saturated and unsaturated FFAs belonging to the class of medium-, long- and very long chain fatty acids (MCFA, LCFA, VLCFA) were selected as target analytes, considering their role in lipid metabolism. Plasma samples were collected from healthy, non-obese volunteers and from patients affected by alterations of their FFAs profile. FFAs were extracted from the samples without any derivatization procedure, and an on-line sample injection enrichment was performed prior to the LC-Direct-EI-MS. The Direct-EI interface was mounted on an Agilent 5975B Inert MSD single quadrupole mass spectrometer (Cappiello et al., Analytical Chemistry, 2007, 79, 5364-5372). Data acquisition was carried out in selected ion monitoring (SIM). The method was validated in term of sensitivity, linearity, precision, and matrix effects evaluation. Preliminary Data: This work represents the first application of Direct-EI-MS in the field of lipidomics. Direct-EI is a miniaturized interface for the efficiently coupling of liquid chromatography with EI mass spectrometry. The current widespread methods for profiling individual plasma FFAs employ gas chromatography coupled to mass spectrometry (GC-MS) after extraction and derivatization of the lipid material. Although protocols based on GC-MS are characterized by satisfactory sensitivity and precision, the complex sample preparation procedures make the determination of individual FFAs a time consuming process. In the last few years liquid chromatography coupled to atmospheric pressure 43


ionization mass spectrometry (LC-API-MS) has gained an increasing importance in the investigation of lipid metabolism. However, most of the current LC-API-MS methods do not represent a valid alternative to GC-MS. In fact, they require derivatization during sample preparation and/or complex postcolumn pH adjustment after chromatography. In addition, quantitative results can be compromised by ion suppression phenomena. Our preliminary results demonstrate that the use of Direct-EI in the analysis of FFAs can offer several advantages towards current GC-MS and LC-API-MS methods: (1) Sample preparation procedure can be carried out without derivatization, reducing the time of analysis (the entire protocol including sample preparation and Direct-EI-MS analysis require about three hours); (2) Reproducible and high-quality NIST library-matchable EI spectra can be obtained, allowing a certain identification of the target compounds; (3) Limits of detection of this method can be comparable with those obtained with GC-MS and LC-APIMS; (4) Direct-EI response of the target compounds is not influenced by matrix effects, which often severely compromise the quantitative performance of LCAPI-MS methods. This assay has been successfully applied to investigate variations of the FFAs profile in patients affected by pathological alterations of lipid metabolism. The results obtained readily show specific changes of the individual FFAs profile between controls and patients. Novel Aspect: A fast and matrix effects free LC-MS method for the determination of fatty acids in biological fluids.

Effect of n-3 and n-6 polyunsaturated fatty acids on liver cell expression Mariella Caputo, Hylde Zirpoli, Gaetano Torino, Mario Felice Tecce Department of Pharmaceutical Science, University of Salerno, Italy Dietary fatty acids are important macronutrient for growth and development of all organisms. They provide substrates for energy metabolism, membranes, and signaling molecules. However quality and quantity of the dietary fatty acids appear to be extremely relevant in the development of several pathologies, like atherosclerosis and cardiovascular diseases [1] . Our previous in vivo data showed hepatic gene modulations in relation to dietary unsaturated fatty acids [2]. This study is being carried out to obtain more detailed information about the action of single nutrients on individual specific genes among those previously resulted as in vivo modulated. We have decided to evaluate the effects of some 44


polyunsaturated fatty acids, like docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA) on the human hepatoma cell line, HepG2. These fatty acids were studied alone and with vitamin E (Vit.E) to overcome their cytotoxic effects and to replicate the situation in which these substances are and have to be found together in diet formulations. We have considered the effects of these nutrients on the stearoyl-CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP-1) expression, which are two of the major factors involved in unsaturated fatty acids synthesis. We have also studied the expression of UDP-glucuronosyltransferase 1A1 (UGT1A1), an enzyme of phase II drug metabolism. DHA, EPA and AA down-regulated SCD and sterol SREBP-1 expression, while vitamin E did not affect these products, both at mRNA and protein level. When added independently to cultured cells, DHA and Vit.E resulted not to affect significantly UGT1A1 mRNA expression. Nevertheless, their combination produced a considerable reduction of this mRNA [3]. EPA and AA strongly down-regulated UGT1A1 mRNA expression, while the combination with vitamin E abolished the down-regulation by EPA, but did not affect that by AA (Figure). Therefore the effect of these nutrients is related to specific gene regulation processes resulting in a cooperation which might be related to their physiological effects as dietary components. The mechanisms could involve heterodimeric nuclear receptors including peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR), retinol X receptor (RXR) and nuclear cofactors. The analysis of these mechanisms could be important to improve the knowledge of metabolic nutritional processes involved in disease risk and prevention, possibly allowing the development of better diagnostic and therapeutic opportunities. [1] P Angerer, C von Schachy (2000). n-3 polyunsaturated fatty acids and the cardiovascular system. Curr Opin Lipidol 11:57-63. [2] D Eletto, A Leone, M Bifulco, MF Tecce (2005). Effect of unsaturated fat intake from Mediterranean diet on rat liver mRNA expression profile: selective modulation of genes involved in lipid metabolism. Nutr Metab Cardiovasc Dis. 15(1):13-23. [3] M Caputo, D 45


Eletto, G Torino, MF Tecce (2008). Cooperation of docosahexaenoic acid and vitamin E in the regulation of UDP-glucuronosyltransferase mRNA expression. J Cell Physiol. 215(3):765-70.

The feed guarantees the quality of the food: an easy way to trace plant species in commercial feedstuffs Anna Paola Casazza, Floriana Gavazzi, Francesco Mastromauro, Silvia Gianì and Diego Breviario Institute of Agricultural Biology and Biotechnology (IBBA) – Italian National Research Council (CNR), Via Bassini 15, 20133 Milan, Italy Tracing food through all stages of production, processing and distribution has been a growing concern over the last decades. The EU’s General Food Law (Regulation n. 178 of 28th January 2002) entered into force in 2005 and makes traceability compulsory for all food and feed businesses. This new regulation not only represents the first step to guarantee a high level of human health safety but also enables consumers to be provided with targeted and accurate information concerning products origin and authenticity. This last point is of great importance particularly for traditional products. Here we present a groundbreaking methodology for the certification of compound feedingstuffs, that satisfies the demands of the Bitto cheesemakers. In fact, to label the Bitto with the PDO (Protected Designation of Origin) mark, the cheese must be made from milk of a certain breed of cow, which have been fed (besides on meadow grasses) only on wheat (W), maize (M), barley (B) and soy (S). The genetic identification of the feedstuff composition is performed using the TBP (TubulinBased Polymorphism) method [1], which is based on the Intron Length Polymorphism (ILP) of the plant β-tubulin gene family. Using TBP we are able to certify the composition of commercial feeds, as required by the Consortium for the protection of Valtellina Casera and Bitto cheese, identifying easily and unambiguously the above mentioned four plant species (W, M, B and S). [1] M. Bardini, D. Lee, P. Donini, A. Mariani, S. Gianì, M. Toschi, C. Lowe, D. Breviario (2004) , Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species. Genome 47: 281-291

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Know what you print, to know what you eat Ilaria Chiusa a, Laura Morello a and Diego Breviario a a Institute of Agricultural Biology and Biotechnology (IBBA) - C.N.R. Milan, Italy Food/feed quality, safety and composition has become an issue of the up most relevance world-wide. Accordingly, there is now a great demand for technologies and tools that can easily, rapidly, conveniently and reliably identify plant species and varieties to certify quality and safety while combating frauds. In order to meet with this request we have recently developed a method for plant species recognition that is based on the Microcontact Printing technique [1,2]. This is a new method by which highly organized structures, such as the tracks of a compact disk, can be used to produce stamps on which a diagnostic molecule (i.e. a short DNA sequence) can be inked. The stamps are then used to print a small surface that then becomes the site of molecular recognition carried out through hybridization methods. The advantages of such a technique are multiple: recognition reactions are carried out at microscale level with the use of a limited amounts of reagents and target molecules, kits made up by multiple target molecules useful for food diagnosis can be produced, diagnostic efficiency is further increased by multiple replicas obtained from the same stamp. We have successfully worked out this technology by printing plant PCR amplification products that are selectively recognized through hybridization with specific fluorescently labelled oligonucleotide probes. The assay, granting species selective recognition, was first developed on rice but its application to the identification of small amounts of undesired plant components in food (i.e allergenic species) is under way. The system was also applied to the identification of DNA of plant origin in DNA isolated from goat milk, through selective hybridization of microprinted PCR reactions (see posters of Ponzoni et al and Mastromauro et al). Yet, this is just the beginning of future developments in the field of plant species recognition in feed or food products (i.e. milk or cheese). [1] S. A. Lange, V. Benes, D. P. Kern, J. K. H. Hörber and A. Bernard (2004) Microcontact Printing of DNA molecules. Analytical Chemistry 76. 1641-1647. [2] C. Thibault, V. Le Berre, S. Casimirius, E. Trévisiol, J. François and C. Vieu (2005) Direct microcontact printing of oligonucleotides for biochip applications. Journal of Nanobiotechnology 3: 7.

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The ancient durum wheat Graziella Ra® (Triticum turgidum durum, Triticeae): a preliminary study Mariastella Colomba1, Armando Gregorini2 1 DiSUAN, University of Urbino, Italy – 2DiPsiTer, University of Urbino, Italy Graziella Ra is a registered accession of durum wheat (Triticum turgidum durum, Triticeae) with rather mysterious and ancient origins. It was found in an old Egyptian grave and brought to Italy in the 70’s by an anonymous archaeologist. Currently, it is organically grown by the Alce Nero Cooperativa in Marche Region (Central Italy) and shows quite low yields, fine pasta-making qualities, good taste and healthy nutritional values. In this study Graziella was compared with four other accessions of durum wheat (Senatore Cappelli, Grazia, Flaminio and Svevo) and Kamut® (considered an ancient relative of modern durum wheat) by several molecular markers generated by RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) and SSRs (Single Sequence Repeats) analyses. Obtained results revealed that Graziella’s genome was strictly related to Kamut (Figure 1). Moreover, we compared the α-gliadin gene sequence from all the accessions under study to reconstruct their phylogenetic relationships when using Kamut as Out Group. Our data – although preliminary – resulted in a Neighbour Joining tree (Figure 2) showing Graziella and the traditional strain Senatore Cappelli in the same cluster (Bootstrap Percentage, BP=100%), thus supporting the hypothesis that Graziella might be an ancient type of durum wheat. Finally, comparative analysis of nutritional values for Graziella and Kamut® (see http://www.kamut.com) revealed that although both wheats shared several features, Kamut® mostly showed higher levels of proteins, aminoacids, minerals and vitamins. On the other hand, Graziella was significantly richer in total lipids and zinc (this latter probably depending on levels of the element in soil). For all these reasons we believe that the two accessions are different and that Graziella might represent one of the most ancient types of durum wheat known up to now, which has survived throughout thousands of years without undergoing any major genetic manipulations. In conclusion, reintroduction of Graziella wheat represents an important step towards the preservation of biodiversity and the conservation of ancestral and/or traditional cereals.

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Figure 1. AFLP UPGMA Cluster using Nei's minimum distance (Bootstrap percentages after 1000 permutations). 0,60

0,45

0,30

0,15

0,00

Kamut 10 0 9 8

Cappelli

48.6 0

10 0

Graziella

Flaminio

45.0 0

Grazia Svevo

Figure 2. Neighbour Joining tree with the Kimura-2 parameters genetic distance model (including transitions+transversions) with rate heterogeneity among sites (gamma distribution, α=1); support for the internodes was assessed by Bootstrap percentages after 1000 resampling steps. 78 44

Svevo Grazia Flaminio Cappelli

100

Graziella Kamut

This research was supported by Regione Marche funds (CIPE 20/04 – DGR 438/2005).

Meat transcriptomics: results from a time course microarray experiment applied to pork Michela Colombo1, Giuliano Galimberti2, Daniela Giovanna Calò2, Annalisa Astolfi3, Vincenzo Russo1 and Luca Fontanesi1 1 DIPROVAL, Sezione di Allevamenti Zootecnici, University of Bologna, Reggio Emilia, Italy, 2Department of Statistics, University of Bologna, Bologna, Italy ,3Dep. of Hematology and Oncology sciences "L. A. Seragnoli", Sant'OrsolaMalpighi Hospital, University of Bologna, Italy In general the belief that post mortem delay critically influences RNA and mRNA stability is still a widely held notion despite contrasting evidences that mRNAs might be stable for variable periods after death of the animals. In molecular biology this legitimacy is often in contrast with the usual work routine that constrains the researchers at extremely programmed sampling and limits the 49


application field of specific techniques. The possibility to carry out the sampling with a significant post mortem delay could contribute to make meat an interesting substrate for the application of transcriptomic techniques. Here we analysed RNA extracted from Semimembranosus muscles collected 20 min, 2, 6, 24 and 48 hours after slaughtering using microfluidic capillary electrophoresis and by microarray analysis with Affymetrix GeneChip® Genome Arrays. RNA integrity number (RIN) and 28S:18S ratio showed no degradation for RNA obtained from muscle samples collected at 20 min, 2 h, 6 h, and 24 h after slaughtering, but degradation occurred in samples collected at 48 h post mortem. Microarray analysis of the first four post mortem time points confirmed these results indicating that gene expression level is not affected by any degradation process. Based on these consideration specific transcriptional profiles obtainable from RNA of muscle until 24 hours post mortem could be useful for the identification of specific meat quality markers, transcriptional profiles useful to define pre (in terms of feeding, stress or pharmacological treatments) and post slaughtering conditions and specific RNA patterns with applications for the authentication of raw materials used for the production of tipical meat products.

When food meets man: counteraction of inflammation Francesca Danesi1, Martin Philpott2, Claudia Huebner2, Alessandra Bordoni1 and Lynnette R. Ferguson2 1 Research Center on Nutrition and Vitamins, Department of Biochemistry “G. Moruzzi” - University of Bologna, Italy; 2 Department of Nutrition, Faculty of Medical and Health Sciences - University of Auckland, New Zealand The gene-specific modulation of inflammatory cytokines by food bioactives represents a possible approach to the nutritional or pharmaceutical prevention and treatment of inflammatory bowel disease (IBD). There is good evidence for a key role of the interleukin-12β1/23 receptor (IL-12 β1/23R) pathway in IBD, and that reduction of the normal expression of the IL-23R gene may provide a therapeutic target for this disease. In this study, we developed an assay to detect chemicals which can reduce the expression of this gene in Kit 225 cells, and investigated the effects of specific dietary bioactives on interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) gene expression, and IL-17 production. This human T cell line has similarities to Th17 cells, a subset of T cells producing IL-17 and TNF-α, and in initial experiments we demonstrated that the cells express 50


both IL-23 receptor subunits, as well as IL-17 and tumor necrosis factor-α (TNF-α) genes. Upon verification that stimulation of Kit 225 cells with 1 ng/ml IL-23 significantly upregulated IL-17 and TNF-α gene expression, and IL-17 production, we supplemented cells with the selected bioactives, caffeic acid phenethyl ester (CAPE), epigallocatechin gallate (EGCG), docosahexaenoic acid (DHA), and linoleic acid (LA), and with phorbol myristate acetate (PMA) and sodium salicylate, used as pro-inflammatory and anti-inflammatory controls, respectively. In both unstimulated cells and after IL-23 stimulation, bioactives modulated the proinflammatory cytokines involved in IBD, confirming the role of foods in this disease. EGCG and DHA, which significantly inhibited both IL-17 and TNF-α expression, appeared particularly interesting.

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C NMR Spectroscopy and quantitative analysis of lipids from fish: comparison between different farming systems Laura Del Cocoa, Paride Papadiaa, Sandra Angelica De Pascalia, Giorgia Bressania, Carlo Storellia, Vincenzo Zonnoa,b, Francesco Paolo Fanizzia. a Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce. bConsorzio Intercomunale Capo S. M. di Leuca, Tricase (Lecce) In recent years there was an increase in demand for seafood products in our country. In response to this request both fishing effort, with consequent fish resources impoverishment, aquaculture development, and fisheries importations from other countries were investigated. If compared to meat, seafood consumption is absolutely lower, notwithstanding its nutritional properties [1]. First of all, fatty acids contained in fish have important functions for the body, in particular the fatty acid named eicosapentaenoic acid (20:5 ω3). Furthermore, seafood fatty acids are different from beef and pork fat, being the latter rich in cholesterol, while fatty acids contained in seafood have the ability to induce a decrease in the level of cholesterol in the blood and are therefore very useful for the prevention of cardiovascular diseases [2]. In particular, seafood fatty acids are critical in the human diet because they are the only source of eicosapentanoic acid (20:5 ω3 or EPA) and docosahexaenoic acid (22:6 ω3 or DHA). The dietary uptake of ω3 fatty acids is essential for growth, the health of the mitochondrial and cell membranes; in fact ω3 are involved in the synthesis of haemoglobin, coagulation mechanisms, capillary fragility and also in other 51


processes such as reproduction. Some breast diseases and abnormalities of the menstrual cycle result from the excessive uptake of saturated fatty acids in relation to ω3/ω6 ratio; finally ω3 develop a better tolerance to carbohydrates in diabetics and are used by as precursors of prostaglandins. There are different fish breeding methods: extensive, intensive in cage, in the sea or tanks, and finally semi-intensive, the latter opening the way to interesting perspectives for quality control of the fish product. In addition to genetic factors, the different type and mode of feeding, the density of population of fishes in tanks or cages, the swimming activities and other environmental factors (temperature, salinity, pH, oxygenation, etc..) can influence the organoleptic characteristics (colour, flavour) and within certain limits, the chemical composition, particularly of the lipid component. The aim of this work was to conduct an evaluation of qualitative and quantitative 1,3 Oleic acid composition of lipid fraction, with  Saturated particular Fatty Acids  Saturated Fatty Acids attention to essential  Oleic acid 2 DHA polyunsaturated  DHA  EPA fatty acids (ω3 and 2 EPA ω6) of commercial size reared     ppm gilthead sea bream Expansion of C NMR spectrum of Sparus aurata lipid fraction (resonances of the carbonilic carbons) (Sparus aurata) specimens, obtained with different methods of fish culture: intensive (from Maribrin s.r.l. aquaculture system, 8 Km south of Brindisi), and semi-intensive (Acquatina basin, Frigole, Lecce). The composition of the lipid fraction in the different farming systems was analyzed: both samples show the same ratio between saturated and monounsaturated fatty acids, while changes in the percentage of polyunsaturated fatty acids and ω3 PUFA were observed. In particular, there is an increase in the ω3 and ω6 fatty acids ratio of Sparus aurata specimens reared in the intensive farming system. Data obtained from NMR analysis reveal a greater amount of ω3 fatty acids in intensive samples when compared to semi-intensive farming ones; the highest percentage of other polyunsaturated fatty acids were found in the semi-intensive system. These data are closely correlated with the different lipid content in the diet of the two breeding systems analyzed. Moreover, the results were compared with those 52


obtained for wild sea breams captured in a coastal area (control). Data obtained suggest that the reared fishes showed (at the end of the trial) a nutritional physiological state comparable to that of wild sea breams. Research financed by Apulian Region POR Puglia 2000-2006 Action 4.13 E. [1] W. Steffens, (1997) Effects of variation in essential fatty acids in fish feeds on nutritive value of freshwater fish for humans, Aquacolture, 151, 97. [2] W. Harris, (2004) Omega-3 fatty acids, thrombosis and vascular disease. (1262) International Congress Series, 680 [3] E. Orban, G. Di Lena, A. Ricelli, I. Paoletti, L. Casini, R. Gambelli, R. Caproni, (2000) Quality characteristics of sharpsnout sea bream (Diplodus puntazzo) from different intensive rearing systems, Food Chemistry, 70, 27-32

Unwanted capture analysis by Nuclear Magnetic Resonance (NMR) Spectroscopy Laura Del Cocoa, Paride Papadiaa, Sandra Angelica De Pascalia, Giorgia Bressania, Carlo Storellia, Vincenzo Zonnoa,b, Francesco Paolo Fanizzia a Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Lecce. bConsorzio Intercomunale Capo S. M. di Leuca, Tricase (Lecce) Bycatch is the accidental capture of unwanted or non-targeted fish or other animals. The practise is increasingly recognized as one of the principal threats to many marine vertebrates, including multiple species of sharks, sea turtles, and seabirds [1]. Marine bycatch can include also species of little or no commercial value, protected or endangered species, species caught ‘out of season’, in closed waters, wrong size (too big or too small) or in excessive numbers, plants and animals such as corals, seagrass, algae and sponges which are floating or have been dislodged from the bottom of the ocean floor, and debris such as rocks and rubbish. Recently, the fishing industry worked hard to develop methods that will prevent as much bycatch as possible. These methods aiming to stop or reduce bycatch depend on the type of fishing gear employed, the animals in question and their behaviour. Many clever modifications that are tailored to the biology or behaviour of unwanted animals have become available and are mandatory in some commercial fisheries. Our laboratory directed attention to the chemical characterization of the unwanted capture biomass using 1D and 2D homo- and heteronuclear NMR spectroscopy ([1H,1H]-COSY, J-resolved, [1H,13C]-HMQC). Analyses were performed directly on the sample extracts without further 53


derivatization. The technique is non-destructive, thus making the sample available for further investigations. In order to investigate about the presence of valuable molecules (metabolites, aminoacids) and fatty acids, NMR experiments on the fraction products (total lipid and organic fractions, respectively, see Figure), obtained according to Bligh and Dyer method, were performed [2]. NMR spectra shown the presence of several aminoacids, including essential aminoacids, and fatty acids mono-and poly-unsaturated with high nutritional value. In addition to the interesting data obtained by NMR spectroscopy, the total absence of pollutants and toxic substances together with the presence of vitamins and micronutrients, measured respectively by GC, HPLC and atomic absorption spectroscopy, suggest a possible applicability of the biomass. Non commercial fishing deriving from bycatch, completely identified, might be thought as a first attempt to find potential application as food or feed [3]. Lipid extract

Organic extract

8.5

7.5

6.5

5.5

4.5

3.5

2.5

1.5

0.5

ppm

Research financed by Apulian Region POR Puglia 2000-2006 Action 4.13 E. [1] M. Finkelstein, V. Bakker, D. F. Doak, B. Sullivan, R. Lewison, W. H. Satterthwaite, P. B. McIntyre, S. Wolf, D. Priddel, J. M. Arnold, R. W. Henry, P. Sievert, J. Croxall. (2008) Evaluating the Potential Effectiveness of Compensatory Mitigation Strategies for Marine Bycatch. PLoS ONE 3(6): 2480 [2] E.G. Bligh, W.J. Dyer, (1959) A rapid method for total lipid extraction and purification. Can.J.Biochem.Physiol. 37: 911-917 [3] N. Eid, B. Dashti, W. Sawaya. (1992) Chemical and physical characterization of shrimp by-catch of the Arabian Gulf. Food Res. Int. 25: 3 181-186.

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The olive oil phenols affect metalloproteinase 9 expression: role of NF-B signaling Mario Dell’Aglia,b, Rossana Fagnania, Germana V. Gallia, Omar Maschia, Federica Gilardia, Stefano Bellostaa, Nico Mitroa, Maurizio Crestania, Enrica Bosisioa,b, Emma De Fabiani, Donatella Carusoa,b a Department of Pharmacological Sciences and b Research centre for the characterization and safe use of natural compounds - Giovanni Galli, University of Milano, Italy Diet is still the preferred option in primary prevention of cardiovascular disease so to elucidate the biological actions of food components is a crucial issue. In particular, the beneficial effects of olive oil consumption on human health have been ascribed to minor components like flavonoids and secoiridoids. In vivo studies suggest that these phenolic component contribute to the antiinflammatory and anti-atherosclerotic actions of olive oil, however the effects in circulating cells have not been fully characterized. Monocytes play a key role in the initiation of atheroma and they contribute to the amplification of the inflammatory process. We investigated the effects of olive oil phenolic extract (PE) and of individual phenolic compounds (IPC) on MMP-9 in THP-1, a human monocytic cell line. TNF-stimulated THP-1 cells were incubated for 6 hrs with 0.5-7.5 μg/ml PE or equivalent concentrations of IPC. The PE and IPC prevented the TNF--induced MMP-9 secretion in THP-1 cells and OleA was mainly responsible for this effect. The reduction of secreted MMP-9 was due to decreased MMP-9 mRNA levels. Since TNF- affects MMP-9 transcription by activating NF-B, we also investigated if the PE and IPC affected this signaling pathway. Increasing concentrations of the PE and of IPC dampened the NF-Bdependent promoter activity, confirming that the effect on MMP-9 expression is due, at least in part, to impair NF-B signaling. In conclusion, our findings provide further evidences on the mechanisms whereby olive oil may reduce the inflammatory burden associated with atherosclerosis. Supported by the FIRST 2007 from University of Milano to D. Caruso

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n-3 and n-6 PUFAs suppress SREBP activity and increase flow of free cholesterol in HepG2 cells Mattia Di Nunzioa, Diederik van Deursenb, Adrie J.M. Verhoevenb, Alessandra Bordonia a Research Center on Nutrition and Vitamins – Dept. of Biochemistry “G. Moruzzi”- University of Bologna Via Irnerio, 48 – 40126 Bologna, Italy. b Department of Biochemistry, Cardiovascular Research School COEUR, Erasmus MC, P. O. Box 2040, NL-3000 CA, Rotterdam, The Netherlands. The plasma lipid lowering effect of PUFAs, one of their main beneficial effects, is considered to be related to the regulation of lipid biosynthesis through transcription factors including sterol regulatory element binding proteins (SREBPs). In this study, we evaluated in HepG2 cells the modifications in lipid fatty acid composition induced by the supplementation with different PUFAs, relating them to SREBP activity. Supplemented PUFAs were not only incorporated but also metabolized to longer and more unsaturated species. These processing activities were higher for n-3 than n-6 PUFAs (p> 18 : 3n-3 = 22 : 6n-3 = 22 : 5n-6 >> 18 : 1n-9. The suppression of SREBP activity greatly depended on the degree of incorporation and transformation of the supplemented PUFA, and correlated positively with the unsaturation index (r= 0.831; p Cifrance x Patty > Sveva x Patty.

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Red microalgae as nutraceuticals: an integrated study Irit Dvir* and Shoshana (Malis) Arad** * Sapir Academic College, D. N. Hof Ashkelon 79165, Israel and **Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Red microalgal biomass comprises a unique combination composed of functional sulfated polysaccharides (included dietary fibers), polyunsaturated fatty acids, zeaxantine, vitamins, minerals, and proteins. Furthermore, a disaccharidefloridoside, that is unique to red algae was recently found to have anticancer and antiviral bioactivities. The red microalgal products have a potential to be health food and therefore a series of feeding experiments have been done in mice and rats to carried out : toxicity; nutritional value; metabolic and morphological effects and mechanism of action of the algal products. The algal materials were added to the diets of the rodents in amounts of 5–10%. The main findings of the feeding experiments were: Significant improvement in cholesterol metabolism: reduction in total serum cholesterol, triglyceride and hepatic cholesterol levels. Increase in HDL/LDL ratio, Fecal excretion of neutral sterols and bile acids. Metabolic changes: Gastrointestinal transit time lowered significantly in biomass-fed rats whereas serum and mucosal cholecystokinin (CCK) levels lower and hepatic HMG-CoA reductase significantly increased in polysaccharide-fed rats. Morphological changes: Small intestine and colon length significantly increased, mucosa and muscularis cross-sectional area of the jejunum increased and hypertrophy of the muscularis layer have seen in polysaccharide-fed rats. These results suggest that red microalgae can be used as potent hypocholesterolemic agents at low concentrations in the diet. The unique combination in the algal biomass of sulfated polysaccharides and unsaturated fatty acids (n-3) is thus of special value. The encouraging results indicate that we should pursue the development of red microalgae as novel nutraceuticals. 1. Yaron, A.; Dvir, I.; Maislos, M.; Mokady, S.; (Malis) Arad, S. The red microalga Rhodella -3 highly unsaturated fatty acid eicosapentaenoic acid. In Food Flavors: Generation, Analysis and Process Influence, Charalambous, G. ed.; Elsevier Science B.V. 1995; pp. 665-674. 2. Dvir, I.; Chayoth, R.; Sod-Moriah, U.; Shany, S.; Nyska, A.; Stark, A. H.; Madar, Z.; Arad, S. M.Soluble polysaccharide and biomass of red microalga Porphyridium sp. alter intestinal morphology and reduce serum cholesterol in rats. Br. J. Nutr. 2000, 84, 58


469-476. 3.Dvir, I.; Stark, A. H.; Chayoth, R.; Madar, Z.; Arad, S. M. Hypocholesterolemic effects of nutraceuticals produced from red microalga (Porphyridium sp.) on rats fed a high-cholesterol diet. Submitted.

Antioxidant Potential of “Visciola” Dried Tart Cherry (Prunus cerasus, L.) Gianna Ferretti1), Tiziana Bacchetti1), Simona Masciangelo1), Letizia Saturni1), Maurizio Antinori2), Alfio Santinelli3), Davide Neri2) 1) Dipartimento di Biochimica, Biologia e Genetica, Università Politecnica delle Marche, Ancona I-60131, ITALY; 2)Dipartimento SAPROV, Università Politecnica delle Marche, Ancona I-60131, ITALY; 3)ASSAM, Ancona I-60131, ITALY Several studies have demonstrated that vegetables, fruits and legumes exert a protective effect against several human diseases such as cardiovascular disease, diabetes and cancer [1,2]. It has been hypothesized that the protective role against development of these diseases could be due to micronutrients and phytonutrients such as antioxidants. These molecules are known to be able to exert a protective role against lipid peroxidation triggered by free radical. Phytonutrients and antioxidants are secondary metabolic products derived from complex pathways. Previous studies have shown that environmental factors such as production, handling and storage could affect their synthesis. The evaluation of the total potential antioxidant (PA), using different methodological approaches, has been widely used to investigate healthy properties of different vegetable foods. Aim of this study was to investigate the potential antioxidant of two different clones (A and B) of “visciola” dried tart cherries Prunus cerasus L. Using ORAC assay and DPPH assay , we compared PA values in fruits harvested at two ripening stages (S1 – early ripen, and S2 – fully ripen). The results showed no statistically differences in the PA values between clone A and clone B of visciola. However ORAC values were lower in S1 samples compared with S2 (9205±2000 and 15933±512 M eqTrolox/100g in S1 to S2 clone A, respectively, p