GF10

Second International Symposium on

Gluten-Free Cereal Products and Beverages

June 8–11, 2010

Tampere Hall

Tampere, Finland

Welcome

Welcome to the ‘GF10 Symposium’. This is the Second International Symposium on Gluten-Free Cereal Products and Beverages, and follows the first held in Cork, Ireland in 2007. We are looking forward to an interesting meeting and are very pleased to see you here in Tampere. The Symposium is organised by the Cereal Technology Group of the University of Helsinki, partly in cooperation with the local Finnish Coeliac Society, which has its main office in Tampere, and organises the International Coeliac Fair 2010 on GF products and services. Tampere is also known for its coeliac science research since there is an internationally well-known coeliac disease study group in the University of Tampere. Cereal products based on wheat, rice, maize and a number of other cereals are staple foods world-wide. However, individuals with gluten intolerance, i.e. coeliac disease, have to avoid wheat and its relative cereals rye and barley, because of a particular protein called gluten, or more precisely prolamin present in the three Triticeae cereals wheat, rye and barley. These three cereals have a major role in bread, pasta and beer, and therefore it is a challenge for food scientists to design alternative, high quality gluten-free products. The prevalence of gluten intolerance appears to be increasing, now reaching up to one or two percent of Western populations. With sympathising family members and friends, as well as other individuals avoiding gluten, the GF food market has a substantial potential, provided technology can be developed to meet not only the tightened regulatory and safety requirements but also consumer expectations regarding sensory properties and price. The GF10 Symposium collects researchers, industry and trade representatives, specialists from regulatory bodies and coeliac societies, and health professionals in a highly multidisciplinary way. A special issue on Gluten-Free Cereal Foods will be published in the journal Quality Assurance and Safety of Crops & Foods, based partly on papers presented in the Symposium. On behalf of the Scientific and Organising committees, it is a great pleasure to welcome all of you from more than 25 countries to this Symposium. I want to thank our supporters and sponsors whose contributions have made the symposium possible. We are grateful to the speakers and to those who brought their posters and thus provide possibilities for detailed discussions and networking. This Symposium will give us all an excellent opportunity to learn of the latest results and developments related to gluten-free cereal science, technology and markets. All food served at the GF10 Symposium is gluten-free.

Hannu Salovaara Chairman of the Scientific and Organising Committees Cereal Technology Group, Department of Food and Environmental Sciences University of Helsinki

I

International Scientific Committee

Professor Hannu Salovaara, University of Helsinki, Finland (Chair)

Dr. Elke Arendt, University College Cork, Ireland

Dr. Don Kasarda, Western Regional Research Center, USDA, USA

Professor Markku Mäki, University of Tampere, Finland

Dr. Katri Kaukinen, University of Tampere, Finland

Professor Frits Koning, University of Leiden, The Netherlands

Professor Michael Gänzle, University of Alberta, Canada

Professor Peter Köhler, German Research Center for Food Chemistry, Germany

Dr. Gerard Downey, Teagasc, Ireland

Dr. Juan Pablo Albar, Centro Nacional de Biotecnología / CSIC, Spain

Professor Kaisa Poutanen, VTT Technical Research Centre of Finland / Healthgrain, Finland

Dr. Jan Willem van der Kamp, TNO / Healthgrain, The Netherlands

Dr. Roland Poms, ICC, Austria

Organising Committee

Professor Hannu Salovaara, University of Helsinki, Finland (Chair)

Dr. Tuula Sontag-Strohm, University of Helsinki, Finland

Dr. Jussi Loponen, University of Helsinki, Finland

Päivi Kanerva, University of Helsinki, Finland

Sanna Luoto, University of Helsinki, Finland

Professor Vieno Piironen, University of Helsinki, Finland

Dr. Christel Lamberg-Allardt, University of Helsinki, Finland

Leila Kekkonen, Finnish Coeliac Society, Finland

II

Scientific Programme

Wednesday, June 9th, 2010 Opening of GF10 Symposium Time: 09:00–9:20 Location: Conference room Rondo, Tampere Hall Session 1: Coeliac disease and the triggering molecules Time: 09:20–10:40 Location: Conference room Rondo Chair: Hannu Salovaara, University of Helsinki, Finland; Jarmo Visakorpi, University of Tampere, Finland 09:20 CURRENT PREVALENCE, DIAGNOSIS AND TREATMENT OF COELIAC DISEASE Markku Mäki, University of Tampere, Finland

09:50 THE TRIGGERING PROTEINS AND PEPTIDES IN COELIAC DISEASE Peter Köhler, German Research Centre for Food Chemistry, Germany

10:20 THREE DAYS ORAL GLUTEN CHALLENGE – RESPONSE IN GLUTEN SENSITIVE INDIVIDUALS WITH AND WITHOUT CD Knut Lundin, Oslo University Hospital, Norway

Coffee break Time: 10:40–11:10 Session 2: Gluten analysis and safety testing Time: 11:10–14:30 Location: Conference room Rondo Chair: Peter Köhler, German Research Centre for Food Chemistry, Germany; Jussi Loponen, University of Helsinki, Finland 11:10 IMPROVING ACCURACY IN DETECTING GLUTEN Päivi Kanerva, University of Helsinki, Finland

11:30 PRELIMINARY RESULTS OF THE COLLABORATIVE STUDY TO VALIDATE THE CHARACTERISTICS OF NEW ELISA KIT Jorge Raul Mujico Fernandez, Leiden University Medical Centre, Netherlands

11:50 SECOND GENERATION TESTING FOR GLIADIN IN COMPLIANCE WITH CODEX ALIMENTARIUS LEVEL Sigrid Haas-Lauterbach, R-Biopharm, Germany

12:05 DETECTION OF TOXIC FRAGMENTS FROM GLUTEN USING A NEW MONOCLONAL ANTIBODY-BASED TEST Richard Fielder, Romer Labs UK, United Kingdom

Lunch break Time: 12:05–13:20

III

Wednesday, June 9th, 2010 13:20 GENOMICS APPROACHES TO ANALYSE CD-TOXICITY FOR THE PRODUCTION OF CD-SAFE WHEAT Luud Gilissen, Plant Research International, Netherlands

13:40 TESTING SAFETY OF LOW GLUTEN FOOD PRODUCTS IN A MOUSE MODEL OF CELIAC DISEASE Tobias Freitag, University of Helsinki, Finland

14:00 GENERAL DISCUSSION ON: SAFETY AND TOLERANCE – ANALYSIS AND REGULATORY STANDARDS – TECHNOLOGY AND VARIETY OF PRODUCTS Hannu Salovaara, University of Helsinki, Finland

Poster hour & Coffee break Time: 14:30–15:30 Location: Winter Garden, Tampere Hall Session 3: Coeliac food market and nutritional requirements, Gluten-free cereals and pseudo-cereals Time: 15:30–17:40 Location: Conference room Rondo Chair: Elke Arendt, University College Cork, Ireland; Kaisa Poutanen, VTT Technical Research Centre of Finland 15:30 NUTRITIONAL REQUIREMENTS FOR GLUTEN-FREE FOODS Tricia Thompson, The Gluten-Free Dietitian, MA, USA

16:00 GLUTEN-FREE FOOD MARKET Markku Mikola, Sennet, Finland

16:20 FOLATES IN GLUTEN-FREE DIETS AND FOODS Vieno Piironen, University of Helsinki, Finland

16:40 CEREALS AND PSEUDOCEREALS FOR GLUTEN-FREE FOODS Regine Schoenlechner, University of Natural Resources and Applied Life Sciences, Austria

17:00 HEALTHY GRAINS FOR ENHANCED GLUTEN-FREE BREADS Eimear Gallagher, Ashtown Food Research Centre, Teagasc, Ireland

17:20 EXPLORING THE USE OF DIETARY FIBRES TO PROVIDE ALTERNATIVES TO A GLUTEN-FREE DIET M. Samil Kök, Abant Izzet Baysal University, Turkey

IV

Thursday, June 10th, 2010 Sesion 4: Gluten detoxification Time: 08:30–10:00 Location: Conference room Rondo Chair: Michael Gänzle, University of Alberta, Canada; Päivi Kanerva, University of Helsinki, Finland 08:30 ENZYMATIC TOOLS FOR GLUTEN DETOXIFICATION Frits Koning, Leiden University Medical Centre, Netherlands

09:00 MANY FACES OF PROLYL OLIGOPEPTIDASE – WHAT WE IGNORE IN MAMMALS AND WHAT WE KNOW IN PLANTS J. Arturo García-Horsman, University of Helsinki, Finland

09:20 ENDOGENOUS CEREAL ENZYMES IN THE ELIMINATION OF PROLAMINS Jussi Loponen, University of Helsinki, Finland

09:40 SYNTHETIC BLOCKING PEPTIDES WITH HIGH AFFINITY TO GLIADIN REDUCE TISSUE TRANSGLUTAMINASE ACTIVITY ON WHEAT GLIADIN IN VITRO Karolina Hoffmann, Chalmers University of Technology, Sweden

Coffee break Time: 10:00–10:30 Session 5: Gluten-free baking and processing I Time: 10:30–12:00 Location: Conference room Rondo Chair: Nanna Mossberg, Fria Bröd, Sweden; Tuula Sontag-Strohm, University of Helsinki, Finland 10:30 OVERVIEW ON THE NEW DEVELOPMENTS IN THE AREA OF GLUTEN FREE FOODS AND BEVERAGES Elke Arendt, University Collage Cork, Ireland

11:00 GLUTEN FREE BAKED PRODUCTS: SOME QUALITY SOLUTIONS William Atwell, Cargill Bakery Category Technology, Plymouth, MN, USA

11:20 FORMATION AND MODIFICATION OF BIOACTIVE COMPOUNDS IN GLUTEN FREE SOURDOUGHS Michael Gänzle, University of Alberta, Canada

11:40 ENZYMATIC PROCESSING OF GLUTEN-FREE FLOURS: A PROMISING TOOL TO IMPROVE THEIR BREAD-MAKING FUNCTIONALITY? Stefano Renzetti, TNO Quality of Life, Netherlands

Lunch break Time: 12:00–13:00

V

Thursday, June 10th, 2010 Session 6: Gluten-free baking and processing II Time: 13:00–14:55 Location: Conference room Rondo Chair: Markku Mikola, Sennet, Finland; Eimear Gallagher, Teagasc, Ireland 13:00 FRIA BRÖD – COMPANY PRESENTATION Nanna Mossberg, Fria Bröd, Sweden

13:15 INFLUENCE OF SELECTED MODIFIED STARCHES AND HYDROCOLLOIDS ON THE RHEOLOGICAL PROPERTIES OF DOUGH AND BREAD BASED ON RICE (ORYZA SATIVA) AND BUCKWHEAT (FAGOPYRUM ESCULENTUM) Stephan Haase, TU München, Germany

13:35 EXOPOLYSACCHARIDE WEISSELLA STRAINS AS STARTER CULTURES FOR SORGHUM AND WHEAT SOURDOUGHS Clarissa Schwab, University of Alberta, Canada

13:55 INFLUENCE OF BETA-GLUCAN FROM DIFFERENT ORIGINS ON THE QUALITY OF GLUTEN FREE BREADS Anna-Sophie Hager, University College Cork, Ireland

14:15 EFFECTS OF TWO-STEP TRANSAMIDATION OF WHEAT FLOUR AND SEMOLINA ON THE TECHNOLOGICAL PROPERTIES OF GLUTEN Mauro Rossi, Institute of Food Sciences, NRC, Italy

14:35 THE EFFECT OF DELETION LINES OF BREAD WHEAT ‘CHINESE SPRING’ ON CELIAC DISEASE STIMULATING EPITOPES AND TECHNOLOGICAL PROPERTIES Hetty Van den Broeck, Plant Research International, Netherlands

Poster hour & Coffee break Time: 14:55–15:55 Location: Winter Garden Session 7: Gluten-free pasta, beer, and industrial perspectives Time: 15:55–17:25 Location: Conference room Rondo Chair: William Atwell, Cargill, MN, USA; Sanna Luoto, University of Helsinki, Finland 15:55 GLUTEN-FREE PASTA: TECHNOLOGY AND QUALITY EVALUATION Manuela Mariotti, Università degli Studi di Milano, DiSTAM, Italy

16:15 PROSO MILLET (PANICUM MILIACEUM L.) A SUSTAINABLE RAW MATERIAL FOR THE MALTING AND BREWING PROCESS Martin Zarnkow, TU München, Germany

16:35 GLUTEN-FREE BEER FROM BARLEY Saara Pöyri, Oy Sinebrychoff Ab, Kerava, Finland

16:45 GLUTEN-FREE WHEAT STARCH – SAFETY AND FUNCTIONALITY ON A NATURAL WAY Maren Wiese, Hermann Kröner GmbH, Germany

VI

16:55 IMPROVING THE TEXTURE AND NUTRITIONAL PROFILE OF GLUTEN FREE BAKED GOODS BY FORMULATION SCIENCE AND SPECIALITY FLOUR TECHNOLOGY: MUFFINS Despina Ioannides, National Starch Food Innovation, Germany

17:05 DEVELOPMENT OF HIGH-QUALITY GLUTEN-FREE BREADS FOR THE EUROPEAN MARKET Valentina Stojceska, Manchester Metropolitan University, United Kingdom

17:15 IMITATED RYE FLOUR – EVALUATION OF PENTOSAN SOURCES Markus Brandt, Ernst Böcker GmbH & Co. KG, Germany

Friday, June 11th, 2010 Session 8: Coeliac disease and gluten-free foods update – Joint session with the Coeliac Fair Time: 08:45–13:00 Location: Small Auditorium, Tampere Hall Chair: Markku Mäki, University of Tampere, Finland; Tuula Sontag-Strohm, University of Helsinki, Finland 09:00 GLUTEN-FREE AND VERY LOW GLUTEN FOODS – ENFORCEMENT OF COMMON EU LEGISLATION Annika Nurttila, Finnish Food Safety Authority EVIRA, Finland

09:45 WHEAT STARCH IN GLUTEN-FREE DIET Katri Kaukinen, University of Tampere and Tampere University Hospital, Finland

10:15 WHEAT STARCH IN GLUTEN FREE BREAD Nanna Mossberg, Fria Bröd AB, Sweden

Coffee break Time: 10:30–11:15 11:15 OATS IN GLUTEN-FREE DIET – CONSIDERATIONS OF A PRAGMATIC POLICY Hannu Salovaara, Päivi Kanerva, Jussi Loponen, Tuula Sontag-Strohm, University of Helsinki, Finland

11:45 TIPS FOR USING OATS IN THE GLUTEN-FREE DIET Sanna Arnala, The Finnish Coeliac Society, Finland

12:15 PURE OATS PRODUCTION-COMPANY PRESENTATION Pirjo Alho-Lehto, Raisio Group, Food Division, Finland

12:30 QUO VADIS, COELIAC DISEASE? Markku Mäki, University of Tampere, Finland

Closing ceremonies of GF10 Symposium Time: 13:00–13:15 Location: Small Auditorium Lunch Time: 13:15–14:30

VII

VIII

Table of Contents

Wednesday, June 9th, 2010

Coeliac disease and the triggering molecules

CURRENT PREVALENCE, DIAGNOSIS AND TREATMENT OF COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . 1Markku Maki, University of Tampere, Finland

THE TRIGGERING PROTEINS AND PEPTIDES IN COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Peter Koehler and Herbert Wieser, German Research Centre for Food Chemistry, Germany

THREE DAYS ORAL GLUTEN CHALLENGE RESPONSE IN GLUTEN SENSITIVE INDIVIDUALS WITHAND WITHOUT CD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

Margit Brottveit, Oslo University Hospital, Norway; Stig Tollefsen, National Veterinary Institute, Norway; MelindaRaki, Ann-Christin R. Beitnes, Frode L. Jahnsen, Centre for Immune Regulation, Norway; Jorunn Bratlie, OsloUniversity Hospital, Norway; Ludvig M. Sollid, Centre for Immune Regulation, Norway; and Knut E. A. Lundin,Oslo University Hospital, Norway

Posters of Session 1: Coeliac disease and the triggering molecules

GLUTEN AS A CAUSE OF GASTROINTESTINAL SYMPTOMS IN PATIENTS WHO DO NOT HAVECOELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

Jessica Biesiekierski, Evan Newnham, Peter Irving, Jacqueline Barrett, Melissa Haines, Monash University, Aus-tralia; James Doecke, CSIRO, Australia; Susan Shepherd, Jane Muir and Peter Gibson, Monash University, Aus-tralia

DIVERSITY OF AVENIN GENES IN OAT (AVENA SATIVA L.) AND CONTENT OF CELIAC DISEASEEPITOPES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Diana Londono, Wageningen UR, Netherlands

ACUTE ADMINISTRATION OF TRITICUM MONOCOCCUM SSP MONOCOCCUM, CULTIVAR ”MON-LIS”:A PHASE II, SINGLE BLIND, CROSS-OVER STUDY IN PATIENTS WITH COELIAC DISEASE. . . . . .

9

Beatrice Petroboni, Barbara Zanini, University of Brescia, Italy; Tarciso Not, University of Trieste, Italy; NorbertoPogna, Research Unit for Cereals, Italy; and Alberto Lanzini, University of Brescia, Italy

DOWN-REGULATION OF γ -GLIADINS IN BREAD WHEAT BY RNA INTERFERENCE (RNAI) . . . . . . 11Javier Gil-Humanes, IAS. CSIC, Spain; Fernando Piston, Universidad de Cordoba, Spain; and Francisco Barro,IAS. CSIC, Spain

GLUTEN EPITOPES IN FINNISH PATIENTS WITH DQ2+ CELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Andrea de Kauwe, University of Helsinki, Finland; Katri Kaukinen, University of Tampere and Tampere UniversityHospital, Finland; Jason Tye-din, Jessica Stewart, The Walter and Eliza Hall Institute, Australia; Kalle Kurppa,University of Tampere and Tampere University Hospital, Finland; Lotta Koskinen, University of Helsinki, Finland;Markku Maki, University of Tampere, Finland; Robert Anderson, The Walter and Eliza Hall Institute, Australia;and Paivi Saavalainen, University of Helsinki, Finland

IX

. . . . .

Session 1:

Gluten analysis and safety testing

IMPROVING ACCURACY IN DETECTING GLUTEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Paivi Kanerva, Tuula Sontag-Strohm, Jussi Loponen and Hannu Salovaara, University of Helsinki, Finland

PRELIMINARY RESULTS OF THE COLLABORATIVE STUDY TO VALIDATE THE CHARACTERISTICSOF THE GLUTEN-TEC ELISA KIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17

Jorge Raul Mujico Fernandez Leiden University Medical Center, Netherlandsand Frits Koning,

SECOND GENERATION TESTING FOR GLIADIN IN COMPLIANCE WITH CODEX ALIMENTARIUSLEVEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19

Sigrid Haas-Lauterbach and Ulrike Immer, R-Biopharm, Germany

DETECTION OF TOXIC FRAGMENTS FROM GLUTEN USING A NEW MONOCLONAL ANTIBODY-BASED TEST. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Richard Fielder, Romer Labs UK, United Kingdom; and Elisabeth Halbmayr, Romer Labs Division Holding,Austria

GENOMICS APPROACHES TO ANALYSE CD-TOXICITY FOR THE PRODUCTION OF CD-SAFE WHEAT 23Luud Gilissen, Hetty C. Van den Broeck, Elma Salentijn, Ingrid M. Van der Meer and Marinus J.M. Smulders,Plant Research International, Netherlands

PRESUMPTIVE SAFETY FOR CELIAC PATIENTS OF BAKED GOODS MADE OF SOURDOUGH FER-MENTED WHEAT FLOUR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

Marco Gobbetti, University of Bari, Italy

TESTING SAFETY OF LOW GLUTEN FOOD PRODUCTS IN A MOUSE MODEL OF CELIAC DISEASE . . 27Tobias Freitag, University of Helsinki, Finland; Yvonne Junker, Harvard Medical School, USA; Paivi Saavalainen,Seppo Meri, University of Helsinki, Finland; and Detlef Schuppan, Harvard Medical School, USA

GENERAL DISCUSSION ON: SAFETY AND TOLERANCE - ANALYSIS AND REGULATORY STAN-DARDS - TECHNOLOGY AND VARIETY OF PRODUCTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

Hannu Salovaara, Paivi Kanerva, Jussi Loponen and Tuula Sontag-Strohm, University of Helsinki, Finland

Posters of Session 2: Gluten analysis and safety testing

DETERMINATION OF THE IMMUNOTOXIC POTENTIAL OF OATS FOR THE SELECTION OF VARIETIESPOSSIBLY SAFE FOR COELIAC PATIENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31

Isabel Comino, Universidad de Sevilla, Spain

RECOGNITION OF GLIADIN AND GLUTENIN FRACTIONS IN FOUR COMMERCIAL GLUTEN ASSAYS 33Laura Allred, ELISA Technologies, Inc., United States

NEW APPROACHES IN GLUTEN ANALYSIS OF PRODUCTS FOR CELIACS BY PROTEOMICS COM-BINED WITH THE R5 ELISA TECHNIQUES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35

Maria Carmen Mena, Manuel Lombardıa, Alberto Hernando and Juan Pablo Albar, Centro Nacional de Biotec-nologia, CSIC, Spain

EFFECTS OF HEATING, REDUCING AND ALCOHOL CONCENTRATION ON PROLAMIN EXTRACTION 37Paivi Kanerva, Tuula Sontag-Strohm, Outi Brinck and Hannu Salovaara, University of Helsinki, Finland

DEAMIDATION OF GLUTEN PROTEINS DRASTICALLY INFLUENCES THE QUANTITATIVE GLUTENANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39

X

Paivi Kanerva, Tuula Sontag-Strohm, Hannu Salovaara and Jussi Loponen, University of Helsinki, Finland

Session 2:

3: Coeliac food market and nutritional requirements, Gluten-free cereals andpseudo-cereals

NUTRITIONAL REQUIREMENTS FOR GLUTEN-FREE FOODS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Tricia Thompson, The Gluten-Free Dietitian, MA, USA

GLUTEN-FREE FOOD MARKET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Markku Mikola, Sennet Oy, Finland; and Esa Wrang, Finpro, Finland

FOLATES IN GLUTEN-FREE DIETS AND FOODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Vieno Piironen, Maija Kinni, Jussi Loponen and Susanna Kariluoto, University of Helsinki, Finland

CEREALS AND PSEUDOCEREALS FOR GLUTEN-FREE FOODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Regine Schoenlechner and Emmerich Berghofer, University of Natural Resources and Applied Life Sciences, Aus-tria

HEALTHY GRAINS FOR ENHANCED GLUTEN-FREE BREADS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Eimear Gallagher, Ashtown Food Research Centre, Teagasc, Ireland

Posters of Session 3: Coeliac food market and nutritional requirements, Gluten-freecereals and pseudo-cereals

ARE COELIACS FOLLOWING A GLUTEN-FREE DIET OR A DIET LOW IN GLUTEN? . . . . . . . . . . . . . . . . . . 51Blanca Esteban, Manuela Marquez and Juan Ignacio Serrano-Vela, Madrid Coeliac Association, Spain

NUTRITIONAL QUALITY OF LINSEED AND OIL HEMP VARIETIES CULTIVATED IN FINLAND WITHSPECIAL ATTENTION TO LIGNAN AND CADMIUM CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

Marketta Saastamoinen, Satafood Development Association, Huittinen, Finland; Juha-Matti Pihlava and MerjaEurola, MTT, Chemical Laboratory, Jokioinen, Finland

ANALYSIS OF THE VARIATION OF HEALTH-PROMOTING COMPOUNDS IN DIFFERENT OAT CULTI-VARS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

Ingrid M. Van der Meer, Hetty C. Van den Broeck, Marinus J.M. Smulders, Plant Research International, Nether-lands; Jurriaan J. Mes, Food and Biobased Research, Wageningen UR, Netherlands; and Ludovicus J.W.J. Gilis-sen, Plant Research International, Netherlands

A SYSTEMATIC LITERATURE REVIEW ON THE NUTRITIONAL ADEQUACY OF A TYPICAL GLUTEN-FREE DIET WITH PARTICULAR REFERENCE TO IRON, CALCIUM, FOLATE AND B VITAMINS . . . . . . .

57

Emma Merrikin, Emily Kirk, Norma McGough, Coeliac UK, Diet and Health, High Wycombe, United Kingdom;Gerry Robins, York Hospitals NHS Foundation Trust, United Kingdom; and Anthony Akobeng, Central Manchesterand Manchester Childrens University Hospital, United Kingdom

TARTARY BUCKWHEAT AS A GLUTEN FREE SOURCE FOR FUNCTIONAL . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Mateja Germ and Ivan Kreft, University of Ljubljana, Slovenia

TEFF (ERAGROSTIS TEF) SUPPLEMENTED GLUTEN-FREE BREADS AS A POTENTIAL PREVENTIONOF IRON-DEFICIENCY ANAEMIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61

Ebtesam Ben-Fayed, Valentina Stojceska and Paul Ainsworth, Manchester Metropolitan University, United King-dom

OAT BETA-GLUCAN AFFECTS THE VISCOELASTIC PROPERTIES OF GASTRIC MUCIN AT PH CONDI-TIONS OF SMALL INTESTINE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63

XI

Reetta Kivela, Sami Hietala, Tuula Sontag-Strohm, University of Helsinki, Finland; Bradley Turner, HarvardMedical School, USA; and Rama Bansil, Boston University, Department of Physics, USA

Session

Gluten detoxification

Frits Koning, Leiden University Medical Centre, Netherlands

MANY FACES OF PROLYL OLIGOPEPTIDASE WHAT WE IGNORE IN MAMMALS AND WHAT WEKNOW IN PLANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67

J. Arturo Garcıa-Horsman, University of Helsinki, Finland

ENDOGENOUS CEREAL ENZYMES IN THE ELIMINATION OF PROLAMINS . . . . . . . . . . . . . . . . . . . . . . . . . . 69Jussi Loponen, Paivi Kanerva, University of Helsinki, Finland; Michael Ganzle, University of Alberta, Canada;Tuula Sontag-Strohm and Hannu Salovaara, University of Helsinki, Finland

SYNTHETIC BLOCKING PEPTIDES WITH HIGH AFFINITY TO GLIADIN REDUCE TISSUE TRANSG-LUTAMINASE ACTIVITY ON WHEAT GLIADIN IN VITRO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71

Karolina Hoffmann, Marie Alminger, Thomas Andlid, Chalmers University of Technology, Sweden; Tingsu Chen,Guangxi Academy of Agricultural Sciences, China; Olof Olsson, Gothenburg University, Sweden; and Ann-SofieSandberg, Chalmers University of Technology, Sweden

Posters of Session 4: Gluten detoxification

DEGRADATION OF GLIADIN PEPTIDES TOXIC FOR COELIAC DISEASE PATIENTS BY PROLYL EN-DOPEPTIDASE SYNTHESIZED BY LACTOBACILLUS ACIDOPHILUS 5E2 AND ASPERGILLUS NIGER

73

Bartosz Brzozowski, Wlodzimierz Bednarski, University Of Warmia And Mazury, Poland; and Barbara Wrob-lewska, Polish Academy of Science, Poland

DETOXIFICATION OF GLUTEN BY GERMINATING CEREAL ENZYMES: IMPLICATIONS FOR NEWTREATMENT OF COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75

Satumarja Stenman, University of Tampere, Finland

DEGRADATION OF IMMUNOGENIC GLUTEN EPITOPES BY PROBIOTIC LACTOBACILLI . . . . . . . . . . . . . 77Maria De Angelis, Raffaella Di cagno, Francesca Gagliardi, Carlo Giuseppe Rizzello, Ruggero Francavilla andMarco Gobbetti, University of Bari, Italy

Greetje Tack, Jolanda Van de Water, VU University Medical Centre, Amsterdam, Netherlands; Maaike Bruins,DSM Biotechnology Centre, Delft, Netherlands; Yvonne Kooy-Winkelaar, Jeroen Van Bergen, Leiden Univer-sity Medical Centre, Netherlands; Gerrit Meijer, Mary von Blomberg, Marco Schreurs, VU University MedicalCentre, Amsterdam, Netherlands; Luppo Edens, DSM Biotechnology Centre, Delft, Netherlands; Chris Mulder,VU University Medical Centre, Amsterdam,, Netherlands; and Frits Koning, Leiden University Medical Centre,Netherlands

AUTO-PROTEOLYTIC AND PHYSICAL ELIMINATION OF PROLAMINS IN ACIDIC SUSPENSIONS OFMALTED WHEAT, BARLEY, AND RYE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

Jussi Loponen, Outi Brinck, Zhongqing Jiang and Hannu Salovaara, University of Helsinki, Finland

HYDROLYTIC POTENTIAL OF MALTS PREPARED OF THREE RYE VARIETIES AND IMPACT ON PRO-LAMIN BREAKDOWN DURING ACIDIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83

XII

Emma Laivisto, University of Helsinki, Finland; Annika Wilhelmson, Arvi Wilpola, VTT Technical Research Centreof Finland, Finland; J. Arturo Garcia-Horsman, Hannu Salovaara and Jussi Loponen, University of Helsinki,Finland

PROLYLENDOPROTEASES FOR GLUTEN DETOXIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Session 4:

CAN PROLYL ENDOPROTEASE ENZYME TREATMENT MITIGATE THE TOXIC EFFECT OF GLUTENIN COELIAC PATIENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

79

Thursday, June 10th, 2010

Gluten-free baking and processing I

OVERVIEW ON THE NEW DEVELOPMENTS IN THE AREA OF GLUTEN FREE FOODS AND BEVER-AGES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85

Elke K. Arendt, University Collage Cork, Ireland

GLUTEN FREE BAKED PRODUCTS: SOME QUALITY SOLUTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87William A. Atwell, Cargill Bakery Category Technology, Plymouth, MN, USA

FORMATION AND MODIFICATION OF BIOACTIVE COMPOUNDS IN GLUTEN FREE SOURDOUGHS . 89Michael Ganzle, Andreas Schieber, Louise Svensson, Januana Teixeira and Victoria McNeill, University of Alberta,Canada

ENZYMATIC PROCESSING OF GLUTEN-FREE FLOURS: A PROMISING TOOL TO IMPROVE THEIRBREAD-MAKING FUNCTIONALITY? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91

Stefano Renzetti, TNO Quality of Life, Netherlands; and Elke K. Arendt, University College Cork, Ireland

Posters of Session 5: Gluten-free baking and processing I

COMPETITIVENESS OF COMMERCIAL STARTERS IN BUCKWHEAT AND TEFF SOURDOUGHS . . . . . . 93Alice V. Moroni, Fabio Dal Bello and Elke K. Arendt, University College Cork, Ireland

DEVELOPMENT OF GLUTEN FREE MUFFIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95Marios Pogiatzis, Weili Li, Russell Ramsden and Charles Brennan, Manchester Metrpolitan University, UnitedKingdom

GLUTEN-FREE SORYZ COOKIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97Cosciug Lidia, Technical University of Moldova, Moldova

METABOLISM AND COMPETITIVENESS OF LACTOBACILLUS SANFRANCISCENSIS AND TING ISO-LATES IN WHEAT AND SORGHUM SOURDOUGHS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

99

Bonno Sekwati-Monang and Michael Ganzle, University of Alberta, Canada

OBTAINING AND CHARACTERIZATION OF GLUTEN FREE FLOUR PRODUCTS ENRICHED WITHDRIED FRUIT AND HIPPOPHAE RHAMNOIDES EXTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

101

Ersilia Alexa, Banat‘s University of Agricultural Science, Romania

OPTIMIZATION OF GLUTEN-FREE FRENCH-STYLE BREAD FORMULATION SUITABLE FOR FROZENDOUGH PROCESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103

Sandra Mezaize, Sylvie Chevallier, Alain Le Bail and Marie De Lamballerie, Oniris, France

RHEOLOGICAL PROPERTIES OF GLUTEN-FREE BREAD FORMULATIONS USING CHESTNUT ANDRICE FLOUR COMBINATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105

Ilkem Demirkesen, Behic Mert, Gulum Sumnu and Serpil Sahin, Middle East Technical University, Turkey

USAGE OF CHUFA FLOUR IN GLUTEN-FREE CAKES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107Elif Turabi, Gulum Sumnu, Serpil Sahin, Middle East Technical University, Turkey; and Mehmet Musa Ozcan,Selcuk University, Turkey

XIII

HETEROPOLYSACCHARIDE PRODUCTION FROM LACTIC ACID BACTERIA IN WHEAT ANDSORGHUM SOURDOUGH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

Sandra Galle, University College Cork, Ireland; Clarissa Schwab, University of Alberta, Canada; Fabio Dal Bello,Elke K. Arendt, University College Cork, Ireland; and Michael Ganzle, University of Alberta, Canada

PROMOTING OF STRUCTURE FORMATION BY HIGH PRESSURE IN GLUTEN-FREE FLOURS. . . . . . . . . 111Katleen. J. R. Vallons, Liam A. M. Ryan and Elke K. Arendt, University College Cork, Ireland

Session 5:

IDENTIFICATION OF LACTIC ACID BACTERIA ISOLATED FROM OAT SOURDOUGHS AND INVESTI-GATION OF THEIR POTENTIAL FOR THE IMPROVEMENT OF OAT BREAD QUALITY. . . . . . . . . . . . . . . . .

113

Edith K. Huttner, Fabio Dal Bello, Emanuele Zannini and Elke K. Arendt, University College Cork, Ireland

PILOT SCALE MANUFACTURE OF HIGHLY CONCENTRATED PROTEIN INGREDIENTS FROM OATSUSING DRY FRACTIONATION TECHNOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115

Juhani Sibakov, Olavi Myllymaki, VTT Technical Research Centre of Finland, Finland; Michael Kuhnen,Hosokawa Alpine AG, Augsburg, Germany; Anu Kaukovirta-Norja, Kaisa Poutanen and Pekka Lehtinen, VTTTechnical Research Centre of Finland, Finland

Gluten-free baking and processing II

INFLUENCE OF SELECTED MODIFIED STARCHES AND HYDROCOLLOIDS ON THE RHEOLOGI-CAL PROPERTIES OF DOUGH AND BREAD BASED ON RICE (ORYZA SATIVA) AND BUCKWHEAT(FAGOPYRUM ESCULENTUM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117

Stephan Haase, TU Munchen, Germany

EXOPOLYSACCHARIDE WEISSELLA STRAINS AS STARTER CULTURES FOR SORGHUM ANDWHEAT SOURDOUGHS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

Clarissa Schwab, University of Alberta, Canada; Sandra Galle, Elke K. Arendt, University College Cork, Ireland;and Michael Ganzle, University of Alberta, Canada

INFLUENCE OF BETA-GLUCAN FROM DIFFERENT ORIGINS ON THE QUALITY OF GLUTEN FREEBREADS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

121

Anna-Sophie Hager, Liam A. M. Ryan, University College Cork, Ireland; John V. O’Doherty, University CollegeDublin, Ireland; and Elke K. Arendt, University College Cork, Ireland

EFFECTS OF TWO-STEP TRANSAMIDATION OF WHEAT FLOUR AND SEMOLINA ON THE TECHNO-LOGICAL PROPERTIES OF GLUTEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

123

Federica Capobianco, Salvatore Moscaritolo, IPALC Research & Development Laboratories, Italy; and MauroRossi, Institute of Food Sciences, NRC, Italy

THE EFFECT OF DELETION LINES OF BREAD WHEAT ’CHINESE SPRING’ ON CELIAC DISEASESTIMULATING EPITOPES AND TECHNOLOGICAL PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125

Hetty C. Van den Broeck, Plant Research International, Netherlands; Hein De Jong, Limagrain Nederland B.V.,Netherlands; Liesbeth Dekking, Leiden University Medical Center, Netherlands; Dirk Bosch, Plant Research In-ternational, Netherlands; Rob Hamer, Laboratory of Food Chemistry, Wageningen UR, Netherlands; Marinus J.M.Smulders, Ludovicus J.W.J. Gilissen and Ingrid M. Van der Meer, Plant Research International, Netherlands

Posters of Session 6: Gluten-free baking and processing II

PHYSICOCHEMICAL PROPERTIES OF OAT VARIETIES AND THEIR POTENTIAL FOR BREAD MAKING 127Edith K. Huttner, Fabio Dal Bello and Elke K. Arendt, University College Cork, Ireland

EFFECTS OF BASIC PROCESS PARAMETERS ON QUALITY OF GLUTEN-FREE RICE BREADS . . . . . . . . 129Gina Jaspers and Markus Brandt, Ernst Bocker GmbH & Co. KG, Germany

XIV

CASEIN NETWORK FORMATION IN GLUTEN FREE BREAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Sheila Kenny, Teagasc, Ireland

EFFECT OF MICROBIAL HOMOPOLYSACCHARIDES ON THE STRUCTURE OF GLUTEN-FREEBREADS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

133

Christine Ruhmkorf, Susanne Kaditzky and Rudi Vogel, TU Munchen, Germany

Session 6:

FERMENTED WHEY-BASED DIARY DESSERT STABILIZED WITH STARCH FROM GLUTEN-FREESOURCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135

Bulgaru Viorica, Technical University of Moldova, Moldova

DEVELOPMENT OF A NEW GLUTEN-FREE BROWN BREAD FLOUR MIX RICH IN FIBER AND HIGHIN NUTRITIONAL CONTENT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137

Virna Lucia Cerne, Dr. Schar GmbH, Italy

SENSORY AND TEXTURAL PROPERTIES OF GLUTEN-FREE BREAD BASED ON RICE/BUCKWHEATFLOUR MIXTURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139

Aleksandra Torbica, Miroslav Hadnadev and Marijana Sakac, Institute for Food Technology, Serbia

THE POSITIVE EFFECT OF AMARANTH SOURDOUGH ADDITION IN GLUTEN FREE BREAD QUAL-ITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141

Andreas Houben, TU Munchen, Germany; Martin Mitzscherling, University of Hohenheim, Germany; and ThomasBecker, TU Munchen, Germany

DISCRIMINATION BETWEEN GLUTEN-FREE BREAD FORMULATIONS USING NEAR INFRAREDIMAGING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

143

Gerard Downey, Teagasc, Ireland

EVALUATION OF PHYSICALLY-CHEMICAL PARAMETERS OF GLUTEN-FREE DUMPLINGS . . . . . . . . . . 145Tatjana Rakcejeva, Ilze Gramatina and Anastasija Fjodorova, Latvia University of Agriculture, Faculty of Foodtechnology, Latvia

NOVELTY FORMULA OF FREE GLUTEN POCKET TYPE FLAT ARABIC BREAD. . . . . . . . . . . . . . . . . . . . . . . 147Hanee Al-Dmoor, Al- Balqa Applied University, Jordan

A GLUTEN-FREE BREAD WITH VISCOUS JAPANESE YAM INSTEAD OF WHEAT FLOUR . . . . . . . . . . . . . 149Masaharu Seguchi, Kobe Women’s University, Japan

EFFECT OF LEGUME FLOURS ON BAKING CHARACTERISTICS OF GLUTEN FREE BREADS . . . . . . . . . 151Begona Minarro, Universidad Autonoma de Barcelona, Spain

EFFECTS OF HIGH PRESSURE AND TEMPERATURE ON BUCKWHEAT STARCH CHARACTERISTICS 153Katleen. J. R. Vallons, Liam A. M. Ryan and Elke K. Arendt, University College Cork, Ireland

RHEOLOGICAL PROPERTIES AND BREAD MAKING PERFORMANCE OF COMMERCIAL WHOLE-GRAIN OAT FLOURS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

Edith K. Huttner, Fabio Dal Bello and Elke K. Arendt, University College Cork, Ireland

GLUTEN-FREE BREAD SUPPLEMENTED WITH CALCIUM THE IMPROVEMENT OF QUALITY ANDTEXTURE PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

157

Ursula Krupa-Kozak, Malgorzata Wronkowska, Maria Soral-Smietana, Agnieszka Troszynska and JadwigaSadowska, Polish Academy of Science, Poland

XV

GLUTEN-FREE PASTA: TECHNOLOGY AND QUALITY EVALUATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159Manuela Mariotti, Carola Cappa and Mara Lucisano, Universita degli Studi di Milano, DiSTAM, Italy

PROSO MILLET (PANICUM MILIACEUM L.) A SUSTAINABLE RAW MATERIAL FOR THE MALTINGAND BREWING PROCESS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161

Martin Zarnkow, Thomas Becker, TU Munchen, Germany; and Elke K. Arendt, University College Cork, Ireland

GLUTEN-FREE BEER FROM BARLEY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163Saara Poyri, Oy Sinebrychoff Ab, Kerava, Finland; Markku Maki, University of Tampere, Finland; Alberto Her-nando, Maria Carmen Mena, Manuel Lombardia, Centro Nacional de Biotecnologia, CSIC, Spain; Pekka Lehto-nen, Alko Oy, Finland; P. Soininen-Tengvall, E. Pajunen, Oy Sinebrychoff Ab, Kerava, Finland; and E. Mendez,Centro Nacional de Biotecnologia, CSIC, Spain

GLUTEN-FREE WHEAT STARCH - SAFETY AND FUNCTIONALITY ON A NATURAL WAY . . . . . . . . . . . . 164Maren Wiese, Hermann Kroner GmbH, Germany

IMPROVING THE TEXTURE AND NUTRITIONAL PROFILE OF GLUTEN FREE BAKED GOODS BY FOR-MULATION SCIENCE AND SPECIALITY FLOUR TECHNOLOGY: MUFFINS . . . . . . . . . . . . . . . . . . . . . . . . . . .

165

Despina Ioannides, Alejandro Perez and Yadunandan Dar, National Starch Food Innovation, United States

DEVELOPMENT OF HIGH-QUALITY GLUTEN-FREE BREADS FOR THE EUROPEAN MARKET . . . . . . . 167Valentina Stojceska and Paul Ainsworth, Manchester Metropolitan University, United Kingdom

IMITATED RYE FLOUR - EVALUATION OF PENTOSAN SOURCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169Markus Brandt and Gina Jaspers, Ernst Bocker GmbH & Co. KG, Germany

Posters of Session 7: Gluten-free pasta, beer, and industrial perspectives

CHANGES IN BIO-ACTIVE COMPOUNDS IN BUCKWHEAT DEPENDING ON GERMINATION CONDI-TIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171

Florian Hubner and Elke K. Arendt, University College Cork, Ireland

PROTEIN CHANGES IN BUCKWHEAT DEPENDING ON THE MALTING CONDITIONS . . . . . . . . . . . . . . . . . 173Florian Hubner and Elke K. Arendt, University College Cork, Ireland

PROTEIN CHANGES DURING MALTING AND BREWING WITH OATS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Christina Klose and Elke K. Arendt, University College Cork, Ireland

OPTIMIZATION OF RHEOLOGICAL PROPERTIES OF GLUTEN-FREE PASTA USING MIXTURE DESIGN 177Virginia Larrosa, Gabriel Lorenzo, Noemı Zaritzky and Alicia Califano, CIDCA, Argentina

CHARACTERIZING SORGHUM-BASED PASTA: A MULTI-DISCIPLINARY APPROACH. . . . . . . . . . . . . . . . . 179Maria Ambrogina Pagani, Francesco Bonomi, Gabriella Bottega, Maria Cristina Casiraghi, University of Milan,Italy; Abd Elmoneim Elkhalifa, Ahfad University for Women, Sudan; and Stefania Iametti, University of Milan,Italy

READY-TO-EAT MEALS BASED ON RICE PASTAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181Virtucio Luis, Pavan, Italy

STARCH CHARACTERIZATION OF RICE PASTA: COMPARISON BETWEEN EXTRUSION-COOKINGAND CONVENTIONAL PASTA-MAKING PROCESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185

Alessandra Marti, University of Milan, Italy; Rosita Caramanico, CRA-SCV, Italy; Koushik Seetharaman, Univer-sity of Guelph, Canada; and Maria Ambrogina Pagani, University of Milan, Italy

XVI

Gluten-free pasta, beer, and industrial perspectives

GLUTEN IN OAT-BASED BEVERAGES AND OATMEAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187Ylva Sjogren, Birgitta Kruse, Monica Ferm, Martin Sandberg and Ingrid Malmheden Yman, National Food Ad-ministration, Sweden

Session 7:

Friday, June 11th, 2010

GLUTEN-FREE AND VERY LOW GLUTEN FOODS ENFORCEMENT OF COMMON EU LEGISLATION . 189Annika Nurttila, Finnish Food Safety Authority EVIRA, Finland

WHEAT STARCH IN GLUTEN-FREE DIET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190Katri Kaukinen, University of Tampere and Tampere University Hospital, Finland

WHEAT STARCH IN GLUTEN FREE BREAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191Nanna Mossberg, Fria Brod AB, Sweden

OATS IN GLUTEN-FREE DIET CONSIDERATIONS OF A PRAGMATIC POLICY. . . . . . . . . . . . . . . . . . . . . . . . . 193Hannu Salovaara, Paivi Kanerva, Jussi Loponen and Tuula Sontag-Strohm, University of Helsinki, Finland

TIPS FOR USING OATS IN THE GLUTEN-FREE DIET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Sanna Arnala, The Finnish Coeliac Society, Finland

PURE OATS PRODUCTION-COMPANY PRESENTATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197Pirjo Alho-Lehto, Raisio Group, Food Division, Finland

QUO VADIS, COELIAC DISEASE? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Markku Maki, University of Tampere, Finland

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

XVII

Session: 8 Coeliac disease and gluten-free foods update - Joint session with the Coeliac Fair

XVIII

Current Prevalence, Diagnosis and Treatment of Coeliac Disease

Markku Mäki

Coeliac Disease Study Group, University of Tampere, Finland

corresponding email: Markku.Maki@uta.fi

1

GLUTEN FREE

2

Notes

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The Triggering Proteins and Peptides in Coeliac Disease

Peter Koehler*, Herbert Wieser German Research Centre for Food Chemistry, Freising, Germany

*corresponding email: peter.koehler@lrz.tum.de Introduction. Coeliac disease (CD) is triggered by the ingestion of wheat, rye, barley, and possibly oat products. During the last decade, many studies have contributed to substantial progress in understanding the general principles that determine the pathogenesis of CD. Toxic proteins. Of the toxic or potentially toxic grains, only proteins from wheat and oats have been extensively studied for CD toxicity. Testing of rye and barley has been rather minimal, the strong similarities of their storage proteins with wheat gluten proteins, however, support their CD harmfulness. The taxonomy of plants might provide useful guidance in dividing grains into safe and unsafe (Kasarda 2001). All toxic grains (wheat, rye, barley) are found in a single tribe, the Triticeae, within the grass family (Poaceae). Considering this, all wheat species including bread and durum wheat, kamut, spelt, emmer, and einkorn or the wheat-rye crossbreed triticale have to be avoided by CD patients. Oats, remaining controversial regarding toxicity, belong to a separate tribe, the Aveneae. The nontoxic cereals including corn, sorghum, millet, and rice are still more distant from the Triticeae and show separated evolutionary lines within the grass family. Plants that did not fall in the grass family such as buckwheat, quinoa, and amaranth have been classified as safe. In vivo and in vitro studies indicated that all gliadin types (α/β-, γ-, ω-type) are coeliac-toxic (reviewed by Shewry et al 1992). Both types of wheat glutenins, HMW-GS and LMW-GS, were not tested until recently. In vivo and in vitro tests revealed that HMW-GS exacerbate CD just as gliadins (Molberg et al 2003; Dewar et al 2006). T-cell stimulation tests on peptides from LMW-GS indicated that this protein type also has the potential to induce a CD-specific immune response (Vader et al 2002). In contrast to wheat, the different storage protein types of rye (HMW-, ω-, γ-40k-, γ-75k-secalins) and barley (D-, C-, B-, γ-hordeins) have not been tested up to now. Based on structural homologies with wheat proteins, it can be assumed that all of them are toxic for CD patients. Toxic peptides. It is generally accepted that peptides derived from the N-terminal region (positions 1–55) of α/β-gliadins (Table 1) trigger coeliac disease. The tetrapeptide sequences PSQQ and QQQP, which are common for active peptides, were considered to be key sequences for further investigations. Synthetic peptides derived from α-gliadins were used for in vivo tests (instillation) in addition to in vitro tests (organ culture) due to the availability of relatively high amounts. Peptide α(206–217) showed toxic effects in two CD patients in remission and no effect in the control group. In vivo challenges using three synthetic peptides from α/β-gliadins showed that peptide α(31–49) caused significant histological changes in four patients, whereas peptides α(202–220) and α(3–21) were weakly active or inactive, respectively. Ala-substituted variants of peptide α(31–49) remained active in the organ culture test, when residues L31 and P36 were substituted, but lost toxicity when residues P38, P39, and P42 were substituted. In vivo testing of two patients demonstrated the toxicity of peptides α(31–43) and α(44–55). Challenge tests with peptide α(56–75) and a negative control peptide from β-casein showed that the gliadin peptide caused intestinal damage, while the casein peptide produced no response. Corresponding repetitive sequences of γ-type and ω-type

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gliadins, secalins, and hordeins have not yet been tested by instillation or organ culture tests, but fit well into the potentially toxic sequences of α/β-gliadins. Table 1. Origin and amino acid sequences of gliadin peptides tested for CD-toxicity. Origin (Position) Sequencea Toxicityb (Test)

α(1 - 30) VRVPVPQLQPQNPSQQQPQEQVPLVQQF + (OC) α(3 - 21) VPVPQLQPQNPSQQQPQEQ - (IN, OC) α(3 - 24) VPVPQLQPQNPSQQQPQEQVPL + (OC) α(25 - 55) VQQQFPGQQQPFPPQQPYPQPQPFPSQQPY + (OC) α(31 - 43) LGQQQPFPPQQPY + (IN, OC) α(31 - 49) LGQQQPFPPQQPYPQPQPF + (IN, OC) α(31 - 49, A31) AGQQQPFPPQQPYPQPQPF + (OC) α(31 - 49, A36) LGQQQAFPPQQPYPQPQPF + (OC) α(31 - 49, A38) LGQQQPFAPQQPYPQPQPF - (OC) α(31 - 49, A39) LGQQQPFPAQQPYPQPQPF - (OC) α(31 - 49, A42) LGQQQPFPPQQAYPQPQPF - (OC) α(31 - 55) LGQQQPFPPQQPYPQPQPFPSQQPY + (OC) α(44 - 55) PQPQPFPSQQPY + (IN, OC) α(51 - 70) SQQPYLQLQPFPQPQLPYSQ + (OC) α(56 - 68) LQLQPFPQPQLPY - (IN, OC) α(56 - 75) LQLQPFPQPQLPYPQPQLPY + (IN) α(202 - 220) QQYPLGQGSFRPSQQNPQA - (IN, OC) α(206 - 217) LGQGSFRPSQQN + (IN) α(247 - 266) CNVYIAPYCTIAPFGIFGTN - (OC) a One-letter-code for amino acids; b OC, organ culture test; IN, instillation test Conclusions. In the case of proteins all types of storage proteins (prolamins + glutelins) of wheat, rye, barley, and possibly oats appear to be involved in activating CD. Concerning the peptides, the investigations carried out so far were, in parts, unsatisfactory with regard to the number of tests, purity of peptides, and accordance of results, but it could be concluded that most of the toxic sequences occur in the repetitive N-terminal domain of α/β-gliadins and mainly consist of Gln, Pro, and aromatic amino acids (Phe, Tyr). References Kasarda, DD. Grains in relation to celiac disease. Cereal Foods Worlds 2001; 46:209-210. Shewry PR, Tatham AS, Kasarda DD. Cereal proteins and coeliac disease. In: Marsh MN,

editor. Coeliac Disease. Oxford: Blackwell Scientific Publications; 1992. p. 305-348. Molberg Ø, Solheim Flaete N, Jensen T, Lundin KEA, Arentz-Hansen,H, Anderson OD,

Uhlen AK, Sollid LM. Intestinal T-cell responses to high-molecular-weight glutenins in celiac disease. Gastroenterol 2003; 125:337-344.

Dewar DH, Amato M, Ellis HJ, Pollock EL, Gonzales-Cinca N, Wieser H, Ciclitira PJ. The toxicity of high molecular weight glutenin subunits of wheat to patients with coeliac disease. Eur J Gastroenterol Hepatol 2006; 8:483-491.

Vader W, Kooy Y, van Veelen P, de Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterol 2002; 122:1729-1737.

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Three Days Oral Gluten Challenge – Response in Gluten Sensitive Individuals with and without CD

Margit Brottveit1,2, Stig Tollefsen3, Melinda Raki4, Ann-Christin R. Beitnes4, Frode L. Jahnsen4, Jorunn Bratlie1, Ludvig M. Sollid4, Knut E. A. Lundin1, 4*

1Oslo University Hospital, 2University of Oslo, 3National Veterinary Institute, Oslo, 4Centre for Immune Regulation – Oslo University Hospital and University of Oslo,

*corresponding email: k.e.a.lundin@medisin.uio.no

Introduction. Gluten induces symptoms in both coeliac disease (CD) patients and in gluten sensitive patients without CD. Both these two groups of patients adhere strictly to a gluten free diet. The effects of gluten challenge may be attributed to induction of innate immune response, adaptive immune response, or non-immunological functions (Sollid and Lundin 2009). We systematically investigated the magnitude of clinical response to gluten challenge in these patient groups and compared this to induction of mRNA for interferon- (IFN- ), TNF- , IL-8, MCP-1, Hsp27 and Hsp 70.

Patients and challenge protocol. Forty-eight study patients were all HLA-DQ2+ on a gluten free diet > 4 weeks. Thirteen were regular, volunteering CD patients. The remaining 35 subjects were investigated for possible CD and had started a gluten free diet without diagnosis. Three of these 35 showed by pre-challenge biopsy to have CD. The study protocol included a three days open challenge with 4 slices of sandwich bread pr day. Symptom scoring 3 days before, during and after challenge using the validated questionnaires: GSRS-IBS (Gastrointestinal Symptom Rating Scale, irritable bowel syndrome (IBS)-version) and SHC (Subjective Health Complaints). Upper endoscopy (EGD) with sampling of small bowel biopsies was performed on all study participants, followed by oral gluten challenge for three days (d1, d2 and d3). The day four (d4), new EGD/biopsy sampling was done. Biopsies were scored according to revised Marsh criteria. Presence of gluten specific T cells in peripheral blood on the 6 day were tested with an HLA-DQ2-gliadin peptide tetramer test (Ráki et al 2007).

mRNA induction. From biopsies RNA was isolated and cDNA synthesised. The relative amount of specific mRNA (cDNA) was quantified using Real Time PCR and expressed as CT values. GAPDH mRNA was used as housekeeping gene. Statistical analysis was performed using paired Wilcoxon signed-rank test on Delta CT (CT cytokine - CT GAPDH) values comparing the estimated relative amount of the different cytokines before and after challenge.

Results. The clinical response was more pronounced among the non-CD patients than the CD patients. Within the time frame studied, CD patients mount a concomitant adaptive (IFN- ) and innate (IL-8 and MCP1) immune response. No significant response in neither Hsp27, Hsp70 nor TNF- . The non-CD patients: Although clinically gluten sensitive: no signs of immune activation.

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Discussion. Our study with open-label gluten challenge in CD and non-CD patients induces clinical responses as measured by GSRS-IBS and SHC questionnaires. The response in the non-CD patients were much stronger than in the CD patients, who by and large did not respond to this challenge clinically. We are aware that this may be caused by a selection bias, as strongly responsive CD patients were not asked to participate in the study. However, when mucosal cytokine mRNA levels were studied, another picture was observed. The CD patients showed a mucosal response including both adaptive and innate cytokines. This was not seen in the non-CD patients. The pathophysiological basis of non-celiac gluten sensitivity remains elusive.

Figures.

1) Median Symptom score according to gluten challenge

Gastrointestinal symptoms: GSRS-IBS General Health Complaints: SHC

Diagnosis Before During After Diagnosis Before During After

Non-CD, n=35 22 37 34 Non-CD, n=35

9 16 12

CD, n=13 24 29 27 CD, n=13 9 9 9

GSRS-IBS: Total score 13-91 SHC: Total score 0-78

2) mRNA induction

Challenge response in 30 non-CD patients and 15 CD patients; showing both an adaptive (IFN- ) and innate immune response (IL-8).

References

Sollid LM, Lundin KE. Diagnosis and treatment of celiac disease. Mucosal Immunol 2009;2:3-7.

Ráki M, Fallang LE, Brottveit M, Bergseng E, Quarsten H, Lundin KE, SOllid LM. Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients. Proc Natl Acad Sci USA 2007;104:2831-6.

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Gluten as a cause of gastrointestinal symptoms in patients who do not have coeliac disease

Jessica R. Biesiekierski, Evan D. Newnham, Peter M. Irving, Jacqueline S. Barrett, Melissa Haines, James D. Doecke, Susan J. Shepherd, Jane G. Muir, Peter R. Gibson

Monash University Department of Medicine & Gastroenterology, Box Hill Hospital, Box Hill, Victoria 3128, Australia

Background: Despite increased prescription of a gluten-free diet for functional gastrointestinal symptoms in those who do not have coeliac disease, there is minimal evidence that gluten is a trigger. The aims were to determine whether gluten ingestion can induce symptoms in non-coeliac individuals and to examine the mechanism.

Methods: A double-blind, randomised, placebo-controlled rechallenge trial was undertaken in patients with irritable bowel syndrome in whom coeliac disease was excluded and who were symptomatically controlled on a gluten-free diet. Participants received gluten or placebo as two bread slices plus one muffin per day with a gluten-free diet for up to six weeks. Symptoms were evaluated by a visual analogue scale and markers of intestinal inflammation, injury and immune activation were monitored.

Findings: 34 patients (29–59 y, four men) completed the study per-protocol. 56% had HLA-DQ2 and/or DQ8. Adherence to diet and supplements was very high. 13 of 19 patients (68%) in the gluten group reported symptoms were not adequately controlled compared to six of 15 (40%) on placebo (p=0·0001; generalized estimating equation). On a visual analogue scale, patients were significantly worse with gluten within one week for symptoms shown in Table 1.

Table 1. Change of symptoms according to the visual analogue scale (mm) after one week of therapy. The results are shown as mean (SEM) (*Independent samples t-test).

Overall Pain Bloating Wind Stool satisfaction

Nausea Tiredness

Gluten (n=19) 27 (7) 29 (7) 26 (7) 25 (8) 24 (7) 16 (6) 25 (6)

Placebo (n=15) 9 (5) 5 (5) 6 (5) 5 (5) 2 (6) 5 (4) -6 (5)

p-value* 0·047 0·016 0·031 0·053 0·024 0·120 0·001

Using the mixed effects model accounting for time, age, sex, and BMI, the severity scores of pain (p=0·02), satisfaction with stool consistency (p=0·03), and tiredness (p=0·001) were higher for those consuming the gluten.

Anti-gliadin antibodies were not induced. There were no significant changes in faecal lactoferrin, levels of coeliac antibodies, highly sensitive-CRP or intestinal permeability. There were no differences in any end-point in those with and without DQ2/DQ8.

Conclusions: ‘Non-coeliac gluten-intolerance’ may exist, but no clues to the mechanism were

elucidated. Clarification of the phenotype of such patients, the mechanisms by which gluten induces symptoms and clinical significance is required.

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Diversity of avenin genes in oat ( Avena sativa L.) and content of celiac disease epitopes

D. LONDONO1,2,*, W.P.C. VAN ‘T WESTENDE1,2,*, E.M.J. SALENTIJN1,2, I.M. VAN DER MEER1,3, L.J.W.J. GILISSEN1,3 and M.J.M. SMULDERS1,2,3

1Plant Research International, Wageningen UR, Wageningen, The Netherlands 2Wageningen UR Plant Breeding, Wageningen, The Netherlands 3Allergy Consortium Wageningen, Wageningen, The Netherlands.

Email addresses: DL Diana.londono@wur.nl WPCW wendy.vantwestende@wur.nl IMM Ingrid.vandermeer@wur.nl EMJS elma.salentijn@wur.nl LJWJG luud.gilissen@wur.nl MJMS rene.smulders@wur.nl

Background Celiac disease (CD) in humans is caused by epitopes from the prolamin fraction of wheat (gliadins), rye (secalins) and barley (hordeins). Symptoms of CD can be avoided by adhering to a strict life-long gluten-free diet, but gluten is difficult to avoid since it is the main component of wheat flour, traditionally used in bakery and pasta industries, and as a hidden ingredient in many other food products as well. Since early 2009, oats (Avena sativa L.) are considered as gluten-free products by the CODEX ALIMENTARIUS, if the amount of contaminating gluten from wheat, rye and barley is below 20 ppm. However, the use of oats by CD patients is still somewhat controversial despite many scientific reports indicating that the consumption of oats for CD patients does not cause harmful symptoms. One reason is the occurrence of gluten contamination of many commercial oat products. In addition, there have been reports on human T-cells reacting to peptides that allegedly were derived from avenins. As only very few avenin sequences are known, it has been impossible to validate such claims. Hence, we set out to generate avenin sequences from the cultivar “ Gigant” and analyse them together with EST sequences obtained from NCBI database. Amplification of all avenins was possible using 5 primer pairs. There are at least 10 functional avenin genes in gDNA of this cultivar but only 7 were present in cDNA. We extended the analysis to 112 EST sequences from the NCBI database, derived from cultivar “ Dancer” , in which 11 avenin genes were found. The expressed avenin sequences from both cultivars formed three groups in the Neighbour-Joining dendrogram. The presence of the presumed avenin epitopes (PYPEQQEPF, PYPEQQQPF), reported as elicitors of T-cell response in some CD patients, was confirmed in 4 and 6 expressed proteins in Gigant and Dancer, respectively. These two highly similar epitopes, with only one amino acid difference, are variants of part of the conserved domains of the protein and occur in two different groups, i.e., a protein may have the one epitope variant, or the other. The expression of these proteins differs between cultivars. The fact that these avenin epitopes are part of a conserved domain of the protein, increases the probability to find them in many Avena sativa cultivars. The well-studied CD inmunoreactive epitopes from wheat α-gliadins, γ-gliadins and glutenins, are not present in any of the oat avenins. We can conclude that avenins are structurally related to alpha-gliadins, which form a large multicopy gene family in wheat, but the avenins have expanded to only a few copies in oat. This research supports the findings of Arentz-Hansen (Arentz-Hansen et al. 2004) about the intrinsic toxicity in oats for some CD patients. References Arentz-Hansen, H., Fleckenstein, B., et al. The molecular basis for oat intolerance in

patients with Celiac disease. Plos Medicine 2004; 1(1): 84-92.

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Acute Administration of Trititicum monococcum ssp monococcum, cultivar “Monlis”:

a Phase II, Single Blind, Cross-over Study in Patients with Coeliac Disease.

Beatrice Petroboni1, Barbara Zanini1, Tarcisio Not2, Norberto Pogna3 Alberto Lanzini1*,1University and Spedali Civili of Brescia, Department of Gastroenterology, Brescia, Italy

2University of Trieste, Department of Pediatric Gastroenterology, Burlo Garofolo, Trieste, Italy

3CRA, Research Unit for Cereals Development, Rome, Italy*corresponding email: lanzini@med.unibs.it

Introduction. Cereals with absent or reduced toxicity are actively researched as alternative therapy to gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum ssp monococcum (Tm) is an ancient wheat with high content of lipids, ash, tocols and carotenoids and with superior bread-making quality for some accessions including Monlis. Several in vitro (Vicentini et al. 2007) and ex vivo (Pizzuti et al. 2006) studies have reported low or no toxicity of gluten derived from Tm. The purpose of our clinical study was to investigate the “in vivo” effect of a single dose of gluten of Tm (cultivar Monlis), in patients with CD in GFD.

Methods. We performed a Phase II, single blind, cross-over study. We selected patients with histologically and serologically proven CD, adherent to GFD for at least 12 months. Each patient was assigned to receive at time 0, 14 and 28 days a single fixed dose of 2.5 grams of one of the following flours: rice flour, Tm flour and Triticum aestivum (Ta) flour mixed with gluten-free pudding. The primary end-point of the study was the change in intestinal permeability (IP) as assessed by changes of urinary lactulose/rhamnose ratio (L/R ratio) measured by HPLC. We also assessed the occurrence of gastrointestinal adverse events including abdominal pain or bloating, constipation, diarrhoea, flatus, nausea, vomiting, heartburn, taste impairing, and we also assessed the occurrence of extra-intestinal symptoms. Adverse events have been graded for intensity and duration according to the WHO scale.Statistics: variables were expressed as mean ± SEM; paired t-test and chi2 test were used as appropriate to compare continue and categorical variables. The study was approved by the local Ethic Committee and each patient signed a written informed consent.

Results. Twelve CD patients were enrolled in the study. The urinary L/R ratio was 0.058 ± 0.03 with rice flour, 0.048 ± 0.02 with Tm and 0.063 ± 0.015 with Ta (figure 1). Differences were not statistically significant. Gastrointestinal adverse events were 11, 8 and 31 with rice, Tm and Ta, respectively. Eight gastrointestinal events occurred during Tm administration, a value similar to that observed with rice (n = 11). In all cases events were graded as “mild” or “moderate”. By contrast 31 adverse events were reported during Ta administration, a value significantly higher than that observed with Tm (p<0.0001) and with rice (p<0.0001, table 1). In 4 cases events during Ta administration were graded “severe” or “disabling”. Among extraintestinal adverse events, headache was reported in 13 cases and aspecific malaise in 3 cases, equally distributed among the 3 flours studied.

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Figure 1: urinary L/R ratio in the three different study days.

Table 1: Gastrointestinal adverse events reported in the three different study daysRice Tm Ta

Abdominal Pain 1 2 7Bloating 2 1 11Costipation - - -Diarrhoea 2 - -Flatus - - -Taste impairing 1 - 2Nausea 5 5 9Vomiting - - 2Heartburn - - -N. of events 11 8 31

Conclusions. In our study administration of a single 2.5 g dose of Tm, cultivar Monlis, to patients with CD adherent to GFD did not cause changes in urinary L/R ratio relative to that observed following administration of rice, the non toxic reference flour. Administration of Ta did not cause a significant increase of L/R ratio that remained similar to that observed with rice and Tm. Tm was well tolerated by all patients, and gastrointestinal and extraintestinal symptoms were few, mild and similar to those observed during rice administration. By contrast Ta caused a significant number of gastrointestinal side effects that in 4 cases were “severe” or “disabling”. The finding that Tm was well tolerated by all patients provides the rationale for further investigation on the safety of this cereal for CD patients. Assessment of intestinal permeability by urinary L/R ratio is not sensitive enough for acute toxicity studies.

Aknowledgments: this study was supported by a grant of “Antica Terra Foundation”, Cigole, Brescia, Italy

References

Pizzuti D, Buda A, D’Odorico A, et al. Lack of intestinal mucosal toxicity of Triticum monococcum in celiac disease patients. Scand J Gastroenterol 2006; 41: 1305-11

Vicentini O, Maialetti F, Gazza L, et al. Environmental factors of celiac disease: Cytotoxicity of hulled wheat species Triticum monococcum, T. turgidum ssp. Dicoccum and T. aestivum ssp spelta. J Gastroenterol Hepatol 2007; 22: 1816-22

Rice

Tm Ta0.00

0.02

0.04

0.06

0.08

0.10

L/R

rat

io

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Down-regulation of γ-gliadins in bread wheat by RNA interference (RNAi)

Javier Gil-Humanes1, Fernando Piston2*, Racha Aouni3, Francisco Barro1

1Instituto de Agricultura Sostenible, CSIC, E-14080, Córdoba, Spain 2Departamento de Genética, Universidad de Córdoba, E-14071, Córdoba, Spain

3Agrasys S.L., E-08028 Barcelona, Spain *corresponding email:b62pipif@uco.es

Introduction. Wheat prolamin proteins largely determine the dough mixing properties of flours and their suitability for breadmaking. These proteins are traditionally classified into glutenins and gliadins, representing about 80% of the total protein in the wheat grain. The high molecular weight glutenin subunits (HMW-GS) have been correlated with differences in the breadmaking quality of wheat. Gliadins are also important because they are mainly associated with the development of coeliac disease, a food-sensitive enteropathy caused by the ingestion of prolamin proteins. There is also clear evidence that α- and γ-gliadins contain clusters of epitopes that are active in coeliac disease (Arentz-Hansen et al., 2000). Plant transformation in combination with RNA interference (RNAi) provides new tools for down-regulating gliadin gene families in wheat.. Methods. RNA interference (RNAi) was used in this work to down-regulate the expression of γ-gliadins. We used an inverted repeat construct (Figure 1) to target γ-gliadins group in two lines of bread wheat cv `Bobwhite´ (BW208 and BW2003) by plant transformation. The gliadin content was evaluated by R5 ELISA assay and Reverse Phase HPLC (RP-HPLC). Results. Gliadin content estimated by R5 ELISA assay did not show significant differences between transgenic and control lines. However, the RP-HPLC showed that γ-gliadins were reduced in all transgenic lines by about 72-98.8%, depending of line (Table 1). Other gliadin fractions (α/β- and ω-gliadins) did not show significant difference relative to control. Figure 1. The sequence of a γ-gliadin gene (AY338388) was used to construct the pDhpg8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. The expression was regulated by an endosperm specific promoter (D-Hordein promoter).

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Table 1. Transgenic plants and gliadin content estimated by R5 enzyme-linked immunosorbent assay (ELISA) and RP-HPLC. ω, ω-gliadins; α/β, α/β-gliadins; γ, γ-gliadins; mAU, multiunit of absorbance at 210 nm; Total, total gliadin content; ND, data non-determined. *, Means are significantly different to control as determined by Dunnett's Multiple Comparisons at P<0.05.

Conclusions. This work demonstrates that RNAi technology can be used to down-regulate groups of proteins encoded by multigene families, such as gliadins. Such material is ideal for elucidating the roles of specific groups of proteins in determining the functional properties of flours and their relative activities in triggering coeliac disease. References Arentz-Hansen EH, McAdam SN, Molberg O, Kristiansen C, and Sollid LM. Production of a panel of recombinant gliadins for the characterisation of T cell reactivity in coeliac disease. Gut 2000; 46: 46-51.

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Gluten epitopes in Finnish patients with DQ2+ celiac disease

Andrea de Kauwe1, Katri Kaukinen2, Jason Tye-din3, Jessica Stewart3, Kalle Kurppa2, Lotta Koskinen1 Markku Mäki2, Robert Anderson3 & Päivi Saavalainen1*

1 University of Helsinki, Department of Medical Genetics, Helsinki, Finland 2 University of Tampere and Tampere University Hospital, Tampere, Finland

3 Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute, Parkville, Australia

*corresponding email: paivi.saavalainen@helsinki.fi

Introduction. The onset of celiac disease requires gene alleles encoding HLA class II molecules DQ2 or DQ8 which are capable to present certain gluten peptides to CD4+ T cells. For the development of successful vaccination or immunotherapy it is necessary to characterize the gluten peptides that are recognised by T cells in celiac disease and determine their consistency between celiac patients. Recently, such epitope mapping has been carried out for patients of British and Australian origin (Anderson et al, 2000&2005) and it is important to validate this further in other populations that may show variation in genetic and dietary factors. Objectives. Aim of the study was to map the gluten epitopes in the Finnish celiac disease patients. Finnish population is a highly interesting cohort in this regard as their diet is more heavily rye-based than that of British and Australian population, which could influence the gluten epitope hierarchy. Methods. DQ2+ patients (n= 58) with celiac disease and DQ2+ healthy controls (n= 9) were recruited to a 3-day challenge with wheat, rye, barley or all 3 grains combined. All patients attested to being strictly GFD compliant (>8 weeks prior to challenge) and were negative for disease-specific IgA antibodies; healthy controls followed a GFD for 4 weeks prior to gluten challenge and were also autoantibody-negative. IFN-gamma response was measured by ELISpot assay from peripheral blood mononuclear cells collected on days 0 and 6 after gluten challenge and stimulated in vitro against a panel of 44 different gluten preparations or synthetic peptides. Results and discussion. 35/58 (60%) of the patients and none of the 9 controls responded to in vitro gluten stimulation after the oral gluten challenge, 28 of the patients being strong and 7 weak responders (Table 1). Responder status of the patients did not associate significantly with age, sex or duration of gluten free diet, although somewhat higher percentage of females was found among responders (83%) than non-responders (61%). Response-rate differed slightly between the challenged cereal groups; the highest response-rate (78%) was seen among wheat challenged and the lowest (47%) among barley challenged patients. However, also in Finnish patients strong responses were demonstrated to the key immunodominant epitopes, the presence and frequency of certain T cell specificities being similar to British and Australian cohorts.

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Table 1. Responder-rates of the gluten-challenged CD patients in IFN-gamma ELISpot assay

Responders (%) Weak responders (%) Non-responders (%) Total (F,M)

Wheat 8 (45%) 6 (33%) 4 (22%) 18 (13F,5M) Rye 7 (50%) 1 (7%) 6 (43%) 14 (11F,3M) Barley 7 (47%) 0 (0% 8 (53% 15 (11F,4M) Combined

6 (55%) 0 (0%) 5 (45%) 11 (8F,3M)

Total (%, F,M) 28 (48%,24F,4M) 7 (12%, 5F,2M) 23 (40%,14F,9M) Conclusions. Responses to gluten epitopes in Finnish DQ2+ celiac disease patients were similar to other cohorts despite the heavier consumption of rye in Finland. Similarity of the disease triggering gluten epitopes throughout populations will ease the development of a universal immunotherapy for celiac disease. References Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AV. In vivo antigen challenge in celiac

disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med 2000; 6:337-42.

Anderson RP, van Heel DA, Tye-Din JA, Barnardo M, Salio M, Jewell DP, Hill AV. T cells

in peripheral blood after gluten challenge in coeliac disease. Gut. 2005; 54:1217-23.

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Improving accuracy in detecting gluten

Päivi Kanerva, Tuula Sontag-Strohm, Jussi Loponen, Hannu Salovaara University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland

*corresponding email: paivi.kanerva@helsinki.fi Introduction. Celiac disease is a common gastrointestinal disease where the prolamin proteins of wheat, rye and barley cause an inflammation of the small intestine. Presently, the only treatment for the disease is a gluten-free diet, which means excluding wheat, rye or barley prolamin containing products from the diet. Wheat prolamins and their role in celiac disease have been studied extensively during past years whereas barley and rye prolamins have gained less attention. As a consequence, the gluten analysis methods are standardized based on the characteristics of wheat prolamins and, therefore, cannot directly quantify gluten from products containing barley or rye. The sample preparation method has a major effect on the prolamin composition of the extract and, therefore, plays an important role in the quantification of gluten. Whether the extraction is done with or without reduction of proteins significantly impacts the extraction yield and the prolamin composition of the extract. The use of reducing agents in the extraction uniformly dissolves all prolamins and increases the yield. Prolamin subgroups have their specific affinities against different prolamin-specific antibodies (Kanerva et al. 2009, Wieser & Seilmeier, 1999). Due to the complex nature of prolamin proteins, the extracted gluten sample is always a mixture of prolamins as are the standards used in gluten assays mixtures of wheat gliadins. Antibodies recognize each prolamin type with a certain affinity, and if the composition of the prolamins in the sample distinctly differs from that of the assay standard, the analysis results may be inaccurate. Modification of proteins by deamidation is used to diversify their functionality. In gluten deamidation, a conversion from glutamine to glutamic acid occurs, which alters the structure of gluten. Deamidated prolamin peptides are also more active in celiac disease (in vitro) than the native peptides (Molberg et al., 1998). Methods. Three alcohols of different aqueous alcohol concentrations were studied for their extraction efficiency with barley, rye and wheat prolamins. Protein concentrations of the extracts were analyzed by a Dumas combustion method. Five different gluten detecting ELISAs were used:

Ridascreen Gliadin Competitive (R-Biopharm AG, Darmstadt, Germany) Ridascreen Gliadin (R-Biopharm AG, Darmstadt, Germany) Biokits Gluten Assay (Tepnel, Flintshire, UK) GlutenTox ELISA Competitive (Biomedal Diagnostics, Sevilla, Spain) Transia Plate Prolamins (Diffchamb SA, Lyon, France)

Deamidated samples were prepared by adding 200ml of 0.1M HCl to 5g of vital gluten and heating at 100°C for 2h. After acid treatment the sample was neutralised with 1M NaOH to pH 8

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and dialysed against distilled water for 24h (MWCO 12-14kD). All contents of the membrane were lyophilized. The prolamin contents of vital and deamidated wheat gluten were determined with the ELISA methods. The samples were extracted as advised by each assay protocol and the protein contents of the extracts were quantified by a Lowry method. Results. The extraction of prolamins with 40% 1-propanol was observed to be the most efficient. The extraction with 60% ethanol, which is commonly used in gluten ELISAs, gave similar results for wheat, but was less efficient solvent for barley and rye prolamins. Heating and reduction increased the yields significantly. The comparison of the R5 and -gliadin antibodies and their cross-reactivity with prolamins of barley, rye and wheat revealed differences between the antibodies. The -gliadin antibody recognized mainly the HMW proteins of barley, rye and wheat. The antibody R5, on the other hand, recognized mainly the prolamin groups having molecular weight of 20-50kD. Quantification of barley from intentionally barley-contaminated oat samples was most accurately measured using a hordein standard instead of the gliadin standard of the assay, indicating substantial differences between barley and wheat. The result showed that when the composition of prolamins in the standard was similar to the composition present in the sample, accurate results were obtained. Deamidation of the prolamin peptides and proteins decreased the ability of antibodies to recognize them. The prolamin-specific antibodies of R5 and G12 had a significantly lower detection of peptides with deamidated glutamine residues compared to the native peptides. The recognition of deamidated wheat gluten was significantly lower than the recognition of the untreated gluten. Conclusions. The sample extraction and the choice of standard are important factors when improving the accuracy in detecting residual prolamin proteins in gluten-free foods. The extraction of all prolamin subgroups from wheat, rye and barley was obtained with 40% 1-propanol with heating and reduction. Standards that are uniformly suitable for prolamins of all three cereals and also for samples containing modified prolamins proteins are needed. Modification of gluten in food processes alters the immunological recognition by prolamin-specific antibodies. This should also be paid attention to when validating the new methods for gluten quantification. References Kanerva PM, Sontag-Strohm TS, Brinck OM, Salovaara HO. Effects of heating, reducing and alcohol concentration on the extractability of prolamins from barley, rye and wheat and the reactivity of prolamins with prolamin antibodies. In Proc 23rd Meet Prol Work Group. Stern M. (Ed.), Verlag Wiss Scripten, Germany, 2009; 75-81. Wieser H, Seilmeier W. Reactivity of gliadin fractions and components from different wheat species in the Skerritt test. In Proc 13th Meet Prol Work Group. Stern M. (Ed.), Tuebingen, Germany, 1999; 11-18. Molberg Ø, Mcadam SN, Körner R et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4(6):713-717.

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Preliminary Results of the Collaborative Study to Validate the Characteristics of the GLUTEN-TEC® ELISA Kit

Jorge R Mujico1, Liesbeth Dekking1, Yvonne Kooy-Winkelaar1, Ron Verheijen2, Piet van Wichen2, Lucia Streppel2, Nermin Sajic2, Jan-Wouter Drijfhout1 and Frits Koning1*

1 Department of Immunology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

2 EuroProxima, Arnhem, The Netherlands *corresponding email: F.Koning@lumc.nl

Introduction: The Leiden University Medical Center, in cooperation with EuroProxima, has developed Gluten-Tec®, a novel competitive ELISA that detects a well characterized T cell stimulatory epitope of α20-gliadin in wheat, and homologue sequences present in barley (hordein) and rye (secalin) (Mitea et al. 2008). Synthetic peptides are used for calibration, which allows an accurate and reproducible standardization. Moreover, not only intact, but also hydrolysed proteins can be detected in one single assay. Objective: The purpose of this study was to test the performance of the Gluten-Tec® ELISA kit through a collaborative study, in accordance to the AOAC guidelines (1995). Methods: 15 laboratories announced their interest in taking part in this study. All laboratories had experience in analyzing gluten in food samples with ELISA techniques. The study included 19 samples, covering a wide range of hydrolysed and/or heat-treated food products (Table 1). The rice baby foods and maize breads were extracted with 60% ethanol and with a new method based on the reducing reagent DTT. The chocolate cake mixes were extracted with a solution of 60% ethanol, containing 5% fish gelatin and 2% PVP. No extraction was performed with the commercial beer (made from barley malt). All samples were diluted 100 times (final dilution) in the buffer supplied with the ELISA kit. In order to generate blind duplicates, each sample was split into two and randomly coded. Two Gluten-Tec® ELISA kits, together with the samples, were sent to each laboratory in a package containing cool packs (2-8ºC). Upon completion of the study, a data reporting form was available to be filled with the 450 nm optical densities. The Gen5 data analysis software (BioTek, USA) was used to plot the calibration curve and to calculate the gliadin concentration of the 19 food samples. Results and Discussion: 12 laboratories sent the results within the established time frame. From these 12 laboratories, results from 2 laboratories were not used because the results did not meet the quality control criteria of the test. Based on the results obtained from the other 10 laboratories, the mean value and the relative standard deviation (RSD) for each sample were calculated (Table 1). Most of the laboratories reported both extracts of the non-spiked rice baby food (samples 1 and 2) as below the lowest point of the standard curve and, therefore, considered as negative. On the other side, both extracts of the highest spiked rice baby food (samples 3 and 4) were always over the highest point of the standard curve. For the rest of the spiked rice baby foods and maize breads, when DTT was used, more gliadins were extracted (between 1.2 and 1.7 times more) and lower RSDs were obtained (11-29% vs 9-42%) as compared to only 60% ethanol extraction.

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Table 1. α20-gliadin content (in parts per billion or ppb) of the 19 samples included in the study (mean and RSD of the results reported by the 10 laboratories submitting valid data).

α20-gliadin Sample Matrix Spiked / Non-spiked Extraction Mean (ppb) RSD (%) 1 Rice baby food Non-spiked EtOH < − 2 Rice baby food Non-spiked DTT < − 3 Rice baby food Spiked 0.4% wheat baby food EtOH > − 4 Rice baby food Spiked 0.4% wheat baby food DTT > − 5 Rice baby food Spiked 0.2% wheat baby food EtOH 706 9 6 Rice baby food Spiked 0.2% wheat baby food DTT > − 7 Rice baby food Spiked 0.1% wheat baby food EtOH 444 23 8 Rice baby food Spiked 0.1% wheat baby food DTT 659 11 9 Rice baby food Spiked 0.05% wheat baby food EtOH 255 32 10 Rice baby food Spiked 0.05% wheat baby food DTT 397 21 11 Rice baby food Spiked 0.025% wheat baby food EtOH 131 40 12 Rice baby food Spiked 0.025% wheat baby food DTT 226 29 13 Maize bread Spiked 44.2 ppm of gliadina EtOH 239 36 14 Maize bread Spiked 44.2 ppm of gliadina DTT 367 27 15 Maize bread Spiked 22.1 ppm of gliadina EtOH 170 42 16 Maize bread Spiked 22.1 ppm of gliadina DTT 211 29 17 Chocolate cake mix Non-spiked EtOH+Gel+PVP 65 51

18 Chocolate cake mix Spiked 0.25% gluten-containing chocolate cake mix EtOH+Gel+PVP 428 18

19 Commercial beer Non-spiked − 179 16 <: Below the lowest point of the standard curve. >: Over the highest point of the standard curve. a Total gliadin (ω-, α- and γ-gliadin) as determined by HPLC. Values expressed in parts per million or ppm. A high RSD value was obtained with the non-spiked chocolate cake mix (sample 17), probably due to the fact that the measured gliadin content was really close to the limit of quantification of the method (25 ppb, dilution factor 100). Nevertheless, the same chocolate cake mix spiked with 0.25% gluten-containing chocolate cake mix (sample 18) was clearly positive (428 ppb, 18% RSD). All laboratories were capable of measuring the low content of hydrolysed hordein present in the commercial beer, with also a very low RSD (16%). Conclusions: Although the data have not been statistically evaluated yet (detection of outliers, determination of repeatability and reproducibility…), the preliminary results of the collaborative study confirm that the method is suitable for the measurement of T cell stimulatory epitopes over a wide range of concentration. If good levels of precision are obtained, and due to the fact that Gluten-Tec® ELISA has additive values over the currently available ELISA kits, it will be presented to the Codex Alimentarius as a preferred method for gluten analysis. References: Mitea C, Kooy-Winkelaar Y, van Veelen P, de Ru A, Drijfhout JW, Koning F, Dekking L:

Fine specificity of monoclonal antibodies against celiac disease-inducing peptides in the gluteome. Am J Clin Nutr 2008;88:1057-1066.

AOAC International guidelines for collaborative study procedures to validate characteristics of a method of analysis. J AOAC Int 1995;78(5):143-160.

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RIDASCREEN® Gliadin competitive - Second Generation Testing for Gliadin in Compliance with

Codex Alimentarius Level

Dr. Ulrike Immer1*, Dr. Sigrid Haas-Lauterbach1

1 R-Biopharm AG, R&D Department, Darmstadt, Germany

*corresponding email: u.immer@r-biopharm.de

Introduction. The Codex Alimentarius recommends the R5-antibody for gluten analysis. The R-Biopharm RIDASCREEN® product line for gluten analysis fulfils this demand. Methods. One of the ready to use assays is the RIDASCREEN® Gliadin, a sandwich ELISA which quantifies wheat as well as rye and barley prolamins. It detects intact prolamin molecules as well as large fragments which contain at least two binding places to form a sandwich. However, smaller fragments of these prolamins, known as peptides, occur e.g. during the hydrolization process of commodities like syrups and starches or during the brewery process. These small fragments bear a risk for coeliacs as they are potentially toxic (Wieser et al. 2006). In processed samples a mixture of intact gliadin molecules, as well as, large and small fragments are present. When using the sandwich assay format for the detection of hydrolysed samples the risk to miss the small potentially toxic fragments is very high. Therefore, the competitive assay format should be used as this is designed especially for the detection of smaller molecules due to the fact that the assay needs only one binding site of the antigen. Results. After the launch of the sandwich ELISAs for gliadin testing, the coeliac associations as well as the beer industry described their needs for an appropriate method to declare gluten free beer. Therefore, the first competitive assay was launched in 2006 also in a ready to use assay format. The RIDASCREEN® Gliadin competitive is a competitive test system to quantify intact prolamins, peptides and small fragments thereof. This assay was calibrated on the toxic peptide QQPFP even knowing that the result could not be recalculated in mg/kg gliadin. Theoretically, such a recalculation is possible by comparing the standard curves of the pure peptide and high purified gliadin, but it does not reflect the normal matrix situation in a sample. The 2nd generation RIDASCREEN® Gliadin competitive will use a mixed hydrolyzed standard made out of hydrolyzed wheat, rye and barley to enable quantification as mg/kg gliadin according to the Codex Alimentarius level. These partially hydrolysed prolamins as references for the quantification of gluten in hydrolysed products has been developed and characterized at the German Research Centre for Food Chemistry in Germany in the working group of Prof. Köhler (Köhler et al. 2008). At present, the use of this “pure” hydrolysed standard to measure peptide levels in food seems to be the most promising compromise for quantification of gluten levels. Conclusions. The results of this new test system will be presented at the Gluten-free Food Conference in June in Tampere, Finland.

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References Wieser H, Hartmann G, Koehler P (2007) Studies on the degradation of gluten proteins during germination of wheat. In: 9th International Gluten Workshop, Gluten Proteins 2006 (Lookhart GL, Ng PKW, eds) AACC International, Inc, ISBN 978-1-891127-57-1, pp 208-212

Koehler P, Geßendorfer B, Wieser H, Preparation of partially hydrolysed prolamins as references for the immunochemical quantification of gluten in cereal-based beverages (Proceedings of the 23rd Meeting of the Working Group on Prolamin analysis and Toxicity, 2008)

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Detection of toxic fragments from gluten using a new monoclonal antibody-based test

Richard Fielder1*, Elisabeth Halbmayr2, Michael Z. Zheng3, Donna Houchins4

1Romer Labs UK ltd, North Wales Business Park, Building Unit 5325 Abergele, LL22 8LJ North Wales, United Kingdom

2Romer Labs Division Holding GmbH, Technopark 1, 3430 Tulln, Austria3Romer Labs Singapore Pte. Ltd., 3791 Jalan Bukit Merah #08-08, e-Centre@redhill

building, Singapore, 1594714 Romer Labs Inc., 1301 Stylemaster Drive, Union, MO 635838600, USA

*corresponding email: richard.fielder@romerlabs.com

Introduction. Celiac disease (CD) is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Celiac disease occurs in genetically predisposed persons and leads to the destruction of the microscopic finger-like projections of the small intestine, called villi. The disease is triggered by the ingestion of peptides from wheat, barley, rye, and in some cases oats. It currently affects roughly 1% of the world´s population, primarily adults. Immunotoxic gluten peptides, such as the fragment called 33-mer, which are resistant to degradation of digestive enzymes appear to trigger celiac syndrome. Homologues of this peptide were found in every food grain (except oats) that is toxic to CD patients, but were absent in all nontoxic food grains (Shan 2002). A monoclonal antibody specific for a sequence occurring three times in the immunotoxic 33-mer was developed (Morón 2008a, 2008b). This work summarizes the results of a new monoclonal antibody used in a lateral flow strip test that specifically recognises the pathogenic fragment of the gliadin protein present in gluten.

Methods. The semi-quantitative immunochromatographic strip test (AgraStrip® Gluten, Romer Labs, Singapore) is based on a sandwich format. The reagents for the test and control line are immobilized on a nitrocellulose membrane. Toxic gluten fragments in the sample extract react with the anti-gliadin 33-mer monoclonal antibody, named G12, which is coupled to coloured microspheres. These are pre-dried on the strip showing a visible line when binding to immobilized anti-gliadin 33-mer monoclonal antibodies on the test line (Morón2008a, 2008b). The mix of conjugate moves through the membrane to the control line where anti-species specific antibodies used for verifying the correct test performance are sprayed.Several gluten-free samples and gluten containing samples were analyzed using the strip test. For confirmation of the results a self established ELISA method using the monoclonal G12 antibody and a commercial available gliadin ELISA test kit were used.

Results. The G12 antibody, specially developed to determine the toxic fractions present in gluten was used for the semi-quantitative immunochromatographic strip test. When food and drinks are hydrolysed or heat processed, the gliadin protein is degraded in small fragments which are difficult to detect with standard available antibodies because their epitopes may be destroyed. The newly developed monoclonal G12 antibody can reliably detect toxic fragments of gluten in hydrolysed food. The outstanding advantage of this new antibody used in the strip test is the possibility to detect the actual toxic fragment of gluten with a very high sensitivity.

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The comparison of the immunochromatographic strip test and ELISA method analyzing different food samples for their gluten content showed correlating results (Table 1).

Table1. Different food samples were analyzed using AgraStrip® Gluten with a 10 ppm cutoff level and ELISAs with the G12 and R5 antibody.

Matrix Results AgraStrip Results ELISACorn starch >10 ppm 18.8 ppmSugar+milk >10 ppm 191 ppmBBQ Spices >10 ppm 23.3 ppm

Paprika <10 ppm <3 ppmWheat starch >10 ppm 166 ppm

Strawberry flavour >10 ppm 12 ppmPudding >10 ppm 36 ppm

Ham flavour <10 ppm <3 ppmGlucose >10 ppm 256 ppm

Rice milk >10 ppm 80 ppmSausage 1 >10 ppm 113 ppmSausage 2 >10 ppm >100 ppm

Cured loin of pork <10 ppm <3 ppmHamburger >10 ppm 96 ppm

Cake <10 ppm <3 ppmAperitive snacks <10 ppm <3 ppm

Baby food >10 ppm 96 ppmBiscuit <10 ppm <3 ppmBread <10 ppm <3 ppm

Chocolate <10 ppm <3 ppmIce cream <10 ppm <3 ppm

Cream <10 ppm <3ppm

Conclusions. Due to current Codex Alimentarius recommendations and Commission Regulation (EC) No 41/2009, food can be labelled gluten free when containing less than 20 mg/kg gluten. The lateral flow test kit (AgraStrip® Gluten, Singapore) using the monoclonal antibody named G12 can detect the toxic fractions of gluten from wheat and other cereals such as barley, rye. The advantage detecting the actual toxic fragment of prolamins helps producers of gluten-free food and beverages to label the gluten content of their products correctly. This leads to more safety for consumers which suffer from celiac disease and have to avoid the intake of gluten to a certain extent.

References

Shan L, Molbergv Ø, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C. Structural basis for gluten intolerance in celiac sprue. Science. 2002 Sep 27;297(5590):2275-9.

Morón B, Bethune MT, Comino I, Manyani H, Ferragud M, López MC, Cebolla A, Khosla C, Sousa C. Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide. PLoS ONE. 2008a May 28;3(5):e2294.

Morón B, Cebolla A, Manyani H, Alvarez-Maqueda M, Megías M, Thomas Mdel C, López MC, Sousa C. Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide. Am J Clin Nutr. 2008b Feb;87(2):405-14.

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Genomics approaches to analyse CD-toxicity for the production of CD-safe wheat

Luud JWJ Gilissen, Hetty C van den Broeck, Elma MJ Salentijn, Ingrid M van der

Meer, Marinus JM Smulders Plant Research International, P.O.Box 619. 6700 AP Wageningen, The Netherlands

Coeliac disease (CD) is a common, food-related inflammatory disorder of the small intestine caused by the ingestion of gluten in genetically predisposed individuals. Currently, a life-long gluten-free diet is the only treatment. This treatment leads to a conflicting situation, since the vast majority of the patients is not aware of having the disease due to large-scale underdiagnosis and wrong diagnosis. For example, in The Netherlands, the Dutch Coeliac Patient Society counts almost 15,000 diagnosed members which form according to epidemiological studies only about 10-15% of the total CD patient population. In the USA, this situation seems to be even worse: about 50,000 individuals have nowadays been diagnosed with CD, but the government estimates that there could be as many as 3 million who are undiagnosed. An extended cohort study over the last 50 years, carried out in the USA among undiagnosed populations of young, mostly male adults, revealed an over 4-fold increased prevalence of CD and a nearly 4-fold increased risk of death (Rubio-Tapia et al, 2009). A Finnish cohort study over 20 years showed a statistically significant doubling of the prevalence among both sexes and in different age-groups to almost 2% of undiagnosed CD (Lohi et al. 2007). These increases in CD over time are mainly ascribed to changes in quantity, quality and processing of wheat in the time period, possibly in addition to a reduction in childhood infections and other life-style changes. Especially the latter factors might stimulate the immune system wrongly towards the development of (auto)immune disorders. Gluten consumption as a major factor of increased CD opens ways for primary and secondary prevention, i.e. intervening before the disease processes have been initiated, and prevention of the occurrence of symptoms, respectively. Especially the option of the reduction of the total CD-toxic gluten load to the entire population, including the undiagnosed celiacs, is challenging. Every reduction in total CD-toxic gluten consumption is considered to contribute to an overall reduction of the prevalence and of symptom severity. This is a huge task to achieve since wheat and its derivatives are ubiquitous in all kinds of foods. A survey of over 10-thousand supermarket items detected wheat in almost 30% of labeled products (Atchison et al. 2010). In these products, the connection to wheat was visible and even celebrated (no problem), or just invisible as in most processed foods and in foods which might not commonly thought of as containing wheat, such as canned vegetables, milk, meat and seafood (big problem). The aim of our study focuses on the overall reduction of the amount of CD-toxic epitopes in wheat gluten proteins. Summarized, the following research questions have been proposed:

• Can we obtain detailed and complete information about the presence of CD-toxic epitopes in gluten proteins of different wheat varieties?

• Can wheat breeding result in low-CD toxic varieties and contribute to decrease the prevalence of CD?

To answer the first question, from hexaploid bread wheat (with the A, B and D genome) and diploid ancestral wheat species, the DNA sequences of over 3000 α-gliadin genes , which group of gluten is considered to be the most toxic to CD patients, have been analyzed to

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determine whether they encode for peptides potentially involved in CD. The results showed that the α-gliadins from the D-genome of wheat are the most antigenic as most include several epitopes, sometimes occurring in multiple and overlapping peptide sequences. The α-gliadins from the B-genome contained the lowest amount of epitopes. Of all the expressed and genomic toxic epitopes found, also natural variants have been identified that have lost their antigenic properties. In addition, there is clear evidence that also γ-gliadins contain CD-toxic peptides with comparable impact to those of the α-gliadins; γ-I is the major epitope. About 1100 γ-gliadin sequences have been analysed and related to their genome of origin, being A, B or D. The highest frequency of expressed γ-I epitopes was found to be present in the D-genome, however, these epitope variants appeared to be not CD-toxic. Regarding the second question, the diversity of gluten proteins for the presence of the major Glia-α9 epitope in modern European wheat varieties was compared with landraces representing the wheat varieties grown up to a century ago. The occurrence of the epitope was measured by immunoblotting using mAbs specifically raised against the epitope Glia-α9. The epitope was found at higher frequencies in the modern varieties, suggesting that current wheat breeding practices have led to an increased exposure to CD-toxic epitopes. On the other hand, some modern varieties as well as landraces have been identified with a low content of the epitope. Such selected lines may serve as a start to breed wheat for the introduction of ‘low CD-toxic’ as a new breeding trait. Large-scale culture and consumption of wheat varieties and their derived gluten with strongly reduced and, on the long term, even completely eliminated CD-toxicity would considerably aid in decreasing the prevalence of CD and would largly increase the quality of life of undiagnosed CD-patients. This prospect should become a new challenge as well as a new responsibility to wheat breeders, research organizations, and governments. References Atchison J, Head L, Gates A. Wheat as food, wheat as industrial substance; comparative

geographies of transformation and mobility. Geoforum 2010;41:236-246 Lohi S, Mustalahti K, Kaukinen K, Laurila K, Collin P, Rissanen H, Lohi O, Bravi E,

Gasparin M, Reunanen A, Mä ki M. Increasing prevalence of coeliac disease over time. Aliment Pharmacol Ther 2007;26:1217-1225

Rubio-Tapia A, Kyle RA, Kaplan EL, Johnson DR, Page W, Erdtmann F, Brantner TL, Kim

WR, Phelps TK, Lahr BD, Zinsmeister AR, Melton LJ, Murray JA. Increased prevalence and mortality in undiagnosed Celiac Disease. Gastroenterol 2009;137:88-93

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Testing Safety of Low Gluten Food Products in a Mouse Model of Celiac Disease

Tobias Freitag1,3, *, Yvonne Junker3, Päivi Saavalainen2, Seppo Meri1 & Detlef Schuppan3

1) Dept. of Bacteriology & Immunology, 2)Dept. of Medical Genetics, Haartman Institute, University of Helsinki, Finland

3)Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

*eMail for correspondence: tobias.freitag@helsinki.fi

Introduction: Celiac disease (cd) is a common small intestinal inflammatory disorder that results from abrogation of tolerance to dietary gluten proteins (Green 2007). We have developed a mouse model of cd that can be used to test the residual immunostimulatory and inflammatory potential of low gluten food products in vivo (Freitag 2009).

Methods: C57BL/6 donor mice are immunized with gliadin (or control antigen) in complete Freund's adjuvant. Spleen cells are harvested from donor mice, and CD4+ memory T cell fractions depleted of (tolerogenic) regulatory T cells (FACS) are adoptively transferred into lymphopenic mice, leading to baseline small intestinal inflammation. Groups of recipient mice are maintained on standardized, gluten-free diet (AIN-76A), or customized diets containing variable amounts of gluten (Table 1).

Results: T and B cell-deficient Rag1-/- recipients of gliadin-presensitized CD4+ CD45RBlowCD25- memory T cells gain less weight and suffer from more severe duodenitis when challenged with oral gluten (2.5g gluten/kg diet) than recipients maintained on a gluten-free diet or recipients of control-presensitized T cells challenged with gluten. This is accompanied by deterioration of mucosal histological features characteristic of cd (mononuclear cell infiltration, crypt hyperplasia, villus atrophy), increased T helper 1 (Th1) and Th17 cell polarization in the duodenum (RT-PCR), and high levels of IFN produced by splenocytes in response to gliadin restimulation (ELISA). Change to a gluten-free diet leads to weight gain, improvement of histological duodenitis and a decrease in duodenal IFN and IL-17 transcripts. B cell-competent nude recipients of gliadin-presensitized memory T cells produce high levels of serum anti-gliadin IgA and IgG only when challenged with oral gluten. Moreover, the dietary gluten dose (0.1g gluten/kg diet, 0.5g/kg, 2.5g/kg) correlates with the production of anti-gliadin IgG and IgG2c subclass (Th1-associated) antibodies.

Conclusions: Adoptive transfer of gluten-reactive memory CD4+ T cells leads to abrogation of gluten tolerance and exacerbation of duodenitis in the small intestine of lymphopenic recipients. This cd mouse model should be useful for testing the residual immunostimulatory and inflammatory potential of low gluten food products in vivo, providing a tool to predict the safety of these products for consumption by celiac patients.

γ

γ

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Table:

Table 1: Animal model of celiac disease. C57BL/6 donor mice are immunized with gliadin or control (e.g. ovalbumin) in complete Freund's adjuvant. Splenic CD3+ T cell fractions from immunized donor mice are stained with fluorescent antibodies against T helper cell marker CD4, naive cell marker CD45RB and cell activation marker CD25. Rag1-/- or nude mice are injected i.p. with 4.5x105 CD4+CD45RBlowCD25- memory T cells, depleted both of CD4+CD25high regulatory T cells and CD4+CD45RBhigh naïve T cells (Facs). After adoptive T cell transfer, recipient mice are either maintained on gluten-free diet (gliadin/gfd control group), or challenged with gluten (gliadin/gluten; ovalbumin /gluten control group).

To test the safety of low gluten food products in this animal model, food products can be mixed into gluten-free standardized diet. Effects of feeding may then be compared with control groups maintained on diets containing defined amounts of gluten. Clinical parameters, histological duodenitis scores and immunological parameters can be used as readout.

References:

Green PH, Cellier C. Celiac disease. N Engl J Med. 2007 Oct 25;357(17):1731-43.

Freitag TL, Rietdijk S, Junker Y, Popov Y, Bhan AK, Kelly CP, Terhorst C, Schuppan D. Gliadin-primed CD4+CD45RBlowCD25- T cells drive gluten-dependent small intestinal damage after adoptive transfer into lymphopenic mice. Gut. 2009 Dec,58(12):1597-605.

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General Discussion on · Safety and tolerance ·

· Analysis and regulatory standards · · Technology and variety of products ·

Hannu Salovaara, Päivi Kanerva, Jussi Loponen, Tuula Sontag-Strohm

University of Helsinki, Department of Food and Environmental Sciences, Helsinki Viikki Food Science, Helsinki, Finland

*corresponding email: hannu.salovaara@helsinki.fi

Introduction. This symposium fundamentally aims at improved quality of foods suitable to individuals with celiac disease. The scope of this symposium is in the cereal based foods which make an essential part of human diets. The challenge is to substitute for the unique and specific technological properties of wheat, rye and barley which are not available when making gluten-free foods because of the harmful prolamins present in these three Triticeae cereals. Safety and tolerance. Safety of the products is the starting point. However, absolute exclusion of the Triticeae prolamins in a GF diet (zero-tolerance) would probably lead to higher prices and more limited product variety, thus reducing compliance to the GF diet, an issue of utmost importance. An obvious pragmatic question is then raised: how much gluten a celiac patent can tolerate without risk, and what level of ‘contamination’ of the Triticeae prolamins could be allowed? The question is tricky; the amount of Triticeae prolamins that is tolerated varies greatly among individuals with CD. Table 1 reviews some studies made in order to clarify the safe gluten threshold, including the recent systemic review by Akobeng and Thomas (2008). Table 1. Proposed safe gluten thresholds in terms of intake (mg/d) and content (mg7kg) Source Safe daily intake of

gluten (mg) * Suggested max gluten conc. (ppm or mg/kg)

Comments and complementary information

Ciclitira et al. (1984) >2.4–4.8 mg/d >12–24 mg/kg Bread, wheat starch, 1 wk , children Catassi et al. (1993) <200mg gluten/d Children, minimal histol changes, 4wks Collin et al. (2004) 30 mg/d 100 mg/kg

if 300g flour/d ‘not to jeopardise the GF diet with inconvenient restrictions’

Ciclitira et al. (2005) 50 mg/d ‘… a consensus suggests…’ Hischenhuber et al. (2006)

>10 mg/d but < 100 mg/d

<100mg/kg for wheat starch

A review.

Biagi et al. (2004) < 1mg/d (3520 mg/kg) 1 patient 1 communion wafer/d,no recover Catassi et al. (2007) <50 mg/d–

>10mg/d Amount tolerated varies among

individuals with CD. Akobeng & Thomas (2008) . Review.

<10 mg/d (20 mg/kg) ‘20 mg/kg means 6 mg/d , which is less likely to induce mucosal damage‘

* Intake of the Triticeae prolamins by a healthy average European is 20–30 g/d (20 000–30 000 mg/d) Only few studies discuss the results from GF food point of view and draw conclusions from amount (milligrams) ingested per day (threshold dose) to gluten content (mg/kg) of food (threshold content). Neglecting product availability and compliance aspects the Akobeng & Thomas study (2008) suggested a 20 ppm (mg/kg) limit for gluten-free foods, and this is actually also the current threshold for GF in the EU Commission regulation (No 41/2009).

29

Analysis and regulatory standards. Analysis is another critical issue not completely solved. New ELISA techniques are available and will be developed for quick determination of the Triticeae prolamins whereas PCR techniques appear to encounter problems with heat-treated processed food samples. Reliable methods are a prerequisite for the regulatory thresholds. It is the harmful polypeptide sequence(s) that needs to be quantified in a reliable and accurate analysis. However, while it is still not yet fully understood which polypeptide sequence(s) actually cause the disease, the current methods more or less detect the presence of the Triticeae prolamins rather than recognize the actual harmful sequence. Analytical methods available for detecting gluten are listed in Table 2. The R5 kits are all based on the same detection antibody (R5) developed by E. Mendez. This antibody identifies a pentapeptide QQPFP present in the Triticeae prolamins. The method is the current official method for detecting gluten in GF products. However, there is some concern of the unequal reaction of the antibody with prolamins from the different cereals (Kanerva et al. 2006), and of defects in reproducibility, particularly in the vicinity of the (low) threshold (20 ppm). ELISA test kits for detecting gluten are available from a number of companies, and they are used by emerging companies offering analytical services, and by research institutes. Table 2. Analytical methods for detection and quantification of gluten in GF foods __________________________________________________________________________________ Method Principle Comments /Accuracy/confidence C Total N <0.05g/100g Kjeldahl N Applied in former Codex stan 118-1979

-gliadin ‘Skerrit’ ELISA Sandwich Commercial kits; rept. 16-33%, repd. 24–33%1

Defect: poor in detecting barley prolamins R5 ‘Mendez’ ELISA Sandwich Commercial kits; rept. 18–20%; repd. 30–32%1 The method is currently endorsed by Codex R5 ‘Mendez’ ELISA Competitive Commercial; applicable for protein hydrolysates G12 anti-gliadin 33mer ELISA Competitive New Stick tests ELISA For use where only limited lab capacity available Other MS, LC, PCR For research, characterization of proteins, DNA 1rept. = repeatability; repd. = reproducibility; in interlaboratory validation reports Technology and variety of gluten-free products help consumers with CD – and their families – by offering an assortment of safe and tasty GF products, thus supporting compliance to the GF diet. Any unnecessary reduction of the regulated limits for GF tends to reduce the number of labelled gluten-free foods available. – A follow-up in the coming years will show if the new EU regulation (20 ppm vs. 200 ppm in the past) will improve the health and life of those with CD. References Akobeng AK, Thomas AG. Systematic review: Tolerable amount of gluten for people with

coeliac disease. Alim Pharmacol Therapeutics 2008;27:1044-1052. Collin P, Thorell L, Kaukinen K, Mäki M. The safe threshold for gluten contamination in

gluten-free products. Can trace amounts be accepted in the treatment of coeliac disease? Aliment Pharmacol Ther 2004;19:1277–83.

Kanerva PM, Sontag-Strohm TS, Ryöppy PH, Alho-Lehto P, Salovaara HO. Analysis of barley contamination in oats using R5 and omega -gliadin antibodies. J Cereal Sci 2006;44:347-352.

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Determination of the immunotoxic potential of oats for the selection of varieties possibly safe for coeliac patients

Isabel Comino1; Ana Real1, Laura de Lorenzo1, 2, Francisco Barros3, Maribel Torres4, Ángel Cebolla5 and Carolina Sousa1*

1 Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain

2 Present address: Centro Nacional de Biotecnología (CNB - CSIC), Departamento de Genética Molecular de Plantas, Madrid, Spain.

3 Departamento de Biología Vegetal y Ecología, División de Fisiología Vegetal, Facultad de Ciencias, Universidad de Córdoba, Córdoba, Spain

4 Departamento de Biología Experimental, Jaén, Spain 5 Biomedal S.L., Sevilla, Spain

* corresponding email: csoumar@us.es

Introduction. Coeliac disease is defined as an alteration of the mucosa of the proximal small intestine, associated with a permanent intolerance to gluten in genetically predisposed individuals, with added environmental factors. In coeliac disease, the mucosa of the upper part of the intestine shows morphological changes, with long pits and complete or partial atrophy of the intestinal villi, following the ingestion of products made with cereals, including those derived from wheat, barley, rye, and probably oats. The main toxic components of wheat gluten belong to a family of closely related proline- and glutamine-rich proteins called gliadins. The disease is triggered by the presence of peptides from the fragmentation of gliadins, which are not digested by human proteases, and are toxic for coeliac patients. One peptide in particular, the 33-mer from gliadin, contains 6 T-cell epitopes, is highly proteolytically resistant, and is a principal contributor to gluten immunotoxicity (Shan et al., 2002). The presence of oats in “gluten-free” food products is controversial, and there are two current theses. Some researchers assert that coeliac patients tolerate oats without signs of intestinal inflammation. In contrast, other studies confirm the toxicity of oats in some types of coeliac patients and the impossibility of consuming oats habitually. In a previous work, we obtained the monoclonal antibodies (moAb) G12 and A1 against the gliadin 33-mer peptide (Morón et al., 2008). We showed that the reactivity of each moAb with a variety of cereal storage proteins correlated with the immunotoxicity of those dietary grains from which the proteins were extracted. With this background, the aim of the present study was to determine whether these monoclonal antibodies is able to detect varieties of oats that are “potentially toxic” for coeliac patients, and distinguish them from those that are possibly safe for coeliacs. Methods. Oat flours from different cultivars were obtained from commercial sources. Oat flour was prepared by grinding the kernels of the oat varieties. The flour samples (6 g) were extracted with 70% v/v ethanol (30 mL) for 24 h with mechanical agitation. The mixtures were then filtered, and prolamines precipitated by the addition of absolute ethanol, as described in Cornell et al. (2002).

G12-HRP moAb concentration is the same as previously reported and G12 competitive ELISA was carried out according to Morón et al. (2008).

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Results. The aim of this work was to study the sensitivity of these antibodies with different varieties of oats showing different capacities to activate coeliac peripheral lymphocytes, and to test whether there is a correlation between their toxicity for a coeliac patient and their reactivity with the antibodies. These studies were carried out using the G12 antibody previously described by Morón et al. (2008), with different varieties of oats. The affinity of the G12 antibody for the different oat varieties was determined by competitive-linked immunosorbent assay (ELISA) with gliadin. The varieties assayed show different affinity for the G12 antibody, which could be a reflection of their greater or lesser toxicity. In order to quantify the affinity of the oat varieties for the G12 antibody, the IC50 and the cross-reactivity were determined for each variety. The IC50 is defined as the concentration that produces a reduction of 50% of the peak signal in the ELISA. The cross-reactivity is determined as (IC50 of the oat variety that presents the greatest affinity for the antibody/IC50 of each variety assayed) x 100.

To detect cereal contamination (wheat, rye, and barley) in the different oat varieties, specific target sequences encoding gliadin (wheat), secalin (rye), hordein (barley), and avenin (oats) were chosen for amplification. Wheat and rice were also used as controls. We used the PCR system for amplification of these prolamine genes. The amplicon length of different cereals varied from 104 bp for oats to 181 bp. In these experiments, negative results were obtained with the PCR specific for wheat, rye, and barley in oat samples; however, positive results were obtained when the oats and 18S specific primer were used. Wheat amplification showed positive results only with the PCR specific for 18S and gliadin. References Cornell HJ, McLachlan A and Cullis PG. Extraction of cereal prolamins and their toxicity in

coeliac disease. J. Biochem. Mol. Biol. Biophys. 2002;6:151-158. Morón B, Cebolla A, Manyani H, Álvarez-Maqueda M, Megías M, Thomas MC, López MC and

Sousa C. Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide. Am. J. Clin. Nutr. 2008;87:405-414.

Shan L, Molberg Ø, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM and Khosla C. Structural

basis for gluten intolerance in celiac sprue. Science 2002;297:2275-2279.

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Recognition of Gliadin and Glutenin Fractions in Four Commercial Gluten Assays

Laura K. Allred, Bruce W. Ritter

ELISA Technologies, Inc., Gainesville, Florida, USA corresponding email: info@elisa-tek.com

Introduction. Gluten sensitivity affects nearly 1% of the population of the United States and Europe. To help these consumers avoid the health issues that result from gluten consumption, the FDA is attempting to establish a definition and testing protocol for gluten-free foods. Establishing this protocol depends on accurate tests that can detect and quantitate gluten. There are multiple immunoassays available for the quantitation of gluten, and most are based on one of two antibodies known respectively as Skerritt and R5. The Skerritt and R5 antibodies were examined through the use of four commercial test kits for their ability to detect the two main components of gluten, known as gliadin and glutenin in wheat Methods. Gliadin and glutenin fractions were separated from wheat flour using the method of DuPont et al. These fractions were then tested separately in four commercial test kits, two based on the Skerritt antibody and two based on the R5 antibody. In addition, 40 processed food samples with unknown gluten content were tested in all four kits. Results. Commercial tests based on the Skerritt and R5 antibodies demonstrated differing affinities for gliadin and glutenin, with the Skerritt-based tests recognizing glutenins more strongly, and the R5 tests recognizing gliadins more strongly. Analysis of 40 processed food samples content revealed differences in gluten detection and quantitation between the Skerritt-based and R5-based assays.

Figure 1. Separation of gliadin and glutenin fractions

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Figure 2. Reactivity of each of the four commercial test kits to the gliadin and glutenin fractions of wheat, expressed as ppm gluten. Conclusions. The discrepancies in gluten test results may be the result of the antibody affinity differences between the Skerritt- and R5-based tests, the solubility differences between gliadins and glutenins, or a combination of these and other factors. References DuPont, F.M., Chan, R., Lopez, R. & Vensel, W.H. J Agric Food Chem 2005; 53:1575-1584 Fasano, A., Berti, I., Gerarduzzi, T., Not, T., Colletti, R.B., Drago, S., Elitsur, Y., Green, P.H.R., Guandalini, S., Hill, I.D., Pietzak, M., Ventura, A., Thorpe, M., Kryszak, D., Fornaroli, F., Wasserman, S.S., Murray, J.A. & Horvath, K. Arch Intern Med 2003; 163, 286-292

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New Approaches in Gluten Analysis of Products for Celiacs by Proteomics Combined with The R5 ELISA

Techniques.

María Carmen Mena1*, Manuel Lombardía1, Alberto Hernando1, Juan Pablo Albar1 1 Proteomics Facility/Gluten Unit, Centro Nacional de Biotecnología, CSIC, Madrid, Spain

*corresponding email: mcmena@cnb.csic.es

Introduction. There are several difficulties associated with proteomic analysis of prolamins and glutelins including the limited number of sequences of wheat, barley and rye that are loaded and registered in public databases and the sample preparation procedure (Qian et al. 2009). Our previous studies have established that protein profiling by MALDI-TOF MS identify a large number of prolamins and glutelins and has become a powerful method for the verification of the presence of gluten in foods. Nevertheless, due to extensive sequence similarities among gluten proteins, their identification based on the analysis of intact proteins is not as exhaustive as required and also the results for hydrolyzed gluten is not enough accurate. Besides, previous studies have used tandem (MS/MS) in order to detect low level of gluten for standard but not in foods (Ferranti et al. 2007). Therefore, in this study we have undertaken the characterization of prolamins and glutelins from Triticum aestivum, Hordeum vulgare and Secale cereale of “gluten-free” foods and ingredients used in food manufacturing. Methods. (1) Prolamins and glutelins from foods were extracted using the new extraction solution developed by our group (UPEX®). This extraction solution leads to fully recover the gluten contained in foods, even when these foods have been heat-treated or hydrolysed during their manufacturing and therefore gluten is more difficult to extract and characterize (Mena et al. 2009). (2) The resulting extracts were in-solution digested with trypsin and purified by a PerfectPure C-18 Tip (Eppendorf) (3) The identification and characterization of the peptides (and by association the original protein) in final fractions was performed using nanoRP-LC-MS/MS and the results were compared with different databases in order to identify proteins. (4) In addition, the extracts were analyzed by the R5 ELISA techniques (Sandwich and Competitive). Results. In Table 1 are presented the results of peptides found in selected wheat starches employed in food manufacturing. Is it observed that the sensitivity of the technique is high enough, demonstrated by positive results even in wheat starch presenting only 6 ppm of gluten. Besides, the peptides correspond to different family of proteins potentially toxic to celiac such as alpha gliadin, gamma gliadin and low molecular weight glutenin proteins. There are some peptides in common between different proteins and different wheat starches types. Also is it observed that trypsin digestion does not offer as much information as needed due to the lack of enough breakdowns aminoacid sequences in prolamins from gluten of this type of protease. As a result the peptides are too much longer to be completely analyzed by the nanoRP LC MS/MS system employed. Conclusions. These results demonstrate the feasibility of the LC-MS/MS proteomics platform as a very useful technique combined with the R5 ELISA to identify and quantify peptides and proteins of gluten in foods.

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References Qian Y, Preston K, Krokhin O, Mellish J, Ens W. Characterization of wheat gluten proteins

by HPLC and MALDI TOF mass spectrometry. J Am Soc Mass Spectrom. 2008;19(10):1542-50.

Ferranti P, Mamone G, Picariello G, Addeo F. Mass spectrometry analysis of gliadins in celiac disease. J Mass Spectrom. 2007;42(12):1531-48. Review.

Mena MC, Hernando A, Lombardía M, Albar JP. The application of proteomics in gluten analysis: Identification and characterization of prolamins and glutelins through mass spectrometry. Working Group on Prolamin Analysis and Toxicity, Verlag Wissenschaftliche Scripten 2009: 23-28. ISBN: 978-3-937524-75-7.

Table 1. Analysis of wheat starches by LC-MS/MS after trypsin digestion.

Wheat starch A (6 ppm gluten)

Protein Mass Peptide1288.66 R.QLAQIPEQFR.C1288.62 R.QPSQIPEQFR.C

Wheat starch B (25 ppm gluten)

Protein Mass Peptide845.44 R.TPFPQTR.G1180.66 R.QLVQIPEQAR.C

gamma gliadin [Triticum monococcum] 1815.84 R.QQCCQPLAQISEQAR.C

1424.72 R.CQAIHNVVESIR.Q1228.66 R.QLAQIPEQFR.C

low molecular weight glutenin subunit[Triticum aestivum]

1228.62 R.QPSQIPEQFR.C

2059.03 K.VFLQQQCSPVAMPQSLAR.S859.49 R.VNVPLYR.T

Wheat starch C (110 ppm gluten)

Protein Mass Peptidealpha-gliadin [Triticum turgidum subsp. durum] 1180.66 R.QLVQIPEQAR.C

1949.93 R.DALLQQCSPVADMSFLR.S2650.18 R.SQAVQPRSCLVMWEQCCQQLK.A1186.64 R.APFASIVAGIGGQ.-2855.61 R.RPLFQLVQGQGIIQPQQPAQLEVIR.S2393.03 R.SDCQVMQQQCCQQLAQIPR.Q1815.84 R.QQCCQPLAQISEQAR.C1424.72 R.CQAIHNVVESIR.Q1288.66 R.QLAQIPEQFR.C2082.04 R.QQQHHQPQQEVQLEGLR.M1949.93 R.DALLQQCSPVADMSFLR.S2650.18 R.SQVVQHSSCLVMWEQCCQQLK.A1244.65 R.QLSQIPEQFR.C1228.62 R.QPSQIPEQFR.C

high-molecular-weight glutenin subunit[Triticum aestivum]

1320.60 R.ELQESSLEACR.Q

2059.09 K.VFLQQQCSPVAMPQSLAR.S

859.42 R.VNVPLYR.Tlow-molecular-weight glutenin subunit group 11[Triticum aestivum]

1732.89 K.VFLQQQCIPVAMQR.C

2059.09 K.VFLQQQCSPVAMPQSLAR.S859.42 R.VNVPLYR.T

s-type low molecular weight glutenin L4-55 [Triticum aestivum]

alpha-gliadin [Triticum turgidum subsp. durum]

low molecular weight glutenin subunit[Triticum aestivum]

s-type low molecular weight glutenin L4-55[Triticum aestivum]

gliadin/avenin-like seed protein [Triticum aestivum]

gamma gliadin [Triticum aestivum]

gamma gliadin [Triticum monococcum]

gliadin/avenin-like seed protein [Triticum aestivum]

gliadin/avenin-like seed protein [Triticum aestivum]

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Effects of heating, reducing and alcohol concentration on prolamin extraction

Päivi Kanerva, Tuula Sontag-Strohm, Outi Brinck, Hannu Salovaara University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland

*corresponding email: paivi.kanerva@helsinki.fi Introduction. Prolamin proteins of barley, rye and wheat are harmful for celiac patients. Despite prolamins of different species are closely related to each other, they have some distinct differences, which need to be considered in quantitative gluten analysis. Gluten is determined with immunological methods using prolamin-specific monoclonal antibodies. The quantification is commonly based on a wheat standard and, therefore, on the properties of wheat prolamins. Barley and rye prolamins differ from wheat prolamins in their composition and extractability, and neglecting these differences in the gluten analysis leads to erroneous results (Kanerva et al., 2006). Therefore, it is important to gain knowledge on the solubility characteristics of barley, rye and wheat prolamins and, thus, to ensure reliable quantitative gluten analysis of gluten-free products. In this study, differences in the extractability of barley, rye and wheat prolamins were studied with different alcohols. The effects of reducing and heating during extraction were also tested. Methods. Grain samples of barley and wheat were pooled from ten cultivars each. Rye sample represented four cultivars that were grown during six years in six different locations in Finland. Milled wholemeal samples were extracted with aqueous ethanol, 1-propanol or 2-propanol in six alcohol concentrations ranging from 20 to 70% (v/v) in ratio of 1:10 for 20 min at 21ºC or 50ºC with continuous shaking. For the reduced samples 1, 2, 4, 6 or 8% (w/v) dithiothreitol (DTT) was added to 50% 1-propanol solution. The protein contents of the extracts were determined by the Dumas combustion method. The relative prolamin content of the samples was determined using an automated electrophoresis system (Experion, BioRad, USA). Results. Raising of the extraction temperature from 21ºC to 50ºC increased the protein yields by 37 to 47%; especially the extractability of high molecular weight prolamins increased. The use of reducing agent (DTT) increased the extraction yield by 20 to 30%. The most efficient solvent for barley and wheat prolamins was 40% 1-propanol (Figure 1) whereas the rye prolamins were extracted most efficiently with 60% 2-propanol. This is explained by the differences in prolamin compositions. Many of the prolamins of barley and wheat need reduction before they dissolve, whereas rye prolamins are nearly completely extracted without a reducing agent. In addition, 2-propanol extracted more efficiently high molecular weight rye proteins.

37

2

Figure 1. Prolamin contents of barley, rye and wheat extracts analysed by Dumas combustion method and automated electrophoresis system. The flours were extracted with six concentrations of ethanol ( ), 1-propanol (---) and 2-propanol ( ). Conclusions. The extraction procedure is a key factor in the analysis of gluten contamination in gluten-free raw materials and products. In this study, 40% 1-propanol was found to be the best solvent for the prolamin extraction. The extraction yield improved by the elevated extraction temperature and the addition of 1% (w/v) DTT was considered sufficient for the extraction. Therefore, the extraction of gluten-free materials with 40% 1-propanol under reducing conditions appears the most suitable sample preparation method for the immunological analysis of gluten-free products with unknown contamination source. References Kanerva PM, Sontag-Strohm TS, Ryöppy PH, Alho-Lehto P, Salovaara HO. Analysis of barley

contamination in oats using R5 and omega -gliadin antibodies. J Cereal Sci 2006;44:347-352.

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Deamidation of gluten proteins drastically influences the quantitative gluten analysis

Päivi Kanerva, Tuula Sontag-Strohm, Hannu Salovaara, Jussi Loponen University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland

*corresponding email: paivi.kanerva@helsinki.fi Introduction. With celiac disease patients, gluten proteins cause an inflammation of the small bowel (Kagnoff 2007). Gluten, due to its unique properties and relatively low price, is widely used ingredient in food industry (Day et al. 2006). A modified derivate of gluten is so called “soluble gluten”, which is produced by chemical or biochemical deamidation. Deamidation improves the solubility and surface activity of gluten and, thus, makes it a versatile food ingredient. Deamidated gluten, however, structurally differs from native gluten, which may influence its quantification by commercial immunoassays. In deamidation, conversion of glutamines to glutamic acids takes place and this alters the protein structure and functionality. This work studied the influence of deamidation on wheat gluten recognition by using R5 antibody. Methods. Deamidated wheat gluten was prepared by adding 200 ml of 0.1M HCl to 5g of vital gluten (Raisio, Finland) and heating the suspension at 100°C for 2h. After the treatment, the suspension was neutralised, dialysed against distilled water (MWCO 12-14kD) and the contents lyophilized. The prolamin concentrations of the vital and the deamidated wheat gluten were determined with a sandwich and a competitive R5 ELISA (Ridascreen, Darmstadt, Germany). The samples were extracted according to the assay protocols, and the protein contents of the extracts were determined by Lowry method. Results. Deamidation of gluten significantly reduced its immunological detection by the prolamin-specific antibody R5. Both gluten samples were analyzed in varying protein concentrations by the sandwich and the competitive R5-ELISA, and compared to the standard curves of each assay (Figure 1). The quantification of vital gluten was accurate (equal as gliadin standard) with the sandwich assay whereas the recognition of the deamidated gluten decreased drastically. The response for the vital gluten was more than 600 times higher than that of the deamidated gluten. When the same samples were assayed by the competitive method, the deamidated gluten, in turn, was recognized with the same intensity as the standard of the assay whereas the affinity of the antibody to the vital gluten apparently was approximately 125 times higher than the deamidated gluten.

39

Figure 1. Comparison of the reactivity of vital and deamidated gluten by a sandwich and a competitive R5 ELISA. Conclusions. This study showed that the deamidation of gluten drastically weakened the antibody recognition compared to the native gluten. This means that if soluble gluten ingredients are used in food products, the gluten content of the products is underestimated. References Kagnoff MF. Celiac disease: Pathogenesis of a model immunogenetic disease. J Clin Invest

2007;117:41-49. Day L, Augustin MA, Batey IL, Wrigley CW. Wheat-gluten uses and industry needs. Trends

Food Sci Technol 2006;17:82-90.

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Nutritional Requirements for Gluten-Free Foods

Tricia Thompson, MS, RD Nutrition Consultant Celiac Disease, Manchester, Massachusetts USA

Corresponding email: tricia_s_thompson@hotmail.com

The gluten-free diet primarily affects food choices from the grain or cereal food group, including bread, pasta, and breakfast cereals. While there is an ever-increasing variety of specially manufactured gluten-free foods, many of these products are made using refined rice and corn flours and starches instead of gluten-free whole grains and pseudocereals, such as teff, millet, sorghum, amaranth, quinoa, or buckwheat. Unlike refined wheat-based foods, refined gluten-free products are not usually enriched with vitamins and minerals. As a consequence many processed gluten-free grain foods have low nutritional value. Only a few studies have been conducted on the nutritional quality of gluten-free grain foods. One study (Thompson 1999) assessed the thiamin, riboflavin, and niacin contents of gluten-free cereal foods. As part of the study, 268 gluten-free products, including breads, pastas, and ready-to-eat breakfast cereals were reviewed for ingredients. Of these, 196 listed a refined grain or starch as the first ingredient and, of these, only 32 were enriched. The study also compared the thiamin, riboflavin, and niacin contents of gluten-free cereal foods to their enriched wheat-containing counterparts. Sixty-one percent of the gluten-free products contained lower amounts of all three nutrients and another 22 percent contained lower amounts of two nutrients. Another study (Thompson 2000) assessed the folate, fiber, and iron contents of gluten-free cereal foods. Of 58 products reviewed for enrichment status, only 12 were enriched. Compared to their wheat-containing counterparts, 77 percent contained lower amounts of iron, 81 percent contained lower amounts of folate, and 31 percent contained lower amounts of fiber. The findings of these studies suggest that gluten-free cereal foods are nutritionally inferior to the wheat-containing foods they are intended to replace. This appears to be due in large part to an over-reliance on refined unenriched gluten-free flours and starches and under-use of gluten-free whole grains. As is illustrated in Table 1 and Table 2, the use of enriched grain over refined grain and whole grain over refined grain would increase the nutritional value of gluten-free grain foods. According to the American Dietetic Association’s Evidence Analysis Library (www.adaevidencelibrary.org), the gluten-free diet as a whole may be high in fat and low in carbohydrates and fiber as well as several other nutrients, including iron, folate, niacin, vitamin B12, calcium, phosphorus, and zinc. In a study conducted in the United States (Thompson et al. 2005), 54 percent of women consumed-below recommended amounts of fiber, 56 percent below-recommended amounts of iron, 69 percent below-recommended amounts of calcium, and 79 percent below-recommended amounts of grain

41

foods. Below-recommended intakes of carbohydrate and grain food may be a contributing factor in low intakes of fiber, iron, and B vitamins. In the United States, grains foods contribute significantly to an adult’s intake of these nutrients. Table 1. Nutrient comparison of unenriched degermed cornmeal and enriched degermed cornmeal Cornmeal, Cornmeal, Percentage unenriched enriched difference ________________________________________________ Amount 1 cup (138 grams) 1 cup (138 grams) ------ Calories 587 587 ------ Folate (DFE) 48.0 549.0 1,144% Riboflavin (mg) .08 .66 825% Niacin (mg) 1.59 8.44 531% Thiamin (mg) .22 .98 445% Dietary Fiber (g) 6.4 6.4 ------ Calcium (mg) 5.0 5.0 ------ Iron (mg) 1.75 6.87 393% Source: Thompson, T. The Gluten-Free Nutrition Guide. New York: McGraw-Hill; 2008. p. 53. Table 2. Nutrient comparison of white rice flour and brown rice flour White rice flour Brown rice flour Percentage difference ________________________________________________ Amount 1 cup(158 grams) 1 cup (158 grams) ------- Calories 578 574 ------- Iron (mg) .55 3.13 469% Riboflavin (mg) .03 .13 333% Thiamin (mg) .22 .70 218% Niacin (mg) 4.09 10.02 145% Dietary Fiber (g) 3.8 7.3 92% Folate (DFE) 6.0 25.0 32% Calcium (mg) 16.0 17.0 6% Source: Thompson, T. The Gluten-Free Nutrition Guide. New York: McGraw-Hill; 2008. p. 52. To improve the nutritional quality of gluten-free processed cereal foods and thus the overall nutritional quality of the gluten-free diet, manufacturers should be encouraged to incorporate gluten-free whole grains into their products and decrease use of unenriched refined flours and starches. If manufacturers do use refined flour and starch, they are encouraged to enrich their products to a similar level as wheat-based products. References Thompson T. Thiamin, riboflavin, and niacin contents of the gluten-free diet: is there cause for concern? J Am Diet Assoc 1999;99:858-862. Thompson T. Folate, iron, and fiber contents of the gluten-free diet. J Am Diet Assoc 2000;100:1389-1396. Thompson T, Dennis M, Higgins LA, Lee AR, Sharrett MK. Gluten-free diet survey: are Americans with celiac disease consuming recommended amounts of fibre, iron, calcium and grain foods? J Hum Nutr Diet 2005;18:163-9.

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Gluten-Free Food Market

Markku Mikola1*; Esa Wrang2 1 Consultant, Sennet Oy, Finland,

2 Leading consultant, Finpro ry, Finland *corresponding email:markku.mikola@alumni.helsinki.fi

Introduction. Gluten-free foods are part of the more general free-from food area which is growing quite rapidly. New markets are opening for gluten-free foods because knowledge of the disease is growing and faster diagnostic tools are being introduced. The gluten free food market is characteristically a strictly regulated segment for a narrow consumer group. However, for various reasons it is going towards a more mainstream market. In this paper we will briefly discuss the development of this market in Europe. Results. Gluten-free foods can be defined strictly as foods not containing gluten. However, in this paper we discuss gluten-free foods as a part of a more general free-from food area. For stores and restaurants it is easier and cheaper to keep products which are free from several allergy or intolerance inducing substances. In addition to the people who have medical reasons to avoid certain ingredients there are more and more people avoiding these because it makes them feel good or someone in their family is already regularly using gluten free products. The global market growth of free-from products was approximately 75% from 2003 to 2008. At the same time the gluten-free market has grown some 125%. The total number of consumers of gluten-free foods is growing due to growth of the number of diagnosed celiacs. The European gluten-free food market is growing and developing rapidly, but of course there are major differences between countries. In Scandinavia generally branded products and local producers seem to dominate the market whereas in UK retail stores and their private label products lead the way. In Germany a lot of gluten-free products are sold through health stores even though lately more products have also appeared in general stores. During the year 2008 in Europe almost 30% of new gluten-free products were launched in UK and 15% in Spain, but the largest gluten-free markets are still Italy and Germany. Currently there is an ongoing harmonisation of European legislation of gluten free foods which will most probably make the gluten-free market more attractive to larger food producers. Association of European Celiac Societies gathers together the national patient organizations all around Europe. These organisations are not all alike but can be important partners for food developers as it is possible to reach a lot of celiacs through them. The Crossed Grain symbol is run by the Societies and is a recognized mark of a safe product for a celiac. Many of the national and local associations do run their own magazines or newsletters and gluten-free foods are partially marketed through them. Since the world is going online also the communication of gluten free products is going there. The internet is not only the web pages of societies and companies, it is also the ever-growing social media. Forums are an ideal place for gluten-free eaters to meet, give ideas, tell about new products and definitely to tell about possible lacks of the products.

43

Conclusion. The gluten-free food market is growing as special foods for celiacs, but also as a part of larger free-from food segment. Due to current and near future changes gluten-free products will become more mainstream and the area in general will become more interesting to large food manufacturers. References Sitra 2009, URL:http://www.sitra.fi; http://www.finpro.fi; ISBN 978-951-563-644-7, Mintel 2008 Euromonitor 2009, Global Market for Food Intolerance Products. http://www.aoecs.org/

44

Folates in Gluten-Free Diets and Foods

Vieno Piironen*, Maija Kinni, Jussi Loponen and Susanna Kariluoto University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland

*corresponding email: vieno.piironen@helsinki.fi Introduction. Cereal products generally contribute significantly to the daily dietary folate intake. In several countries mandatory fortification of wheat flour is practiced to ensure adequate folate intake. However, also non-fortified cereal products are important folate sources accounting for example in Finland for 36 and 32% of the total dietary folate intakes among men and women, respectively (Paturi et al. 2007). In gluten-free (GF) diets, wheat, barley and rye are avoided, and GF cereal foods are frequently made using refined GF flours or starches. Moreover, these raw materials are usually not fortified. Therefore, GF cereal foods may be significantly poorer folate sources than their gluten-containing counterparts. Differences in food choices, e.g. reduced consumption of cereal-based foods, and impaired absorption may further lower folate status of celiac patients. In this presentation, the aim is to evaluate GF cereal products and their raw materials as folate sources in GF diets. Possibilities to increase folate intake by raw material selection and processing are discussed. Methods. Folate levels in GF foods and diets are discussed based on our own study and published studies (Thompson 2000; Yazynina et al. 2008; Lee et al. 2009). We determined folate contents in flours and other selected raw materials used in GF baking and in samples taken from industrial GF baking processes. In addition, laboratory scale baking was carried out using two GF sourdoughs (rice and quinoa based) and different fermentation times. A microbiological assay was used to measure total folate contents (Kariluoto et al. 2004). Results and discussion. Commonly used GF flours and starches contain clearly lower amounts of folate than their gluten-containing counterparts. We showed that two GF flour mixes contained ca. 5 µg/100 g fw (wheat starch based mix) and 10 µg/100 g fw (mix composed of wheat starch, rice flour and non-cereal ingredients) of folate whereas folate contents in wheat and rye flours ranged from ca. 17 µg/100 g (wheat flour with low ash content) to 45 µg/100 g (whole meal flours) (Vuorisalo 2007). Accordingly, Yazynina et al. (2008) reported that starches and low protein flours, commonly used as main ingredients in GF products, were poor folate sources. Only traces of folate vitamers were detected in maize starch, potato starch and GF flour mix. Thus, without fortification these raw materials provide much less folate than common non-fortified flours. Clearly better folate sources are found among other GF cereal raw materials for baking. The total folate contents of whole meal oat flour and oat flake samples in our study were ca. 30 µg/100 g fw. In different buckwheat flours the range of the total folate contents was 31–54 µg/100 g fw and that in other buckwheat milling products 24–56 µg/100 g fw. The total folate contents of rice, maize and potato based flakes and flours were 23–55 µg/100 g. Utilization of other celiac-safe cereals (e.g. millet) and pseudocereals (in addition to buckwheat e.g. quinoa and

45

amaranth) could further diversify assortments of GF products. Thompson (2000) reported that the folate contents of quinoa, amaranth and millet were 49-85 µg/100 g. According to Lee et al. (2009), substituting the grain or starch portion of three servings (in established standard GF dietary pattern) with oats, brown rice and quinoa increased the total folate intake from grain products from 23 to 151 µg.

Dough fermentation can be used to enhance folate contents of bakery products. Yeasts contain significant amounts of folate. Furthermore, during fermentation yeasts and bacteria may synthesize folate. Laboratory-scale fermentation for 23 hours increased the folate content of dough, made using oat flour, up to 2-fold. Bread samples, from bakeries practicing GF baking, were good folate sources containing up to 70 µg/100 g fw of folate when buckwheat was used as raw material. Conclusion. Utilization of alternative cereal raw materials, instead of starches and refined flours, can significantly increase the folate levels of non-fortified GF foods and diversify the assortments of GF products. Oats, buckwheat and other celiac-safe cereals as well as pseudo-cereals should be studied further in GF baking. Fermentation can further enhance folate levels in GF products. References Kariluoto S, Vahteristo L, Salovaara H, Katina K, Liukkonen K-H and Piironen V. Effect of

baking method and fermentation on folate content of rye and wheat breads. Cereal Chem. 2004; 8:134–139.

Lee AR, Ng DL, Dave E, Ciaccio EJ, Green PHR. The effect of substituting alternative grains in the diet on the nutritional profile of the gluten-free diet. J Human Nutr Diet 2009; 22:359-363.

Paturi M, Tapanainen H, Reinivuo H, Pietinen P. The National Finndiet 2007 study. Publications of the National Public Health Institute 2007; B23/2998.

Thompson T. Folate, iron, and dietary fiber contents of the gluten-free diet. J Am Diet Assoc 2009; 100: 1389-1396.

Yazynina E, Johansson M, Jägerstad M, Jastrebova J. Low folate content in gluten free cereal products and their main ingredients. Food Chem 2008; 111: 236-242.

Vuorisalo L-M. Folates and tocols in cereal products. Master’s thesis. 2007. EKT-series 1393.

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Cereals and pseudocereals for gluten-free foods

Regine Schoenlechner*, Emmerich Berghofer University of Natural Resources and Applied Life Sciences (BOKU), Department of Food

Sciences and Technology, Institute of Food Technology, Vienna, Austria *corresponding email: regine.schoenlechner@boku.ac.at

Introduction Principally there are two possibilities for production of gluten-free products. One is the removal of gluten from gluten-containing raw materials. This possibility is often applied for wheat in order to receive a gluten-free wheat starch. The second possibility is the use of gluten-free raw materials, mainly gluten-free cereals, pseudocereals, legumes or roots. Gluten-free cereals Under the term gluten-free cereals all cereal species are summarised, which are not considered to contain gluten or any other prolamins causing coeliac disease. Namely, these are rice, maize, millet, sorghum and oat. All these raw materials have in common that due to their lack of gluten they do not contain any network-forming proteins. In order to produce breads, bakery or pasta products, in most cases additional ingredients or additives are necessary. Rice Rice is mainly used as milled (white) rice, although brown rice has better nutritional value. Rice flour is one of the major ingredients in many gluten-free baking mixes in the Western countries. Due to its bland taste, white colour, digestibility and hypoallergenic properties it is the most suitable cereals grain flour for the production of gluten-free products. Rice protein shows a unique pattern of albumin, globulin, prolamin and glutelin content among the cereals, with a high concentration of glutelins and a low concentration of prolamins (called orycin in rice). Due to this composition rice proteins have relatively poor functional properties for food processing. The low concentration of prolamins results in the lack of formation of a protein network when rice is kneaded with water. A different approach to the production of gluten-free bread is to use rice flour blended with other flours and different starches (Rosell and Marco, 2008). Although rice flour alone is not suitable for bread production, it can be very well used to produce noodles. Rice noodles are long known, traditional products in Asia. Maize Maize itself is gluten-free, but it has no dough forming properties per se. By soaking of the kernels in lime water and consequent "alkaline-cooking" the maize protein is chemically and physically changed in such a way that a dough (masa) can be formed, which can be formed and baked to flat bread. The dominant protein class in maize is the prolamins, which are called zeins in maize. Isolated zeins are available commercially and are mainly used for coatings on food products. They have been found to be able to form viscoelastic dough when mixed at high temperatures, but for the production of gluten-free products this has not been explored yet (Schober and Bean, 2008). The use of maize starch in food applications (e.g. extruded snacks, breakfast cereals) is widespread. Yet, little research has been done on the production of breads from maize flour or maize starch. Own investigations have shown that by addition of maize flour to gluten-free flour blends bread quality (volume, texture, etc.) was decreased drastically. Gluten-free maize

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noodles can be found on the market, which are produced from 100% maize flour, but no information about its processing method can be found in the literature. Millet and Sorghum All sorghum and millet species are gluten-free. Prolamins (kafirin) are the dominant type of protein in sorghum and most millet species. Unlike in other cereals, protein digestibility of sorghum decreases upon cooking, apparently due to increased cross-linking. Millet and sorghum can be used solely for bread baking, which traditionally is done in some African countries. These breads have more the character of flat bread. The production of Western-style bread based on millet and sorghum has been summarised by Taylor et al. (2006). To produce wheat-free sorghum or millet bread, bread quality is increased by the addition of native or pre-gelatinised starches, hydrocolloids, fat, egg or rye pentosans. However, specific volumes are lower than those for wheat bread or gluten-free breads based on pure starch, and in many cases, breads tend to stale faster. In own investigations it was found that within a gluten-free flour blend millet addition showed good ability to increase bread quality, although final bread quality still has to be optimised. Oats Oats are now recommended to be included as a part of the gluten-free diet. Special oat brands have been introduced in which the cross-contamination of oats with other cereals is minimised by careful control through the whole production chain Oats deliver a typical cereal character into products and can be included into various products to diversify the diet of patients with celiac disease (Sontag-Strohm et al., 2008). Pseudocereals The three main used pseudocereals are amaranth, quinoa and buckwheat. Botanically they are dicotyledonae. Unlike the true cereals, pseudocereals contain only little amounts of prolamins, 2-3% in amaranth, 0.8% in quinoa and 0-4% in buckwheat (Schoenlechner et al., 2008), which are so far known to be non-toxic to coeliac disease. Unfortunately these raw materials do not possess dough forming or baking properties. The production of bread and bakery products from pseudocereals alone cannot be carried out without further ingredients or processing adaptation. Easier is the production of noodles from 100% pseudocereal flour. Own trials have shown that by using a flour blend of all three pseudocereals (amaranth, quinoa and buckwheat) and adapting the recipe (lower moisture content, addition of albumen, emulsifier and enzymes) gluten-free noodles could be produced with good textural quality. Still some open questions remain to be improved like sensory properties, elasticity or colour of the noodles. References Rosell CM, Marco C (2008), Schober TJ, Bean SR (2008), Sontag-Strohm T., Lehtinen P, Kaukovirta-Norja A (2008), Schoenlechner R, Siebenhandl S, Berghofer E (2008) in: Arendt E, Dal Bello F: Gluten-free cereal products and beverages. Academic Press, Elsevier. Taylor JRN, Schober TJ and Bean SR (2006): Novel food and non-food uses for sorghum and millets. J. Cer. Sci. 44 (2006) 252-271

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Healthy Grains for Enhanced Gluten-free Breads

Eimear Gallagher1*, Laura Alvarez 1,2 and Elke Arendt2

1 Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland.2 Dept. of Food and Nutritional Sciences, National University of Ireland, Cork, Ireland.

*corresponding email: Eimear.Gallagher@teagasc.ie

Introduction: Gluten-free breads, as well as being safe for coeliacs, should have a nutritionalvalue equivalent to that of the gluten-containing breads they are intending to replace.However, the nutritional quality of gluten-free products currently in the market has beenreported to be of concern, and there is a need for an improvement in their formulations(Thompson, 2009). In general, gluten-free breads and bread mixes are formulated usingrefined flours and/or starches and, in contrast to their wheat-containing counterparts, gluten-free products are not normally fortified. Pseudocereals do not belong to the grass family butstill produce flours and seeds which can be used as flour. They are naturally gluten-free, andare characterised by high protein, fibre and micronutrient contents. In the present study, anumber of nutritional and bioactive properties of pseudocereal flours were studied, along withtheir suitability as ingredients in gluten-free breads.

Methods: Buckwheat, amaranth and quinoa flours replaced potato starch in a control gluten-free bread formulation (C), based on rice flour and potato starch. Standard baking tests wereconducted on the resulting breads. Macronutrient and mineral analyses were completed on theflours (pre-baking) and breads (post-baking). Total antioxidant capacity of the pseudocerealgrains and breads was determined by the DPPH method and the FRAP assay. Total phenolcontent was assessed by FCR assay.

Results: Loaf volumes were increased (P<0.05) for buckwheat (1.63ml/g) and quinoa(1.4ml/g) breads in comparison to the control (1.3ml/g). No difference in volume was foundfor breads containing amaranth. The crust colour of the loaves was significantly darkenedfollowing the inclusion of all pseudocereal flours (P<0.01). Crumb texture was significantlyimproved, with all pseudocereal-containing breads having a softer (P<0.05) and morecohesive (P<0.01) texture in comparison with the control. The C-Cell image analysis systemrevealed that the crumb grain of the breads with pseudocereals was improved (Figure. 1). Inparticular, breads containing buckwheat had the highest number of evenly distributed cells,and the control had the highest number of holes. An overall increase in the nutrient content ofall pseudocereal breads was observed. In particular, the protein, fibre and Mg levelssignificantly increased (P<0.05). Levels of total antioxidants in the buckwheat flour weresignificantly higher than the other samples. Antioxidant levels were significantly reduced(although still present for buckwheat breads) after baking, whereas those present in wheatbreads were too low to measure. Similarly, significantly higher levels of total phenols werefound in buckwheat flours and breads. An example of some of these results is seen in Table 1.

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Figure 1. C-Cell imaging of the gluten-free breads containing amaranth, buckwheat andquinoa, versus the control formulations.

Grains

Total phenol asgallic acid equivalent(mgGAE/100g dwb)

FRAP assay(mgTE/100g dwb)

Amaranth 21.2 ± 2.3 55.3 ± 1.6

Quinoa 71.7 ± 5.5 92.1 ± 1.7

Buckwheat 323.4 ± 14.1 436.3 ± 12.8

Wheat 53.1 ± 2.8 109.5 ± 4.7

Breads

Amaranth (A) 13.8 ± 0.0 60.6 ± 6.2

Quinoa (Q) 30.7 ± 0.3 71.4 ± 2.8

Buckwheat (B) 64.5 ± 3.1 147.7 ± 4.6

Wheat control (WC) 29.1 ±0.6 81.7 ± 1.6

GF control (GFC) 8.8 ± 1.0 47.6 ± 3.3

100% quinoa (100%Q) 55.2 ± 0.9 87.0 ± 5.2

Sprouted buckwheat (SpB) 116.2 ± 1.8 263.6 ± 3.6

Table 1. Total phenol content and antioxidant capacity of the methanolic extracts (seeds,sprouts and breads).

Conclusions: The pseudocereals buckwheat, amaranth and quinoa are practical ingredients inthe formulation of gluten-free breads with good baking properties, sensory scores and crumbtexture. Their presence also significantly enhances the nutritional attributes of the breads.

References:Thompson, T. The Nutritional Quality of Gluten-free Foods. In: Gallagher, E, editor. Gluten-

free Food Science and Technology. Oxford: Wiley-Blackwell; 2009. p. 42-51.

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Are Coeliacs following a Gluten-free Diet or a Diet Low in Gluten?

Blanca Esteban, Manuela Márquez, Juan I. Serrano-Vela* Madrid Coeliac Association, Madrid, Spain

*corresponding email: nachoserrano@celiacosmadrid.org Introduction. The only existing successful treatment for coeliac disease (CD) is a strict long life gluten-free diet (GFD). The GFD should be based on fresh foods that are naturally gluten free such as milk, meat, fish, eggs, fruits, vegetables, legumes and gluten-free cereals (corn, rice, millet, sorghum, etc.). The consumption of manufactured foodstuffs involves potential risks, since a given product may bear the term “ gluten free” when the gluten content does not exceed 20 mg/kg in the food as sold to the final consumer, according to the Regulation 41/2009 made by the European Commission concerning the composition and labelling of foodstuffs suitable for people intolerant to gluten. However, most of coeliac patients think that the content of gluten is zero when a product is labelled as "gluten free" or when it appears in the gluten free food and drink directory provided by the corresponding coeliac association. Following low gluten diets leads to the persistence of positive antibody levels in serum as well as the persistence of symptoms and in some cases the onset of autoimmune diseases. Methods. CD patients showing positive antibody levels and/or persistent symptoms despite following a GFD were referred to the Dietetics Service of the Madrid Coeliac Association (DSMCA) by clinicians, in order to conduct a review of the diet and confirm whether the GFD was correct. They or their parents were asked to respond a survey indicating all data about the foodstuffs, drinks and drugs consumed during a week. Subsequently, the DSMCA analyzed the responses and checked whether the diet was correct or if the patient consumed gluten, either voluntarily or involuntarily, through ignorance. In order to determine the degree of compliance with the diet, the DSMCA studied whether the foodstuffs consumed by the patient appeared in the gluten free food and drink directory, and if the special gluten free foodstuffs were certified by the quality mark created by the Spanish Federation of Coeliac Associations (FACE), whereby the foodstuff must not contain more than 10 mg gluten/kg. In addition, the following parameters were analyzed: i) if the patient chose the foodstuffs based on reading the label; ii) if the patient did transgressions and how often; iii) if the consumption of manufactured foodstuffs was excessive or appropriate; iv) if the consumption of naturally gluten free foods was adequate or low; and v) how often the coeliac made his/her meals away from home and where (schools, businesses, restaurants, etc.). On the other hand, the family was advised to change bad eating habits when the diet was considered nutritionally not adequate. Results. The DSMCA has been revising the diet of coeliac patients who showed a persistent enteropathy despite the treatment since 2003. 302 patients’ diet has been evaluated so far: 99 males, 203 females, 227 children and 75 adults. The main reasons for the revision of the diet were the persistence of positive levels (or even the increase) of serum autoantibodies after a sufficient time in GFD, the persistence of anaemia and the persistence of gastrointestinal symptoms (Figure 1). In almost all cases it was found that patients consumed incorrect foodstuffs such as: i) foodstuffs specially made for coeliacs lacking any warranty or

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certification; ii) foodstuffs for normal consumption made from corn or rice and not specially produced, prepared and/or processed to meet the special dietary needs of coeliacs; iii) organic or biological foodstuffs made from corn or rice in which the absence of gluten was not certified; iv) baby food made from corn or rice but not specific for coeliacs; v) foodstuffs that seemed not to contain gluten according to the label; vi) foodstuffs removed from the gluten free foods and drinks directory; and vii) foodstuffs containing gluten, what was unknown to the patient. In all these cases, the DSMCA recommended a much stricter GFD, reducing the consumption of manufactured foodstuffs, and eliminating the incorrect foodstuffs from the diet. When making these changes, almost all patients reached negative antibody levels in a time between 3 and 12 months. It was also found that some adults followed an incorrect GFD when they ate outside, by ignorance and by not adequately informing the catering staff. Interestingly, most of the diet reviews of children who ate at school daily, the diet provided was correct; however gluten was consumed at home. Conclusions. It is necessary to get a correct labelling of gluten-free foodstuffs and better control the quality of such products to ensure the correct feeding of coeliacs. On the other hand, it would be desirable to improve the nutritional education of the general population to promote healthy eating habits and thus be able to select appropriate food for all the family, coeliacs or not.

Persistence

or

increased of

antibodies

Low iron Persistent

symptoms

Persistence Low iron Persistence of positive of symptoms antibody levels

10% 5%

85%

Figure 1. Main reasons for diet revision.

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Nutritional quality of linseed and oil hemp varieties cultivated in Finland with special attention to lignan and

cadmium contents

Marketta Saastamoinen1*, Juha-Matti Pihlava2, Merja Eurola2

1 Satafood Development Association, Huittinen, Finland, 2 MTT Agrifood Research Finland,

Jokioinen, Finland *corresponding email: marketta.saastamoinen@satafood.net

Introduction. Celiac disease is difficult condition hindering the usage of cereal containing gluten proteins in the diet. Only maize, rice, and millet from monocotyledonous cereals can be used by all celiac patients. Most celiac patients can use also pure oats. Dicotyledonous crops can be used as a component of bread mixtures containing flour from different crops. Linseed (Linum usitatissimum L.) is very well suitable as part of the mixture in bread making. Some 2000 ha of linseed are cultivated annually in the southern part of Finland but large amounts of linseed are also imported to Finland every year. Linseed contains nutritional oil with high a high linolenic acid content, good quality protein and especially high lignan content. Lignans are phytoestrogens inhibiting hormonal cancer development in women and men. Linseed has, however, the ability to accumulate cadmium from the soil to the seeds. Cadmium, a heavy metal, activates estrogen receptor causing estrogen- like effects in vitro and in vivo (Johnson et al. 2003) causing breast cancer in humans (McElroy et al. 2006). Hemp (Cannabis sativa L.) is a highly variable crop with both fibre and oil hemp varieties. Seed production of high THC cannabinoid content of hemp limits its cultivation. There are, however, varieties with very low THC cannabinoid content. In Finland there exists Finola variety intended for hemp oil production with no, or very low, THC cannabinoid content. Material and methods. Linseed and oil hemp seed samples were collected from farms in 2005-2009. These included samples from two linseed varieties, Helmi and Laser. A replicated variety trial was established at MTT station in Piikkiö in South-western Finland in 2009. The linseed varieties in the trial were Helmi, Heljä, Laser, Abacus and Sunrise and the fibre flax varieties Martta and Belinka. Oil and protein and contents were analysed from the samples. Linseed lignan contents, secoisolariciresinol diglycoside (SDG) lignan content was analysed by liquid chromatography after oil separation and basic hydrolysis. Cadmium and lead contents of the samples were analysed by ICP mass spectrometer after wet digestion. Lignan, cadmium and lead analyses were carried out at the MTT chemical laboratory. Results. The quality of linseed and oil hemp samples was good. Both crops are oil crops containing high levels of oil and protein (Table 1). Oil content of linseed varieties varied from 41.3 % to 50.3 % from dry matter. The Helmi variety had lower oil content than other linseed varieties. Two flax varieties, Martta and Belinka, had the lowest oil contents in 2009. Flax varieties had higher protein contents of 24.8 and 25.0 % compared to the linseed varieties. In oil crops oil and protein are very often negatively correlated characteristics. The Finola oil hemp had lower oil content and slightly higher protein content than linseed varieties. Linseed absorbed rather a lot of cadmium from the soil, the cadmium content of the seed varying from 0.36-1.11 mg/kg. Cadmium content of Finola oil hemp was very low, 0.017 and 0.023 mg/kg.

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It seem that Helmi may take up a little more cadmium but there was more local variation in the cadmium content of farm samples. Lead contents were low in all crops and samples. SDS lignan contents were higher in Helmi than in Laser in farm and in trial samples in all years (Table 1). In the trial Laser had the lowest SDG lignan content. Heljä and Helmi linseed varieties and Martta and Belinka flax varieties had the highest SDG lignan contents in the trial. Lignans are plant estrogens, which are decreasing the development of hormonal cancers. Cancer development inhibiting compound is enterolactone, which is developed from lignans in intestinal by microbial process. It appears that Laser is not as healthy as other linseed varieties. Table 1. Oil and protein content of linseed, flax and oil hemp varieties, levels of cadmium and lead, and the SDS lignan content of linseed in different years and samples

Crop Sample Variety Year Number Oil Protein Cadmium Lead SDG of samples content content content content lignan n % d. m. % d. m mg/kg mg/kg mg/kg

Linseed Farm Helmi 2007 3 41,3 0.73 0.051 7400

Helmi 2008 3 42.9 22.6 1.11 0.028 7550 mean 42.1 22.6 0.92 0.040 7470 Laser 2007 8 44.4 0.43 0.052 4990 Laser 2008 3 47.2 22.2 0.36 0.055 4840 mean 45.8 22.2 0.39 0.054 4920

Linseed Trial Helmi 2009 1 45.2 22.3 0.61 0.009 9070 Heljä 2009 1 48.7 21.7 0.61 < 0.007 8910 Laser 2009 1 48.4 18.5 0.48 0.008 5320 Abacus 2009 1 48.8 19.4 0.46 0.010 7120 Sunrise 2009 1 50.3 20.8 0.54 0.016 7690

Flax Martta 2009 1 41.4 24.8 0.57 0.007 9560 Belinka 2009 1 41.8 25.0 0.42 0.007 8190

Oil hemp Farm Finola 2005 1 34.7 0.023 0.011 2007 1 35.4 0.017 0.027

2009 2 35.8 24.6 Conclutions. Linseed and oil hemp are highly suitable dicotyledonous crops for the diet of celiac patients. There are differences in SDG lignan content of linseed varieties. Linseed absorbs a lot of cadmium from soil. The local differences in cadmium content are, however, pronounced. Small differences may exist between varieties, too, in regard to taking up cadmium from the soil. Cadmium may interfere with the efficiency of lignans as a cancer inhibiting agent. It is important to produce linseed at clean, high pH soils. Lead levels in linseed were low. Oil hemp had low cadmium and lead levels in Finland. Acknowledgement. The present work was financed by EU-European Agricultural Guidance and Guarantee Fund through the Centres for Economic Development, Transport and Environment in Finland. References Johnson MD, Kenney N, Stoica A et al. Cadmium mimics the in vivo effects of estrogen in

the uterus and mammary gland. Nat Med 2003; 9: 1081-4. McElroy JA, Shafer MM, Trentham-Dietz JM et al. Cadmium exposure and breast cancer

risk. J Nat Cancer Inst 2006; 98:869-73.

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Analysis of the Variation of Health-Promoting Compounds in Different Oat Cultivars

Ingrid M. van der Meer1 , Hetty C. van den Broeck1, Marinus J.M. Smulders1,

Jurriaan J. Mes2, Ludovicus J.W.J. Gilissen1, 1Plant Research International, Wageningen UR, PO Box 16, 6700 AA Wageningen, The

Netherlands 2Food and Biobased Research, Wageningen UR, PO Box16, 6700AA Wageningen

Wageningen, The Netherlands corresponding email: Ingrid.vanderMeer@wur.nl

Introduction. Oat has a high nutritional value and contains many health-promoting compounds compared to other cereals. It contains a higher protein level, more poly-unsaturated fatty acids and a higher level of dietary fibers compared to wheat, barley and rye. Furthermore, oat can be tolerated by most patients suffering from celiac disease (CD). Oat would not only be beneficial for CD patients to be included in the daily diet, but because of the health-promoting compounds, it would also be beneficial for all consumers (Andon and Anderson, 2008; Butt et al., 2008). In this study we analyzed the level and the variation between different cultivars of oat for several interesting high value, health-promoting compounds. Objectives. To analyze and study variation in levels of health-promoting compounds in 21 different oat cultivars, in order to select cultivars high in specific metabolites for breeding and marketing. Methods. First, genetic variation between the different cultivars was analyzed which would justify the analysis of putative differences in levels of oat metabolites. This was verified by studying total protein patterns of protein extract form grains of the different oat cultivars. Further, oat grains from a selection of these cultivars were subjected to various analytical chemical methods for the quantitative analysis of the following compounds: total saturated fatty acids, mono-unsaturated fatty acids, poly-unsaturated fatty acids, protein, starch, sugar, dietary fibres, vitamin E, polyphenols, and beta-glucans. The bioactivity of some of these compounds was analyzed for immune stimulatory and anti-oxidant activity. Results and Discussion. The different oat cultivars studied showed genetic diversity based on total protein pattern as analyzed by SDS-PAGE (Figure 1). This stimulated to further investigate putative variation in levels of health-promoting compounds. The cultivars tested did not show significant variation in the levels of ‘common’ compounds, such as starch, total protein, sugars and total fatty acids. However, we could detect significant variation in the levels of specific metabolites, such as vitamin E (varying a factor 3), mono- and poly-unsaturated fatty acids (factor 2), polyphenols, and beta-glucans. Coupled to some of these metabolites, we could also detect variation in bioactivity such as antioxidant activity and immune-modulating activity.

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Figure 1. SDS-PAGE analysis (silver stained) of proteins extracted from de-hulled grains of different oat cultivars. Table 1. Qualities of different oat cultivars. Antioxidant capacity is given as mol TE/100g. Bread quality was measured based on different parameters; ranking is given from 1 (best) to 13 (worst) bread baking quality.

Cultivar anti- oxidant

fibres %

vit E g/g

M+PUFA % of fat

bread quality

yield (t/ha)

glucan mg/g

1 1150 10 9.7 3.8 2 3.3 81 2 967 10.3 11.4 5.1 2 4.5 114 3 1053 10 11.3 5.1 1 4.7 162 4 939 10.5 9.1 6.2 2 3.9 113 5 1129 9.2 8.9 6.1 5 5.1 150 6 1243 10.8 7.3 6.6 10 3.6 192 7 1403 12.3 3.6 7.2 8 3.2 90 8 1229 11.2 9.1 6.5 8 4.0 89 9 1180 10.3 7.3 6.1 5 5.4 81 10 1463 10.3 8.7 6.9 10 5.9 87 11 1592 10.4 8.1 6.4 5 4.0 144 12 1109 10.8 8.7 6.8 12 4.3 98 13 1214 11.7 6.1 6.7 13 3.3 184

Conclusions. Different oat cultivars show variation in levels of putative health-promoting compounds, such as dietary fibers, vitamin E, polyphenols, beta-glucans and poly-unsaturated fatty acids. We are now combining these results to baking quality and agronomical characteristics of these cultivars. Knowledge on levels and variation of health-promoting compounds in oat can be used for further breeding and marketing. References Andon, M.B. and Anderson, J.W. The oatmeal-cholesterol connection: 10 years later. American J of Lifestyle Medicine (2008) 2: 51-56. Butt, M.S., Tahir-Nadeem, M., Khan, M.K.I., Shabir, R. and Butt, M.S. Oat: unique among the cereals. Eur. J. Nutr (2008) 47:68-79.

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56

A systematic literature review on the nutritional adequacy

of a typical gluten-free diet with particular reference to

iron, calcium, folate and B vitamins

Emma Merrikin1; Emily Kirk, Norma McGough, Gerry Robins

2, Anthony Akobeng

3

1 Coeliac UK, Diet and Health, High Wycombe, UK

2 York Hospitals NHS Foundation Trust, UK

3 Central Manchester and Manchester Children’s University Hospital, UK

Introduction. Coeliac disease (CD) is a life-long autoimmune disease affecting 1 percent of

the UK population.i

To date, the only effective treatment for CD is strict adherence to a

gluten-free (GF) diet, which involves eliminating the cereals wheat, rye, barley (and in some

cases oats) from the diet. GF staple products including GF bread, GF pasta and GF flour are

available in place of standard staples. There is legislation which covers the nutrient

composition of wheat flour in terms of fortification with calcium and iron, and the B vitamins

thiamin and nicotinic acid. For GF substitute products, this is not the case. The nutritional

adequacy of the GF diet is not well established. In addition, the nutritional status of those

with CD may be affected by a complex range of factors. The aim of this research project was

to carry out a systematic review to assess the evidence base on the nutritional adequacy of the

GF diet.

Methods. This systematic literature review searched a series of databases, followed by hand

searching of reference lists and a search for unpublished research. All first authors of

identified papers, key professional bodies and researchers and individual manufacturers were

contacted. A quality assessment of each paper was carried out by two independent reviewers.

Results. There were eleven papers included in this systematic review; 10 case-control studies

and 1 cohort. All papers were found to have moderate or high risk of bias. Most papers

concluded that individuals with CD following a GF diet had the same nutritional intake as the

general population. Where the results were found to differ between these groups, there was

often no values recorded to demonstrate statistical significance making it difficult to comment

on these results.

57

Table 1. Papers included in systematic review

No Author Date Title

1 Collins et al

1986 Dietary history and nutritional state in treated coeliac patients

2

Dickey et al 2008 Dickey, W. Ward, M., Whittle, C.R., Kelly, M.T., Pentieva, K., Horigan,

G., Patton, S. and McNulty, H. (2008) Homocysteine and related B vitamin

status in coeliac disease. Effects of gluten exclusion and histological

recovery. Scand J Gastro, 43(6), p682-8.

3 Grehn et al

2001 Dietary habits of Swedish adult coeliac patient treated by a gluten-free diet

for 10 years

4 Hallert et al

2002 Evidence of poor vitamin status in coeliac patients on a gluten-free diet for

10 years

5 Hopman et al 2006 Nutritional management of the gluten-free diet in young people with

celiac disease in the Netherlands

6 Kemppainen et al

1995 Intakes of nutrients and nutritional status in coeliac patients.

7 Kinsey

2007 A dietary survey to determine if patients with coeliac diease are

meeting current healthy eating guidelines an how their diet compare

to that of the British general population

8 McFarlane

1995 Subclinical nutritional deficiency in treated coeliac disease and nutritional

content of the gluten-free diet.

9

Robins et al 2008 Coeliac patients on a gluten-free diet consume a disproportionate amount

of milk intrinsic sugars

10 Storsrud

2003 Beneficial effects of oats in the gluten-free diet of adults with special

reference to nutrient status, symptoms and subjective experiences.

11 Thompson et al

2005 Gluten-free diet survey: are Americans with coeliac disease consuming

recommended amounts of fibre, iron, calcium and grain foods?

*** contact made. Author confirmed only 1 participant on GF for less

than 6 months (4 months). Supps excluded from analysis.

Conclusions. At the moment, there is not enough evidence to support fortification of GF

staple substitute products. However, there is an argument for legislation on fortification of GF

flours used in the production of GF substitute staple products so that nutritional composition

is comparable to gluten-containing staple products. More robust research is required,

ensuring adequate sample sizes, to investigate further the nutritional adequacy of the GF diet

in people medically diagnosed with CD. There is no conclusive evidence to suggest that

nutritional deficiency is a significant problem in individuals diagnosed with CD, established

on a GF diet. However, these conclusions may reflect the paucity of data, rather than a

genuine absence of nutritional deficiencies in people following a GF diet.

References

i. West J, Logan RFA, Hill PG, Lloyd A, Lewis, S, Hubbard, R. Seroprevalence, correlates

and characteristics of undetected coeliac disease in England. Gut. 2003 52: 960-965.

58

Tartary Buckwheat as a Gluten Free Source for Functional Food

Mateja Germ1; Ivan Kreft2*

1University of Ljubljana, Biotechnical Faculty, Department of Biology, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

2University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Večna pot 111, SI-1000 Ljubljana, Slovenia

*corresponding email: ivan.kreft@guest.arnes.si Introduction. Buckwheat is a pseudocereal, its grain are according to De Francischi et al. (1994) safe as a gluten-free food source. Two buckwheat species with edible grain are known, common buckwheat (Fagopyrum esculentum Moench) and tartary buckwheat (Fagopyrum tataricum Gaertn.). Tartary buckwheat has been widely grown as a crop in Europe since the beginning of the 19th century. Compared with common buckwheat and other crops, it is more resistant to conditions at high altitude (up to about 1,200 m in Slovenia). Additionally, it is resistant to grazing by wild or domestic animals, and to limiting soil and weather conditions. Because tartary buckwheat is an undemanding crop, it can be grown in climate change conditions. Growing areas of common and tartary buckwheat started to decrease in Europe in the middle of 20th century, as cereals and other more yield intensive crops covered more fields. Tartary buckwheat is now grown in Europe mainly in Luxemburg, and outside Europe around the Himalayas and in China. Recently, it was found that the somewhat bitter taste of tartary buckwheat grain is due to the high concentration (even in comparison to common buckwheat) of polyphenols, and the especially the high concentration of rutin. Tartary buckwheat contains relatively high amounts of fibres, vitamins B1, B2 and B6, and proteins with a balanced amino acid composition and high biological value (Bonafaccia et al. 2003). Recently, as a result of the interest in environmentally friendly plant-growing, functional foods and the market demand for special food products (including products for patients with celiac disease, and products rich in anti-oxidants and fibre), interest in growing common and tartary buckwheat, and for producing and consuming their products, has been revived. Buckwheat (tartary and common) is appropriate for production of many food products with high nutritional value. Dehusked grains (named ‘kasha’) are traditionally used in Slovenian cousine (and many others like Italian, Polish, Chinese, Japanese) for making dishes with vegetables or meat, as well as for soups, sausages and desserts. Different types of dough are prepared out of buckwheat flour used for sweet and main dishes. Pap, or in Slovenian, močnik, is a soft floury dish traditionally made from buckwheat flour. Močnik was traditionally perceived as a food of the poor. Yet milky buckwheat močnik has an attractive smell, its taste is good and pleasing. As buckwheat is believed to keep the body warm, milky buckwheat močnik can make a delicious meal on long winter evenings. Though rarely used, buckwheat bread is a good every day source of nutritionally important components. It can be produced out of buckwheat flour solely when using without any admixture with wheat or other cereal flour, when suitable technique is adopted (Kreft 2007). Interest in the cultivation, consumption and research of tartary buckwheat is increasing internationally and novel products have started to appear. The present work investigated tartary buckwheat as a functional food source for gluten free diets.

59

Methods. Buckwheat samples, tartary buckwheat from Luxemburg and common buckwheat Siva from Slovenia, milled in a traditional stone mill; milling yields: flour 56%, bran 25% (% dry-weight basis) were used. Samples (250 mg), were extracted with 5 ml methanol/water (67:33) at room temperature, by shaking for 40 min. HPLC was performed using a Spectra-Physics (Mountain View, California, USA) instrument Spectra System P4000, Hibar – LiChrospher 100, RP-18 column (E. Merck, Armstadt, Germany, 250 mm · 4 mm). The solvents for HPLC were A acetonitrile and methanol (1:2), and B 0.75% aq. H3PO4. Initial condition was 100% B. The samples were run on a linear gradient to 60% A and 40% B in 20 min; and then a linear gradient to 100% A and 0% B for a further 20 min, and finally 10 min equilibration (100% B). The compounds were detected at 380 nm and identified by comparison of the retention time with the retention time of the standard solutions. All analyses were performed in triplicate in three independent samples. The data were analyzed statistically using STATG (Statgraphics 5.0, Statistical Graphics Corporation, USA).

Results. Tartary buckwheat milling fractions have much higher concentration of rutin in comparison to common buckwheat (Table 1) , additionally to high concentration of dietary fiber.

Table 1. Rutin and dietary fibre concentration in milling products of tartary buckwheat (domestic variety from Luxemburg) and common buckwheat Siva. Data for dietary fibre are adapted from Bonafaccia et al. (2003).

Rutin Dietary fibre (% dry weight) mg/g total soluble insoluble % sol. Bran of tartary buckwheat 17.61±0.20 24.8 1.2 23.6 4.77 Bran of common buckwheat 0.280±0.015 26.4 0.9 25.5 3.45 Flour of tartary buckwheat 11.82±0.24 6.3 0.5 5.8 8.27 Flour of common buckwheat 0.225±0.014 6.8 0.9 5.9 12.99

Conclusion. This research showed that main tartary buckwheat grain milling products are not only basis for gluten-free cereal-like food, but they, especially tartary buckwheat bran, are due to their high concentration of rutin and dietary fiber, valuable functional food materials, which are basis of diversification of diets for celiac patients. References

Bonafaccia G, Marocchini M, Kreft I. Composition and technological properties of the flour

and bran from common and tartary buckwheat. Food Chem. 2003;80:9-15. De Francischi MLP, Salgado JM, Da Costa CP. Immunological analysis of serum for

buckwheat fed celiac patients. Plant Foods Human Nutr 1994;46:207-211. Kreft I. Nä hrwert des Buchweizens: Grü tze - Mehl - Brot - Gemü se - Tee - Essig - Honig. In:

Kreft I, Ries C, Zewen C, editors: Das Buchweizen Buch: mit Rezepten aus aller Welt. 2. ü berarbeitete und erweiterte Aufl. Arzfeld: Islek ohne Grenzen EWIV; 2007. p. 92-104.

60

Teff (Eragrostis tef) Supplemented Gluten-Free Breads as a Potential Prevention of Iron-Deficiency Anaemia

Ebtesam Ben – Fayed, Valentina Stojceska* and Paul Ainsworth The Manchester Metropolitan University, Department of Food and Tourism Management,

Hollings Faculty, Old Hall Lane, Manchester, M14 6HR, UK.

*corresponding email: V.Stojceska@mmu.ac.uk

Introduction. Coeliac disease (CD) is a genetically based autoimmune enteropathy caused by a permanent sensitivity to gluten (Hamer et al, 2005) and very common food intolerance in the UK population (Coeliac, UK). Among common deficiencies associated with a gluten-free diet is iron deficiency occurring in 33% of men and 19% of women probably due to the predominant site of mucosal damage (Harper et al., 2007). Teff is a little-known gluten-free cereal grain traditionally grown in Africa with a rich source of bioavailable iron which may be attributed to its low phytate content (Umeta et al., 2005). Bread made with Tef enjera contains around 30mg of iron per 100g and up to 35mg when the food is fermented. The prevalence of iron deficiency anaemia is relatively low in Ethiopia which may be attributed to Eragrostis tef forming a staple part of the diet.

The objective of this work was to develop gluten-free breads rich with iron by incorporating Teff flour and to study the textural and sensory characteristics of the finished products.

Methods. The new GF recipe using wheat starch, skimmed milk powder, glucono-delta-lactone, sodium bicarbonate, soy flour, xanthan gum, egg whites and bread improver was developed and used as a control. Teff flour was added at the levels 10 and 20% and its effect on loaf volume (rapeseed replacement method), hardness (TA – XT2 texture analyser (Stable Micro Systems Ltd, Godalming, UK)), shelf life over eight days, bread structure were studied. Five panellists from Manchester Coeliac Association, UK were asked to assess the control, 10 and 20% Teff breads in the terms of flavour, uniformity, mouth feel, moistness, aftertaste, and to mark 10 cm line in accordance with their opinion.

Results. The results revealed that increasing the Teff flour decreased the loaf volume (Figure 1A, B). There was a significant (P<0.05) increase in staling for all the breads during the six but not at seven and eight days (Figure 1C). Compared to control and 10% Teff breads, 20% Teff breads showed significant (P<0.05) increase in staling while 10% Teff breads were comparable to control ones. The overall acceptability gave a mean score of 8.8 for control, 8.72 for 10% Teff and 7.7% for 20% Teff breads (Figure 1D). The addition of 20% Teff flour increased the level of iron for 45% comparing to the control sample.

61

Figure 1. Some textural characteristics (loaf volume (B), shelf life (C) and sensory evaluation(D)) of gluten-free Teff breads made with 0, 10 and 20% Teff flour (A)

Conclusion: Iron-deficiency anaemia is a very common symptom of coeliac disease. Teff (Eragrostis tef) is a gluten-free cereal rich with iron. New Teff supplemented gluten-free breads with improved dietary iron level have been developed in this study. The results revealed that up to 20% Teff flour could be incorporated in breads formulation resulting with the good texture and structure of baked breads.

References. Hamer., R.J. (2005) Coeliac disease: background and biochemical aspects, Biotechnology Advances 23 (6), pp. 401–408. Umeta M, West CE and Fufa H. (2005). Content of zinc, iron, calcium and their absorption inhibitors in foods commonly consumed in Ethiopia. Journal of Food Composition and Analysis 18, 803-817. Harper JW, Holleran SF, Ramakrishnan R, Bhagat G, Green PH. 2007. Anemia in celiac disease is multifactorial in etiology. American Journal of Hematology, 996-1000.

62

Oat -glucan affects the viscoelastic properties of gastric mucin at pH conditions of small intestine

Reetta Kivelä1*, Sami Hietala2, Tuula Sontag-Strohm1, Bradley Turner3, Rama Bansil4

1 University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland

2 University of Helsinki, Department of Chemistry (polymer chemistry), Helsinki, Finland 3Beth Israel Deaconess Medical Center and Harvard Medical School, Division of

Gastroenterology, Boston, Massachusetts, USA 4Boston University, Department of Physics, Boston, Massachusetts, USA

*corresponding email: reetta.kivela@helsinki.fi

Introduction. For people with celiac disease, pure oats can provide a beneficial source of dietary fibre that often lack from their diet. The most important fibre of oat is the water-soluble (1 3), (1 4) - -D-glucan, usually referred to as -glucan. Native oat -glucan has a high molar mass (1-3x106g/mol) partially due to its aggregative nature in aqueous matrix, and it forms highly viscous water solutions (Lazaridou and Biliaderis, 2007). The viscous behaviour is closely related to the health benefits of -glucan. The viscous layer in the intestine has been suggested to slow down the absorption of low molecular weight compounds, such as glucose and bile acids, thus balancing the insulin and cholesterol metabolisms. However, low molecular weight -glucan is also nutritionally beneficial (Naumann et al., 2006). As -glucan is extracted in stomach from the food matrix, it will meet mucin, which is a glycoprotein of gastrointestinal mucus covering the whole digestive tract. Mucin occurs as a solution at pH 6 i.e. in the conditions of small intestine. In stomach, during digestion and resting (pH 2 and 4), mucin occurs as a gel. This behaviour has been suggested to be based on the breakage of salt bridges of the protein in mucin at low pH, and has in the function to protect the digestive tract in the large pH gradient and shear stresses during digestion (Celli et al., 2007). Materials. Mucin was purified from pig mucus in Boston University. The unique purification method enabled to study the non-degraded mucin, which differs from commercial mucin materials (Celli et al., 2007). Solutions of 1.4% of mucin in buffers with pH 2 and 6 were prepared. Native -glucan was extracted (30 min, 40 C) from an oat bran concentrate (Oat Well 14%, Swedish Oat Fibre, Våröbacka, Sweden) and a crude beta-glucan extract, containing 40% beta-glucan (MW 1.4x106g/mol) and 15% of proteins of its dry matter, was used as a sample solution. Purified oat -glucan (98% beta-glucan, MW 0.35x106g/mol) was purchased from Megazyme International and 1% solution was prepared by wetting -glucan with 99% ethanol and hydrating it for 3 hours at 80 C. The solution of low molar mass beta-glucan is called here LMM-BG. Mucin- -glucan solutions were prepared by shaking the solutions gently 15 hours at room temperature and the pH was controlled to be in the range of pH6±0.2 and pH2±0.2. Methods. The rheological measurements were performed with cone and plate geometry (35mm/2° and 40 mm/2°, respectively) at 20 C. For the oscillatory frequency sweep tests, value of applied stress was chosen based on a stress sweep at a constant angular frequency (6.3 rad/s) within the linear regime. Flow properties of mucin and mucin- -glucan were determined as steady shear flow with the TA instrument rheometer. Flow curves of different beta-glucans were determined with the ThermoHaake rheometer with shear rate range of 0.03-300-0.03 1/s.

63

Results. As expected, mucin behaved as a gel at pH 2 and as a viscoelastic liquid at pH 6. As the beta-glucan solutions were combined with mucin solution, the gelling properties were affected moderately at pH 2 and more significantly at pH 6. At pH 2, low molar mass beta-glucan (LMM BG) apparently weakened the mucin gel at low frequencies, but however slightly strengthened the viscous part of the viscoelastic mucin gel at higher frequencies (f>1rad/s). Native beta-glucan had similar effect at pH 2, but increased the viscous modulus at low frequencies as well. At pH 6, purified mucin showed gel-like behavior at high frequencies (f>1rad/s) but when combined with LMM beta-glucan, the interactions of the system were disrupted and it behaved as a liquid at the whole frequency range studied (0.05-100 rad/s). Vice versa, the addition of native beta-glucan strengthened the system and it behaved as a weak soft gel through the whole frequency range. The similar effect was obtained in the flow properties: both the beta-glucan solutions could increase the viscosity at pH 2, but only native beta-glucan could enhance the viscosity at pH 6 (Fig. 1).

Figure 1. A steady shear flow curve of mucin and mucin combined with two different beta-glucan solutions at pH 2 and pH 6. The mucin consentration was constant and the initial viscosity of beta-glucan similar (0.050±0.0010 Pa·s at 10 1/s) in all the experiments. Conclusions. At the pH conditions of small intestine (pH 6), beta-glucan associated with mucin and strengthened its gel-like behaviour. However, molecular or solution properties of beta-glucan clearly affected the association so that native and unprocessed high molar mass beta-glucan strengthened, whereas low molar mass, highly purified beta-glucan actually weakened the interactions of mucin. At pH condition of stomach (pH 2), where mucin itself interacts more strongly, addition of beta-glucan affected only moderately. These findings open new views to understand the differences and protective effects of dietary fibres such as beta-glucan in intestine. References Celli, J.P.,Turner, B.S., Nezam H. Afdhal N.H., Ewoldt, R.H., McKinley, G.H., Bansil R. and Erramilli, S. (2007).

Rheology of Gastric Mucin Exhibits a pH-Dependent Sol-Gel Transition. Biomacromolecules Lazaridou, A. and Biliaderis, C. G. (2007). Molecular aspects of cereal -glucan functionality: Physical properties,

technological applications and physiological effects. J. Cereal Sci. 46, 101-118. Naumann, E., van Rees, A. B., Onning, G., Oste, R., Wydra, M. and Mensink, R. P. (2006). -Glucan incorporated

into a fruit drink effectively lowers serum LDL-cholesterol concentrations. Am J Clin Nutr 83, 601-605.

mucin

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64

Prolylendoproteases for Gluten Detoxification Frits Koning

Department of IHB, LUMC, Leiden, The Netherlands corresponding email: f.koning@lumc.nl

Celiac disease (CD) is an intestinal disorder caused by intolerance to gluten, proteins present in wheat and related cereals. CD only develops in individuals with a certain genetic background: they express HLA-DQ2 and/or DQ8 molecules. Recent advances have revealed the molecular basis for the association between CD and HLA-DQ2/DQ8. Due to enzymatic degradation and modification of gluten proteins in the gastrointestinal tract gluten peptides are generated that can bind to HLA-DQ2/8 with high affinity and subsequently trigger inflammatory T cell responses. These T cell responses lead to the typical symptoms associated with CD: malnutrition, diarrhea and osteoporosis. Consequently, the inflammation and associated symptoms disappear when gluten in removed from the diet. However, symptoms quickly re-emerge when gluten is introduced again. A lifelong gluten-free diet is therefore the standard treatment for CD but due to the widespread use of gluten in the food industry such a diet is hard to adhere to. There is therefore a presently unmet need for an alternative treatment. Gluten is a complex mixture of gliadin and glutenin proteins. We have identified and characterized a series of gliadin and glutenin peptides that are involved in the disease process. These peptides are characteristically glutamine- and proline-rich and due to the presence of proline they are resistant to degradation in the gastrointestinal tract, a property that is linked to their disease-inducing properties. It has therefore been suggested that prolyl oligopeptidase might be exploited to accelerate the degradation of the proline-rich gluten molecules in the gastrointestinal tract. Indeed, such enzymes have been shown to effectively degrade gluten peptides. In addition the generation of smaller gluten fragments improves the subsequent degradation of gluten peptides by brush border enzymes. However, the gluten proteins and peptides must be degraded before they reach the small intestine and the prolyl oligopeptidases investigated were not active under the conditions found in the stomach. These enzymes are thus not suitable for oral supplementation as an alternative treatment for CD. Recently, we have described a prolyl endoprotease from Aspergillus niger (AN-PEP). This enzyme was found to efficiently degrade gluten peptides and intact gluten proteins. Moreover, the pH optimum of the enzyme is compatible with that found in the stomach while the enzyme itself is resistant to degradation by pepsin. To test if the enzyme might be suitable for in vivo degradation of gluten we have tested the efficiency of gluten degradation under near in vivo conditions with the use of a dynamic, multicompartimental in vitro system mimicking the conditions in the human gastrointestinal tract. The result of these studies demonstrate that within two hours AN-PEP was capable of degrading all T cell stimulatory epitopes of gluten present in a complex meal. Co-administration of AN-PEP with a gluten containing meal thus appears a feasible approach to detoxify gluten before it can do harm in the small intestine of CD patients. A phase I clinical trial conducted in 2009 has demonstrated that the enzyme is safe for oral administration in humans. To determine efficacy, a phase II trial is in preparation.

65

Gluten analysis on surfaces and in food

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66

Notes

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Many faces of prolyl oligopeptidase – What we ignore in mammals and what we know in plants

J. Arturo García-Horsman

University of Helsinki, Division of Pharmacology and Toxicology, Helsinki, Finland email: Arturo.Garcia@helsinki.fi

Prolyl oligopeptidase (POP) is a very well conserved protease found in bacteria to human. POP cleaves at the C-side of proline with high specificity (Garcia-Horsman et al, 2007). While in lower prokaryotes it is able to digest long peptides, in higher eukaryotes it has evolved to have specificity only for short peptides under around 30 amino acids. In bacteria and protozoa, POP is secreted and it has been implicated the process of infection by degrading host surface proline rich proteins, otherwise resistant to most of the other proteases. Although POP like activity has been detected in all biological fluids in mammals, POP is considered cytoplasmic protein also found, under certain circumstances, associated to membranes. POP has been considered to participate in the metabolism of neuroactive peptides and hormones, and thus it has been assigned a role in central nervous system processes like memory and learning. Moreover, POP inhibitors have been suggested as protective from neuronal damage and apoptosis. However, despite the large research conducted mainly about pharmacology of inhibitors, but also about POP role in several biological processes, we ignore almost completely the physiological role of this peptidase and its implication in disease (Garcia-Horsman et al, 2007). We have studied POP in mammalian systems aiming to answer basic questions in order to suggest a function for this peptidase. Although considered a house-keeping enzyme, expressed in all tissues, we have established that POP expression varies from tissue to tissue and among different cells of the same tissue (Myöhänen et al 2009). We also have described, that POP expression is regulated during cell development, maturation and aging, and that its cell location responds to the status of the cell cycle (Moreno-Baylach, 2008). We have done efforts to identify the physiological substrates of POP, as well as to determine the biological processes associated to its activity, by genomic analysis. We have found that POP is implicated in angiogenesis, cancer and senile plaque deposition. With this information, along with the research carried out in other labs, we suggest that POP has a possible role in proliferation, neurogenesis, inflammation, and mitochondrial function through still ignored molecular mechanisms (Fig. 1.). Plant prolyl oligopeptidase has been scarcely studied. It has been isolated from carrot and several genes have been described in few plant species as Arabidopsis, Linum usitatissimum, Daucus carota, and spinach. Recent studies suggest that POP might be implicated in seed development by controlling plant signalling peptides as leginsulin (Gutierrez et al 2008). We have detected POP activity in ray malt in consistency with its role in seed development (Fig. 2.). A considerable amount of research on plant POP is waiting to be done in the future.

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References Gutierrez L, Castelain M, Verdeil JL, Conejero G, Van Wuytswinkel O. A possible role of

prolyl oligopeptidase during Linum usitatissimum (flax) seed development. Plant Biol (Stuttg). 2008 May;10(3):398-402.

Myohanen TT, Garcia-Horsman JA, Tenorio-Laranga J, Mannisto PT. Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among mRNA, protein levels, and enzymatic activity. J Histochem Cytochem 2009; 57: 831-848.

Moreno-Baylach MJ, Felipo V, Mannisto PT, Garcia-Horsman JA. Expression and traffic of cellular prolyl oligopeptidase are regulated during cerebellar granule cell differentiation, maturation, and aging. Neuroscience 2008; 156: 580-585.

Fig. 1. Schematic representation of the brain and sites where POP might have a function.

Fig. 2. Localization of POP transcripts (Lu04072) by in situ hybridisation on 15 DAF flax embryo sections. A purple signal, indicating the Lu04072 transcripts, is localised in the whole embryo. A stronger hybridisation signal is observed at the root cap and cotyledon levels, with a higher intensity toward the top end and the adaxial side of the cotyledon. (taken from Gutierrez et al 2008).

BBB damage

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Endogenous cereal enzymes in the elimination of prolamins

Jussi Loponen1*, Päivi Kanerva1, Michael Gänzle2, Tuula Sontag-Strohm1, Hannu Salovaara1

1University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland 2University of Alberta, Department of Agricultural, Food & Nutritional Science, Edmonton, Canada

*corresponding email: jussi.loponen@helsinki.fi Prolamins of wheat, barley, and rye are the primary triggers of celiac disease (CD). They also are the major storage proteins of cereals, which during the seed germination are hydrolyzed from compact structures into free amino acids by germination-induced cereal peptidases. In other words the prolamins are the natural substrates for endogenous cereal enzymes.

In wheat and barley, cysteine endopeptidases are the predominant peptidase group that hydrolyse prolamins (Jones 2005). In rye, the aspartic endopeptidases are equally dominant with cysteine endopeptidases (Brijs et al 2002) which differentiates rye from wheat and barley. In addition, an arsenal of other endopeptidases and, especially, (partially) proline-specific serine carboxypeptidases play a significant role in the prolamin hydrolysis that takes place in the nature. A pivotal feature is that the most dominant peptidases operate at low pH (Table 1).

Table 1. The predominant endogenous cereal peptidases and their pH-optima

Peptidase class pH optimum

Aspartic endopeptidases 3.5

Cysteine endopeptidases 4

Serine carboxypeptidases 4.5

The hydrolysis of prolamins is a potential way to eliminate gluten. An approach relying on a papaya extract was introduced already decades ago (Messer et al 1964) but more recently new approaches have emerged. Two main philosophies to commit the elimination are the medicinal approach where gluten elimination takes place after ingestion in stomach and the food-technological approach where the gluten elimination occurs during food processing. It has been noted that an efficient hydrolysis of prolamins requires enzymes that can hydrolyse proline-rich fragments of prolamins. A proline oligopeptidase and a proline endopeptidase, both of microbial origin, were shown to cleave toxic prolamin structures (Shan et al 2004, Stepniak et al 2006). The endogenous cereal enzymes have also paid attention, and a recombinant barley cysteine endopeptidase and a natural pool of germination-induced cereal enzymes hydrolyzed prolamin fragments as well (Bethune et al 2006, Hartmann et al 2006).

We have developed a food technological approach to eliminate prolamins. When germinated grains of wheat and rye were used as a raw material in sourdoughs, and the doughs acidified with lactobacilli or chemically, the extent of prolamin hydrolysis in wheat and rye systems were 95% and 99.5%, respectively (Loponen et al 2007, 2009). This means that the prolamin hydrolysis was so extensive that the immunoassays using a R5 antibody (neither sandwich nor competitive) were unable to detect any prolamins. This strongly indicates that the prolamins had lost their immunoreactivity, because the competitive assay accurately detected a 9-mer

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prolamin peptide (unpublished). In addition, the SE-HPLC analysis of proteins and peptides revealed that the hydrolysis products were smaller than the 9-mer peptide. Germinated rye seemed to be the most potent candidate for the elimination of prolamins in acidic malt suspensions compared to wheat and barley malt (Figure 1 and 2).

Figure 1. SE-HPLC analysis of total proteins (prolamins cover roughly 50% of total) extracted from acidic suspensions of wheat, barley, and rye malts. Samples were taken in the beginning, after 6-hr, and after 24-hr fermentation. The elution of marker prolamin peptides (33-mer, 19-mer, 9-mer) indicated in up-down arrows.

Figure 2. Prolamin contents of malt suspension that were incubated under acidic conditions for 24-hr. References Jones BL 2005. Endoproteases of barley and malt. J. Cereal Sci. 42:139-156. Brijs K., Trogh I., Jones BL. Delcour JA 2002 Proteolytic enzymes in germinating rye grains. Cereal Chem. 79:423-428. Messer M, Anderson CM and Hubbard L 1964. Studies on the mechanism of destruction of toxic action of wheat gluten in celiac disease by crude papain. Gut. 5:295-303. Shan L., Molberg Ø., Parrot I., Hausch F., Filiz F., Gray G.M., Sollid L.M. and Khosla C. 2002. Structural basis for gluten intolerance in celiac sprue. Science. 297:2275–2279. Stepniak D., Spaenij-Dekking L., Mitea C., Moester M., de Ru A., Baak-Pablo R., van Veelen P., Edens L. and Koning F. 2006. Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease. Am. J. Physiol. Gastrointest. Liver Physiol. 291:621-629. Bethune M.T., Strop P., Tang Y., Sollid L.M. and Khosla C. 2006. Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease. Chem. Biol. 13:637-647. Hartmann G., Koehler P. and Wieser H. 2006. Rapid degradation of gliadin peptides toxic for coeliac disease patients by proteases from germinating cereals. J. Cereal Sci. 44:368-371. Loponen J., Sontag-Strohm T., Venäläinen J., Salovaara H. 2007. Prolamin hydrolysis in wheat sourdoughs with differing proteolytic activities. Journal of Agricultural and Food Chemistry, 55, 978–984 Loponen J, Kanerva P, Zhang C, Sontag-Strohm T, Salovaara H, Gänzle M.G. 2009. Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods. J Agric Food Chem, 57, 746–753

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Synthetic Blocking Peptides with High Affinity to Gliadin Reduce Tissue Transglutaminase Activity on Wheat

Gliadin in Vitro

Karolina Hoffmann1*, Marie Alminger1, Thomas Andlid1, Tingsu Chen2, Olof Olsson3, Ann-Sofie Sandberg1

1Chalmers University of Technology, Department of Chemical and Biological Engineering, Food Science, Gothenburg, Sweden

2Guangxi Academy of Agricultural Sciences, Microbiology Institute, Nanning, Guangxi, China

3Gothenburg University, Department of Cell and Molecular Biology, Gothenburg, Sweden

*corresponding email: karolina.hoffmann@chalmers.se

Introduction: The enzyme tissue transglutaminase (tTG) plays an important role in celiac disease development as it is involved in generation of potent epitopes from gluten peptides which leads to an increased stimulation of CD4+ T-cells and triggers the immune responses that result in the intestinal inflammation (Fleckenstein et al. 2002). In this work we investigated whether tTG-catalyzed modification of gliadin could be reduced in vitro by synthetic blocking peptides selected for high affinity to gliadin. Methods: Gliadin was extracted from wheat gluten. Gliadin binding peptides were selected with phage display technique from a 12-Phage Display Peptide Library kit and synthesized with >95% purity. Three blocking peptides denoted P61, P64, and P22 respectively, a peptide pool (P61, P64, P22 in1:1:1:1 weight ratio), and a control peptide without affinity to gliadin PC31, were used in the experiment (Table 1). Transglutaminase activity assay with guinea pig liver transglutaminase according to Skovbjerg (Skovbjerg et al. 2002) in which time resolved fluorescence of Europium is measured was performed with gliadin as a coated substrate for tTG, in the presence and absence of the blocking peptides and the control peptide. Table 1. One-letter amino acid code sequences of gliadin blocking peptides used in the tTG experiment.

Peptide Sequence

P61

P64

WHWRNPDFWYLK

WHWTWLSEYPPP

P22 LETSKLPPPAFL

PC31 AYYPQNHKSNAE

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Results: Blocking peptides significantly reduced the tTG activity detected as reduced ability to integrate a biotinylated substrate with coated gliadin. The tTG activity reduction was ~36% for P22, ~33% for P64, ~31.4% for P61 and ~30% for the peptide pool as compared to tTG activity in the absence of blocking peptides (Table 2). The control peptide PC31 did not cause any significant changes in tTG activity which demonstrated the absence of unspecific blocking (Hoffmann et al. 2009). Table 2. Tissue transglutaminase (tTG) activity reduction [%] in the presence of blocking peptides P61, P64, P22, and a peptide pool.

Peptide Reduction of tTG activity [%]

P61

31.4 ±17.3 (P<0.001)

P64

32.82±14.3 (P<0.001)

P22

36.21±12.8 (P<0.001)

Pool

29.95±17.6 (P<0.004)

Conclusions: This work showed that blocking peptides may reduce the tTG processing of gliadin in vitro. Such blocking peptides have a potential for gluten detoxification and could be evaluated as additives in designing new food products with low gluten amount in the future, provided that peptide complexes with gliadin are stable during the passage through the GI tract and that the blocking works in real food systems. References: Fleckenstein B, Molberg O, Qiao SW, Schmid DG, von der Mulbe F, Elgstoen K, Sollid LM. Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 2002; 277:34109-16. Hoffmann K, Alminger M, Andlid T, Chen T, Olsson O, Sandberg A-S. Blocking peptides decrease tissue transglutaminase processing of gliadin in vitro. J Agric Food Chem 2009; 57:10150-10155. Skovbjerg H, Noren O, Anthonsen D, Moller J, Sjostrom H. Gliadin is a good substrate of several transglutaminases: possible implication in the pathogenesis of coeliac disease. Scand J Gastroenterol 2002; 37:812-817.

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Degradation of gliadin peptides toxic for coeliac disease patients by prolyl endopeptidase synthesized by

Lactobacillus acidophilus 5e2 and Aspergillus niger

Bartosz Brzozowski1*, Włodzimierz Bednarski1, Barbara Wróblewska

2

1 Department Of Food Biotechnology, University Of Warmia And Mazury, Olsztyn, Poland 2 Department of Immunology and Food Allergens, Institute Of Animal Reproduction And

Food Research, Polish Academy of Science, Olsztyn, Poland *corresponding email: bartosz.brzozowski@uwm.edu.pl

Introduction. Prolyl endopeptidases (PEP, EC 3.4.21.26) are a family of proteases with the unique ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue. Although these enzymes are expressed in several mammalian tissues, their absence from gastric or pancreatic secretions, or from the intestinal brush border membrane, highlights the lack of a role for PEP activity in the assimilation of dietary proteins in mammals. The immunogenic gliadin peptides, which are rich in proline residue, can be readily cleaved by bacterial PEPs suggesting a strategy for detoxifying gluten. The aim of this work was application of prolyl endopeptidases synthesized by Lb. acidophilus 5e2 (LB PEP) and A. niger (AN PEP) to hydrolysis wheat prolamins and immunoreactive synthetic (31-43)-gliadin. Methods. Prolamins were isolated from commercially available wheat flour from Młyny

Szczepanki Sp. z o.o. (Łasin, Poland) using 60% ethanol after removing albumins and globulins. The synthetic α(31-43)-gliadin with sequence LGQQQPFPPQQPY were from JPT Peptide Technologies GmbH (Berlin, Germany). The enzyme LB PEP was obtained from biomass of Lb. acidophilus 5e2 after ultrasonication (Sonics Vibre Cell, Meryin, Switzerland) and ultrafiltration (5kDa and 100kDa cut-off membranes, Pellicon XL, Millipore Sp. z o.o., Poland). The enzyme AN PEP was from DSM Food Specialties (Warszawa, Poland). Both enzymes were analyzed for resistance for pepsine digestion (10 mM HCl, pH 2.0, 60 min, 37 ) and next for trypsin digestion (65,7 mM Na2HPO4, 1 M NaOH, pH 7.8, 120 min, 37 ). The activity of PEPs was expressed as µmol of pNa released from Z-Gly-Pro-pNa after 60 min, at 37 in pH 7.0. The LB PEP and AN PEP were used to hydrolysis gliadins and immunoreactive peptide α(31-43)-gliadin at 30 and 37 in pH 4.0 and pH 6.0. Hydrolysates obtained from gliadins ware separated by free zone capillary electrophoresis (BioRad). The contents of toxic peptide with sequence QQPFP were analyzed by competitive ELISA (Ridascreen, Darmstadt, Germany) in peptides released from gliadin. Results. The in vitro digestion of intracellular prolyl endopeptidase synthesized by Lb. acidophilus 5e2 demonstrated that the enzyme was inhibited in simulating gut-conditions. Results obtained showed that the LB PEP demonstrated 11% of initial activity after treatment by pepsin in acidic conditions (pH 2.0) and trypsin in basic conditions (pH 7.8). Both enzymes were able to hydrolyze gliadin. The highest hydrolysis ratios of gliadin were obtained in pH 4.0 at 30 and 37 for AN PEP. Whereas, gliadin degradation performed with use LB PEP showed the highest hydrolysis ratio in pH 6.0 at 30 . The content of epitopes reacted with R5 antibodies which recognize sequence QQPFP was influenced by the source of PEP, pH and temperature. The lowest content of toxic peptide (11,4 µg/mL) was

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obtained after gliadin treatment by LB PEP in pH 6.0 at 37 (Table 1). There wasn't any correlation between gliadin hydrolysis ratio and content of toxic peptide. Both enzymes hydrolyzed (31-43)-gliadin peptide. The highest hydrolysis ratio of (31-43) peptide was obtained in pH 4.0 at 37 for LB PEP (Figure 1). Table 1. The contents of toxic peptide with sequence QQPFP in gliadin hydrolyzed by LB PEP and AN PEP. The means followed by different upper-case letters are significantly

different (P<0.05, ANOVA, Benferroni test).

Hydrolysis conditions Peptide contents

(µg/mL) Temp.(°C) Acidity (pH)

Control - 6.0 27,3±0,43 C,D

LB PEP 30 6.0 26,5±0,59 D

37 6.0 14,0±0,46 F

AN PEP 30 6.0 29,8±0,26 A

37 6.0 28,2±0,56 B,C

Control - 4.0 26,5±0,38 D

LB PEP 30 4.0 16,0±0,20 E

37 4.0 11,4±0,09 G

AN PEP 30 4.0 30,5±0,10 A

37 4.0 30,4±0,04 A

Figure 1. Peptide (31-43)-gliadin electropherogram after 0, 1, 2 and 3h hydrolysis with LB PEP at 37 in pH 4.0. Conclusions. This study showed that the prolyl endopeptidases obtained from A. niger is more resistance for pepsine/trypsin digestion, then Lb. acidophilus 5e2 ones. Both enzymes were able to hydrolyze gliadin decreasing their immunoreactivity and to degrade (31-43)-gliadin peptide, toxic for coeliac patients. Acknowledgments. This study was financially supported by the Ministry of Science and Higher Education, from Research Project no. N312 066 31/3701.

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Detoxification of gluten by germinating cereal enzymes: Implications for new treat ment of c oeliac disease

Satumarja Stenman1, Katri Lindfors1, Jarkko I Venäläinen2, Anne Hautala1, Pekka T

Männistö3, Anu Kaukovirta-Norja 4, Shuo-Wang Qiao5, Ludviq M Sollid5, Markku Mäki1,6 and Katri Kaukinen1,7

1Medical School, University of Tampere, Finland

2Department of Pharmacology and Toxicology, University of Kuopio, Finland

3Division of Pharmacology and Toxicology, University of Helsinki, Finland 4Technical Research Centre of Finland, Espoo, Finland

5Rikshospitalet University Hospital and University of Oslo, Norway 6Department of Pediatrics, Tampere University Hospital, Finland

7Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Finland

Introduction. The unique composition of cereal prolamins in wheat, barley and rye renders them resistant to gastrointestinal proteolytic enzymes. This is due mainly to a high content of glutamine and proline residues which leads to incomplete degradation of these proteins during normal human digestion (Shan et al. 2002). Such partial degradation is thought to be one crucial factor in the activation of the immune response in the small-bowel mucosa and the progression of coeliac disease (CD) in genetically susceptible persons. Currently the only treatment for coeliac disease is avoidance of gluten prolamins (gliadin, hordein and secalin). However, there exist several overwhelming problems with the restricted diet, thus alternative treatment strategies are warranted. Enzyme supplements have been proposed as a novel treatment for the disease in order to accelerate the complete breakdown of gluten epitopes in the gut in advance of their absorption in the small-bowel mucosa (Shan 2002). Gluten prolamins are storage proteins which provide nitrogenous nutrients to grains serving major support for the growth of a young seedling in a process termed germination. During the germination these prolamins are cleaved into single amino acids by a variety of grain own enzymes, evolutionarily selected for total degradation of the cereal storage proteins (Shewry 1995). Objectives. In the current study, we investigated the efficacy of germinating cereal enzymes to hydrolyze wheat gliadin and rye secalin into short fragments and whether gluten-induced harmful effects in coeliac disease in vitro can be reduced by such pre-treatment. Methods. Efficacy of germinating oat, wheat and barley enzymes to hydrolyse gluten prolamins was analysed in the HPLC-MS and SDS-PAGE. Toxicity of enzymatically pre-treated, pepsin and trypsin-digested gliadin and secalin were then further assessed in vitro in the human intestinal epithelial cell culture, CD patient-derived T cell assay as well as ex vivo in the human small-intestinal mucosal organ culture biopsies of CD patients.

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Results. Proteases from germinating cereals were particularly efficient in degradation of the gluten prolamines. Of those, germinating barley enzymes showed superior cleavage of gliadin and secalin (Figure. 1).

Gliadin + PTGliadin + wheat enzymes + PTGliadin + PTGliadin + wheat enzymes + PT

Figure 1. Degradation of wheat gliadin (A, C) and rye secalin (B, D) by germinating cereal enzymes.

Unlike unprocessed gluten prolamins, enzymatically pre-treated gliadin and secalin did not increase epithelial permeability, induce actin cytoskeleton rearrangement or changed tight junctional protein expressions in intestinal Caco-2 cells. In addition, the pre-treatment diminished proliferation of CD-specific T cells in vitro and enhanced production of autoantibodies from the small-intestinal biopsies of CD patients. Conclusions. Germinating cereal enzymes are particularly efficient in the degradation of gluten prolamins. In the future these enzymes might be utilized as a novel medical treatment for coeliac disease or in food processing in order to develop high-quality coeliac-safe food products.

References Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C. Structural

basis for gluten intolerance in celiac sprue. Science 2002;297:2275-9. Shewry PR, Napier JA and Tatham AS. Seed Storage Proteins: Structure and Biosynthesis.

The Plant Cell. 1995;7:945-956.

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Degradation of Immunogenic Gluten Epitopes by Probiotic Lactobacilli

Maria De Angelis1*, Raffaella Di Cagno1, Francesca Gagliardi2,

Carlo Giuseppe Rizzello1, Ruggero Francavilla2, Marco Gobbetti1

1 University of Bari, Department of Plant Protection and Applied Microbiology, Bari, Italy

2 University of Bari, Department of Pediatrics, Bari, Italy *corresponding email: m.deangelis@agr.uniba.it

Introduction. Celiac disease (CD) is an inflammatory disorder of the small intestine that affects genetically predisposed individuals when they ingest gluten from any Triticum species and similar proteins of barley and rye, and their crossbred varieties. The high concentration of glutamine and, especially, proline prevents the complete degradation by human gastric and pancreatic enzymes, and results in the build up of oligopeptides in the small intestine which are resistant to further proteolysis and toxic to genetically predisposed CD patients. A strict, lifelong gluten-free diet is the only accepted treatment of CD. Alternative therapeutic options include the proposed oral supplementation with microbial oligopeptidases (Pyle et al., 2005). This work was aimed at showing the capacity of probiotic lactobacilli to hydrolyze of gliadins and glutenins under simulated gastro-intestinal conditions. The mechanism of hydrolysis of various Pro-rich immunogenic epitopes (containing high level of proline residues) by peptidases of probiotic lactobacilli was highlighted.

Methods. Thirty strains of commercial probiotic lactobacilli were used. Hydrolysis of Pro-rich synthetic epitopes was investigated under simulated gastro-intestinal conditions. Hydrolysis was also investigated on baker’s yeast bread previously treated with digestive enzymes which mimicked the activity of probiotic lactobacilli during gut colonization. Hydrolysis of Pro-rich synthetic epitopes, gliadins and glutenins was determined by complementary electrophoresis, chromatography, mass spectrometry and immunology analyses (Di Cagno et al., 2007; De Angelis et al., 2005; Rizzello et al., 2007). Cytokines interferon gamma (IFN-γ) and interleukin 2 (IL-2) and interleukin 10 (IL-10) assays on duodenal biopsies from CD patients were used to determine the toxicity of hydrolysed Pro-rich synthetic epitopes and gluten (De Angelis et al., 2010).

Results. After 180 min of incubation, the combination of at least six different probiotic lactobacilli totally hydrolyzed the 33-mer (50 mM) into free amino acids. The same results were found for other immunogenic epitopes such as the fragments 57-68 of α9-gliadin, 62-75 of A-gliadin and 134-153 of γ-gliadin. As shown by electrophoresis, chromatography, mass spectrometry and immunology analyses, several peptides from gliadins and glutenins persisted after treatment of baker’s yeast bread by pepsin and pancreatin. The signal of all these immunoreactive Pro-rich peptides disappeared after further treatment by a pool of six probiotic lactobacilli. The in vivo digestion was simulated and proteins/peptides extracted from the pepsin-trypsin (PT) digest after hydrolysis with six strains of probiotic lactobacilli induced the expression of IFN-γ, IL-2 and IL-10 at levels comparable to the negative control.

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Conclusions. The findings of this study provide the evidence that the immunogenic sequences of gliadins/glutenins were not present after simulated gastro-intestinal digestion when selected probiotic lactobacilli were added. This study showed that a combination of probiotic lactobacilli may have an importance after gut colonization, to degrade gliadin contaminants in gluten freeproducts. References Pyle GG, Paaso B, Anderson BE, Allen DD, Marti T, Li Q, Siegel M, Koshla C, Gray GM Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac spue. Clin Gastroenterol Hepatol 2005;3:687-694. Di Cagno R, De Angelis M, Auricchio S, Greco L, Clarke C, De Vincenzi M, Giovannini C, D’Archivio M, Landolfo F, Parrilli G, Minervini F, Arendt E, Gobbetti M. Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients. Appl Environ Microbiol 2004;70:1088-1096. De Angelis M, Rizzello CG, Fasano A, Clemente MG, De Simone C, Silano M, De Vincenti M, Losito I, Gobbetti M. VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for celiac sprue. BBA – Mol Basis Dis 2005;1762:80-93. Rizzello CG, De Angelis M, Di Cagno R, Gianfrani C, Silano M, Losito I, De Vincenzi M, De Bari MD, Palmisano F, Maurano F, Camarca A, Gobbetti M. Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease. Appl Environ Microbiol 2007;73:4499-4507. De Angelis M, Cassone A, Rizzello CG, Gagliardi F, Minervini F, Calasso M, Di Cagno R, Francavilla R, Gobbetti M. Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum L. var. durum by sourdough lactobacilli and fungal proteases. Appl Environ Microbiol 2010;76:508-518.

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Can prolyl endoprotease enzyme treatment mitigate thetoxic effect of gluten in coeliac patients?

Greetje Tack1, Jolanda van de Water1, Maaike Bruins2, Yvonne Kooy-Winkelaar3,Jeroen van Bergen3, Gerrit Meijer1, Mary von Blomberg1, Marco Schreurs1, Luppo

Edens2, Chris Mulder1, Frits Koning3

1 VU University Medical Centre, Amsterdam, The Netherlands2 DSM Biotechnology Centre, Delft, The Netherlands

3 Leiden University Medical Centre, Leiden, The Netherlands

Introduction: Celiac disease (CD) is characterized by a small intestinal immune response toproline/glutamine-rich gluten peptides. Previous in vitro studies showed that the Aspergillusniger prolyl endoprotease (AN-PEP) enzyme is highly effective in degrading these immuno-toxic epitopes into harmless fragments under gastric conditions (Stepniak 2006, Mitea 2008).

Methods: To assess whether AN-PEP enzyme could mitigate the immunogenic effects ofgluten in CD patients, a randomised, double-blind placebo-controlled pilot study wasperformed (Figure 1).

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All 16 subjects (18-70 yrs) consumed 5 pieces of toast (~7 g gluten) with AN-PEP topping for2 weeks (AN-PEP phase). Subjects who did not relapse (increase in 2 Marsh grades) were,after a two-week washout period, randomised to a 2-week gluten with either AN-PEP (n=7)or placebo (n=7) (randomisation phase). Measurements included duodenal mucosaimmunohistology (Marsh scores, T-cells, anti-tissue transglutaminase-IgA (tTG-IgA)deposits), CD-specific serum T-cells and antibodies (tTG-IgA, dual anti-deamidated gliadin-related peptides and anti-tTG IgA (DGP/tTG-IgA), anti-gliadin-IgA and IgG (AG-IgA andAG-IgG), and anti-endomysium (EM-IgA)), self-reported CD-specific health-related qualityof life questionnaire (CDQ, subcategories; disease-related worries, emotional, social, andgastrointestinal problems), and physician-reported health complaints.

Results: Of the 16 subjects enrolled in the study, 2 were excluded after the AN-PEP phasebecause of histological deterioration of 2 Marsh grades although serum antibodies remainednegative. Fourteen subjects did not show any deterioration on gluten plus AN-PEP andentered the randomisation phase and all completed the study. Only minor deteriorations wereobserved on gluten intake in both AN-PEP and placebo group for all parameters. Nosignificant differences in change from baseline between the groups were observed for any ofthe parameters; neither for serology, nor for histology. tTG-IgA antibodies did not rise after

79

gluten challenge, neither in the placebo group, nor in the AN-PEP group. EMA-IgA remainednegative in all subjects during the study. AG-IgA rose to positive levels in 2 of 7 patients onplacebo and AG-IgG in 1 placebo and 1 AN-PEP patient. DGP/tTG-IgA became positive in 1placebo patient. Mucosal IgA-tTG deposit staining increased in 4 subjects on placebo and 1on AN-PEP as compared to baseline. Total CDQ scores and subcategory scores wererelatively high and did not significantly change in time and differ between groups. The CDQgastrointestinal subscale was rated high throughout the study (score range 37-38 out of 49)and did not differ between groups. Mild to moderate gastrointestinal complaints were reportedwithout difference between groups. Posthoc analysis revealed significant negative correlationsbetween patients’ years on GFD and response to gluten by Marsh, tTG-IgA, DGP/tTG-IgA,and AG-IgG (p=.03, .04, .02, <.0001). When patients <10 years on GFD were selected foranalyses, the median, and sum of ranks was lower after AN-PEP than placebo treatment forall parameters, which was significant for Marsh and tTG-IgA (Table 1).

Parameter Median (IU/mL) Sum of ranks p-value

AN-PEP Placebo AN-PEP (n=4) Placebo (n=4)

Marsh 1.0 2.0 13 23 <.05

tTG-IgA 0.00 0.75 13 23 <.05

AG-IgA 1.50 1.85 15 21 NS

AG-IgG 2.15 6.00 14 22 NS

Table 1. Sum of ranks of serological and histological parameters after 2 weeks of gluten intake with placebo orAN-PEP

Conclusion: Little clinical deterioration was observed in CD patients consuming a dailyamount of 7 g gluten for 2 weeks, neither in the placebo, nor in the AN-PEP group. Therefore,longer gluten intake is needed to demonstrate an effect of AN-PEP enzyme. Furthermore,selecting patients that are on a GFD for only a few years may increase their responsiveness toa gluten challenge, and thereby increase the chance of finding a treatment-related effect.

References

Stepniak D, Spaenij-Dekking L, Mitea C, Moester M, de RA, Baak-Pablo R, et al. Highlyefficient gluten degradation with a newly identified prolyl endoprotease: implicationsfor celiac disease. Am J Physiol Gastrointest Liver Physiol 2006;291:G621-G629.

Mitea C, Havenaar R, Drijfhout JW, Edens L, Dekking L, Koning F. Efficient degradation ofgluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliacdisease. Gut 2008;57:25-32

80

Auto-proteolytic and physical elimination of prolamins in acidic suspensions of malted wheat, barley, and rye

Jussi Loponen*, Outi Brinck, Zhongqing Jiang, Hannu Salovaara

University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland *corresponding email: jussi.loponen@helsinki.fi

Introduction. Cereal malts contain an arsenal of germination-induced peptidases and a vast majority of these operate under acidic conditions (Jones 2005). These enzymes are dedicated to hydrolyse prolamins, their natural substrates. An extensive proteolysis can be utilized to breakdown the toxic structures of prolamins and, hence, to reduce their immunogenic properties (Sollid and Khosla 2005). Such elimination of prolamins could facilitate the inclusion of barley, wheat, and rye containing products in GF-diets for improved sensory and nutritional properties. This study investigated the auto-proteolytic potential of barley, wheat, and rye malts i.e. how intensively the malt peptidases hydrolyzed the prolamins of malts. Experimental. Germinated and gently dried seeds (enzyme malts) of wheat, barley, and rye (Laihian Mallas) were incubated (DY 600) at pH 3.6–4.1 for 24 h. Protein hydrolysis was studied by analysing the lyophilized suspensions (total) and the extracts of the suspensions (soluble) as well as heat-treated and ultra-filtrated (MWCO 3,000) soluble fractions. A prolamin peptide calibrated size-exclusion chromatography (SE-HPLC) analysis (Figure 1) and the amino nitrogen content of the samples determined the extent of protein breakdown. A competitive prolamin immunoassay (R-biopharm) quantified the residual prolamins.

Hydrolysisproducts

33-mer

19-mer

9-mer

Polymericproteins

Monomericproteins

Figure 1. A tandem column SE-HPLC system with Superdex Peptide and Superdex 200, separated proteins and peptides. The system was calibrated with synthetic prolamin peptides and used UV-detection at 210 nm. Results and Discussion. Rye malt was the most potent raw material for the auto-proteolytic elimination of prolamins. The the final prolamin levels (dry matter) in rye, wheat, and barley suspensions were hundreds, thousands, and more than 10,000 mg/g, respectively (Figure 2A). This means that the prolamin contents of rye suspensions decreased approximately 99% while wheat and barley malt had the respective decreases of 95% and 75%. SE-HPLC analysis showed the same and further that in all systems most of the protein hydrolysis products were

81

smaller than a 9-mer prolamin peptide. Accordingly, the formation of protein hydrolysis products and, thus, was most intensive with rye (Table 1).

0

5 000

10 000

15 000

20 000

25 000

pH 3.6 pH 3.8 pH 4.1

Prolamin content of total fraction after 24 h malt incubation

BARLEY WHEAT RYE

0

200

400

600

800

1 000

1 200

1 400

1 600

pH 3.6 pH 3.8 pH 4.1

Prolamin content in the soluble fraction of malt suspensions

BARLEY WHEAT RYE

A B

Figure 2. Prolamin contents of malt suspensions after 24 h incubation under acidic pH conditions. The Y-axis of total fraction (A) is the prolamin content in lyophilized suspensions (mg/kg) and that of the soluble fraction (B) is the prolamin content in the supernatant of suspension (mg/L). With barley, most of the prolamins were absent from the soluble extracts of malt suspensions, which means that the prolamins detected in the total sample were mostly insoluble and, thus, removable by centrifugation (Figure 2B). The opposite phenomenon was evident with wheat malt systems, where practically all of the detected prolamins remained in the extract fraction and, thus, were soluble, though, boiling of wheat suspensions prior to the centrifugation resulted in 50% lower prolamin contents. Moreover, the ultra-filtration efficiently removed the residual prolamins from all malt extracts. Table 1. Mean formation rates of amino nitrogen in malt suspensions during 24 h incubations

Amino nitrogen mg kg-1 h-1 pH 3.6 pH 3.8 pH 4.1 Barley 57 63 47 Wheat 16 31 40 Rye 73 76 69

Conclusion. These results imply that rye malt was the most potential raw material for the auto-proteolytic elimination of prolamins. Physical methods (centrifugation, precipitation, filtration) can be utilized to remove residual prolamins from the malt hydrolysates. References Jones BL. Endoproteases of barley and malt. J Cereal Sci 2005;42:139-156. Sollid L.M. and Khosla C. 2005. Future theraupetic options for celiac disease. Nat. Clin. Pract. Gastroenterol. Hepatol. 2:140-147.

82

Hydrolytic potential of malts prepared of three rye varieties and impact on prolamin breakdown during acidification

Emma Laivisto1, Annika Wilhelmson2, Arvi Wilpola2, Arturo Garcia-Horsman3, Hannu Salovaara1, Jussi Loponen1*

1 University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland 2 VTT Technical Research Centre of Finland, Espoo, Finland

3 University of Helsinki, Faculty of Pharmacy, Helsinki, Finland *corresponding email: jussi.loponen@helsinki.fi

Introduction. Rye malt is a prospective raw material for the production of novel low-gluten cereal products (Loponen et al 2009). Rye malt contains high levels of germination-induced hydrolytic enzymes including peptidases (Brijs et al 2002). The aim of this work was to evaluate whether malts prepared of different rye varieties differ in their hydrolytic potential.

Experimental. Rye varieties Evolo, Reetta, and Riihi (Boreal) were germinated with a Joe White malting system. Germination samples were collected after steeping (moisture 46%) and after 1, 2, and 4 days of germination. The samples were lyophilized, milled and extracted. Azocasein (Megazyme) hydrolysis determined the general proteolytic activity (pH 4.9, 40°C). Proline oligopeptidase (POP) activity was determined (pH 7.0, 37°C) using Z-Gly-Pro-AMC. Amylolytic activities were determined with alfa-amylase (Ceralpha) and beta-amylase (Betamyl) assays (Megazyme). Auto-proteolytic degradation of prolamins (i.e. hydrolysis of malt prolamins by malt peptidases) was investigated in sourdoughs and chemically acidified doughs (DY 240, 24 h, 34°C). The prolamins were quantified with a competitive immunoassay (R-biopharm).

Results and Discussion. Rye malts, prepared of three rye varieties, differed in their enzyme activities (Figure 1). Reetta had highest alfa-amylase, proline-oligopeptidase, and general proteolytic activities. Evolo, in turn, had the lowest activities among the studied rye varieties. Alfa-amylase activity increased strongly whereas beta-amylase retained the same activity level throughout the germination. The general proteolytic activity as well as the POP activity increased during the germination. No hydrolysis of the POP-substrate occurred at pH 4, which indicates that the measured activity originated from POP rather than from cysteine endopeptidases. Rye malt prolamins were efficiently hydrolyzed during chemical and microbial acidification. Chemically acidified and fermented doughs had prolamin concentrations (dry matter) of 300 mg/kg and 500 mg/kg, respectively (Figure 2). Compared to the prolamin concentration of untreated rye malt this indicates that 99% or more of the prolamins were hydrolysed during acidification.

Conclusions. Rye malts prepared of different varieties substantially differed in their measured enzyme activities. The differences in the proteolytic activities, however, had no drastic impact on the prolamin hydrolysis that took place during 24 h acidifications. For instance, regardless of the variety, in all dough acidifications the extent of prolamin hydrolysis was at the same range i.e. 99% or more of the prolamins were hydrolyzed. This confirmed that rye malt acidification is an efficient process for the elimination of rye prolamins, and that small variation in the proteolytic activity had no impact on the final prolamin levels after a long processing time. On the other hand, malts with higher enzyme activities might commit the elimination faster.

83

Figure 1. Enzyme activities of grain samples after steeping and 1-3 day germination.

Figure 2. Residual prolamin concentrations of 24 h acidified rye malts (bars) and the relative decrease of prolamin content (%-values above the bars) References Loponen, J., Kanerva, P., Zhang, C., Sontag-Strohm, T., Salovaara, H. Gänzle, M.G. Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods. J Agric Food Chem 2009; 57:746–753 Brijs K., Trogh I., Jones BL. Delcour JA 2002 Proteolytic enzymes in germinating rye grains. Cereal Chem. 79:423-428.

REETTA

RIIHI

EVOLO

0,0

50,0

100,0

150,0

200,0

250,0

300,0

350,0

After steeping

1-day 2-day 4-day

Act

ivit

y, U

g-1

(dry

mat

ter)

A alfa-amylasep-nitrophenyl maltoheptaoside (blocked)

REETTARIIHI

EVOLO

0,0

100,0

200,0

300,0

400,0

500,0

600,0

After steeping

1-day 2-day 4-day

Act

ivit

y, U

g-1

(dry

mat

ter)

B beta-amylasep-nitrophenyl maltopentaoside

REETTA

RIIHI

EVOLO

0,0

25,0

50,0

75,0

100,0

125,0

150,0

175,0

200,0

After steeping

1-day 2-day 4-day

Act

ivit

y, n

mol

AM

C m

in-1

g m

alt-1

(dry

mat

ter)

C proline-oligopeptidaseZ-Gly-Pro-AMC

REETTA

RIIHI

EVOLO

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

After steeping

1-day 2-day 4-day

AU

D proteolytic (general)azo-casein

84

Overview on the new developments in the area

E. K. Arendt

School of Food and Nutritional Sciences, University College Cork, Ireland. Correspondence: Tel: +353 21 490 2064; Fax: +353 21 427 0213; E-mail: e.arendt@ucc.ie.

The incidences of celiac disease or other allergic reactions / intolerances to gluten are

increasing largely due to improved diagnostic procedures and changes in eating habits. This

creates a high demand for high quality gluten-free products. The majority of the gluten free

cereal products currently on the market are lacking structure flavour and are very often of

poor sensory quality. This presentation gives and overview on novel approaches for the

development of gluten free cereal products focusing on the gluten free bread. The areas

covered in the presentation are the detailed characterisation of gluten free cereals and the

assessment of these cereals as potential ingredients for gluten free breads. The

characterisations ranges form a detailed chemical characterisation, to rheological evaluation

of the resulting doughs, structural properties of the doughs and breads using advanced

microscopical methods as well as pilot-scale baking trials and sensory evaluation. Methods to

improve the quality of cereal products will also be introduced; one example being the use of

specially selected Lactic acid bacteria with properties such as antifungal activity,

exopolysaccharides production and enzyme production. A full characterisation of the selected

strains is provided including the isolation and characterisation of specific antifungal

compounds. The formation of structure is important for high quality gluten free products. The

influence of a range of enzymes such as transglutaminase, glucose oxidase and protease on

wide range of gluten free cereals will be shown. An in-depth understanding of the

interactions of transglutaminase with the various proteins will be explained with the help of

cereal proteomics. Novel processing such as high pressure processing will be introduced as a

means to create ingredients for gluten free cereal products.

of gluten free foods and beverages

85

In addition to the cereal products and overview of the recent advances in the production of

gluten free malt and beer will also be covered. The technology traditionally used for the

production of malt and beer made from barley can not be applied for gluten free cereals. It is

therefore essential to optimise the processing conditions for every gluten free cereal. This

presentation gives and overview on novel approaches for the development of gluten free malt

and beer. The presentation will focus on a number of different grains such sorghum,

buckwheat, and oats (oats can be tolerated by most celiac patients, even that it is not

considered gluten free). The areas covered in the presentation are the detailed characterisation

of gluten free cereals and the assessment of these cereals as potential ingredients for gluten

free malts. The optimisation of the malting and brewing process is discussed in detailed.

Results of mathematical modelling approaches to optimise both the malting and brewing

process are given. Advanced microscopy has been used to determine the ultra structural

changes taking place during the malting of gluten free cereals, where as the proteomic

approach was used to explain the protein changes taking place during malting. A detailed

analysis including nutrition analysis of the various malts and beer will also be presented.

86

Gluten Free Baked Products: Some Quality Solutions

William A. Atwell Cargill Bakery Category Technology, Plymouth, MN, USA

bill_atwell@cargill.com Gluten is a functional ingredient in all wheat containing baked products but it is more functional in some products than in others. For example, in cookies gluten helps to set the structure and stop a cookie from spreading during the baking process. Gluten contributes to batter emulsification and viscosity, and hence contributes to gas retention and cell structure during cake baking. In bread baking gluten plays multiple roles. These include providing cohesiveness to bread dough during processing, retaining leavening gases, setting the crumb structure, and imparting elasticity (i.e., chewiness) to the texture. The difficulty of replacing gluten and formulating gluten free baked products is directly related to the amount of functionality gluten plays in the corresponding wheat based product. Hence high quality gluten free cookies are easier to formulate than high quality gluten free breads. This is readily apparent in the gluten free baked product market where gluten free breads are fairly low quality when compared to their wheat based counterparts. Cookies, however, are more similar to wheat containing controls. Over the past five years Cargill Bakery Technology has conducted research and development leading to high quality solutions for all types of gluten free baked products. Our quality target has not been simply to be better than other gluten free baked products on the market. Our goal is to formulate products that are equivalent in all quality parameters to their wheat based counterparts. During this presentation the product development and patent pending technology supporting recently introduced gluten free cookie, batter-based and bread products will be discussed.

References

Atwell, WA, Engleson, JA, Muroski, AR, Finnie, SM, Smith, SA. Gluten-free baked products and methods of preparation of same. US patent application 20090092716

Engleson, JA, Lendon, CA, Atwell, WA. System for gluten replacement in food products. US patent application 20080038434

Engleson; JA, Lendon, CA, Hope, J, Casper, JL. System for gluten replacement in food products. US patent application 20090098270

87

N a t u r a l F l a v o u r

Ernst Böcker GmbH & Co. KG PO Box 217032427 Minden/GERMANY

Natural Sourdoughs, Preferments & Best Fermentations

Phone +49 (571) 8 37 99-0Fax +49 (571) 8 37 99-20www.sauerteig.de

88

Notes

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Formation and modification of bioactive compounds in gluten free sourdoughs

Michael G. Gänzle, Andreas Schieber, Louise Svensson, Januana Teixeira, and Victoria McNeill

University of Alberta, Department of Agricultural, Food and Nutritional Science, Edmonton, AB, Canada

Introduction: The formulation of gluten free bread recipes typically results in products with low contents of micronutrients and dietary fibre. The incorporation of non-toxic cereals, pseudocereals, or legume flours in gluten-free recipes may increase the content of fibre and micronutrients in gluten free breads (1). However, the high levels of polyphenols and oligosaccharides in red sorghum and legumes, respectively, are considered antinutritive factors, and lactic fermentation is employed to improve their palatability and digestibility. This study aimed to characterise metabolism of sorghum polyphenols and pulse oligosaccharides during sourdough fermentations with sorghum and pulse flours, respectively. Material and Methods: Polyphenol metabolism. Red sorghum (Sorghum bicolor) flour PAN3860 was fermented with binary strain combinations (Lactobacillus plantarum and L. casei, or L. fermentum and L. reuteri). Phenolic acids and flavonoids were extracted with aqueous methanol followed by liquid-liquid extraction, and characterised and quantified by LC-DAD-MS. Raffinose metabolism. Fava bean (Vicia faba) flour or field pea (Pisum sativum) flour was fermented with the α-galactosidase positive L. reuteri LTH5448 or the α-galactosidase negative L. sanfranciscensis LTH2590. Isogenic strains lacking levansucrase activity (L. reuteri LTH5448 ΔftfA and L. sanfranciscensis LTH2590 ΔlevS) were used for comparison (2, 3). Carbohydrates were quantified by HPAEC-PAD. Results: L. reuteri LTH 5448 metabolised raffinose, stachyose, and verbascose by levansucrase activity with concomitant accumulation of galacto-oligosacchrides, followed by their internalisation and hydrolysis (Figure 1A). Oligosaccharide metabolism in the levansucrase-negative mutant strain was slower, and the extracellular accumulation of intermediates was not observed. The galactosidase negative L. sanfranciscensis LTH2590 accumulated melibiose and corresponding higher galacto-oligosacchrides whereas the isogenic levansucrase-negative strain did not grow in pulse flour, and did not metabolise any of the pulse oligosaccharides (Figure 1B). Metabolism of phenolic acids, glycerol esters of phenolic acids, as well as flavonoids and flavonoid glucosides were quantified in doughs prepared from the red sorghum variety PAN 3860. Caffeoylglycerol, dicaffeoylglycerol, coumaroyl-caffeoylglycerol and coumaroyl-feruloylglycerol have to date not yet been identified in sorghum. In chemically acidified doughs, phenolic acids were partially released from the corresponding glycerol esters, and flavonoid glucosides were hydrolysed to the corresponding aglycones. The hydrolysis of glycerols esters of phenolic acids, and the hydrolysis of flavonoid glucosides was virtually quantitative in sorghum doughs fermented with lactobacilli. Analysis of polyphenol metabolism by single strains in laboratory media indicated that one strain in each binary strain combination metabolised phenolic acids by decarboxylase and / or reductase activity. L. plantarum additionally hydrolysed naringenin glucoside to naringenin.

89

Conclusion: Polyphenolic compounds and oligosaccharides are bioactive food components which, depending on the type of compounds and their concentrations, are considered anti-nutritive factors or beneficial micronutrients. This study provides insight into the conversion of the bioactive compounds by lactic fermentation to improve the nutritional and sensory quality of gluten-free bread. References

(1) Gallagher, E., T.R. Gormley, E.K. Arendt. Recent advances in the formulation of gluten-free cereal-based products. Trends Food Sci. Technol. 2004, 15, 143-152.

(2) Tieking, M., M.A. Ehrmann, R.F. Vogel, M.G. Gänzle. Molecular and functional characterization of a levansucrase from Lactobacillus sanfranciscensis. Appl. Microbiol. Biotechnol., 2005, 66, 655-663.

(3) Schwab, C., J. Walter, G.W. Tannock, R.F. Vogel, M.G. Gänzle. Sucrose utilization and impact of sucrose on glycosyltransferase expression in Lactobacillus reuteri. 2007. System. Appl. Microbiol. 30, 433-443.

A

Elution time (min)

8 10 12 14

dete

ctor

sig

nal (

nC)

20

40

60

80

100

B

8 10 12 1420

40

60

80

100

Figure 1. Separation of oligosaccharides in fava bean flour fermented with L. reuteri or L. sanfranciscensis. Melibiose, sucrose, raffinose, stachyose, and verbascose were identified on the basis of internal standards, the identity of 6’galactosylmelibiose and 6’6’digalactosylmelibiose were inferred on the basis of the enzymatic activities of the cultures used. Panel A, Fermentation with L. reuteri LTH5448 after 8 h (upper trace) fermentation with L. reuteri LTH5448ΔftfA after 8h (middle trace) and after 24h (lower trace). L. reuteri LTH5448 also completely metabolised all oligosaccharides after 24h (data not shown). Panel B. Fermentation with L. sanfranciscensis LTH2590ΔlevS after 24 h (upper trace), and fermentation with L. sanfranciscensis LTH2590 after 24 h (lower trace). Data are representative for two independent experiments. Comparable results were obtained in field pea flour (data not shown).

melibiose galactosylmelibiose sucrose digalactosylmelibiose

verbascose stachyose raffinose melibiose

galactosylmelibiose sucrose digalactosyl- melibiose

90

1

Enzymatic processing of gluten-free flours: a promising

tool to improve their bread-making functionality?

S. Renzetti1* and E.K. Arendt

2

1 T�O Quality of Life, Zeist, The �etherlands

2Department of Food and �utritional Science, University College Cork, Ireland

*corresponding email: stefano.renzetti@tno.nl

Introduction. Gluten-free (GF) flours are unsuitable for the production of bread as their

proteins do not possess the visco-elastic properties typically found in gluten. Therefore, the

development of GF breads with good textural quality is technologically difficult. Enzymes are

a specific bio-processing tool which offers the possibility of modifying the structure-

functionality of GF flour components in order to improve the bread-making performance of

the flours. The present work investigated the impact of various cross-linking enzymes, i.e.

transglutaminase, glucose oxidase and laccase, and of a protease on the bread-making

performance of several GF flours. More specifically, the molecular effects of the enzymes

were related to the rheological and textural properties of batters and breads, respectively.

Methods. A simplified dough system consisting of flour/water mixture with no addition of

functional ingredients, e.g. hydrocolloids, was used for all flours. The enzymes used were: a

trasnglutaminase (100 units/g, Ajinomoto Co., Hamburg, Germany) a glucose oxidase

(Gluzyme Mono 10000 BG, Novozymes, Baegsvaard, Denmark) containing 10000 GO

units/g; a laccase (NS26021, Novozymes) containing 1000 LAC units/g; and a protease

(Neutrase 1.5 MG, Novozymes) containing 1.5 AU-NH/g. Fundamental rheology, confocal

laser scanning microscopy, texture profile analysis, SE-HPLC and SDS-PAGE were mostly

used to understand the enzymatic effects from molecular to macroscopic level.

Results. The use of cross-linking enzymes has been suggested in GF systems in order to

promote protein networks and increase the visco-elastic behaviour of GF batters, thus

resulting in improved textural quality of breads. The cross-linking enzymes showed positive

effects on buckwheat, brown rice, corn and oats. In general, they produced batters which were

stiffer and more elastic than the control (Table 1). The resulting breads showed lower specific

volumes but improved crumb texture (Table 1). This was beneficial when crumb defects were

present, as in the case of buckwheat (Figure 1). However, the impact of the enzymes was very

much dependent on the flour (protein/non-starch polysaccharides) source.

On the other hand, traditional GF breads such as injera and kisra rely on protein hydrolysis

from lactic acid bacteria fermentation, which increase the viscosity of the starch phase.

Protein hydrolysis and, in the case of oats, β-glucan depolymerisaion by side enzymatic

activity resulted in batters with increased deformability and elasticity (Table 1). The resulting

breads showed increased loaf volume (Figure 1) and improved texture and softness of the

crumb (Table 1).

Conclusions. The results of this study suggest that the most promising improvements in the

bread-making functionality of GF flours can be achieved by a depolymerisation mechanism,

91

2

which results in increased deformability and elasticity of batters. On the other hand, a

polymerisation mechanism might be beneficial to promote good crumb texture.

The improved flour functionality can help reducing the amount of additional ingredients in

GF formulations (e.g., starches, hydrocolloids).

Table 1. Impact of enzymatic processing on the bread-making functionality of GF flours. Enzyme Beneficial for Batter rheology Bread properties Molecular effects

Transglutaminase

EC 2.3.2.13

Buckwheat

Brown rice

Increased

resistance to

deformation and

degree of

elasticity

Decreased specific

volume

Improved crumb

texture

Cross-linking of

major protein

fractions

Entrapment of

LMW proteins in

macromolecular

complexes

Strengthened

protein network

Glucose

oxidase

EC 1.1.3.4

Corn Increased

resistance to

deformation and

degree of

elasticity

Increased specific

volume and lower

crumb hardness

Polymerisation α-

zein

Laccase

EC 1.10.3.2

Oats Increased

deformability

and elasticity at

small and large

deformation (in

combination

with side β-

glucanase

activity of

commercial

preparation)

increased loaf

volume and

softened crumb

Slight

polymerisation of

globulins

Insoluble β-glucan

degradation due to

side enzymatic

activity

Enhanced stability

of starch phase

Proteinase

EC 3.4.11-19

Oats Brown rice

Increased deformability

and elasticity

Higher specific volume, softer

crumb

Opening up of macromolecular

protein complexes,

release of LMW

proteins

Enhanced

continuity and

stability starch

phase

In oats: insoluble β-

glucan degradation

due to side

enzymatic activity

Figure 1. Effect of cross-linking and proteolytic enzymes in GF breads. Buckwheat bread

control (A) and with 10 U (g of protein) of transglutaminase (B). Oat bread: control (C) and

with 0.001% (flour basis) protease (D).

References

S. Renzetti, C.M. Courtin, J.A. Delcour and E.K. Arendt, 2010. Oxidative and proteolytic

enzyme preparations as promising improvers for oat bread formulations: rheological,

biochemical and microstructural background. Food Chem. 119(4), 1465-1473.

S. Renzetti, J. Behr, R. Vogel and E.K. Arendt, 2008. Transglutaminase polymerisation of

buckwheat (Fagopyrum esculentum Moench) proteins. J. Cereal Sci., 48(3), 756-763.

S. Renzetti, F. Dal Bello and Elke K. Arendt, 2008. Microstructure, fundamental rheology and

baking characteristics of batters and breads from different gluten-free flours treated with a

microbial transglutaminase. J. Cereal Sci., 48(1), 33-45.

C D B A

92

Competitiveness of commercial starters in buckwheat and teff sourdoughs

Alice V. Moroni, Fabio Dal Bello, and Elke K. Arendt Department of Food and Nutritional Sciences,

University College Cork, Ireland Introduction The application of sourdough (SD) technology in gluten free (GF) baking has been recently proven as a potential tool to improve the quality of GF bread (Moroni et al., 2009). However, to date only little information on the applicability of commercially available starters in GF sourdough fermentations is available. The aim of this study was to assess the competitiveness of two commercial starters, i.e. SA and SB, for the production of GF sourdoughs from the GF flours buckwheat or teff. Materials and Methods The starter SA was used for type A sourdough, which was incubated at 25 ° C with 10% (w/w) refreshment every 12 hrs; whereas starter SB was applied for type B fermentation, carried out at 35 ° C with 10% (w/w) refreshment every 24 hrs. Sourdoughs were propagated till a stable biota was established. Lactic acid bacteria (LAB) and yeasts constituting the dominant microbial community were isolated and identified by sequencing of the 16S rDNA and 28S rDNA, respectively. In addition, the dynamics of the microbial community were followed by specific PCR-denaturing gradient gel electrophoresis (PCR-DGGE) (Meroth et al., 2003). Results and Discussion Analysis of the microbial community revealed that not all the starters strains were competitive in the sourdough fermentation of buckwheat and/or teff flours. PCR-DGGE analysis revealed that most of the dominant LAB were already detected at the start of the fermentation and the intensity of the corresponding bands did not vary through the fermentation process (Fig. 1). Remarkably, none of the starters yeasts could survive the fermentation process. Furthermore, autochthonous LAB and yeasts, e.g. Lactobacillus brevis and Weissella cibaria in buckwheat and Saccharomyces cerevisiae in teff, could associate and dominate with some of the starters strains. Under otherwise identical fermentation conditions, the fermentation substrate played a key role in determining the competitiveness of the starters strains. For example, among the species of LAB present in SB, Lactobacillus plantarum, Lactobacillus paralimentarius and Leuconostoc argentinum persisted in buckwheat but not in teff sourdough, whereas Lactobacillus pontis and Lactobacillus reuteri were dominant in teff but not in buckwheat sourdough. Conclusion Given that the persistence and dominance of the starters strains is a pre-requisite for the successful application of sourdough starters, we conclude that commercial starters are not ideal for the fermentation of buckwheat and teff. Investigations on the

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spontaneous biota of GF flours will help developing novel starters which are adapted and competitive for the production of GF sourdough bread. Figure 1. PCR-DGGE analysis of the LAB biota during type A fermentation of buckwheat. L, identification ladder; LAB profile at the beginning (start) and after 12 h (12h), 1 (1d), 2 (2d), 3 (3d), 5 (5d) and 8 (8d) days of fermentation. References Meroth, C.B., Walter, J., Hertel, C., Brandt, M.J., Hammes, W.P. 2003. Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, 69: 475-482. Moroni, A.V., Dal Bello, F. and Arendt, E.K., 2009. Sourdough in gluten-free baking: An ancient technology to solve a novel issue?. Journal of Food Microbiology, 26: 676-684.

start 12h 3d 2d 1d 5d 8d L

c

a

b

e

d

b c

d

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Development of Gluten Free Muffin

Pogiatzis M., Li W., Ramsden R. and Brennan C.

Manchester Metropolitan University, Department of Food, Tourism and Management

Introduction. In recent years, there has been a rapid increase in the demand of gluten free product for the customers with coeliac disease (Arendt et al 2008). This project focuses on the development of gluten free muffin product with a replacement of wheat flour. In the study, wheat flour of a control muffin sample was replaced and the formulation of the wheat flour replacement was optimized through trials with the starches of waxy maize, tapioca, and potato and the flours of rice, soybean and quinoa individually and in selective combination.

Methods. The waxy maize starch (National Frigex), and the tapioca starch (Novation 3600) were from National Starch and the potato starch was from Farina. The soybean flour and quinoa flour were from Goods Organic and the rice flour was from Doves Farm Est. 1978. The muffins were made by following a standard method in the laboratory of the Department of Food and Tourism Management, Manchester Metropolitan University. The firmness and the crumb structure of muffins were measured using a TA.XT plus texture analyser (AACC 74-09) and a C-Cell imaging system respectively. The height, specific density and volume of muffins were also measured using the seed displacement method. The sensory test was also carried out on the gluten free and control muffins to evaluate overall acceptability of gluten free muffin (Angelika P., 2008). The results were statistically analyzed using one-way analysis of variance (ANOVA), by SPSS.

Table 1. The quality parameters of control muffin and the muffin with wheat flour replacements

Trials Firmness (g)

Height (mm)

Volume (ml)

Cell Diameter(mm)

Number of cells

Control 531 ± 42 49 ± 2.5 155 ± 4 2.6 ± 0.1 1364 ± 79.5

Maize 112 ± 32 51 ± 3.1 177 ± 2 2.7 ± 0.1 1692 ± 41.1

Quinoa 852 ± 76 43 ± 2 125 ± 4 2.4 ± 0.2 1296 ± 51.4

Tapioca 309 ± 26 52 ± 3.9 156 ± 6 2.4 ± 0.2 1719 ± 25.5

Rice 1283 ± 2.5 40 ± 3.3 130 ± 4 1.9 ± 0.2 1401 ± 42.5

Rice-Tapioca-Quinoa 1073 ± 92 42 ± 0.9 130 ± 0.8 2.1 ± 0.1 1579 ± 74.7

Tapioca-Rice-Quinoa 610 ± 28 45 ± 0.9 154 ± 1.4 2.2 ± 0.2 1602 ± 31.5

Rice-Maize-Quinoa 536 ± 72 43 ± 1.7 144 ± 3.3 2.1 ± 0.1 1583 ± 79.2

Maize-Rice-Quinoa 305 ± 12 44 ± 1.2 137 ± 6.1 1.9 ± 0.1 1652 ± 33.1

Rice-Tapioca-Maize-Quinoa 400 ± 11 46 ± 1.7 145 ± 2 2.3 ± 0.1 1664 ± 3.6

Rice-Maize-Tapioca-Quinoa 334 ± 38 45 ± 0.9 145 ± 2.5 2.2 ± 0.2 1558 ± 17.7

Tapioca-Rice-Maize-Quinoa 520 ± 57 43 ± 2.6 113 ± 2.5 1.9 ± 0.1 1644 ± 82.5

Rice-Tapioca-Maize-Potato-Soybean 493 ± 69 48 ± 2.3 131 ± 2 1.9 ± 0.1 1661 ± 141

Results. The results in Table 1 summarize the characteristics of muffins, in which the wheat flour was replaced by the gluten free ingredients individually or in a selective combination. Clearly, there

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were significant changes in the characteristics of muffin when the wheat flour in muffin formulation was replaced by the single gluten free ingredient. The muffins with waxy maize starch and tapioca starch both had too soft texture (a lower firmness), but a good volume, whilst the muffins with the rice flour and the quinoa flour both had a small volume and a hard and dense texture. Based on specific properties of individual ingredient and its contribution to the characteristics of muffin observed, the gluten free ingredients were grouped in a selective combination and used as a replacement of wheat flour in muffin formulation. It was found that the development of muffin characteristics can be controlled by varying the combination ratio of ingredients. With the combination of Rice-Tapioca-Maize-Potato-Soybean at a ratio of 37.5%, 34.5%, 13%, 10% and 5% respectively, the gluten free muffin had a finer texture with a smaller average size of gas cells compared the control muffin (at p≤0.1), but there no significant differences in firmness and volume between them (at p≤0.1). Furthermore, the results obtained in sensory test (Figure 1) show the very good acceptability of the gluten free muffin as there were no significant differences in firmness, chewiness, moistness, aftertaste and overall rating quality (at p≤0.05) compared with control

muffin. Although a significant difference existed in the cohesiveness of mass attribute (p≤0.05), a slight preference to the gluten free muffin was observed.

CONTROLGLUTEN FREE

MUFFIN SENSORY

109876543210HAND SPRINGINES

FIRMNESS

CHEWINESS

MOISTNESS

MOUTHFEEL

COHESIVENESS

AFTERTASTE

OVERALL RATING

Figure1. The sensory test quality parameters of gluten free muffin and control muffin

Conclusion: The results obtained in this study show that the replacement of wheat flour in the muffin formulation with a single selected gluten free ingredient resulted in the significant changes in characteristics of muffin, which closely linked to the properties of individual ingredient. However, the characteristics of gluten free muffin can be developed by varying the combination ratio of gluten free ingredients with specific properties. References: Arendt E., Rasetti S. and Moore M., 2008, Novel approaches in the design of gluten free cereal products, Food science and Technology, 22 (1) Angelika P., Monika R., Lorna L., and Carlo L., 2008, Sensory Evaluation of processed wheat from a defined field-trial 16th IFOAM organic world congress. Modena Italy, June 16-20, 2008

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Gluten-free soryz cookies Cosciug Lidia, Dupouy Eleonora, Bulgaru Viorica

Technical University of Moldova, Department of Food Science and Nutrition 168 Stefan cel Mare boulevard, MD-2004, Republic of Moldova

tel: (+373 22) 509 959, e-mail: viorica.bulgaru@yahoo.com Introduction The prevalence of celiac disease is increasing around the world. The number of persons with intolerance to gluten is increasing rather rapidly, at the same time growing the geographic area of spread. At present at the department of Food Science and Nutrition of the Technical University of Moldova are carried out a series of investigations on the technological and nutritional properties of soryz (Sorghum Oryzoidum), a gluten-free cereal obtained locally in the country, in view of its application in the production of gluten-free foods. It was investigated the possibility to use the soryz flour for the obtaining of a variety of gluten-free cookies, that can be recommended in the nutrition of the persons with metabolic deficiency in gluten assimilation. Objectives The investigations presented in this paper have the following objectives: To investigate the possibility to use the soryz flour for the obtaining of a variety of

gluten-free cookies ;

To evaluated the sensorial indices of quality of the gluten-free cookies.

To evaluated the physico-chemical indices of quality of the gluten-free cookies.

Metods The sensorial and physico-chemical indices of quality of the gluten-free cookies were determined by standard methods [1]. Results and discussion It were evaluated the sensorial and physico-chemical indices of quality of gluten-free cookies. As reference for quality indices were used the cookies from wheat flour. The substitution of wheat flour with soryz flour has influenced most of all the colour of cookies and less other indices of quality. In order to mask the grey color, that may be considered as a quality defect, in the flour of some varieties of cookies were added such ingredients as the cacao powder, puree of vegetables, that along with improving the color, have increased in the final product the content in vitamins, minerals and dietary fibers. The flavour and the taste of soryz сookies is specific and pleasant, the vanilla arome and sweet taste in them are slightly diminished. Physico-chemical quality indices (weight loss at the baking, specific volume and weight, humidity, content of lipids, sugar and ash, alkalinity, water hydratation capacity ) of reference and test samples have close values. The weight loss at the baking of gluten-free soryz cookies varies with about (+4,5%) in comparison with the respective value for the reference sample. This result correlates with a higher level (over 5%) of humidity of cookies with puree of vegetables. The content of lipids, sugar and ash in studied samples does not

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differ essentially, their values ranging respectively 24,3…28,3%, 13,7…17,9% and

1,4...2,2%. Water-holding capacity of soryz cookies increased in comparison with reference sample that will positively influence their digestibility. Conclusions The sensorial and physico-chemical indices of quality of investigated samples have proved the possibility of the utilization of soryz flour for the production of gluten-free cookies. The production of gluten-free cookies from local raw materials will facilitate the provision of appropriate food to more categories of consumers with special dietary requirements. The production of gluten-free cookies from soryz flour is beneficial in economic terms taking into account that the local raw material is available at low costs. That will ensure the production of non-expensive gluten-free cookies and will provide the enlargement of gluten-free pastry products on the local food market. The availability of gluten-free foods, produced from the local raw materials, on the food market in Moldova will contribute to the personalization of the nutrition of the group of population intolerant to gluten and thus contribute to the improvement of public health and life quality in the country. References

Ловачева Г, Мглинец А, Успенская Н. Стандартизация и контроль качества продукции.

М.: Экономика,1990.

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Metabolism and competitiveness of Lactobacillus sanfranciscensis and ting isolates in wheat and sorghum

sourdoughs

Bonno Sekwati-Monang and Michael G. Gänzle University of Alberta, Department of Agricultural, Food and Nutritional Science, Edmonton,

AB, Canada

Introduction: The use of sourdough as baking aid improves textural, sensorial, and nutritional aspects of gluten-free breads (1). Culture selection for gluten free sourdoughs is based on metabolic properties with beneficial impact on bread quality; additionally, the competitiveness of cultures in gluten free is an important criterion. The microbiota of traditional sourdoughs propagated in Triticale cereals, wheat or rye, does not exhibit characteristic differences, however, the use of other cereal flours or pseudocereals selects for fermentation microbiota that differs from wheat and rye sourdoughs (2). This study aimed to compare metabolism and competitiveness of Lactobacillus sanfranciscensis in wheat and sorghum sourdoughs to isolates from traditional sorghum sourdoughs. Material and Methods: L. sanfranciscensis LTH 2590, a wheat sourdough isolate, and four isolates from traditional sorghum sourdoughs (ting) in Botswana, L. casei FUA3166, L. harbinensis FUA3199, L. parabuchneri FUA3169, and L. coryniformis FUA3307, were inoculated in sorghum sourdoughs, sorghum sourdoughs supplemented with 2% maltose, or whole wheat sourdoughs. Fermentations at 34°C were characterised by determination of cell counts, pH, and the quantification of metabolites. Neutral and acid aseptic doughs were used as control. To determine the competitiveness of strains, wheat and sorghum sourdoughs were inoculated with equal cell counts of L. sanfranciscensis, L. parabuchneri, and L. casei, fermented at 28°C or 34°C, and propagated by back-slopping every 24h. Results: Maltose was the main carbon source in wheat sourdough whereas glucose was the dominant carbon source in sorghum. Moreover, glycerol, released from hydrolysis of phenolic acid esters, was present in sorghum but not in wheat (data not shown). L. sanfranciscensis grew in wheat but not in sorghum sourdoughs, or sorghum sourdoughs supplemented with 2% maltose. The metabolism of the ting isolates, L. casei, L. harbinensis, L. parabuchneri, and L coryniformis did not exhibit characteristic differences when sorghum and wheat sourdoughs were compared (Figure 1 and data not shown). Ting isolates were overgrown by L. sanfranciscensis after four propagations in wheat sourdough independent of the incubation temperature but they prevailed in sorghum sourdoughs. Conclusions: The selection of cultures for gluten-free sourdoughs requires strains that are highly adapted to the cereal substrate employed. Isolates from traditional fermentations with non-toxic cereals are a suitable source of strains for use in gluten-free baking. References (1) Moroni, AV, Dal Bello, F, Arendt, EK. Sourdough in gluten-free bread-making. An

ancient technology to solve a novel issue? Food Microbiol. 2009, 26, 676-674.

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(2) Vogelmann, SA, Seitter, M, Singer, U, Brandt, MJ, Hertel, C. Adaptability of lactic acid bacteria and yeasts to sourdoughs prepared from cereals, pseudo-cereals and cassava and the use of competitive strains as starter cultures. Int. J. Food Microbiol. 2009, 130, 205-212.

L. coryniformis

Lact

ate

Acet

ate

Etha

nol

1,3

Prop

aned

iol

010203040

80

120

160L. parabuchneri

Lact

ate

Acet

ate

Etha

nol

1,2

Prop

aned

iol

[met

abol

ites]

(m

Mol

kg-1

)

010203040

80

120

160

Sorghum flourSorghum flour +maltoseWhole wheat flour

Figure 1. Metabolite formation during growth of L. parabuchneri and L. coryniformis after 24h fermentation at 34°C in sorghum sourdoughs, sorghum sourdoughs supplemented with 2% maltose, and whole wheat sourdoughs. Data are representative for three independent experiments.

Whole wheat sourdough

fermentation time (h)

0 24 48 72 96

cell

coun

ts (

cfu

/ mL)

107

108

109

Sorghum sourdough

0 24 48 72 96

107

108

109

Figure 2. Cell counts of L. sanfranciscensis (black symbols), and L. casei + L. parabuchneri (open symbols) during growth in wheat and sorghum sourdoughs. Sourdoughs were inoculated with 107 cfu / g of each of the three strains, incubated at 34°C( ) or 28°C(Δ), and back-slopped every 24h with a 10% inoculum. Backslopping is indicated by an arrow. Data are representative for three independent experiments.

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Obtaining and Characterization of Gluten Free Flour Products Enriched with Dried Fruit and Hippophae

Rhamnoides Extract

Ersilia Alexa1*, Daniela Stoin1, Teodor-Ioan Trasca1, Georgeta Pop2, Monica Negrea1,Dorin Pop2

1 Banat`s University of Agricultural Science, Faculty of Agro-Food Technology, Timisoara, Romania

2 Banat`s University of Agricultural Science, Faculty of Agriculture, Timisoara, Romania*corresponding email: ersilia_alexa@yahoo.com

Introduction. Romania official statistics show that three Romanian of one hundred are born with Coeliac disease. Unfortunately, in present in Romania, flour diet food production is not sufficiently developed, are very few companies which produce diet foods, especially for those people affected by Coeliac and Phenylketonuria disease.In this paper are presented and characterized 3 types of enriched gluten free flour products obtained in the Milling and Bakery Laboratory in the Faculty of Food Processing Technology enriched with fruits and Hippophae rhamnoides. Hippophae rhamnoides is a fruit-bearing shrub known as being part of Romania's spontaneous flora, which is used both in the food industry, forestry, pharmacy and as ornamental plants. Hippophae rhamnoides fruit contains twice as much vitamin C than maces and 10 times more than citrus and have a high antioxidant capacity (Parvulescu, 2006). The benefit of this plant are known since ancient times. In China traditional medicine recommends treatment of digestive diseases.

M ethods: Experimental were obtained 3 types of gluten free products: flours premix based on corn and rice flours, dried raisins and figs, gluten free pasta obtained from rice flour in mixture with corn starch, gluten free crackers obtained from rice flour, Hippophae rhamnoides extract, nuts, eggs and vegetal fats.Receipt setting was made considering the gluten free composition of the raw material and taking into account the technological scheme for obtaining of nutritive flour, classical biscuits and pasta. It was determined the ash content (6000C), lipids (Soxhlet method), protein (STAS 6283-4-84/Kjeldahl method) and vitamins B (HPLC-DAD). Microelements content (Fe, Mn, Cr, Cu, Zn) of the obtained products was determined according to SR EN 14082: 2003 (AAS).

Results. Gluten free products obtained in the Milling and Bakery Laboratory in the Faculty of Food Processing Technology are shown in Figure 1.The experimental results indicate that physical-chemical parameters of gluten free products (moisture, protein, lipids) fall within the range of values according to actual standards.Products lipid content, in which recipes have used fat addition, is higher. The addition of nuts and Hippophae rhamnoides increase the content in minerals (1,37%), while fruits intake in gluten free flours increases vitamin content of the final products (0,322 mg/100g B1, 0,151 mg/100g B2).

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Figure 1. Gluten free products

The obtained products have a good content of microelements (Cu, Cr, Mn, Zn, Fe) comparing with other types of gluten free flouring products. The iron content range between 15,6 – 21,20 µg/g, Zn and Mn content is higher in cackes with Hippophae rhamnoides extract 9,01 µg/g Zn, respectively 7,82 µg/g). Gluten free products obtained have been analyzed in terms of gluten content, all had a gluten content below 20 ppm, calculated by the ELISA method (Arendt, 2008).

Table 1. Gluten free products physical-chemical characterization

Table 2. Microelements composition of gluten free products

Cu

(µg/g)

Cr

(µg/g)

Fe

(µg/g)

Zn

(µg/g)

Mn

(µg/g)Flour 2,21 0,19 16,86 7,70 6,21Pasta 3,10 0,37 21,20 8,75 2,94

cackes 1,70 0,25 15,60 9,01 7,82

Conclusions. The addition of dried fruit, nuts and Hippophae rhamnoides extract improved mineral content, microelements composition, vitamins and also sensorial characteristics.

ReferencesPârvulescu L, Gergen I., Rujescu C., Bordean D., Poiana M.A., Researches about antioxidant capacity, ascorbic acid and polyphenols content in some vegetables, Bulletin of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Agriculture/Horticulture, 2006:62: 323-328.Arendt E.K., Dal Belio, F.Gluten-Free Cereal Products and Beverages, Academic Press.2008: 58-60.Acknowledgement: This work was funded by Romanian Ministry of Agriculture Forest and Rural Development (MAPDR) and World Bank through Contract No.141529/2008, AG 142044/02.10.2008: The implementation of modern technology systems to obtain dietary flouring products.

Flour Pasta Cackes

Moisture (%) 9,5 9,7 3,6Lipid (%) 1,11 3,48 9,58Protein (%) 6,99 6,66 5,94Ash (%) 0,8 0,9 1,37Vitamine B1 (mg/100g) 0,210 0,110 0,322Vitamine B2 (mg/100g) 0,122 0,120 0,151

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Optimization of Gluten-free French-style Bread Formulation suitable for Frozen dough process

Sandra Mezaize, Sylvie Chevallier, Alain Le Bail, Marie de Lamballerie* ONIRIS - Nantes-Atlantic National College of Veterinary Medicine, Food Science and

Engineering, Site de la Géraudière, GEPEA UMR 6144, BP 82225, 44322 Nantes cedex 3, France

*corresponding email: marie.de-lamballerie@oniris-nantes.fr

Introduction. Staling rate in gluten-free breads is higher than for their wheat counterparts, mainly due to their high starch content. Solutions in terms of formulations are currently investigated in order to slow down this staling rate (Nunes et al. 2009). The use of freezing process could be another way to circumvent the staling: the consumer could bake the gluten-free bread at his convenience at home. Nevertheless, freezing has negative impact on dough properties, as it has been shown on wheat bread for years (Berglund et al. 1991), and thus leads to decrease the final product quality. The present study investigates, by experimental design, the optimization of a gluten-free formulation for French-style bread (characterized by an important specific volume, a soft crumb, a well-coloured crust and a heterogeneous bubbles size distribution; (Mezaize et al. 2009)) and suitable for the frozen dough process. Methods. Gluten-free formulas contained rice, corn and buckwheat flours, corn and potato starches, inulin, guar gum and / or HPMC, salt, sunflower oil, compressed yeast and water. Combinations of water levels and proportions of HPMC versus guar gum were used following a three-level, full factorial design for 2 factors (Table 1). This experimental design was conducted for (i) conventional and (ii) frozen dough breadmaking process. In frozen dough process, dough was frozen at - 30 ° C for 30 min to reach - 18 ° C in the middle of the product and then stored at - 18 ° C for one week before being proofed and baked. Responses measured were: bread specific volume, crumb hardness (maximum force of a compression test), gas cell distribution, crust colour and bread dry matter. Datasets were analysed together using a multifactor ANOVA; water quantity, HPMC quantity, and process as defined factors. Table 1. Composition of the tested formulas and the coded levels for water and HPMC. Factors Levels

-1 0 +1 Water (%) 85 90 95 HPMC (%) (Guar gum (%)) 0 (3) 1 (2) 2 (1) Results. Freezing has an important impact on gluten-free bread quality: breads have a smaller specific volume, a harder crumb, a more homogeneous bubbles size distribution and a crust colour slightly modified, when obtained by frozen dough process than conventional breadmaking. Formulation has also an impact on gluten-free bread quality (Table 2).

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Table 2. Effects of water and HPMC levels on the gluten-free bread characteristics. Bread Characteristics Effects

Water level HPMC level Specific volume � - Crumb hardness � � Gas cells heterogeneity � � Crust lightness � - Bread dry matter � � To obtain gluten-free bread with an important specific volume, a soft crumb, the best formula is the one which contains 90 % of water, 2 % of HPMC and 1 % of guar gum. Nevertheless, 2 % of HPMC included in formula do not allow to maintain heterogeneity in gas cells size distribution. In this case, to get closer to the French bread characteristics, the formula containing 90 % of water, 1 % of HPMC and 2 % of guar gum is a good compromise: the bread specific volume and the crumb hardness criteria are improved, even if in a lesser proportion, and the bubbles size distribution is heterogeneous. Conclusions. This study showed that there is a negative impact of the frozen storage of gluten-free dough on the bread characteristics. Formulation can be helpful to limit this negative impact. Several combinations of water and HPMC levels are particularly efficient to obtain gluten-free bread produced by the frozen dough process with characteristics more similar to the bread reference, the French bread. References Berglund P, Shelton D, Freeman T. Frozen bread dough ultra structure as affected by duration

of frozen storage and freeze-thaw cycles. Cereal Chem 1991 ; 68: 105-107. Mezaize S, Chevallier S, Le Bail A, de Lamballerie M. Optimization of gluten-free

formulations for French style breads. J Food Sci 2009; 74: 140-146. Nunes MHB, Moore MM, Ryan LAM, Arendt EK. Impact of emulsifiers on the quality and

rheological properties of gluten-free breads and batters. Eur Food Res Technol 2009; 228: 633-642.

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Rheological Properties of Gluten-Free Bread Formulations

Using Chestnut and Rice Flour Combinations

Ilkem Demirkesen; Behic Mert, Gulum Sumnu, Serpil Sahin

Middle East Technical University, Department of Food Engineering, Ankara, Turkey

Introduction: Celiac disease, also called as gluten-sensitive enteropathy is triggered by the

response of the body’s immune system to the proteins in certain grains (Bower et al., 2006).

When people with celiac disease consume gluten, their immune system generates antibodies

against this protein causing damage to the tiny hairlike projections in the small intestine.

Therefore, they can not absorb nutrients and they must avoid all the gluten containing food

products for their entire life. Flours such as rice, corn, cassava and chickpea flours have been

used to make gluten free breads. Chestnut flour can also provide reasonable gluten free flour

due to its nutritional and health benefits. It contains low amount of protein and fat and high

amount of starch, sugar, and dietary fiber, Vitamin E and B group vitamins, potassium,

phosphorous, and magnesium.

Rheological information is critical to optimize acceptability, stability and textural properties

of baked products. The aim of the present work was to analyze the rheological behavior of

different dough formulations prepared with different chestnut to rice flours ratios.

Methodology: Basic dough recipe on 100 g flour basis contained 8% sugar, 8% shortening,

and 2% salt. On flour basis, the amount of water (30°C) added to dough varied between 150

%-210 % for the different chestnut to rice flour ratios (0:100, 10:90, 20:80, 30:70, 40:60,

50:50, and 100:0). The rheological measurements were conducted using a TA rheometer (RA

2000ex, Sussex, UK). All measurements were conducted at 25°C, using parallel plate

geometry (40 mm diameter and 2 mm gap). The dough sample was placed between the plates

and the edges were carefully trimmed with a spatula. The flow experiments were conducted

under steady-shear conditions with shear rate ranging from 1 to 50 1/s. For the relaxation of

the residual stresses, the dough was rested at room temperature for 20 minutes before testing.

In the case of the dynamic oscillatory experiments, first the linear viscoelastic region of the

samples was determined. Then, frequency sweep experiments were carried out at 0.5% strain

rate between 0.1 to 10 Hz. Finally, elastic (G′ ) and loss (G ′′ ) modulus were obtained. All the

rheological experiments were performed at least twice and their averages were reported in the

study.

Results and Discussion: The flow curves of the samples having chestnut to rice flour ratios

of 0:100 and 10:90 couldn’t be obtained properly since the rice flour particles quickly

sedimented during rheological measurement. All the other dough formulations were found to

fit Herschel-Bulkley model. The flow behavior index ( n ) of the samples at 25°C ranged from

0.52 to 0.87 and the consistency indices (K) of the samples were between 3.6 and 79 Pa

s n (Table 1). In addition, the yield stress ( 0σ ) values of the samples ranged from 4.8 to 85.9

Pa. The higher consistency index, yield stress and apparent viscosity values were obtained

from the dough samples containing higher amount of chestnut flour. Fibrous structure of

chestnut flour was the main reason of the increase in the consistency index, apperant viscosity

and yield stress of dough. Entanglement of fibers creates additional resistance to flow and

105

causes increased consistency index, yield stress and apparent viscosity values. Furthermore,

the hydroxyl groups available in fiber structures can bind more water through hydrogen

binding mechanism which, in turn reduces the amount of available water for plasticizing

effects (Nelson, 2001). Oscillation measurements showed that there was also a significant

increase in both elastic modulus (G′ ) and viscous modulus (G ′′ ) with increasing the chestnut

flour content. Entanglement of fibers in chestnut flour again appears to be responsible for the

high elastic moduli values of the dough samples.

Table 1. Herschel-Bulkley model constants of the dough samples at 25°C.

Chestnut to Rice Flour Ratio

K (Pa.sn)

n

0σ (Pa) r

2

100:0 79.0 0.52 85.9 0.99

50:50 41.0 0.56 59.2 0.99

40:60 8.4 0.59 18.1 0.99

30:70 3.6 0.75 8.6 0.99

20:80 1.7 0.87 4.8 0.99

Conclusions: All dough formulations followed Herschel-Bulkley model. Both flow and

oscillation measurements showed that, the higher viscosity and viscoelastic modulus were

obtained from the dough samples supplemented with higher amount of chestnut flour.

Therefore, usage of chestnut flour can be recommended to obtain the desired rheological

properties for gluten-free breads. In order to determine the optimum chestnut to rice flour

ratio, bread quality should also be evaluated as a future study.

References: Bower SL. What is Celiac Disease. In: Bower SL., Sharrett MK, & Plogste S, editors. Celiac

disease: a guide to living with gluten intolerance. NY: Demos Medical Publishing; 2006.

p. 1-9.

Nelson AL. Baked goods and extruded applications. In: Nelson AL, editors. Fiber Ingredients.

MN: AACC ; 2001. p. 44-61

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Usage of Chufa Flour in Gluten-free Cakes

Elif Turabi1, Gulum Sumnu

1, Serpil Sahin

1, Mehmet Musa Ozcan

2

1Middle East Technical University, Department of Food Engineering, 06531, Ankara, Turkey

2 Selcuk University, Faculty of Agriculture, Department of Food Engineering,42030 Konya,

Turkey

Introduction: Chufa (Cyperus esculentus L.), also known by various other names such as

tigernut, earth nut, groundnut, rush nut and edible galingale is an edible nut-like tuber. It was

discovered more than 4000 years ago. Chufa is used as a human food source in several

countries around the Mediterranean Sea, particularly Spain and Egypt, as well as Nigeria, and

has been promoted in recent years by vegetarian organizations in European countries, USA

and Israel. The best-known application of chufa in food technology is the production of

‘horchata de chufa’ (milk of chufa) and it is also used as a flavoring agent in ice cream. Flour

of roasted chufa is added to biscuits and other bakery products due to its unique sweet taste.

Besides some beneficial properties such as high content of dietary fiber, potassium and

magnesium, chufa is also gluten-free and may be used as an alternative in producing gluten-

free bakery products, especially in combination with other gluten-free cereal flours.

Understanding the rheological and physical properties is important for determination of the

quality of the bakery products. Therefore, the aim of this study was to determine of chufa

flour on the bakery products.

Methods: The cakes were composed of different flours (only rice flour, only wheat flour or 10

% chufa flour in combination with 90% of rice flour), sugar, egg white powder, salt, baking

powder, shortening and water. Rheological properties of cake batters were investigated by

using parallel plate rheometer that was worked at steady shear rate between 0-100 1/s. Cake

sample (100 g) was baked in conventional oven at 175 o

C for 30 min. As quality parameters,

weight loss, specific volume and firmness of the cakes were determined.

Results: All the cake batters showed shear-thinning behavior according to the results of

rheological measurements. Herschel Bulkley model was found to be the most suitable model

fitted to the data. Addition of chufa flour to the formulation increased consistency coefficient

(9.56-32.80) and decreased flow behavior index (0.620-0.694). Rice flour containing cakes

lost more water than cakes containing wheat flour (Figure 1). Chufa flour addition to the

formulations decreased the moisture loss. There was no significant difference in specific

volume of cakes when chufa flour was added to rice flour containing cakes (Figure 2). Chufa

addition in rice flour cake gave higher specific volume. Rice flour containing cakes gave

higher hardness values. On the other hand, chufa flour addition gave softer cakes (Figure 3).

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Figure 1. Weight loss values of cakes containing different flour types

Figure 2. Specific volume of cakes containing different flour types

Figure 3. Firmness of cakes containing different flour types

Conclusions. This study showed that chufa flour can be used as an alternative for gluten-free

bakery products when used in combination with other gluten-free flours such as rice flour.

Addition of chufa flour had some positive effects on quality of gluten-free cakes such as

decrease in weight loss and increase in specific volume.

References Coskuner, Y., Ercan, R., Karababa, E., Nazlican, A.N. Physical and chemical

properties of chufa (Cyperus esculentus L) tubers grown in the Cukurova region of Turkey. J.

Sci Food Agric., 2002; 82:625-631.

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Heteropolysaccharide production from lactic acid bacteria in wheat and sorghum sourdough

Sandra Galle1,2,3, Clarissa Schwab1, FabioDal Bello2,3, Elke Arendt2 Michael Gä nzle1

1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton Canada; 2Department of Food and Nutritional Science and 3Biotransfer Unit, University

University College Cork, Ireland

*Corresponding e-mail address: e.arendt@ucc.ie

Introduction: Hydrocolloids improve the machinability of dough, and the volume, texture and shelf life of bread. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough fermentation can replace hydrocolloids (Tieking and Gä nzle 2005). Past studies focussed on EPS produced by glycansucrase activity from sucrose (Schwab et al. 2007). It was the aim of this study to determine whether heteropolysaccharides (HePS), which are synthesised intracellularly from sugar-nucleotides by glycosyltransferases, are produced in wheat and gluten free sorghum sourdough.

Experimental: HePS-producing strains of Lactobacillus casei FUA3185, L. paracasei FUA3186 and Lactobacillus parakefiri FUA3154 were used; Weissella cibaria 10M which produces no EPS in the absence of sucrose served as EPS negative control strain. Monomer composition of EPS was analysed after EPS purification from culture supernatants and acid hydrolysis. The relative molecular weight (MW) of EPS was determined by size exclusion chromatography and their flow behavior in broth was investigated by measuring the shear viscosity. These strains were used to ferment wheat and sorghum flours, and the resulting sourdoughs were characterized with respect to microbial cell counts and organic acid formation. Frequency sweep was performed in order to determine the viscoelastic behavior of wheat and sorghum sourdoughs. Glucosytransferase genes were amplified by PCR using specific primers (Provencher et al. 2003), whereas gene expression was determined by PCR amplification of copy DNA prepared from mRNA isolated from the doughs.

Results: The polysaccharides formed by L. casei FUA3185, L. paracasei FUA3186 and L. parakefiri FUA3154 peaked at 104-105 Da in the size exclusion chromatography (Table 1). Analysis of monosaccharide composition showed that the polymers synthesized by L. casei FUA3185 and L. paracasei FUA3186 are composed by four different monosaccharides, whereas the polysaccharide from L. parakefiri contains only 3 different sugars (Table 1). Supernatants from cultures of L. parakefiri FUA3154 in mMRS showed the highest viscosity at low shear rate. Overall the presence of HePS influenced the rheological properties of sorghum but not wheat sourdoughs. No difference in |G*| was exhibited between sorghum sourdough fermented with the control strain W. cibaria 10M and sourdough fermented with L. casei FUA3185 and L. paracasei FUA3186. However, sorghum sourdough fermented with L. parakefiri FUA3154 showed the lowest |G*| compared to the control, which indicates a decrease in resistance to deformation. At the same time an increase in tan δ indicated a decreased degree in elasticity.

Conclusion: This study provides the first report for hetero-EPS production in wheat and sorghum sourdoughs. Hetero-EPS significantly influenced the rheology of sorghum sourdoughs whereas the rheology of wheat sourdoughs remained unaffected. The use of LAB

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producing hetero-polysaccharides expands the variety of cultures as well as the diversity of polysaccharides produced by sourdough starter cultures for use in gluten-free baking.

Table 1. Strain used in this study and characterization of the polysaccharides

Strain Origin Monosaccharide composition a

Size [Da] Gene Expression in sorghum and wheat dough

L. casei FUA3185

oatdrink Galactose, Rhamnose, Glucose, one unidentified

104 - 105 GTF +

L. paracasei FUA3186

oatdrink Galactose, Rhamnose, Glucose, one unidentified

104 - 105 GTF +

L. parakefiri FUA3154

breaddrink Galactose, Glucose, one unidentified one

104 - 105 GTF +

Weissella cibaria 10M

sourdough Glucose 2*105 - 5*106 Dextransucrase ND

ND...not detected

References:

Provencher C, LaPointe G, Sirois S, Van Calsteren MR, Roy D. Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group. Appl. Environ. Microbiol. 2003; 69:6:3299-3307.

Schwab C., Walter J, Tannock GW, Vogel RF, Gä nzle MG. Sucrose utilization and impact of sucrose on glycosyltransferase expression in Lactobacillus reuteri. Syst. Applied Microb.2007; 30:433-443.

Tieking M, Gä nzle MG. Exopolysaccharides from cereal associated lactobacilli. Trends in Food Sci. Technol. 2005; 16:79-84.

110

Promoting of Structure Formation by High Pressure in Gluten-Free Flours

K. J. R. Vallons; L. A. M. Ryan, E. K. Arendt*

Department of Food and Nutritional Sciences, National University of Ireland, University College Cork, College Road, Cork, Ireland

*Corresponding email: e.arendt@ucc.ie Introduction. The use of gluten-free flours in the development of breads is technologically difficult, as their proteins do not possess the visco-elastic properties typically found in gluten. There is a growing interest in physical modification of cereal flours using innovative processing techniques such as high pressure treatment to improve their baking performance. In order to evaluate the potential of high pressure treatment to improve the functional properties of gluten-free flours, the effect of high pressure on the rheological properties of white rice, buckwheat and teff batters was investigated. Methods. Buckwheat, white rice and teff batters (40 %) were treated for 10 min at 200, 400 or 600 MPa. Changes in the microstructure of the batters after HP-treatment were observed using scanning electron microscopy (SEM). Fundamental rheology (temperature sweep) and Lab-on-a-Chip capillary gel electrophoresis were used to evaluate the pressure susceptibility of the major flour components starch and protein. Results. Similar to heat treatment, high pressure treatment of starch induced gelatinisation, as revealed by the pasting profiles (Figure 1). The consistency of the starch suspension increased with increasing pressure of the pre-treatment. The pasting profiles of control and pressurized white rice batters are shown in Figure 1. The profiles of other flours were similar and therefore, not shown.

Figure 1: Pasting profile of control and HP-treated white rice batters, with the complex modulus (G*) and temperature given as function of time.

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Lab-on-a-Chip capillary gel electrophoresis revealed protein polymerisation by thiol-/disulphide-interchange reactions in white rice and teff batters. However, the presence of free sulfhydryl groups is required to catalyse the mechanism, as the reaction occurs via the nucleophilic attack of a disulphide bond by the ionised S--form of an SH group (Funtenberger et al., 1997). As cysteine residues in buckwheat globulin exists as disulphide linkages rather than as free SH-groups, other mechanisms must be responsible for the decreased buckwheat protein extractability after high pressure treatment. A reduced accessibility of the proteins to the extraction buffers due to the presence of gelatinised starch and/or or starch-protein interactions was suggested. White rice- and buckwheat-based batters showed a decrease in tanδ (Table 1) with increasing pressures, indicating an increasing contribution of the elastic compound. On the contrary, teff showed two different rheological responses to the applied pressures (Table 1). When the treatment was performed at pressures up to 200 MPa, the structure weakened (increased tanδ). When treated at pressures above 200 MPa however, the pressures became more and more elastic (decreasing tanδ). Table 3: Effect of pressure on rheological parameters of flour-water suspensions at ω = 7.84 Hza. Damping Factor (1) White Rice Buckwheat Teff 0.1 MPa 0.153a 0.219a 0.178a 200 MPa 0.124ab 0.216a 0.245b 400 MPa 0.122b 0.179b 0.190a 600 MPa 0.074c 0.078c 0.092c a Mean values of at least triplicates; values followed by the same letter in the same column are not significantly different (p < 0.05).

Conclusions. This study has shown that high pressure treatment has the potential to improve the functionality of gluten-free breads in terms of their baking performance. By inducing starch gelatinisation and protein cross-linking by disulphide bonds (depending on the substrate and its amount of free sulfhydryl groups), HP caused an increase in visco-elastic properties of buckwheat, brown rice and teff batters. Renzetti et al. (2008) previously reported that protein polymerisation can improve the bread making performance of gluten-free floursw by enhancing elastic-like behaviour of the batters. Furthermore, positive effects of pre-gelatinised starch such as improved texture and shelf-life of baked goods, have been described (Hü ttner et al. 2009). References Renzetti S, Dal Bello F, Arendt EK. Microstructure, fundamental rheology and baking

characteristics of batters and breads form different gluten-free flours treated with a microbial transglutaminase. J Cereal Sci 2008, 48:33-45.

Hü ttner EK, Dal Bello F, Poutanen K, Arendt EK. Fundamental evaluation of the impact of high Hydrostatic Pressure on oat batters. J Cereal Sci 2009, 49:363-370.

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Identification of Lactic Acid Bacteria Isolated from Oat Sourdoughs and Investigation of their Potential for the

Improvement of Oat Bread Quality

Edith K. Hü ttner1, Fabio Dal Bello1, Emanuele Zannini1 and Elke K. Arendt1,*

1Department of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, Ireland

*corresponding email: e.arendt@ucc.ie Introduction. Recently, new consumer demands have emerged for food products with improved nutritional quality and health benefits, posing new challenges for the baking industry. Oat (Avena sativa) represents an ideal raw material for the production of bread since it contains high amounts of valuable nutrients. Moreover, oats are tolerated by the majority of people suffering from celiac disease. Yet, the utilization of flours alternative to wheat is restricted due to their low baking quality as a consequence of the absent gluten network (Arendt et al. 2008). The use of sourdough in breadmaking is common practice since it has the advantage of improving i.e. texture, nutritional value and shelf-life of wheat and rye breads. Consequently, exploitation of sourdough could be a new frontier for the development of oat bread with enhanced quality and additional features, i.e. improved aroma and shelf-life. Methods. Two different sourdoughs were prepared from wholegrain oat flour (dough yield 200) without addition of starter cultures and continuously propagated at 28 (SD 28) or 37°C (SD 37) until the composition of the lactic acid bacteria remained stable. The dominant lactic acid bacteria were identified by sequence analysis of the 16S rDNA isolated from pure cultures. The isolated lactic acid bacteria were further used as starter cultures for the production of oat sourdough. SD 28* was prepared with addition of a starter culture containing each LAB isolated from oat SD 28 and SD 37* was inoculated with the strains isolated from SD 37. In addition, rheological properties as well as changes in the protein profile of SD 28* and SD 37* were investigated. Moreover, the so produced oat sourdoughs were utilized for the production of oat sourdough bread. Results. Identification of the dominant lactic acid bacteria revealed differences in the microbiota of SD 28 and SD 37. Depending on the fermentation temperature, different lactic acid bacteria species became dominant. Leuconostoc argentinum, Pediococcus pentosaceus and Weissella cibaria were the dominant LAB species isolated from SD 28 (28°C ). In contrast, Lactobacillus coryniformis became dominant in SD 37 (37°C ). Rheological analysis of oat sourdoughs produced with the isolated strains as starter cultures revealed a softening of the sourdoughs as indicated by the decreased G* (Figure 1) and changes in the pasting properties compared to a non-acidified control produced without sourdough. The results obtained for the acidified control were between the values obtained for the sourdoughs and non-acidified control (Figure 1). Overall, rheological properties of SD 28* and SD 37* were not significantly different. Capillary electrophoresis was used to investigate changes in the protein profiles and the protein solubility due to sourdough fermentation. The protein size distribution in the sourdoughs and the controls was the same.

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10000

100000

0.1 1 10 100Angular frequency (1/s)

G*

(Pa)

Figure 1: Frequency sweep complex modulus (G*) of non-acidified control (× ), chemically acidified control CA 28* (●) and CA 37* (-) as well as SD 28* (∆) and SD 37* (□). Bread analysis revealed that addition of sourdough yielded breads with greater specific loaf volume, a more open crumb structure and even distribution of gas cells when compared to either the non-acidified or chemically acidified control (Figure 2). Textural parameters monitored over a 5-day storage period, were not significantly different of the sourdough breads compared to the non-acidified and the chemically acidified breads and changes were overall only minimal for all bread types.

Figure 2: Pictures of oat bread made without sourdough (A), oat bread made with 40 % SD 28* (B) or 40 % SD 37* (C) and CA breads CA 28* 40 % (D) and CA 37* 40 % (E). Discussion. The lactic acid bacteria isolated from the oat sourdoughs varied depending on the fermentation temperature and were different compared to the common microbiota of wheat and rye sourdoughs. Thus, the differences in the dominant lactic acid bacteria were affected by technological parameters as well as the raw material. The potential of the isolated strains for the improvement of oat bread quality was also investigated. The baking tests clearly showed that incorporation of oat sourdough can enhance loaf volume which can be influenced by direct impact of pH on dough structure, the effect of acid on cereal enzymes and indeed, CO2 production by heterofermentative LAB (Clarke et al. 2004). Overall it was concluded that the combination of gas production by the heterofermentative LAB, softening of the doughs and changes in the starch pasting properties brought about the observed differences. References. Arendt EK, Morrissey A, Moore MM, Dal Bello F. Gluten-free breads. In: Arendt EK, Dal Bello F (eds) Gluten-Free Cereal Products and Beverages. Academic Press, MA, 2008; p. 289-311 Clarke CI, Schober TJ, Dockery P, O’Sullivan K, Arendt EK. Wheat sourdough fermentation: effects of time and acidification on fundamental rheological properties. Cereal Chem 2004; 81:409-417

A C B D E

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Pilot scale manufacture of highly concentrated proteiningredients from oats using dry fractionation technology

Juhani Sibakov1*, Olavi Myllymäki1, Michael Kuhnen2, Anu Kaukovirta-Norja1,Kaisa Poutanen1, Pekka Lehtinen1

1 VTT Technical Research Centre of Finland, Espoo, Finland2Hosokawa Alpine AG, Augsburg, Germany*corresponding email: juhani.sibakov@vtt.fi

Introduction. Oats are special among the cereal grains, because they may be included in agluten-free diet (Sontag-Strohm et al. 2008). In the present study we investigated theenrichment of oat fraction with high protein concentration using low-energy technologies,such as milling and air classification. The separation of the protein-rich fraction extends fromthe oat -glucan dry fractionation technology of Kaukovirta-Norja et al. (2008). The shortageof plant-based proteins, other than soy protein, will be a challenge in the future. The oatprotein concentrate obtained could serve as a potential alternative to soy protein.

Methods. Dehulled oat grains were obtained from Raisio plc (Kokemäki, Finland). Super-critical CO2-extraction was used to remove the lipids at 2 000 kg scale at NateCO2 GmbH(Wolnzach, Germany). The extraction temperature was 40 ºC, pressure 290 bar and time 13 h.Defatted oats were milled with CW Contraplex pin disc mill at Hosokawa Alpine AG(Augsburg, Germany). The rotation speeds of mill discs were 11 200 / 5 600 rpm (cw / ccw).Oat endosperm flour was separated from the coarse, -glucan rich bran fraction by airclassification using 315 ATP air classifier (rotor speed 2 200 rpm). Thereafter, the highlyconcentrated protein fraction was separated from endosperm flour either by pilot scale50 ATP air classifier or by industrial scale 200 ATP-NG air classifier. Protein concentrationwas calculated as N x 6.25, where nitrogen content was analyzed using a Kjeldahlautoanalyzer.

Results. A protein-enriched fraction was separated from endosperm, and the proteinconcentration varied between 23-73 %, depending on the mass yield of the protein-enrichedfraction (Table 1). The mass yield was adjusted by changing the rotor speed and air-inlet ofthe air classifier. The maximum protein concentration obtained was 73 % with a mass yield of5.0 %, calculated from the whole oat kernels. The particle size distribution of the mostenriched fraction was in the range of 0.5-10 µm, with at least 95 % of the particles between0.5-7 µm (Fig. 1).

Discussion. Protein-enriched fractions would have versatile food applications. Oat proteinsare considered more nutritious, with higher content of essential amino acids, than in othercereals, but are used less for human consumption than e.g. wheat and corn protein (Mohamedet al. 2009). Similar protein fractions with over 70 % of protein have been previously reportedto be produced from oats by wet milling processes (Wu et al. 1973) or by air classification(Wu and Stringfellow 1995). However, the current process overcomes the energy-consumingdrying operations related to wet milling and obtains remarkably higher mass yield of protein-enriched fraction than what has been reported with air classification. The process presentedhere produces cereal protein suitable for a wide range of foods also in a gluten-free diet.

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Table 1. Mass yields and protein concentrations of oat flours and protein-enriched fractions.The type of air classifier is shown in brackets.

Description of the sample Mass yield (%) * Protein concentration (%)

Oat flour without defatting 100 17Oat flour, with lipid removal by CO2 100 17

Protein fraction 1 (50 ATP) 47 23Protein fraction 2 (50 ATP) 30 34Protein fraction 3 (50 ATP) 21 38Protein fraction 4 (50 ATP) 3.0 65

Protein fraction 5 (200 ATP-NG) 5.0 73

*) Mass yield was calculated from the whole oat kernels

73 % proteinconcentration

Particle size ( m)

Dis

trib

utio

n de

nsit

y

Sum

of d

istr

ibut

ion

(%)

73 % proteinconcentration

Particle size ( m)

Dis

trib

utio

n de

nsit

y

Sum

of d

istr

ibut

ion

(%)

Figure 1. Particle size distribution of 73 % protein fraction with all the particles below 10 m.

Conclusions. This study showed that combined milling and air classification of defatted oatflour enabled the separation of protein-enriched fraction from oat flour. The maximal obtainedprotein concentration 73 % was reached with a relatively high mass yield of 5.0 %.

References

Kaukovirta-Norja A, Myllymäki O, Aro H, Hietaniemi V, Pihlava J-M. Method forfractionating oat, products thus obtained, and use thereof, WO 2008/096044 A1.

Mohamed A, Biresaw G, Xu J, Hojilla-Evangelista MP, Rayas-Duarte P. Oats protein isolate:Thermal, rheological, surface and functional properties. Food Res Int 2009;42:107-114.

Sontag-Strohm T, Lehtinen P, Kaukovirta-Norja A. Oat products and their current status inthe celiac diet. In: Arendt EK and Dal Bello F (eds.) Gluten-Free Products andBeverages. Elsevier, Amsterdam, the Netherlands, 2008, pp. 191-202.

Wu YV, Cluskey JE, Wall JS, Inglett GE. Oats proteins concentrate from a wet millingprocess: Composition and properties. Cereal Chem 1973;50:481-488.

Wu YV and Stringfellow AC. Enriched protein and -glucan fractions from high-protein oatsby air classification. Cereal Chem 1995;72:132-134.

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Influence of selected modified starches and hydrocolloids on the rheological properties of dough and bread based on rice

(Oryza sativa ) and buckwheat (Fagopyrum esculentum)

Stephan Haase*; Andreas Houben; Thomas Becker

Technische Universität München, Center of Life and Food Sciences Weihenstephan, Institute of Brewing and Beverage Technology,

Work group: Cereal Process Engineering, Freising, Germany

*corresponding email: Stephan.haase@wzw.tum.de

Introduction. Celiac disease is caused by gluten intolerance, in particular by the gliadin fraction as well as the glutenine fraction of wheat and by prolamins of rye and barley. The only way to prevent celiac disease is a lifelong gluten-free diet (Gallagher et al., 2004). Due to improved diagnostic capabilities this disease can be detected more frequently. As a consequence the number of patients and thus the need for gluten-free baked goods increases. An alternative to improve taste of rice and maize based products, quite poor in taste by nature, could be the use of pseudocereals. They are more flavorful and they provide nutritional benefits related to several constituents. However, these pseudocereals possess due to the fact that they are gluten-free no ability to produce high quality baked goods. Gluten is known as a structural protein (Gallagher et al., 2004) able to produce an extensible dough with gas-retention capacity and final good crumb properties. Gluten-free baked goods show mostly small volumes, they are more compact and contain often crumbly crumbs, weak colors and quality losses during bread staling. For years, the bakery sector knows that gluten functionality can be increased or even replaced with special thickening agents to obtain the necessity of gas-retention (Rotsch, 1954). In modern food technology modified starches as thickening agents are already used on a large scale for various foods. These additives seem to have a positive effect on the rheological properties, like gas binding capacity, of rice /buckwheat based doughs and breads. In the present study, the influence of various selected modified starches and hydrocolloids was investigated.

Methods. For the baking tests, a flour mixture consisting of 80% rice flour (Ziegler & Co. Naturprodukte GmbH, Germany) and 20% wholemeal buckwheat flour (Schälmühle, Germany) was used. Three hot soluble modified starches (further called 1, 2, 3), one cold soluble modified starch (4), and one special starch for texture strength (5) were taken. In addition to the modified starches exopolysaccharides (EPS), obtained from the Department of Technical Microbiology, TU München and hydroxypropylmethylcellulose (HPMC) K4M (Dow Chemicals, Midland, USA), were also used. The addition of modified starches to the recipe was varied in practical quantities. Thus, each 1%, 2% and 3% relative was added to the flour. All rheological parameters of the prepared doughs and the changes during storage of the final bread were investigated.

Results. Loaf volume was related to the concentration of tested additives, but data were different. Increasing concentration of the modified starches1-4 resulted in low increase only, whereas the special modified starch for texture (5) had a sharp increase especially at the highest concentration tested. The same was to EPS, HPMC, and one hot soluble modified starch (3), but on a common level (Figure 1).

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Figure 1: Influence of the concentration of additives (%) based on the amount of used flour on the bread volume (mL)

based on 200g taken dough

Hardness of bread loafs changed during shelf life in relation to the used additives. Just after loaf cooling, results of all variants were almost the same, but a significant difference of increasing values could be observed during staling. The special starch for texture (5) had the most pronounced hardness after 7 days, whereas the cold soluble modified starch (4) resulted in a small reduction of hardness. Figure 2 presents data of a 2% addition.

Figure 2: Influence of the storage time (d) on the hardness (N) of bread loafs (2% additives)

Conclusions. The studies have noted differences between the various additives. It could be shown that two modified starches produced larger volumes with increasing concentrations. The addition of the cold soluble starch (4) (2%) resulted in a significant decrease in strength during storage and therefore in a poor suitability for the production of bread. In contrast, the special modified starch for texture (5) was superior to all other starches. Further studies are needed to clarify whether a combination of different modified starches has a combined effect on the investigated parameters.

References Gallagher, E., Gormley, T.R., Arendt, E.K.: Recent advances in the formulation of gluten-free cereal-based products, Trends in Food Science & Technology 15, 2004: 143-152

Rotsch, A.: Chemische und technische Untersuchungen an künstlichen Teigen, Brot und Gebäck, 1954: 8

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Exopolysaccharide forming Weissella strains as starter cultures for sorghum and wheat sourdoughs

Clarissa Schwab1, Galle, Sandra1,2,3; Elke Arendt2 and Michael Gä nzle1 1University of Alberta, Department of Agricultural, Food and Nutritional Science, Edmonton,

AB, Canada 2University College Cork, Department of Food and Nutritional Science, Cork, Ireland; 3University College Cork Biotransfer Unit, Ireland, Cork, Ireland

Introduction: The addition of sourdough fermented with lactic acid bacteria (LAB) synthesizing organic acids, and oligo- and exopolysaccharides (EPS) from sucrose enhances texture, nutritional value, shelf life and machinability of wheat, rye and gluten-free bread (Arendt 2007, Schwab et al. 2008). This study compared acetate, mannitol, and oligosaccharide formation of EPS producing strains of Weissella and Leuconocstoc spp. to a traditional sourdough starter L. sanfranciscensis LTH2590.

Material and Methods: Formation of organic acid and EPS of LAB was tested in MRS supplied with sucrose or sucrose and maltose. Sourdoughs were prepared with sorghum and wheat flour with addition of 15% sucrose at a dough yield of 200. Organic acids and mannitol were determined by HPLC with an Aminex HPX-87 column (Bio-Rad, Mississauga, Canada). Sugars were analysed with a CarbopacPA20 column (Dionex, Oakville, Canada). Oligosaccharides formed in MRS were directly analyzed from culture supernatant. Oligosaccharides synthesized in dough were extracted with H20 at 80 ° C for 2 h. EPS was precipitated form aqueous dough extracts with ethanol, dialyzed against distilled water and lyophilized. EPS was analyzed by size exclusion chromatography using a Superdex 200 Column (GE Healthcare, Baie d’Urfe, Canada).

Results: In broth, Leuconostoc strains formed acetate and mannitol whereas strains of Weissella formed only small amounts of acetate and no mannitol in presence of sucrose. In the presence of sucrose and maltose Weissella and Leuconostoc strains synthesized EPS and glucooligosaccharides. Strains of Weissella were successfully employed as starter cultures for wheat and sorghum sourdough and formed 0.8 – 8 g kg-1 EPS, and glucooligosaccharides, but only low amounts of acetate and mannitol (Figure 1). In contrast, formation of EPS from sucrose led to production of high amounts of acetate and mannitol by L. sanfranciscensis LTH2590. A correlation of acceptor sugars present in the flour and oligosaccharide formation was found. Higher levels of glucose in sorghum sourdoughs lead to the production of isomaltooligosaccharides by both Weissella species. In comparison, these strains formed in wheat flour long chain glucooligosaccharide consisting of panose linked with glucose (Figure 2).

Conclusion: This study indicates that Weissella strains are suitable starter cultures for wheat and sorghum sourdoughs and efficiently produce EPS without strong acid production. Depending on the substrate, starter culture can be chosen to enhance texture, nutritional value shelf life and machinability of the dough by formation of EPS, oligosaccharides, and the formation of organic acids.

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Figure 1. Utilization of glucose (○) and maltose (◊) and formation of lactate (●), acetate (▲), and ethanol (■) during wheat (A) and sorghum (B) sourdough fermentation by W. kimchii F28.

Figure 2. Formation of oligosaccharides in wheat (A) and sorghum (B) sourdough by W. kimchi F28 (1) and W. cibaria MG1 (2). suc sucose, mal maltose, pan panose, pan-(glu)n glucosylated panose, n degree of polymerization, IM isomaltose, IM3 isomaltotriose, IMO isomaltooligosaccharides

References Arendt EK, Ryan LAM, Dal Bello F. Impact of sourdough on the texture of bread. Food

Microbiol 2007;24:165-174. Schwab C, Mastrangelo M, Corsetti A, Gä nzle MG. Formation of oligosaccharides and

polysaccharides by Lactobacillus reuteri LTH5448 and Weissella cibaria 10M in sorghum sourdoughs. Cereal Chem 2008;85:679-684

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Influence of Beta-glucan from Different Origins on the Quality of Gluten Free Breads

Anna-Sophie Hager1; Liam A.M Ryan1, John V. O’Doherty2, Elke K. Arendt1*

1Department of Food and Nutritional Sciences, University College Cork, Ireland 2School of Agriculture, Food Science and Veterinary Medicine, University College Dublin,

Ireland *corresponding author email: e.arendt@ucc.ie

Introduction. Celiac disease is one of the most common food intolerances worldwide. At presence the gluten-free diet remains the only suitable treatment. (Thompson, 2000) reported that in general celiac patients have an unbalanced diet, relating to an excessive consumption of energy, proteins, and fats, and a reduced intake of fibre. Thusly, the incorporation of dietary fibre into gluten-free baked products is critical. Among the possible sources of fibre, the neutral cell wall polysaccharide beta-glucan has outstanding functional and nutritional properties. Different physiological effects of beta-glucan are related to its viscosity: lowering of serum cholesterol levels as well as attenuation of postprandial plasma glucose and insulin responses (Skendi et al. 2002). The aim of the present study was to investigate how beta-glucan, originating from oats, yeast and brown algae, could affect baking quality parameters of a gluten-free bread formulation. Methods. In this study, the farinograph was found to be unsuitable for the determination of the water addition level required for the various enriched gluten free batters. Thusly, the level of water addition was determined based on small deformation rheological measurements at constant strain (0.01%) and frequency (10Hz) as previously reported by Nunes et al. (2009). The resultant water levels were utilized for the production of gluten free bread. The loaf volume was determined using the rapeseed displacement method. The crust colour was measured with a Chroma Meter (Minolta CR-300, Japan) and expressed as value according to the CIE L*a*b* colour system. Texture profile analysis (TPA) was performed using a TA-XT2i texture analyser (Stable Micro Systems, Surrey, UK) equipped with a 25 kg load cell and a 20 mm aluminium cylindrical probe. The settings used were a test speed of 5 mm/s with a force of 0.98 N to compress the middle of the breadcrumb to 50 % of its original height. Results. Water is an essential factor affecting the rheological behaviour of gluten free batters. This is most evident during proofing, where expansion and gas holding capacity is dependent on batter elasticity and resistance to deformation. Furthermore, water content influence the quality characteristics of the final bread, namely texture, visual appearance, flavour and staling behaviour. It is well known that dietary fibres can bind high amounts of water. Therefore, the water level of the gluten free batter had to be adjusted after the addition of inulin or beta-glucan respectively, resulting in the water levels shown in table 1.

Dietary fibre Level of dietary fibre [%] Water level [%] Control 0.0 90.0Inulin 9.0 83.0Oat beta-glucan 5.6 132.0 Yeast beta-glucan 0.6 92.7 Algal beta-glucan 0.6 91.3

Table 1. Level of water and dietary fibre addition to the gluten free batters given in % of flour/starch base.

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The four added dietary fibres had variable effects on the quality parameters of the gluten free breads. The colour of the gluten free bread crust varied significantly. The addition of inulin as well as yeast beta-glucan led to darkening of the crust. On the contrary, the addition of oat beta-glucan resulted in a crust lighter than the control. Finally, the addition of beta-glucan originating from algae did not show an effect. Figure 1 shows the crumb hardness values of breads at day 0, 2 and 5 of storage. It can be seen that incorporation of oat beta-glucan led to a significant softening of the bread, as indicated by lower crumb hardness values. On the contrary, the addition of inulin resulted in higher crumb hardness. Supplementation with yeast and algal beta-glucan did not significantly affect crumb hardness. Moreover, the addition of dietary fibre did not have a significant effect on loaf volume. Interestingly, loaves with beta-glucan from algae showed a slight increase in volume.

0

5

10

15

20

25

30

35

40

45

day 0 day 2 day 5

crum

b ha

rdne

ss [

N]

Figure 1. Crumb hardness of breads enriched with ß-glucan from various origins over 5-days storage period: yeast ß-glucan (horizontal lines bars); algal ß-glucan (diagonal line bars); oat ß-glucan (solid white bars); inulin (solid gray bars); control (vertical lines bar). Conclusions. The inclusion of beta-glucan from different sources can have positive impact on the nutritional value of gluten free bread as well as its quality. This study clearly showed that beta-glucan isolated from various sources can successfully be included into gluten free baked products, enhancing the dietary fibre content without having a diminishing effect on bread quality parameters. In the case of oat beta-glucan, incorporation results in an improvement of baking characteristics. The commonly used dietary fibre inulin increased crumb hardness. Whereas the addition of oat beta-glucan showed the opposite effect and resulted in breads with a softer crumb and a lower rate of staling. Overall the use of beta-glucan seems to be a promising way to increase dietary fibre intake of gluten free baked products. References Nunes M.H.B, Ryan L.A.M, Arendt E.K. Effect of low lactose dairy powder addition on the

properties of gluten-free batters and bread quality. European Food research and technology 2009; 229:31-41

Thompson T. Folate, iron and dietary fibre contents of the gluten-free diet. Journal of The American Dietetic Association 2000; 100:1389-1395

Skendi A, Biliaderis C.G, Lazaridou A, Izydorczyk M.S. Structure and rheological properties of water soluble β-glucans from oat cultivars of Avena sativa and Avena bysantina. Journal of Cereal Science 2003; 38:15-31

122

Effects of Two-step Transamidation of Wheat Flour and Semolina on the Technological Properties of Gluten

Federica Capobianco1, Salvatore Moscaritolo1, Mauro Rossi2*

1 IPALC Research & Development Laboratories, Frigento-AV, Italy. 2 Institute of Food Sciences, National Research Council, Avellino, Italy.

*corresponding email: mrossi@isa.cnr.it Introduction. Celiac disease (CD) is characterized by activation of intestinal gluten-specific CD4+ T cells. In particular, gluten becomes a better T cell antigen following deamidation catalyzed by tissue transglutaminase (tTG) (Molberg et al. 1998). We reported that a preventive transamidation of gliadin by a single incubation of wheat flour with microbial TG (mTG) and lysine methyl ester (K-CH3), completely inhibited the IFN-γ expression of intestinal gliadin-specific T cell lines from CD patients (Gianfrani et al. 2007). More recently, we showed that a protracted intake of this transamidated gluten was tolerated in a subset of CD patients (Mazzarella et al. 2009). The present work investigated the effects of a two-step transamidation process of wheat flour and semolina on gluten properties. Methods. T. aestivum flour and T. durum semolina were from IPAFOOD srl (Frigento-AV, Italy). Their dry gluten content was determined according a standard method (ICC 137). Food-grade microbial transglutaminase (mTG) was from Ajinomoto Foods (Hamburg, Germany; ACTIVA®WM; 81-135 U/g); K-CH3 was from SISCO (Sisco Research Laboratories Pvt. Ltd, Mumbai, India). Flour or semolina was suspended in two volumes of water containing 8 U/g flour mTG and 20 mM K-CH3. Incubation was performed in two steps: first step, 2 hr at 30°C; second step, 3 hr at 30°C with fresh enzyme and K-CH3. The suspension was centrifuged and dough recovered. A bread baking procedure was tested with 500 g of dough mixed with 3.0 g table salt, 1.0 g olive oil and 10.0 g baker's yeast. A pasta making machine was used to make pasta (spaghetti) in a size of 200 mm long, 1.75 mm thick. Pasta was dried according to the following schedule: 90°C, 83% relative humidity (rh) 3 hrs; 63°C, 73 % rh 1 hr; 40°C, 70% rh 90 min, room temperature 48 hrs. The prolamin content in transamidated products was determined by R5-ELISA (Istituto Ricerche Agrindustria. Modena, Italy). Results. We found that the gluten content in transamidated bread, determined by R5 ELISA, drastically decreased from 1102.7 mg/kg after the single step to 5.8 mg/kg after the two-step reaction (Table 1). A similar result was obtained for transamidated semolina (Table 1), suggesting that prolamins were extensively masked following a two-step transamidation process. The transamidated wheat bread had wheat-like flavour, brown crust color and crumb structure similar to control bread (Figure 1A). However, the specific volume was found lower than in control bread (2.21 vs. 2.69 ml/g, transamidated vs. control). Similarly, a dried pasta was produced with transamidated semolina. The water uptake of transamidated pasta following cooking was found comparable to that of untreated pasta (146% vs. 149%; transamidated vs. control).

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Table 1. Gluten concentrations of final products.

Gluten (mg/Kg) untreated wheat bread (ICC137) 72000 + 1000

one-step transamidated bread (R5-ELISA) two-step transamidated bread (R5-ELISA)

1102.7 + 34.0 5.8 + 0.8

untreated semolina (ICC137) transamidated semolina (R5-ELISA)

115000 + 1150 15.0 + 2.3

Figure 1. A), bread prepared by using untreated (left) or two-step transamidated wheat flour (right). B) dried pasta (spaghetti) prepared using a two-step transamidated wheat semolina. Conclusions. This study showed that the two-step transamidation process of wheat flour or semolina was able to completely block the immune recognition of wheat prolamins by R5 monoclonal antibody. This treatment did not hamper the main technological properties of gluten, as good quality bread and dried pasta were produced. The safety for all celiac patients of a two-step transamidated wheat is currently under investigation. References Molberg O, McAdam SN, Korner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H,

Noren O, Roepstorff P, Lundin KE, Sjostrom H, Sollid LM. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4:713-717.

Gianfrani C, Siciliano RA, Facchiano AM, Camarca A, Mazzeo MF, Costantini S, Salvati VM, Maurano F, Mazzarella G, Iaquinto G, Rossi M. Transamidation inhibits the intestinal immune response to gliadin in vitro, Gastroenterology 2007;133:780–789.

Mazzarella G, Salvati VM, Iaquinto G, Capobianco F, Stefanile R, Giardullo N, Malamisura B, Rossi M. Transamidation of wheat flour: a new enzyme strategy to block gluten toxicity in a subset of celiac disease patients. GASTRO ’09, London, UK. Gut 2009; 58 (Suppl II) A80.

124

The Effect of Deletion Lines of Bread Wheat ‘Chinese Spring’ on Celiac Disease Stimulating Epitopes and

Technological Properties

Hetty C. van den Broeck1∗∗∗∗, Hein C. de Jong2, Liesbeth Dekking3, Dirk Bosch1, Rob J. Hamer4, Marinus J.M. Smulders1, Ludovicus J.W.J. Gilissen1, Ingrid M. van der Meer1

1Plant Research International, Wageningen UR, PO Box 16, 6700 AA Wageningen, The Netherlands

2 Limagrain Nederland B.V., P.O. Box 1, 4410 AA Rilland, The Netherlands 3Leiden University Medical Center, PO 9600, 2300 RC Leiden, The Netherlands

4Laboratory of Food Chemistry, Wageningen UR, PO Box 8129, 6700 EV Wageningen, The Netherlands

∗corresponding email: hetty.busink@wur.nl

Introduction: Celiac disease is a T-cell mediated inflammatory response of the small intestinal mucosa occurring in genetically susceptible individuals after ingestion of specific gluten proteins from wheat, rye and barley. Gluten proteins comprise several protein families and are encoded by 15 major multigene loci present on the homoeologous chromosomes 1 and 6 of the three homologous genomes (A, B and D) of hexaploid bread wheat (Triticum aestivum). The HMW-GS are encoded by the Glu-1 loci on the long arm of group 1 chromosomes. The LMW-GS are mainly encoded by the Glu-3 loci on the short arms of group 1 chromosomes and are tightly linked to the loci encoding the γ-gliadins (Gli-1) and ω-gliadins (Gli-3). Most α/β-gliadins are encoded by the Gli-2 loci on the short arms of group 6 chromosomes. Deletion lines of T. aestivum cv. Chinese Spring (CS) were selected having specific deletions on the short arms of group 1 and 6 chromosomes (Endo and Gill, 1996; Qi et al., 2003) Objectives: Develop wheat low in T-cell stimulatory epitopes while retaining its technological properties. Selection of deletion lines reduced in T-cell stimulatory epitopes and relatively good technological properties to perform crossing experiments to obtain accumulation of deletions for further reduction of T-cell stimulatory epitopes. Use flour of deletion lines to test the possibility to restore the technological properties by addition of oat avenins, which are similar to wheat gliadins but not CD-stimulating. Methods: The effect of deleting individual gluten loci on both the reduction of the amount of T-cell stimulatory epitopes and the technological properties of wheat dough was analyzed. The reduction of T-cell stimulatory epitopes was analyzed by immunoblotting using the monoclonal antibodies Glia-α9 and Glia-α20 that recognize important α-gliadin epitopes. The deletion lines were technologically tested with respect to dough mixing properties (2g-mixograph), dough stress relaxation (Advanced Rheometer AR2000), and dough extensibility (TA.XT2i texture analyzer). Deletion lines 1DS-5 and 1BS-19/6DS-4 were used in crossing experiments. Results and Discussion: Immunoblotting showed that deletion lines 1DS-5 and 1BS-19/6DS-4 missed diverse gluten proteins containing the T-cell stimulatory epitopes Glia-α9 and Glia-α20 (Figure 1).

125

B

CS 31 2 54 6

C

CS 31 2 54 6

A

CS 31 2 54kDa

116.3-97.4-

66.2-

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ω-gliadins

D-type LMW-GS

B-, C-LMW-GS

α/β-,γ-gliadins

HMW-GS

6

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CS 31 2 54 6

C

CS 31 2 54 6

A

CS 31 2 54kDa

116.3-97.4-

66.2-

45.0-

31.0-

ω-gliadins

D-type LMW-GS

B-, C-LMW-GS

α/β-,γ-gliadins

HMW-GS

ω-gliadins

D-type LMW-GS

B-, C-LMW-GS

α/β-,γ-gliadins

HMW-GS

6

Figure 1. Analysis of Chinese Spring deletion lines. (A) SDS-PAGE gel (10%) stained with PageBlue. (B) Immunoblot using mAb Glia-α9. (C) Immunoblot using mAb Glia-α20. CS is Chinese Spring wild type. Deletion of the short arm of chromosome 1D, containing loci encoding ω-gliadins, γ-gliadins, and LMW-glutenins, reduced the number of T-cell stimulatory epitopes and increased dough elasticity. Deletion of the short arm of chromosome 6D, containing the α-gliadin locus, resulted in a significant decrease in T-cell stimulatory epitopes, and in a change in technological parameters. The elasticity of the dough decreased, but, in contrast to the poor baking quality of CS wt, the dough strength improved. This decreased elasticity could be related to the deletion of loci encoding α-gliadins. Compensation of this loss of α-gliadins by addition of non-CD-toxic monomeric proteins from oat to the flour increased dough strength. Crosses were performed between deletion lines. The progeny plants carrying both 1DS-5 and 6DS-4 deletions showed a reduction of many T-cell stimulatory epitopes (Figure 1). New deletion lines were obtained from the crossing experiment: 6DS-2 and 1BS-19. Their technological properties are under investigation. Conclusions: The results demonstrate that a breeding strategy towards CD-safe bread wheat with good technological properties is feasible by deletion of the gluten proteins encoded by the Gli-D1/Glu-D3 loci on the short arm of chromosome 1D and the Gli-D2 locus on chromosome 6D. These deletions can remarkably improved dough quality. Addition of oat avenins allowed to compensate for the loss of wheat gliadins and was observed to improve dough strength. References Van den Broeck HC, van Herpen TJWM, Schuit C, Salentijn EMJ, Dekking L, Bosch D,

Hamer RJ, Smulders MJM, Gilissen LJWJ, van der Meer IM. Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines. BMC Plant Biol 2009;9:41.

Endo TR, Gill BS. The deletion stocks of common wheat. J Hered 1996;87:295-307. Qi L, Echalier B, Friebe B, Gill B: Molecular characterization of a set of wheat deletion

stocks for use in chromosome bin mapping of ESTs. Funct Integr Genomics 2003;3:39-55.

126

Physicochemical properties of oat varieties and their potential for bread making

Edith K. Hü ttner1, Fabio Dal Bello1 and Elke K. Arendt1,*

1Department of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, Ireland

*corresponding email: e.arendt@ucc.ie Introduction. Oat (Avena sativa) is an important cereal crop all over the world which is primarily used as livestock feed. Oats have received increased interest for human nutrition, as a consequence of their dietary benefits, their low allergenicity and their suitability for most celiac patients (FDA 1997; Welch 1995). Thus, the development of oat bread could enhance oat consumption, satisfy the consumer demand for novel and healthy foods and increase the range of products suitable for people suffering from celiac disease. However, to date, oat varieties have not been developed specifically for the production of bread. Consequently, a principal understanding of oat properties is necessary to achieve the desired bread quality. The objectives of this study were to establish whether certain oat varieties yield better quality bread than others and to determine the physicochemical properties essential for oat bread making. Methods. Six different spring oat varieties (Typhon, Ivory, Buggy, Nord 08/311, Energie, Zorro) were selected for examination. After milling, the flours were characterised by measuring moisture, ash, protein, starch, amylose, fat, dietary fibre and β-glucan content. Starch damage and water hydration capacity as well as enzymatic activities were also established and the flour pasting properties were investigated using a Rapid Visco Analyser. Moreover, capillary gel electrophoresis was for measuring the protein profiles of the different oat varieties. In order to determine the bread making potential of the varieties a simple wheat-free recipe was developed. The rheological properties of the oat batters were studied by applying small amplitude oscillatory shear measurements within the linear visco-elastic region and the breads were analysed using standard bread analysis methods. Table 1: Characterisation of flours produced from the different oat varieties. Typhon Ivory Buggy Nord 08/311 Energie Zorro

Ash (% db) 2.01 ± 0.04 2.13 ± 0.05 1.99 ± 0.03 2.17 ± 0.05 2.19 ± 0.11 1.97 ± 0.04

Moisture (%) 13.60 ± 0.04 13.26 ± 0.03 13.33 ± 0.03 12.84 ± 0.06 12.38 ± 0.01 13.93 ± 0.03

Protein (% db) 13.14 ± 0.11 13.40 ± 0.03 10.61 ± 0.25 14.71 ± 0.25 16.49 ± 0.31 12.38 ± 0.13

Fat (% db) 3.81 ± 0.09 6.16 ± 0.20 6.07 ± 0.17 6.12 ± 0.01 10.43 ± 0.15 5.07 ± 0.09

Total starch (% db) 71.87 ± 2.78 67.50 ± 1.49 67.64 ± 0.96 64.44 ± 1.08 56.99 ± 0.91 63.49 ± 0.53

Starch damage (% db) 2.12 ± 0.01 1.92 ± 0.02b 1.95 ± 0.08b 2.15 ± 0.05 1.54 ± 0.00c 2.58 ± 0.03

Amylose (% db) 32.98 ± 0.11 26.93 ± 0.41 27.27 ± 0.41 25.67 ± 0.23 24.14 ± 0.32 25.12 ± 0.27

β-glucan (% db) 4.15 ± 0.07 3.43 ± 0.04 3.69 ± 0.13 4.14 ± 0.17 4.22 ± 0.14 4.14 ± 0.19

Dietary fibre (% db) 14.84 ± 0.24 14.28 ± 0.38 12.60 ± 0.38 16.95 ± 1.05 14.37 ± 0.63 15.33 ± 1.13

Water hydration capacity (ml/g)

0.74 ± 0.01 0.63 ± 0.02 0.68 ± 0.01 0.77 ± 0.01 0.71 ± 0.01 0.76 ± 0.00

127

Results. Flour analysis revealed significant differences in the ratio of the flour constituents (Table 1). Overall, the oat varieties varied considerably in their starch, fat and protein content. However, the protein size distribution was the same of all oat varieties. Similarities in the pasting properties were observed for Buggy, Energie and Zorro, as well as Typhon, Ivory and Nord 08/311. Rheological analysis revealed that oat batters made from Buggy, Energy and Zorro were softer compared to Typhon, Ivory and Nord 08/311. Bread analysis showed differences in breads made from the oat varieties (Figure 1). According to the visual appearance of the crumb Buggy, Energie and Zorro showed an even gas cell distribution and consequently good bread quality. In contrast, breads made from Typhon, Ivory and Nord 08/311 had a hole in the centre of the crumb and accordingly poor quality (Figure 1).

Figure 1: Pictures of breads made from oat variety Typhon (A), Ivory (B), Buggy (C), Nord 08/311 (D), Energie (E), Zorro (F). Discussion. This study was designed to compare different oat varieties under standardised conditions and to provide a system for understanding the basis of oat bread quality. Altogether, the largest difference in bread quality among the oat varieties was found in the crumb characteristics (Figure 1). Oat varieties showing lower batter resistance to deformation such as Buggy, Energie and Zorro resulted in better bread quality. Moreover, oat varieties with low protein content such as Buggy and Zorro were established to be suitable for the production of oat bread. High amounts of proteins in oat flour interfere with starch by disrupting the uniformity of the starch gel during baking. Interestingly, the variety Energie showed desirable bread characteristics, although it had the highest protein content. However, Energie also had the highest fat content which indicates that oat lipids positively affect the bread making properties of oat varieties. In addition, starch pasting properties such as high setback and final viscosity as observed for Buggy, Energie and Zorro were found to be essential in order to obtain superior oat bread quality. Due to the fact that the oat varieties were not heat treated enzymatic activities were investigated. Overall, no correlation was found between oat bread quality and enzymes such as β-amylase, β-glucanase, protease or peroxidase. However, α-amylase activity negatively affected oat bread quality. References Welch RW. The Oat Crop. Production and Utilization. Chapman and Hall, London.1995 p. 433-471 FDA. Food labelling: health claims. Federal Register 1997: 62, 3583-3601.

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128

Effects of Basic Process Parameters on Quality of Gluten-free Rice Bread

Gina Jaspers, Markus J. Brandt*

Ernst Böcker GmbH & Co. KG, Minden, Germany

*corresponding email: markus.brandt@sauerteig.de

Introduction. Recipes of gluten-free breads are often complex: They are based on starches and

hydrocolloids or fibre components for water binding; include one or more protein sources

(e.g. soy, milk or egg) and sometimes additional emulsifiers are in use. It was the aim of our

study to investigate the effects of basic process parameters (salt, dough yield, hydrocolloid

concentration) on a simple bread recipe based on rice flour.

Methods. The basic baking recipe consists of rice flour, yeast (3%), salt, xanthan and water. If

a sourdough was used it was started with a commercial rice starter containing Lactobacillus

plantarum, L. fermentum, L. paracasei, L. helveticus, L. paralimentarius, Leucostonoc

argentinum and Saccharomyces pastorian0us. The production process standard conditions

were: Dough mixing with a spiral kneader, proofing for 30 minutes at 31°C and 85% relative

humidity, bread baking at 230°C in a deck oven. After 24h of cooling, bread crumbs were

characterised by texture-profile-analysis (TPA): Test speed 0,80 mm/sec, crumb compression

of 20%, bread slice thickness 1,5 mm. The specific volume was measured by rapeseeds

displacement.

Results. Pure rice breads did not show that typical bread characteristics, comparable to the

crumbs of wheat or rye bread. The crumbs were too brittle and sticky. Therefore we added

buckwheat flour in varying proportions. The effects on the bread crumb are depicted in Figure

1.

buckwheat flour (%)

0 20 40 60 80 100

cru

mb h

ard

ness (

N)

0

10

20

30

rice flour (%)

020406080100

cru

mb e

lasticity (

%)

80

85

90

95

100

Figure 1. Effects of varying amount of buckwheat concentrations on bread crumb hardness

(-▲-) and crumb elasticity (-●-).

129

The combination with buckwheat resulted in more elastic bread crumbs. Therefore further

experiments were performed with a mixture of 70% rice flour and 30% buckwheat flour.

High salt concentrations inhibited the baker’s yeast metabolism whereby the specific bread

volume decreased. The effect of xanthan depends mainly on the available water. As expected,

for xanthan concentration >1% higher dough yields are necessary to obtain satisfactory bread

quality. For breads without xanthan, the structure is very instable (Figure 2).

0

2

4

6

8

10

12

14

190

200

210

220

230 0,00,5

1,01,5

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ha

rdn

ess (

N)

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[Xanthan] (% on flo

ur)

Figure 2. Combined effects of xanthan and dough yield on crumb hardness of rice /

buckwheat breads (70/30)

Conclusion. Complex recipes for an acceptable gluten-free bread quality are not always

necessary. Especially for artisan bakeries, simple bread recipes using flour of gluten-free

cereals instead of starches, the right amount of water and sourdough for an improved flavour

may be an alternative.

130

Casein Network Formation in Gluten Free BreadSheila Kenny1*; Eimear Gallagher2, Mark A.E. Auty1, Brendan T. O’ Kennedy1

1Moorepark Food Research Centre, Teagasc, Fermoy, Co. Cork, Ireland2Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15.

*corresponding email: sheila.kenny@teagasc.ie

IntroductionCasein and gluten form gel networks with covalent (disulphide) and co-ordination (withcalcium) links, respectively. Under the correct conditions of pH and ionic strength, casein canform aggregated casein networks (Stathopoulos and O’Kennedy 2008). The aim of this workwas to investigate the conditions required to produce a casein network with properties similarto gluten in a gluten free dough system.

MethodsAcid casein was used in gluten free dough formulations to give a protein level of at a level of8.3% (w/w). Calcium levels were 0mg, 17mg and 34mg calcium/g casein. pH levelsinvestigated were 5.8, 6.2, 6.6 and 7. pH and calcium level were controlled by appropriateaddition of calcium hydroxide, calcium chloride and sodium hydroxide. Standard baking testswere performed and confocal scanning laser microscopy was used to determine the extent ofprotein network formation in bread. Dynamic oscillation tests were carried out on yeastlessdoughs in the temperature range 25 to 90°C .

ResultsConfocal scanning laser micrographs showed no visible protein network in formulations with0mg calcium/g casein (Fig 1a). In contrast, good protein network formation was visible with17mg calcium/g casein and a dough pH of 6.2 (Fig 1b). The elastic modulus, G', of the 17mgcalcium pH 6.2 formulation increased in the range from 35 to 55°C, indicating the presence ofa protein network that strengthens on heating and is capable of gas retention (Fig. 2).Increasing calcium level to 34mg calcium/g casein resulted in a decrease in G' in the rangefrom 35 to 55°C, indicating a weakening of the protein network on heating. Formulations with0mg calcium had lower G' than 17mg calcium formulations and G' increased on heating. 0mgcalcium doughs were sticky and difficult to handle whereas formulations with 17mgCalcium/g casein were most cohesive.

Figure 1.Confocal scanning laser micrographs (a) 0mg calcium/g casein, pH 6.2. (b) 17mg calcium/gcasein, pH 6.2.

(a) (b)

Proteinnetwork

Starch

131

Figure 2.Elastic Modulus (G') profiles of gluten free dough formulations

1.00E+04

1.00E+05

1.00E+06

1.00E+07

25 35 45 55 65 75 85

Temperature (°C)

Ela

stic

Mod

ulus

G'(

Pa)

0mg calcium, pH 6.217mg calcium, pH 5.817mg calcium, pH 6.234mg calcium, pH 5.834mg calcium, pH 6.2

ConclusionsUnder optimum conditions of pH and calcium concentration, casein aggregates and forms aprotein network capable of retaining gas in gluten free dough. The formulation with 17mgcalcium and pH 6.2 had an extensive protein network and dynamic oscillation testingindicated strengthening of this network on heating. Decreases in G' observed by reducing thepH to 5.8 and increasing calcium level to 34mg calcium/g casein indicate that theseconditions result over-aggregation of casein and a reduction in gas retention properties of thecasein network. The G' profile of the 0mg calcium formulation indicates that sodium caseinateacts as a stabiliser in gluten free dough.

References

Stathopoulos, C.E. and O'Kennedy, B.T. A rheological evaluation of concentrated caseinsystems as replacement for gluten: calcium effects. International Journal of Dairy Technology2008; 61: 397-402

Stathopoulos, C.E. and O'Kennedy, B.T. The effect of salt on the rheology and texture of acasein based ingredient intended to replace gluten. International Journal of Dairy Technology2008; 63 430-433

132

Effect of microbial homopolysaccharides on the structure

of gluten-free breads

Christine Rühmkorf1, Susanne Kaditzky

1* and Rudi F. Vogel

1

1Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Germany,

Freising

*corresponding email: susanne.kaditzky@wzw.tum.de

Introduction. Due to the lack of viscoelastic properties, hydrocolloids and further additives are

added to gluten-free batters. Microbial exopolysaccharides (EPS) are able to serve as

biothickeners and can be added to several food products (Waldherr and Vogel, 2009).

Especially homopolysaccharides, composed of either glucose or fructose, are known to

improve the quality of wheat bread. So far, only potential effects of EPS on gluten-free breads

are reported. Therefore, in this study three different types of EPS, one levan and two dextrans,

were analysed for their impact on the structure of gluten-free bread.

Methods. Doughs were prepared from rice and buckwheat flour with a dough yield of 208 and

1 % (flour base) EPS was added. The EPS had been purified from fermentations with

L. sanfranciscensis (levan), L. reuteri (dextran) and L. curvatus (dextran). As control, doughs

with and without xanthan were made. All doughs were chemically acidified. The breads made

of the doughs were analyzed by TPA and the specific volume was determined. Furthermore a

sensory analysis was done.

Results. The pore distribution of the crumb was best with added xanthan. Still, breads with

EPS from L. curvatus showed the lowest specific volume of the breads with solved EPS, and

the best pore distribution and the least crumb hardness. The mode of application of the

purified EPS appeared as crucial with respect to the baking results observed. EPSs added

lyohilized produced non-uniform doughs with small hardly dissolving nuggets, and the

resulting breads did not differ much from those without EPS addition. Dissolving and

swelling EPSs prior addition to the dough produced a more uniform crumb with fewer holes.

All of the three added EPS caused a juicy clumping mouthfeel. The surface of the breads with

EPS was splintered. Furthermore, all the three strains were able to grow in buckwheat and

rice sourdoughs, except for L. sanfranciscensis, that only grew in buckwheat doughs.

Conclusions. The EPS from L. curvatus shows the best hydrocolloid character and is a promising candidate to replace or reduce the amount of hydrocolloids in gluten-free breads.

Dissolved and swollen EPS was more effective than freeze dried preparations. Therefore in

situ production of EPS in predoughs appears to be the most promising way to cheaply and

effectively apply bacterial EPS, and advertise “clean label” products.

References

Waldherr F, Vogel RF. 2009. Commercial exploitation of homo-exopolysaccharides in non-

dairy food systems. In: Ullrich, M. (ed.) Bacterial polysaccharides – current innovations

and future trends. 313-344.

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Notes

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Fermented whey-based diary dessert stabilized with starch from gluten-free source Bulgaru Viorica, Dupouy Eleonora

Technical University of Moldova, Department of Food Science and Nutrition, 168 Stefan cel Mare boulevard, MD-2004, Chisinau, Republic of Moldova,

tel: (+373 22) 509 959, e-mail: viorica.bulgaru@yahoo.com

Introduction

In response to the existent need to increase the availability of foods for consumers sufferring from the celiac disease, the food industry is developing new assortments of foods from various gluten-free sources. Since starch is widely applied in food industry as a functional ingredient, in the present paper is investigated the possibilities to use the soryz starch isolated from a gluten-free source, Sorghum Oryzoidum grains, as a stabiliser in the production of a whey-based diary dessert. Sorghum Orysoidum is a sorghum hybrid obtained in the Republic of Moldova. The starch isolated from the Sorghum Oryzoidum grains is a gluten-free ingredient that makes possible to recommend it for the applications in the production of gluten-free foods for celiacs.

Objectives

The investigations presented in this paper have the following objectives: To investigate the possibility to use soryz starch as a functional stabilizing ingredient;

To determine the quality indices of the dairy whey-based dessert stabilized with soryz starch;

To develop the assortment of new gluten-free stuffs for the food industry.

Metods

The quality indices of the whey-based dairy dessert stabilized with soryz starch were determined by standard methods [1,2].

Results and discussion

Sensorial and physico-chemical indices of quality of the whey-based dairy dessert samples with different concentrations of soryz starch were analysed and the receipt with the best characteristics was identified. The following characteristics and indices of whey-based dairt dessert stabilized with soryz starch were determined: appearance and consistency, taste and

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flavour, colour, pH, total acidity, viscosity, dry substance, inverted sucrose, fat. The total acidity of the fermented whey-based dairy dessert with soryz starch increased gradually over the 5 days. This occurs since the starch is a favourable nutritive substrate for the development of the lactic bacteria in the product. The presence of soryz starch contributes to a more viscous, better consistency and tasty product – characteristics well appreciated by consumers.

Tabelul 1. Sensorial and physico-chemical characteristics of the whey-based dairy dessert stabilized with soryz starch

Indices

Fermented whey-based dairy dessert stabilized with soryz starch

Appearance and consistency Homogenous, semifluid consistent, uniform

Flavor and taste Sweet taste, specific flavor whey

Color Yellow

pH 4,26

Total acidityoT (period of validity) 34-39

Viscosity, s-1 1,38

Dry substances, % 9,88

Inverted sucrose, % 13,37

Fat, % 0

Conclusions

Soryz starch, along with the fact that it is obtained from a gluten-free source, has the advantage of being a food ingredient with useful functional properties, fact demonstrated in the present work on obtaining of a fermented whey-based dairy dessert. The soryz starch has influenced positively both the formation of the consistency and the structure of the product, as well as the term of the product validity duet to the favourable evolution of titrable acidity. The fermented whey-based dairy dessert stabilized with soryz starch is recommended in the celiacs diets due to the product’s curative properties and the presence of a functional

ingredient from gluten-free source.

References

Costin G., Florea T. Produse lactate fermentate. Galati, Romania. Academica. 2007.

Guzun V. Tehnologia laptelui şi a produselor lactate. Lucrări de laborator şi practice. Chişinău.

Civitas. 1998.

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Development of a new gluten-free brown bread flour mix rich in fiber and high in nutritional content

Virna Cerne and Ombretta Polenghi

Schär R&D Centre, AREA Science Park, Padriciano 99,I – 34012 Trieste The object of this study was the development of a new gluten-free brown bread flour mix that is rich in fibre and high in nutritional value, for people who are gluten-intolerant. Special emphasis was placed on the sensorial quality of the final product (taste, aroma, aspect and texture), as well as for the ease of its preparation at home by consumers. A number of raw materials were selected for testing due to their nutritional composition, for example the quantity of fibre and the presence of Omega-3 fatty acids. Among these, the raw materials chosen were those that from the baking tests performed resulted in bread having the best taste and aroma, and, at the same time, granting the best characteristics for its preparation. The final flour mix obtained provides for the best bread in terms of its softness, taste and aroma. The new brown flour mix for bread has been characterized, and is compared with the product from a wheat-rye flour mix, by analysis of the dough’s consistency (Mixolab-Chopin), analysis of the proofing properties of the dough (Rheofermentometer-Chopin), analysis of nutritional values, analysis of the textures (Texture Profile Analysis- TA-XT2), image and sensorial analyses of the breads. The results from these analyses are presented and indicate the validity of this brown bread flour mix in the diet of those who are gluten-intolerant.

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Notes

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Sensory and textural properties of gluten-free bread based on rice/buckwheat flour mixtures

Aleksandra Torbica1*; Miroslav Hadnañev1, Tamara Dapčević1, Marijana Sakač1

1Institute for Food Technology, Novi Sad, Serbia *corresponding email: aleksandra.torbica@fins.uns.ac.rs

Introduction. Gluten represents a major protein component of some cereals that is responsible for flour processing characteristics in bakery industry. However, gluten must be eliminated from the diet of patients suffering from celiac disease because its ingestion causes serious intestinal damage (Sciarini et al. 2008). Food product obtained using ingredients that do not contain prolamins from wheat or all Triticum species such as spelt, kamut or durum wheat, rye, barley, oats or their crossbred varieties with a gluten level not exceeding 20 ppm can be labelled as gluten-free food ( Gallagher et al. 2004). The present work investigated the sensory and textural properties of gluten-free bread samples prepared from rice/buckwheat flour mixtures without addition of hydrocolloids. Methods. Mixtures of rice flour (RF) and unhusked buckwheat flour (UBF) as well as of rice flour and husked buckwheat flour (HBF) were prepared. In both types of mixtures the ratio of rice flour to buckwheat flours was 90:10, 80:20, 70:30, respectively. Textural properties, bread firmness (AACC (74-09), of the final gluten-free products were investigated using Texture analyzer TA.XPplus (Stable Micro System, UK). Textural properties were determined 2 hours after baking and storage at room temperature and the obtained results were expressed as the hardness of the final product. Sensory analyses of gluten-free bread were carried out 2 h after baking by 10 trained panellists. The following sensory attributes were evaluated: taste, appearance, softness and flavour. For each parameter nine-point hedonic scale was used, ranging from 1 (dislike extremely) to 9 (like extremely). Products were found acceptable if their mean scores for the acceptability were above 5 (Lazaridou et al. 2007). Results. The obtained values of the works of compression for the products containing different proportions of RF and both buckwheat flours (Table 1) were not significantly higher for the samples prepared with UBF than for those containing HBF (p>0.05). Also, the resulted works of compression increased but not significantly (p>0.05) with increasing the BF content in the final product. Therefore, the increase in BF addition did not significantly affect the textural properties of the final product. Increase in the amount of UBF in the tested mixtures led to decrease in the scores for the taste and flavor. However, by increasing the amount of HBF from 10% to 20% taste properties significantly (P≤0.05) increased, due to the intensity of aromatic taste characteristic for HBF. Unlike the UBF, which possesses bitter taste predominantly found in the husk which is mainly removed during the processing, HBF containing products expressed more pleasant flavor and taste. Generally, samples containing HBF were scored better than the samples with UBF (Fig. 1). However, since all the gluten-free samples were scored by the mean number much higher than 6, their sensory properties were found to be more than acceptable

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Table 1. Textural properties of final gluten-free products containing husked buckwheat (HF) or unhusked buckwheat (UBF) flour A

Type of buckwheat flour

10% 20% 30%

Area (g.sec) HBF 3261±190a 4266±297ab 4439±247abc UBF 4121±348ab 4706±403bc 5597±823c

AValues represent the means± standard deviation; n=5. Values in table followed by different lower-case letters are significantly different from each other (p ≤ 0.05).

Figure 1. Sensory evaluation of the final gluten-free products containing: a) husked buckwheat flour, b) unhusked buckwheat flour and breadcrumb structure of the final gluten-free product

Conclusions. This study showed that it is possible to create gluten-free bread using the mixture of rice and buckwheat flour that does not require the addition of the hydrocolloids (xanthan, guar gum, HPMC etc.) for the dough structuration effect. Increasing both BF content resulted in minor increase of hardness value. According to sensory analysis increasing the amount of HBF in flour mixtures resulted in improved sensory properties. However, lower scores for the UBF containing mixtures were due to bitter compound present in the husk of the buckwheat seeds. Nevertheless, all samples were found to be acceptable according to results of the sensory analysis.

References

Gallagher E, Gormley RT, Arendt KE. Recent advances in the formulation of gluten-free cereal-based products. Trends Food Sci Tech 2004;15:143– 152. Sciarini SL, Ribotta DP, Leó n EA, Pérez TG. Influence of Gluten-free Flours and their Mixtures on Batter Properties and Bread Quality. Food Bioprocess Tech 2008; DOI 10.1007/s11947-008-0098-2 2008. Lazaridou A, Duta D, Papageorgiou M, Belc N, Biliaderis CG. Effects of hydrocolloids on dough rheology and bread quality parameters in gluten-free formulations. J Food Eng 2007;79:1033–1047.

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1

The positive effect of amaranth sourdough addition in gluten free bread quality

Andreas Houben1*; Martin Mitzscherling2, Thomas Becker1 1 TU München, Department of Beer and Beverages, Freising, Germany

2 University of Hohenheim, Department of Process Analysis and Cereal Technology, Stuttgart, Germany

*corresponding email: houben@wzw.tum.de Introduction. The worldwide increasing amount of people with celiac disease raises the need for gluten free products (Mustalathi et al. 2002). Especially in the world of baking this is still a big challenge for the bread manufactures. In most cases the used recipes are based on rice and maize flours. But even these gluten free flours do not really aim in all in bread production expected quality parameters. Next to these baking disadvantages the finally bread is also quite poor in its nutrition level. A way to increase nutrition can be the addition of pseusocereals. Pseudocereals are plants that do not belong to the family of grasses. Their seeds however, are handled like cereals. The most famous pseudocereals are amaranth, buckwheat and quinoa. One of their advantages is their high nutrition content from nature. Amaranth for example possesses a high-value amino acid combination for humans (Singhal, Kulkarni, 1988). The handling properties of pseudocereals are difficult; there baking quality is also poor by nature. There is no structure forming ingredient included in these pseudocereals. So it is not possible to produce high-volume loaf breads with only pseudocereals. Another reason, their use is quite low in bread production could be as well their always very special taste. A rheological, technological and sensorial interesting way to solve these disadvantages in use can be the fermentation of pseudocereals by stable and repeatable growing lactobacilli strains. In the present work the influence of these sourdoughs on dough rheological and gluten free bread quality was investigated. Methods. Rice flour, maize flour, maize starch and amaranth kernels were received from Davert GmbH (Senden, Germany). The amaranth kernels were milled into full corn flour of a maximum particle size of 250 µm. For fermentation used starter cultures were: Lactobacillus plantarum AL30 and L. paralimentarius AL28 from the Food Microbiology Department, University of Hohenheim, Germany. All sourdoughs were prepared at 30°C for 24 h using dough yield of 200. The basic recipe for dough and finally bread production includes 50% rice flour, 25% maize flour and 25% maize starch, dry yeast, salt, water, margarine, water and HPMC. Influence of the sourdoughs was measured via dough rheology and baking tests. Out of the baking tests there are sensorical tests done with a trained sensoric panel. Results. Both used sourdoughs showed more or less equal behavior. Up to an addition of 40% depending on the amount of flour amaranth fermented did not show a negative effect on the reached bread volume (figure 2). During storage tests the elasticity of the bread stayed more or less the same, the hardness of the bread decreased by the use of sourdough. In the same time the cohesion of the bread slices increased by increasing amount of sourdough (figure 2). In the sensorical tests it could be shown, that small amounts of sourdough were able to increase taste and acceptance of the bread up to the amount were the sour taste more or less covered any other taste.

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2

Figure 1. Influence of the addition of amaranth fermented by L. paralimentarius to the basic recipe in gluten free bread production during a storage test of 7 days

Figure 2. Total volume of the resulting bread after addition of different amounts of amaranth fermented by Lactobacillus plantarum (L.plant) and L. paralimentarius (L. para). Conclusions. In this study it was shown, that the use of fermented pseudocereals like amaranth could be a way to increase the quality and taste of gluten free bread. Next to the volume and elongation of the shelf life, even some benefits could be detected in the still existing disadvantages of gluten free bread. References Singhal, R.S., Kulkarni: Review: Amarants – an underutilized resource. International Journal

of Food Science and Technology 1988; 23: 125-139. Mustalathi, K.; Lohiniemi, S.; Collin, P.; Vuolteenaho, N.; Lappala, P.; Mäki, M.: Gluten-free

diet and qualità of life in patients with screen-detected celiac disease. Effective Clin. Practise 2002; 5; 105-113

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Discrimination between gluten-free bread formulations using near infrared imaging

Gerard Downey1,2*; Carlos Esquerre1,2, Eimear Gallagher1, Laura Alvarez1, Colm

O’Donnell2, Aoife Gowen2 1 Teagasc, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland

2 University College Dublin, Belfield, Dublin 4, Ireland *corresponding email: gerard.downey@teagasc.ie

Introduction. Gluten-free breads have undergone considerable development in recent years. Previous work (Alvarez et al., 2010) has reported the development and characterisation of a range of such breads based on rice and pseudocereal (amaranth, quinoa and buckwheat) formulations. As part of a larger investigation into the structure and proofing behaviour of these bread formulations, this initial study has focused on the possibility of discriminating between baked breads using near infrared (NIR) hyperspectral imaging. This technique enables the rapid and simultaneous collection of spectral and spatial information from a sample and is emerging as a powerful analytical tool which has potential for on- or at-line applications in the food industry. Methods. Four bread formulations were investigated; the control recipe involved rice flour and potato starch as dry ingredients while, in the other breads, potato starch was replaced by either buckwheat, quinoa or amaranth flour. Details of the baking procedure and sources for the raw materials are reported in Alvarez et al. (2010). Hyperspectral diffuse reflectance images were obtained using a pushbroom line-scanning instrument operating in the near infrared wavelength range (950-1650 nm, spectral resolution=7 nm, spatial resolution= 320 pixel x 450 lines (DV Optics Ltd, Padua, Italy)). Full details are given by Gowan et al. (2008). Results. Mean spectra for each of the 4 bread types are shown in Figure 1 with or without standard normal variate (SNV) pre-treatment. It is obvious that the main difference between these mean spectra are offsets which are reduced by the SNV treatment. Principal component analysis of the individual pixel spectra showed almost total overlap between scores of the individual bread types (data not shown). Discrimination of the hyperspectral images of the bread types was attempted using partial least squares (PLS) regression and a dummy Yvariable for each bread type. Using raw spectral data and low numbers of latent variables, effective models were developed to separate both quinoa and buckwheat breads from the control formulation; these results are shown in Figure 2 on the basis of predicted pixel values. In Figure 2, the bread type being modelled is given an arbitrary Y value equal to 1 with all other breads being given a value of zero. Clear separation of control and amaranth breads was not possible using this approach. In an extension, moderate segregation between these two bread types alone was achieved using PLS discriminant analysis of SNV-treated spectra. The explanations for this behaviour are currently under study. Conclusions. This preliminary work has shown the potential of NIR hyperspectral imaging to discriminate with varying degrees of success between four gluten-free bread types. This performance may be further improved by on-going chemometric analysis. Examination of the

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regression vectors for each of the models developed will facilitate interpretation of the molecular basis for this discrimination. References Alvarez, L.A., Arendt, E.K. and Gallagher, E. (2010). Baking properties and microstructure of pseudocereal flours in gluten-free bread formulations. European Food Research and Technology, 230 (3), 437-445. Gowen, A.A., O’Donnell, C.P., Taghizadeh, M., Cullen, P.J., Frias, J.M. and Downey, G. (2008). Hyperspectral imaging combined with principal component analysis for bruise detection on white mushrooms (Agaricus bisporus). J. Chemometrics, 22, 259-267.

Figure 1. Mean raw and SNV-treated reflectance spectra of four bread types (quinoa – magenta; buckwheat – green; control – red; amaranth – blue)

Figure 2. PLS regression models for binary classifications of selected bread type versus the rest (a) buckwheat – 3 latent variables; (b) quinoa – 5 latent variables; (c) control – 3 latent variables and (d) amaranth – 3 latent variables.

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Evaluation of Physically-Chemical Parameters of Gluten-Free Dumplings

Tatjana Rakcejeva*, Ilze Gramatina, Anastasija Fjodorova Latvia University of Agriculture, Department of Food Technology, Jelgava, Latvia

*corresponding email: tatjana.rakcejeva@llu.lv Introduction. Celiac disease is an immune-mediated disease, triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. The only treatment for celiac disease is a strict gluten-free diet for life (Pulido et al. 2009). Therefore, the main purpose of the food producers is to make new tasty gluten-free products with elevated nutritive value to enrich the menu of celiac cases. One of such products will be dumplings with chicken as stuffing. Dumplings are based on flour, potatoes, bread, and may include meat, fish, or sweets. They may be cooked by boiling, steaming, simmering, frying, or baking. Ingredients may be as a part of a filling, or mixed throughout the dumpling. Dumplings may be sweet, spicy or savoury. They may be eaten alone, in soup, with gravy, or in many other presentations (Ang et al. 1999). The main cereal ingredients for gluten free dumpling dough production can be corn and rice flour. Since rice flour is made from broken milled rice, their chemical composition is the same as that of whole rice. There are, however, varietals differences in protein, lipid, starch content, and the amylose and amylopectin ratio in starch (Bor 1991). However, the main products from dry-milled corns are corn grits, cornmeal and corn flour. The composition is typically 77– 79% starch, 7– 8% protein, less than 1% fats, ash and fiber (Smith et al. 2004). For the better water absorption extruded corn or rice flour can be used. Methods. White rice, yellow extruded corn flour from Joint Stock Company “ Ustukių Malūnas” (Lithuania), salt, egg powder, potato starch and water were used for gluten free dumpling preparation. Traditional dumplings were prepared for quality and technological properties comparison; as main ingredients wheat flour, egg powder, salt and water were used. As dumpling stuffing a sausage-chicken meat, salt, black pepper and onion were used. Traditional dumpling preparation technology was applied. For the quality control of dumpling dough total sugar content (Bertran method, the combustion of keton group boiling solution with Felling reagent), total protein content (ISO 5983), total fat content (ISO 6492), colour differences (in the colour system CIE L*a*b* were determined by means of “ ColorTec-PMC” equipment). For the quality control of stuffing total sugar (Bertran method), total protein (ISO 5983) and total fat content (ISO 6492) was evaluated. Results. It is known, that the main component for traditional wheat dough making is gluten. Gluten is often equated with the proteins of the wheat that are insoluble in water. It is a fact that glutenin and gliadin are the main constituents of gluten in terms of quantity and determines its basic character (Popper et al. 2006). Dough prepared for dumplings should be white, bright, transparent, and smooth and have cooking resistance (does not brake during boiling). These futures are largely dependent on the flour quality used for production (Ang et al. 1999). White rice flour and yellow corn flour have no gluten. Therefore, as results of our experiments show, it is not possible to produce springy dough using only flour and water. Therefore for better water absorption and elasticity potato starch and extruded corn flour were

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used. During experiments it was ascertained that total protein and fat content in both dumpling dough samples was similar (Table 1). However significant differences in total sugar content were found – the total sugar content of control dumpling dough was 2.14 times higher than in gluten free dumpling dough sample, it could be explained with higher sugar content in wheat flour. Chicken stuffing quality testing results show, that the total sugar content was 0.49%, total protein – 22.32% and total fat content – 1.80% in dry matter corresponding good meat quality. Table 1. Physically – chemical parameters of dumpling dough.

Color differences Dough sample

Total sugar content, %

Total protein content, %

Total fat content, % L* a* b*

Control 0.45 0.03 8.98 0.2 0.21 0.01 76.10 3.49 -0.83 0.73 15.51 1.91 Gluten free 0.21 0.03 8.90 0.2 0.23 0.01 64.94 1.33 0.54 0.41 20.73 1.97 The control dumpling sample having higher brightness value (Table 1) can be evaluated may be as favourable and marketable product with response to colour quality, however, darker colour of gluten-free dumplings can be explained with darker colour (the mild yellowness value was higher) of added yellow extruded corn flour. But for the consumers who prefer corn flour in diet the darker colour of dumplings will be acceptable too.

Figure 1. Dumplings with chicken meet stuffing: control (left) and gluten-free (right). Conclusions. This study shows that a combination of gluten-free white and yellow extruded corn flour can be used for gluten-free dumpling dough production. Significant changes in total sugar content was found – the total sugar content of control dumpling dough was 2.14 times higher than in gluten free dumpling dough sample. Total protein and fat content in both dumpling dough samples was similar, respectively 8.98 0.02% and 0.21 0.01%. The control dumpling sample has higher brightness at the same time the mild yellowness value of gluten-free dumplings was higher. This research has been prepared within the framework of the ESF Project „ Formation of the research group in food science” , Contract Nr. 2009/0232/1DP/1.1.1.2.0/09/APIA/VIAA/122” . References Ang KYW, Liu KS, Huang YW, Asian Foods: Science & Technology. Tchnomic publication;

1999. p. 100-103. Bor SL, Rice utilization. Volume 2. Second edition. ISBN 0-442-00485-0; 1991. p. 10-11. Popper L, Schä fer V, Freund W, Future of Flour. AgriMedia; 2006. p. 6-7. Pulido OM, Gillespie Z, Zarkadas M, Dubois S, Vavasour E, Rashid M, Switzer C, Benrejeb S.

Introduction of Oats in the Diet of Individuals with Celiac Disease: A Systematic Review. J. Advances in Food and Nutrition Research 2009;57:235-285.

Smith WC, Betrà n J, Runge ECA, Corn. Origin, history, technology, and production. Wiley; 2004. p. 867-870.

±±± ±

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146

Novelty Formula of Free Gluten Pocket Type Flat Arabic Bread

Hanee M. Al-Dmoor

Al-Balqa Applied University, Faculty of Technological Agriculture, Department of

Nutrition and Food Processing, Al-Salt, Jordan-mail: dmour@bau.edu.jo

Introduction; Middle Eastern countries consumed with the entire food pocket flat bread. A correct balance of visco-elastic properties is an important during flat bread making. Objectives; The aim of the study was formulating a wheat flour substitute for production of free-gluten pocket flat bread. Methods; Deferent trials carried out for setting up the best formula. The formula composed of 25% rice flour ,42 % corn starch , 5 % potato flours , 10 % potato starch, 5 % milk , 5 % whey powder, 2 % guar gum, 1.5 % yeast, 2 % salt, 1.5 % sodium bicarbonate and 1% plant oil. Water was add in amounts 35 % of mixture weight to optimize the dough making and mixed for a 15 min to obtain the parameters that required during the handling of dough before baking which takes 2 mints. Results and Discussion; Free gluten pocket flat Arabic bread sensorial parameters evaluated (flavor, appearance, crumb texture, crust color, satisfaction and keeping quality) by both of Celiac disease patients and normal customers. The results was bread have a uniform crumb with well-distributed cells, satisfaction crust color, flavor and good keeping quality compared with wheat pocket flat Arabic bread. Conclusion; Production and consumption of pocket type flat Arabic bread were preferable by Celiac disease patients.

Key-words: Celiac disease, gluten-free, pocket, flat, Arabic bread

References;

1. Sanchez, H. D., Osella, C. A., & de la Torre. Optimization of gluten-free bread prepared from cornstarch, rice flour and cassava starch. Journal of Food Science, 2002; 67, 416–419.

2. Toufeili, I., Dagher, S., Sadarevian, S., Noureddine, A., Sarakbi, M., & Farran, M. T. Formulation of gluten-free pocket-type flat breads: Optimization of methylcellulose, gum Arabic and egg albumen levels by response surface methodology. Cereal Chemistry, 1994; 71, 594–601.

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Figure 1. Free gluten pocket type flat Arabic bread

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A Gluten-Free Bread with Viscous Japanese Yam Instead

of Wheat Flour Masaharu Seguchi

Kobe Women’s University, Kobe, Japan *corresponding email: seguchi@suma.kobe-wu.ac.jp

Introduction. We newly designed a gluten–free bread for Celiac Disease sufferers who do not have a wheat bread. Japanese Yam (Dioscorea japonica) Tuber(1) instead of wheat flour was used in the gluten-free bread. Methods. Powder of Japanese Yam (Dioscorea japonica) tuber, wheat starch, sugar, compressed yeast, and water were mixed for 18min, fermented at 40℃ for 20min, and resulting bread dough was baked at 210℃ for 10min. Japanese yam was dialyzed against water, and separated to nondialyzable fraction (higher molecular weight (HMW) fraction) and dialyzable fraction (lower molecular weight (LMW) fraction). Sugars and peptides groups in LMW fraction were obtained by paper (grade 590) chromatography (Pyridine-BuOH-water=4:6:3). Results. We could obtain a gluten-free bread with powder of Japanese yam tuber, and which had similar breadmaking properties such as bread height (mm) and specific volume (cm3/g) to wheat bread. Japanese yam tuber was dialyzed against water, and separated to nondialyzable fraction (higher molecular weight (HMW) fraction) and dialyzable fraction (lower molecular weight (LMW) fraction). They were dried, and were subjected to bread making in the same manner, respectively. The results indicated that bread baked with sole HMW and LMW fraction showed a poor breadmaking propertes, however, HMW plus LMW fractions gave a remarkable bread. Next, the LMW fraction was separated into sugars and peptides groups by a paper chromatography. Peptides group gave a remarkable breadmaking properties when mixed with HMW fraction. Figure 1. Appearance of Japanese Yam bread

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Conclusions. Powder of Japanese Yam (Dioscorea japonica) tuber instead of wheat flour could give a remarkable gluten-free bread. Yam tuber was separated to HMW and LMW fractions, and LMW fraction was further separated into sugars and peptides groups. Remarkable bread was obtained when HMW fraction and peptides group in the LMW fraction were mixed. References (1) Journal of the Japanese Society for Horticultural Science 76(3) pp.230-236 20070700.

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Effect of Legume Flours on Baking Characteristics of Gluten Free Bread

B. Miñarro, E. Albanell, M.Capellas

Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), CeRTA, XiT, Departament de Ciència Animal i dels Aliments. Edifici V - Campus UAB

Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Introduction. Gluten is an important protein in wheat bread making process, giving to bread dough its elastic and extensible properties. Due to the important roles of gluten in bread making, formulation of gluten free breads with good sensory characteristics presents big difficulties and results a challenge (Gallagher et al., 2004). In recent years, some studies have been done by different authors, mainly involving the approach of incorporation of starches, dairy proteins and hydrocolloids into a gluten-free flour base to try to obtain good quality gluten free products. To our knowledge, no detailed scientific study has been undertaken to evaluate the influence of legume flours, which are rich in protein. This works investigates the effect of pea, chickpea and carob germ proteins on baking characteristics of gluten-free bread. Methods. Three sources of protein were evaluated in gluten free breads: Chickpea flour (21.4% protein), carob germ flour (47.7% protein) and concentrate pea protein (82.5% protein). All recipes were standardised to 10% of protein by adjusting the amount of protein source. Main ingredients of the tested formulations were: pea (600.8 g of starch and 12.2 g of concentrate pea protein); chickpea (566 g of starch and 47 g of chickpea flour) and carob germ (591.9 g of starch and 21.1 g of carob germ flour). Constant ingredients in all formulations were: water (45.25%), sugar (2.5%), emulsifier (2%), shortening (2%), yeast (2%), salt (1%), baking powder (1%), xanthan gum (0.9%). Loaf volume was measured in duplicated by the method of displacement of millet seeds. Specific volume was calculated using the formula: specific volume (cm3/g) = volume (cm3) / weight (g). The initial batter weight and the weight of bread after cooling was measured and the bake loss was calculated using the formula: Bake loss (%) = (initial weight of batter - weight of bread after cooling) x 100 / initial weight of batter. Texture profile analysis of the crumb was performed on three slices taken from the centre of each loaf. Texture profile analysis was carried out using a TA-TX2 texture analyzer (Stable Micro Systems, Surrey, UK) equipped with a 25 kg load cell and a 20 cm diameter aluminium cylindrical probe. Probe speed was set to 2 mm/s to compress the centre of the bread crumb to a 40% of its original height. Crust and crumb colour of bread samples were measured with a Hunter Lab colorimeter miniScan XTE (Hunter Associates Laboratory INC, Reston, Virginia, USA). CIE L*, a* and b* values were measured with an illuminant of D65 and a standard observer of 10º. Results. Carob germ formulation had lower specific volume and bake loss values than chickpea and pea recipes, and showed the highest hardness values during seven days of storage in modified atmosphere packaging at room temperature. Chickpea bread obtained the lowest values in hardness and the highest in bake loss and specific volume. No

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differences were found in cohesiveness and springiness values (Table 1). Feillet et al. (1998) found a decrease in extensibility and sweeling index when carob germ was added at 1%, which seems to agree with the high hardness and low volume of our bread (Figure 1). Although pea and chickpea belong both to pulse family they vary in essential amino acids. Boye et al. (2010), reported higher foam expansion and stability values for chickpea, compared to pea. These differences, attributed to different proteins fractions, could explain the higher volumes obtained in chickpea and their influence in low hardness values. Table 1. Baking characteristics of three different recipes

Carob Germ Pea Chickpea

Hardness 732,53 ± 51,84 518,05 ± 102,56 367,13 ± 82,61

Cohesiveness 0,54 ± 0,01 0,55 ± 0,02 0,55 ± 0,02

Springiness 0,96 ± 0,01 0,95 ± 0,03 0,96 ± 0,01

Bake loss 9,8 ± 0,64 11,92 ± 0,38 12,94 ± 0,40

Specific volume 2,78 ± 0,17 2,86 ± 0,05 2,98 ± 0,02

L* crust 51,80 ± 2,73 53,07 ± 2,48 49,52 ± 3,13

Water activity 0,979 0,979 0,978

Figure 1. Different breads obtained Conclusions. This preliminary study shows that chickpea, pea and carob germ flours are suitable to produce gluten free products of high quality. All formulations had acceptable bread characteristics and appearance. However, some further research is needed, including sensory analysis, rheology, microstructure, etc to deeply understand the role of these legume flours in the bread system and its behaviour over the time. References Gallagher E, Gormley TR, Arendt EK. Review: Recent advances in the formulation of

gluten-free cereal-based products. Trends Food Sci & Tech 2004;15:143-152. Feillet P, Roulland TM. Caroubin: a gluten-like protein isolated from carob bean germ.

Cereal Chem 1998;75:488-492. Boye J, Zare F, Pletch A. Review: Pulse proteins: Processing, characterization, functional

properties and applications in food and feed. Food Res Int 2010;43:414-431.

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Effects of High Pressure and Temperature on Buckwheat Starch Characteristics

K. J. R. Vallons; L. A. M. Ryan, E. K. Arendt*

Department of Food and Nutritional Sciences, National University of Ireland, University College Cork, College Road, Cork, Ireland

*Corresponding email: e.arendt@ucc.ie Introduction. Buckwheat is a non-glutinous pseudo-cereal that has a long and traditional history as a food source. High pressure treatment is a promising new processing method that has been investigated with growing interest as an alternative to heat treatment in the development of foods with novel textures. As the physicochemical properties of buckwheat starch will affect the functional properties of foods containing buckwheat, the understanding of high pressure induced gelatinisation of starch is vital for the development of new buckwheat applications using high pressure. The objective of this study was to evaluate the effect high pressure treatment on rheological and structural properties of buckwheat starch and to compare it to the one obtained by temperature treatment. Methods. Pressure-induced gelatinisation of buckwheat starch suspensions (25 % w/w) was studied and compared to heat-induced gelatinisation. Due to limitations of the measuring equipment, the pressure dependence of the starch gelatinisation could not be determined by continuous steady increase of the pressure. Therefore, starch-water suspensions were pre-treated with either pressure (200 - 600 MPa) or temperature (60 – 95 oC) for 10 min and subsequently analysed for changes in their properties by a heating process, using either differential scanning calorimitry (DSC) or rheology (temperature sweep). The gelatinisation temperature and pressure ranges, the degree of gelatinisation and the pasting properties were determined. The results obtained by these two methods were evaluated using scanning electron microscopy (SEM; structural properties) and confocal laser scanning microscopy (CLSM; loss of birefringence) Results. The correlation between the degree of gelatinisation (as determined by DSC) and treatment pressure followed a sigmoidal-shaped curve with a sharp increase between 300 and 500 MPa (Figure 1A). Heat induced gelatinisation of buckwheat starch resulted in a similar sigmoid gelatinisation curve with most of the crystals melting between 60 and 70 oC (Figure 1B). The gelatinisation temperature and pressure ranges obtained by CLSM (loss of birefringence) and rheology (consistency increase) were similar. Furthermore, the CLSM pictures suggested partial preservation of granular structure after treatment with 600 MPa, while heating at 75 oC seemed to cause the loss of integrity of most granules (Figure 2A). This difference between HP-treatment and heating was investigated further with SEM. Although most granules appeared swollen and deformed, the majority of granules retained some degree of integrity after treatment with 600 MPa. However, heating a buckwheat starch suspension to 75 oC caused almost all granules to lose their structure completely and a regular sponge-like structure was clearly visible (Figure 2B).

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Better preservation of the granular structure upon pressurization compared to heating, resulted in stronger gels for the former. This led to the assumption that the viscosity of the paste was determined by the swollen granule and the granule-granule interaction. Entanglement of leached amylose seemed to play a minor role. Consequently, disintegration of the granules when heated above 65 oC resulted in a weaker gel matrix.

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Figure 2. CLSM (A) and SEM (B) images of buckwheat starch suspensions after treatment for 10 min at different pressures and temperatures. As pre-treatment with high pressure as well as temperature changed the granular structure and crystallinity, it changed the behaviour of the starch upon subsequent pasting. Restricted swelling, restricted granule disintegration and a decreased rapid integration of the leached amylose showed that buckwheat starch granules can be stabilized by pre-treatment with high pressure or temperature, to make them more resistant to breaking apart under the influence of additional heat. Conclusions. High pressure as well as temperature caused gelatinisation of starch within the ranges 300-500 MPa and 60-70 oC, respectively. However, better preservation of the granules and stronger gels were observed for high pressure treatment. Furthermore, both high pressure and temperature seemed to make the buckwheat starch more resistant to destruction by further heating.

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Rheological properties and bread making performance of commercial wholegrain oat flours

Edith K. Hü ttner1, Fabio Dal Bello1 and Elke K. Arendt1,*

1Department of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, Ireland

*corresponding email: e.arendt@ucc.ie Introduction. The nearly ubiquitous consumption of bread places it in a position of global importance in human nutrition. Wheat (Triticum aestivum) is the most important crop for bread making due to its supreme baking performance compared to other cereals. However, the interest in alternative grains is increasing due to the consumer demand for novel and healthy foods. Oat (Avena sativa) is one of the most adventurous cereal grains for human diet since it contains naturally high amounts of valuable nutrients such as soluble fibres, proteins, unsaturated fatty acids, vitamins, minerals and phytochemicals. Moreover, recent studies have shown that oats can be tolerated by most people suffering from celiac disease. However, the effects of oats on dough properties and bread quality have been studied mainly on composite breads made from wheat and oats. In these studies the bread making potential of oats was masked by the outstanding effect of wheat gluten and such breads are not suitable for celiac patients. Consequently, the objectives of this study were to investigate the bread making properties of commercial oat flours without addition of wheat flour and to identify the physicochemical factors responsible for good oat bread quality, which could be used to establish flour quality standards for the production of good quality oat bread. Methods. Commercially available wholegrain oat flours from Ireland (WOI), Finland (WOF) and Sweden (WOS) were used for this study. Bread recipe and baking procedure were established in preliminary trials to obtain the most appropriate conditions for bread making. Small amplitude oscillatory shear measurements within the linear visco-elastic region were used to study the rheological properties of the oat batters. The flours were characterised by measuring moisture, ash, protein, starch, amylose, fat, dietary fibre and β-glucan content as well as starch damage and water hydration capacity. Rapid Visco Analyser (RVA) analysis was applied to determine the pasting properties of the flours and capillary gel electrophoresis was used to investigate the protein profile of the commercial oat flours. Results. The use of different commercial oat flours resulted in breads with varying quality (Figure 1). Breads made from WOS and WOI flour conferred desirable oven spring, resulting in a soft and well-aerated crumb and therefore high loaf specific volume and good bread quality while bread made from WOF showed the lowest values and thus poor bread quality.

Figure 1. Pictures of breads made from (A) WOF, (B) WOI and (C) WOS flour.

A B C

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Rheological analysis revealed that batters prepared with WOI and WOS flour resulted in softer batters as indicated by lower G*, G' and G''. Compositional analysis showed significant differences (p<0.05) in protein, starch, damaged starch and β-glucan content of WOF flour compared to WOI and WOS flour (Table 1). Additionally, the water hydration capacity of WOF flour was twofold higher, compared to the other two wholegrain oat flours. The flour pasting profile of WOI was significantly different compared to that of WOF and WOS flour. The commercial wholegrain oat flours also showed variations in their particle size distribution due to differences in the milling techniques. Table 1. Chemical composition and water hydration capacity of commercial WO flours.

WOF WOI WOS

Moisture (%) 10.63 ± 0.02 10.99 ± 0.03 14.06 ± 0.08

Ash (% db) 2.25 ± 0.07 1.95 ± 0.05 2.13 ± 0.03

Protein N × 6.25 (% db) 17.06 ± 0.15 12.27 ± 0.15 11.95 ± 0.34

Fat (% db) 6.43 ± 0.08 6.06 ± 0.01 5.24 ± 0.29

Total starch (% db) 62.31 ± 0.41 67.67 ± 0.17 65.38 ± 1.04

Amylose (% db) 28.83 ± 0.04 31.63 ± 0.51 28.87 ± 0.38

Starch damage (% db) 9.19 ± 0.20 6.73 ± 0.01 1.60 ± 0.01

Total dietary fibre (% db) 19.22 ± 0.07 17.84 ± 0.77 19.06 ± 0.07

β-glucan (% db) 4.47 ± 0.07 3.75 ± 0.03 4.18 ± 0.03

Water hydration capacity (ml/g) 1.48 ± 0.01 0.73 ± 0.02 0.70 ± 0.02 Discussion. Commercial wholegrain oat flours are readily available on the market, even though no information exists about the properties of oat flours which are required in order to bake good quality bread. In this study significant differences were found in the bread making properties of commercial oat flours. Overall, low batter viscosity had positive effects on bread quality as observed for WOI and WOS. On the other hand, WOF batters which showed high viscosity resulted in poor bread quality. Batter consistency can be influenced by the water hydration capacity of the flour. WOF flour showed the highest water hydration capacity which was influenced by small particle size, high amount of damaged starch as well as high protein content, thus explaining the poor bread making quality. Small flour particle size results in increased water hydration capacity due to easy swelling of all components. Negative effects of high levels of damaged starch granules on bread quality can be explained by their rapid hydration, which leads to increased batter viscosity. High amounts of proteins in wholegrain oat flour interfere with starch by disrupting the uniformity of the starch gel during baking. Yet, the commercial oat flours showed the same protein profile which therefore did not affect the bread making properties. Pasting properties did not affect the bread making properties of the commercial oat flours. Conclusions. Based on the data collected we could show that in order to achieve high quality oat bread wholegrain oat flour should present the following properties: low batter viscosity, low flour water hydration capacity, starch content of above 65 %, protein content of about 12 %, low starch damage and coarse particle size.

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Gluten-Free Bread Supplemented with Calcium – the Improvement of Quality and Texture Properties

Urszula Krupa-Kozak, Małgorzata Wronkowska, Maria Soral-Śmietana, Agnieszka

Troszyńska, Jadwiga Sadowska

Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland

*corresponding email: u.krupa-kozak@pan.olsztyn.pl Introduction. Celiac disease is a gluten-sensitive entheropathy. Later studies suggest an increasing prevalence of that disease, likely due to the development of more sensitive methods of screening (Sampson et al. 2005). The reaction to gluten ingestion by patients suffering from that chronic disease is inflammation of the small intestine leading to the malabsorption of several important nutrients, vitamins and minerals, especially of calcium and iron. Osteopenia and osteoporosis are a frequent complication accompanying coeliac disease. Bosscher et al. (2006) in the review describe studies in animal models which have shown increased calcium availability with inulin and oligofructose in the diet; also a beneficial effect of inulin-type fructans on the delay of osteoporosis and accumulation of bone mineral and formation of improved trabecular network structure. The only effective treatment for coeliac disease is a long-life strict adherence to a gluten-free diet. Up to now, research studies related to gluten-free bakery products have been focused on the design of gluten-free matrixes by combining different starches and gluten-free cereals. However, no special attention has been paid to the use of those products as carriers or vehicles of microelements, which are necessary for celiac patients. Objectives. The aim of the research was to design of gluten-free formula fortified with organic calcium sources and inulin destined to bake a bread. Methods. The basic formula of gluten-free bread consisted of corn and potato starches, pectin, and inulin according to the procedure described in Polish patent specification P 386253 (Krupa et al., 2008). The calcium supplement (2%) constituted two calcium salts: caseinate (CaCA) and citrate (CaCI). Basic chemical composition and size-related parameters of gluten-free bread were assessed. Texture properties of crumbs were measured by using a compression device of Instron 1011 (Instron Ltd., High Wycombe, England). Quantitative descriptive analysis (QDA) was used to determine differences in the sensory characteristics of the breads. QDA were carried out by a panel consisting of 8 members who evaluated the intensity perceived for each sensory attribute on unstructured graphical scales. A semi-consumer panel of 30 members has made hedonic evaluation of the samples. In the test, each panelist was asked to assess the breads for overall quality. Results and discussion. Addition of both calcium supplements affected beneficially the specific volume of bread. The enriched in proteins and minerals was noted. The effects of calcium supplementation of gluten-free formula on the overall quality of breads are shown in Figure 1. The average overall quality of scores for supplemented breads ranged from 4.7 units to 5.4 units, whereas the control obtained 3.5 units (in the scale of 10 units). It suggests that calcium supplements might contribute to the improvement of the sensory properties of gluten-

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free bread. Based on the sensory evaluation, bread fortified with equal amount of calcium caseinate and citrate (1%CaCA/1%CaCI) was selected as the best.

Figure1. Overall quality of gluten-free bread. The texture profile analysis of fresh experimental gluten-free bread crumbs included the parameters of fracture point, i.e., fracture strain and stress, and toughness (Table 1). Examined control bread crumb was crumbly. Both calcium supplements increased the value of fracture strain and decreased toughness. Table 1. Texture properties of crumbs of fresh gluten-free bread supplemented with calcium.

Bread Df [%] Stress [kPa] Toughness [kPa] Control 14,51d ±1,99 14,19a ±4,18 1,105a ±0,38 2%CaCA 21,99a ±2,99 7,93c ±2,42 1,068a ±0,39 2%CaCI 16,16bcd ±2,25 8,39bc ±2,39 0,765bc ±0,26 1%CaCA/1%CaCI 15,72cd ±2,28 8,42bc ±2,60 0,784abc ±0,20 1.3%CaCA/0.7%CaCI 17,04bc ±2,32 10,50b ±2,21 1,022ab ±0,29 0.7%CaCA/1.3%CaCI 18,39b ±2,84 6,65c ±1,43 0,724c

±0,13 Conclusions. Summarizing, the application of calcium supplements such as calcium caseinate and citrate in order to fortified gluten-free bread with calcium influence beneficially its sensory and nutritional properties simultaneously enhancing the structure and texture of bread. *Research was partly supported by the Ministry of Science and Higher Education grant No NN312 3450 33. References Sampson M., Zhang L., Yazdi F., Mamaladze V., Pan I., McNeil J., Mack D., Patel D., Moher

D. (2005): The prevalence of coeliac disease in average-risk and at-risk Western European populations: A systematic review. Gastroenterology, 128(suppl 1): 57-67.

Bosscher D., Van Loo J., Franck A. Inulin and oligofructose as functional ingredients to improve bone mineralization. International Dairy J., 2006,16, 1092-1097.

Krupa U., Wronkowska M., Soral-Śmietana M. (2008). Mieszanka bezglutenowa (eng. Gluten-free formula). Polish Patent Specification No P 386253 (in Polish).

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Gluten-free Pasta: Technology and Quality Evaluation

Manuela Mariotti, Carola Cappa, Mara Lucisano Università degli Studi di Milano, DiSTAM (Dipartimento di Scienze e Tecnologie Alimentari

e Microbiologiche), Via G. Celoria 2, Milan, Italy *corresponding email: mara.lucisano@unimi.it

Introduction. Gluten forming proteins are fundamental for the production of a great variety of food, including pasta, most appropriately made from durum wheat. The replacement of gluten network, in order to produce gluten-free pasta (GFP), is a major technological challenge, and ingredients that imitate the viscoelastic properties of gluten are always required. The first attempts in the search for substances able to imitate the viscoelastic properties of gluten exploited starch gelatinization and retrogradation phenomena, modifications that can be obtained during the technological process or using pre-gelatinized starches or starchy flours as raw materials. Later on, other ingredients have been considered in GFP formulation: rice and corn flours, flours from pseudocereals, starches from different sources, vegetable proteins, emulsifiers, hydrocolloids. The technological process also plays an important role on the final quality of GFP. The traditional process for making rice noodles involves the presence of many heating and cooling phases aimed at reorganizing the starchy matrix; the batch process can be switched to a continuous one with the use of the extrusion technology performed at high temperature; the same process and the same equipment employed in the production of durum wheat pasta can be used if pregelatinized materials are used. In consideration of such a large variety of formulations and technologies, the aim of this study was to evaluate the characteristics of as much as possible commercial GFP (spaghetti shape), in order to get a wide view of what is actually available on the Italian market. The study regarded the chemical, biochemical and physical characterization of the samples, focusing the attention on starch and protein organization. Cooking behaviour and textural characteristics of cooked pasta (at different cooking times) were also evaluated. Methods. Fourteen commercial brands of GF spaghetti (GFS) were collected. The uncooked GFS were characterized for gluten content, color, chemical composition, protein solubility and thiol accessibility, starch accessibility, starch pasting properties (Brabender Micro-Visco-Amylograph, MVA), spaghetti fracture properties (TAHDplus Texture Analyser); the cooked GFS were evaluated for water absorption, cooking loss, dimensional changes (Image Analysis), and textural properties (compression test, creep test) at different cooking times, in order to determine the kinetics of all these phenomena. Results. Only some of the results obtained are here reported. On the basis of their ingredients the 14 GFS were identified as: rice spaghetti (coded: R1, R2, R3, R4), corn spaghetti (C1, C2, C3), corn starch based spaghetti (CS1, CS2, CS3, CS4; some of them containing also potato flour, rice flour, corn flour, pea protein isolate, lupin flour, lupin proteins), and spaghetti (M1, M2, M3) obtained from a mixture of rice flour, corn flour and other ingredients (tapioca flour, yeast, buckwheat flour, sunflower flour). These products were characterized not only by a different chemical composition (protein: 5-11% d.b.; starch: 79-90% d.b.) but also by a different protein and starch organization (starch accessibility: 8.1-16.0% d.b.; MVA peak viscosity: 167-404 BU). All these factors influenced the cooking quality of the different GFS. Some quality parameters (at the optimum cooking time, OCT), are reported in Table 1. R3,

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obtained according to the oriental technology, exhibited high water absorption, low cooking loss and adhesiveness, differently from the others “R” samples. Various behaviours were also observed in legume-based GFS, due not only to dissimilar properties of proteins in botanically related species but also to different organizations of the protein network in these samples. Table 1. Quality parameters of the GFS, at their OCT (*, compression test)

Sample Water absorption (%)

Diameter increase (%)

Cooking loss (g/100g d.b.)

Young Modulus* (N/mm2)

Adhesiveness* (·10-3J)

R1 100.0 44.2 ± 8.2 12.4 ± 0.1 0.39 ± 0.02 5.21 ± 0.72 R2 112.5 45.3 ± 7.9 8.5 ± 0.1 0.37 ± 0.01 2.47 ± 0.48 R3 162.5 41.0 ± 7.6 3.2 ± 0.2 0.26 ± 0.01 0.67 ± 0.05 R4 112.2 41.5 ± 4.5 5.6 ± 0.3 0.36 ± 0.01 2.65 ± 0.60 C1 125.0 52.6 ± 4.5 6.2 ± 0.1 0.36 ± 0.01 0.90 ± 0.10 C2 117.1 39.8 ± 2.9 5.3 ± 0.2 0.34 ± 0.01 1.48 ± 0.13 C3 117.1 51.2 ± 4.7 5.0 ± 0.1 0.34 ± 0.01 1.01 ± 0.16 CS1 122.0 47.0 ± 5.2 5.1 ± 0.1 0.28 ± 0.01 1.99 ± 0.69 CS2 107.3 42.8 ± 4.4 6.8 ± 0.1 0.33 ± 0.01 1.09 ± 0.17 CS3 142.5 54.3 ± 5.3 3.9 ± 0.1 0.27 ± 0.01 3.82 ± 0.18 CS4 125.0 47.7 ± 5.1 6.7 ± 0.1 0.31 ± 0.01 1.38 ± 0.26 M1 134.1 61.5 ± 4.7 5.1 ± 0.1 0.31 ± 0.01 1.89 ± 0.25 M2 122.0 39.2 ± 3.2 7.5 ± 0.1 0.36 ± 0.001 1.34 ± 0.07 M4 120.0 53.0 ± 4.6 2.5 ± 0.1 0.30 ± 0.01 1.87 ± 0.48

A creep test applied to the GFS cooked at their OCT (Figure 1) proved to be very useful in describing the viscoelastic characteristics of the cooked products: very different patterns were observed, even in the same product category (e.g. C1 vs. C3, R1 vs. R3), underlying the importance of the technological process adopted besides the role of the formulation. Figure 1. Viscoelastic behaviour (creep test) of the GFS, at their OCT. Conclusions. The overview on GFS samples presented in this study highlighted the wide variety of raw materials and technologies adopted in this sector, indicating the on-going research of solutions providing for the gluten-network absence. At the same time, it came out how all these factors can directly influence the final structure and quality of the products. Phenomena related to starch retrogradation certainly have a central role on the final texture of the products, but also the origin of the protein included in the formulation plays an important role in the definition of the protein-protein interactions, especially in those samples including proteins from different vegetable sources.

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Proso Millet (Panicum miliaceum L.) a Sustainable Raw Material for the Malting and Brewing Process

Martin Zarnkow1, 2*; Thomas Becker1, Elke K. Arendt2 1 Technische Universität München, Lehrstuhl für Brau- und Getränketechnologie, Freising,

Germany 2 University Collage Cork, Department of Food and Nutritional Science,

Cork, Ireland *corresponding email: Martin.Zarnkow@wzw.tum.de

Introduction. The objective of this work was to optimise the malting, mashing and fermentation conditions for proso millet (Panicum miliaceum L.) using various methods. P. miliaceum shows promising potential as an alternative food ingredient, especially in regions where the growing conditions for cereals such as wheat and barley, among others, are poor. Furthermore, proso millet is gluten-free. These cereals have received more attention recently as part of ongoing efforts to create a fermented beverage for those suffering from coeliac disease can enjoy. Methods. The main objective of this thesis was to develop processing procedures for the production of good quality malt and beer based on proso millet. The specific objectives were as follows: 1) to examine the effect of germination parameters on the quality attributes of proso millet malt in order to achieve high amylolytic activity using response surface methodology; 2) to observe the microstructural changes in proso millet kernels during the malting process using scanning electron and confocal scanning microscopy techniques; 3) to evaluate the influence of different varieties of proso millet on malt quality attributes; 4) to apply the research results previously aquired in order to develop an optimal malting regimen for the proso millet variety selected and to develop an optimal mashing procedure using size exclusion chromatography; 5) and finally, to study the fermentation performance of different yeast strains during the fermentation of the optimised substrate, wort made with proso millet malt. Results. Based on the results of these studies, it was concluded that the optimal malt quality is achieved after the 5th germination day with moisture content of 44% and a set temperature of 22°C for steeping and germination. The predicted values for the quality parameters were 65.3% extract, 1.367 mPa × s viscosity, 74.3% AAL, 106 U/g α-amylase activity, and 105 U/g β-amylase activity. An early visible degradation (after 24 h) of starch granules located in the floury endosperm near the embryo, was observed. Confocal scanning laser microscopy was used to document this degradation, which was evident in the form of a less dense particle spatial arrangement in this part in the endosperm. A detailed analysis of the data revealed that the variety Braune Wildform proved to be the best proso millet variety for brewing purposes. α-Amylase showed its highest activity in proso millet malt wort at a temperature of 60°C and at a pH of 5.0, whereas β-amylase exhibited an optimum at 40°C and at a pH of 5.3. The limit dextrinase activity reached its maximum at 50°C and at a pH of 5.3. On the basis of the above results, it was possible to create an optimised mashing regimen (Figure 1). There were strong similarities in the fermentation performance of the various Saccharomyces yeast strains used in these trials. Brettanomyces yeast strains showed significantly faster fermentation rates in

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the fermentation trials, based on the pH reduction, increase in alcohol content and sugar conversion, conducted at 18°C, the higher of the two fermentation temperatures. The analysis of the flavour compounds was performed using an established GC method. One of the aims of the aroma compound analysis was to establish aroma compounds which are specific for proso millet, rather than the yeast used. A summary of the aroma compound analysis is given in Table 1. A detailed analysis of the results revealed that different amounts of aroma compounds were identified for the various yeasts and the fermentations conditions (12 and 18°C), but it was not possible to identify proso millet specific flavour compounds.

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Table 1: Obtained attributes of aliphatic and aromatic alcohols, esters and fatty acids after

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[µg/L] at 12°C in proso millet malt beer S 208 S 250 S 262 S 290 B 20 barley malt lager

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Sum of aromatic alcohols 8053 8467 6591 4946 3840 10069–30461

Sum of esters 1217 402 2543 497 188 210–1000 [µg/L] at 18°C proso

millet malt beer S 208 S 250 S 262 S 290 B 20 barley malt lager

Sum of aliphatic alcohols 158 78 61 267 137 45–120

Sum of aromatic alcohols 11844 10653 2437 8569 7685 10069–30461

Sum of esters 1284 395 1068 580 1149 210–1000

Conclusions. All in all, this research indicates that proso millet has great potential as a new raw material for malting and brewing purposes. Additionally, proso millet could be utilized as a novel ingredient for a wide range of food and beverage products designed specifically for those who suffer from coeliac disease. Lastly, it is a readily available raw material, which already enjoys acceptance among consumers, exhibiting properties comparable to barley malt in all aspects of malting and brewing.

162

Gluten-Free Beer from Barley

S. Pöyri*4, M. Mäki1, A. Hernando2, M. Mena2, M. Lombardia2, P. Lehtonen3, P. Soininen-Tengvall4, E. Pajunen4, E. Mendez2

1University of Tampere, Tampere, Finland 2Unidad de gluten, Centro Nacional de Biotecnologia, Madrid, Spain

3Alko Oy, Vantaa, Finland 4Oy Sinebrychoff Ab, Kerava, Finland

*corresponding email: Saara.Poyri@sff.fi

Introduction. Barley malt is the natural raw material for beer production. During malt and beer processing the storage proteins (hordeins) of barley are hydrolysed by the natural proteolytic enzymes of grains. Normal beer contains variable amounts of prolamins (gluten) and the hydrolysed peptides. The prolamin content and the susceptibility to coeliac disease is depending on the beer type, raw materials and processing methods used. Materials and Methods. Several European beers were analyzed by western blots and further, the prolamins were extracted with ethanol or cocktail solution and analyzed by sandwich and competitive R5-ELISA, a method developed to detect gluten in processed foods. Results. Western blot analysis detected that the European beers contain hydrolysed gliadins and hordeins. Gluten was detectable in only 11/93 beers by the sandwich R5-ELISA method whereas the competitive method showed >20 ppm gluten in 62 of these beers. The prolamin levels of some of the Finnish barley malt beers were well below 20 ppm. The observation has led to a controlled brewing process, where beers brewed from malted barley repeatedly show prolamin and gluten levels well below the 20 ppm level, the level for natural gluten free products and even below the detection limit. Discussion. The natural barley malt can be used as raw material for high quality gluten free beer with controlled removal of gluten during beer processing. The sandwich R5-ELISA method is not suitable for detecting hydrolyzed prolamins in beers, instead a competitive R5-ELISA method should be applied.

163

Gluten-Free Wheat Starch - Safety and Functionality on a Natural Way-

Maren Wiese Hermann Kröner GmbH, Ibbenbüren (Germany) corresponding email: wiese@kroener-staerke.de

Introduction. Due to the excellent baking qualities and the aromatic taste wheat is commonly used in bread and bakery products. Because of the existence of gluten, wheat is not suitable for persons with gluten intolerance. To use these positive properties of wheat a special quality of wheat starch, suitable for persons with gluten-intolerance, has been available on the market for several years. In the past it was not possible to produce this starch with a gluten-content that corresponds with the content in naturally gluten-free products. Objectives. The intension was to improve the quality of gluten-free wheat starch significantly. Without application of enzymes and chemical additives, the company KRÖNER-STÄRKE aimed for specifying a high quality product with maximum 20mg gluten/kg wheat starch. According to the new EC-regulation No.41/2009 this quality can be labelled as “gluten-free”. The production-process of gluten-free wheat starch is demonstrated and the continuous improvement process of the qualities is shown relating to the recommendations of the Codex-Alimentarius. Methods. Enzyme-linked Immunoassay (ELISA), R5 Mendez Method (according to Codex Alimentarius 118-1981 revised 2008) Results and discussion. Based on current technical developments the company KRÖNER-STÄRKE could improve the quality of their gluten-free wheat starch SANOSTAR significantly. A quality of gluten-free wheat starch with max. 20mg gluten/kg starch is feasible by pure natural processing. This quality considerably exceeds the requirements for wheat starch-containing products intended for people with gluten intolerance. According to the current EC-regulation a maximum content of 100mg gluten/kg food as sold to the consumer is permitted. Special advantages of this gluten-free wheat starch compared to naturally gluten-free products are the excellent baking characteristics and the aromatic taste. Furthermore, the problem of GMO does not exist in wheat products. Conclusions. Due to technical developments, the production of a high functional gluten-free wheat starch without additives is possible. The wheat starch fulfils the same limits of gluten, specified for naturally gluten-free products, combined with the advantages of excellent baking and sensory properties. SANOSTAR is a high functional ingredient with the safety of natural gluten-free products. References. Codex Alimentarius 118-1981 and Codex Alimentarius 118-1981 revised 2008 EC regulation No.41/2009

164

Improving The Texture And Nutritional Profile of Gluten

Free Baked Goods by Formulation Science And Speciality

Flour Technology: Muffins

Despina Ioannides (EU)*, Alejandro J Perez (US), Yadunandan Dar (US)

National Starch Food Innovation

* corresponding email: despina.ioannides@nstarch.com

Introduction: Gluten-free bakery products are often considered of low eating quality because

of their unappealing texture due to the lack of gluten network. They also have a deficient

nutritional profile since they lack protein and fibre. Manufacturers may de-prioritize the

nutritional aspects by using high levels of sugars and fats to address the textural and flavour

challenges encountered in these applications. Additionally, formulators do not have

alternatives to functional modified and other non-label-friendly ingredients to create gluten-

free products. The native flours and gums commonly used for this purpose lack functionality

and versatility.

Objective: Develop a functional and clean-label gluten-free flour replacement solution with

excellent taste, texture and enhanced nutritional profile to match those of traditional gluten-

containing counterparts. Further enhance this offering to achieve wholegrain and “source of

fibre” claims.

Methods: Using the DIAL-IN™ Texture Technology approach through the employment of

techniques such as descriptive sensory analysis of texture, design of experiments (DOE) and

their formulation science expertise, the bakery experts at National Starch Food Innovation

were able to design a gluten-free flour-based system, Homecraft CreateTM

GF 10, that

contributes the desired specific textural profile (Fig.1) to the end product. As a flour based

system, this ingredient has also an enhanced nutritional profile compared to that of starches

and gums, which are generally of low protein content. Additionally, Hi-maize® wholegrain

flour, the highest fibre-containing gluten-free wholegrain flour (Fig. 2); was additionally

evaluated to determine its impact on further improving the nutritional profile of the muffin.

Results: Homecraft CreateTM

GF 10was developed based on extensive screening of functional

flour ingredients. Descriptive sensory analysis techniques along with bakery science

formulation and evaluation methods were used to optimize the flour composition as well as

the texture profile of the muffins. For muffins, the most relevant textural attributes were

observed to be openness and structure of the crumb, cohesiveness, the overall shape, colour,

and roughness of the mass during chewing. Homecraft CreateTM

GF 10 outperformed regular-

grade rice flours from different sources alone or in combination with starches and/or

hydrocolloids in terms of ease in process-ability, dough rheology, appearance, texture, shelf

life, and clean label ingredient profile. Furthermore, addition of 8g of Hi-maize® wholegrain

flour per serving resulted in a formulation that successfully supports the “contains

wholegrain” claim, having also a Total Dietary Fibre (TDF) value of 3.7g/100g of product

and thus meeting the criteria for the “source of fibre” claim without significant negative

impact on the overall eating experience (Fig. 1).

165

Discussion: Gluten-free muffins are

generally perceived as drier and grainier

versus their gluten-containing counterparts.

The absence of a gluten network results in

relatively closed crumb structures, with poor

viscoelastic and expansion properties. The

increased staling rates and water mobility

found in traditional gluten-free applications,

due to the lack of a gluten network, limits

their shelf-life, rendering them mostly

dependant on their packaging to prevent

water release. Homecraft CreateTM

GF 10

was developed as a gluten-free flour replacer. Its composition and physical properties assure

uniform moisture distribution throughout the different processing steps of bakery

applications. Its molecular characteristics prevent lumping formation, offer improved water

hydration and enhanced emulsifying properties. The gluten-free flour system combines the

benefits of speciality rice and tapioca flours with no impact on grittiness, and enhancing the

overall consistency of the product during the chew down. A proprietary physical process was

used to functionalize the flour system while preserving the native flour label.

Gluten-free replacements for cereals and baking mixes are often made up of a combination of

regular-grade corn starch, potato starch, tapioca starch and/or white or brown rice flour. The

nutrient density of such ingredients is low compared to that of whole grains. Hi-maize®

wholegrain flour has a fibre content of minimum 25%, the highest content among other

gluten-free whole grains (Fig. 2).

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

Amaranth Buckw heat Millet Brow n Rice Quinoa Sorghum Teff Wild Rice Hi-maize Whole

Grain Corn f lour

% Fibre Content

Figure 1. Hi-maize® wholegrain flour versus other gluten-free whole grains.

Conclusion: A gluten-free muffin containing wholegrains and 3.7% TDF was developed

utilising Homecraft CreateTM

GF 10 specialty flour system and incorporating Hi-Maize®

whole grain flour- a highly beneficial wholegrain ingredient. Based on the gluten-free muffin

case study presented here, it is possible to improve the eating quality of gluten-free bakery

products to the extent of almost matching their gluten containing counterparts. Work

continues to evaluate the performance of the newly designed Homecraft CreateTM

GF

speciality flour systems to achieve similar results across a wider gluten-free portfolio.

Smooth

Dry

Crumby

Grainy

Moist

Chewy

C

2

M

Smooth

Dry

Crumbly

Grainy

Moist

Chewy

22

M1

M2

M1: NSFI – Gluten-free prototype

M2: NSFI – Wholegrain Gluten-free prototype

Commercial gluten-containing muffin

Commercial gluten-free muffin

Smooth

Dry

Crumby

Grainy

Moist

Chewy

C

2

M

Smooth

Dry

Crumbly

Grainy

Moist

Chewy

22

M1

M2

M1: NSFI – Gluten-free prototype

M2: NSFI – Wholegrain Gluten-free prototype

Commercial gluten-containing muffin

Commercial gluten-free muffin

M1: NSFI – Gluten-free prototype

M2: NSFI – Wholegrain Gluten-free prototype

Commercial gluten-containing muffin

Commercial gluten-free muffin

Figure 1. Sensory differentiation in gluten-free muffin formulations.

Smooth

Dry

Crumby

Grainy

Moist

Chewy

C

2

M

Smooth

Dry

Crumbly

Grainy

Moist

Chewy

22

M1

M2

M1: NSFI – Gluten-free prototype

M2: NSFI – Wholegrain Gluten-free prototype

Commercial gluten-containing muffin

Commercial gluten-free muffin

Smooth

Dry

Crumby

Grainy

Moist

Chewy

C

2

M

Smooth

Dry

Crumbly

Grainy

Moist

Chewy

22

M1

M2

M1: NSFI – Gluten-free prototype

M2: NSFI – Wholegrain Gluten-free prototype

Commercial gluten-containing muffin

Commercial gluten-free muffin

M1: NSFI – Gluten-free prototype

M2: NSFI – Wholegrain Gluten-free prototype

Commercial gluten-containing muffin

Commercial gluten-free muffin

Figure 1. Sensory differentiation in gluten-free muffin formulations.

166

Development of High-Quality Gluten-Free Breads for the European market

Valentina Stojceska and Paul Ainsworth

The Manchester Metropolitan University, Department of Food and Tourism Management, Hollings Faculty, Old Hall Lane, Manchester, M14 6HR, UK.

*corresponding email: V.Stojceska@mmu.ac.uk

Introduction. Village Bakery Nutrition (VBN) is the largest gluten-free bread factory in Europe, established 2007. Currently, it has been involved in 27 months Knowledge Transfer Partnership (KTP) programme with the Manchester Metropolitan University. KTP is a part of government-funded activity to encourage collaboration between businesses and universities in the United Kingdom. The main aim of this KTP programme has been to achieve the successful operation of gluten-free production, optimise the physical and sensory characteristics of gluten-free bakery products and initiate a programme of new products development. Today, VBN factory uses a £3m facility to produces over 2,500 loaves every hour, which is equivalent to 4 million loaves a year. The gluten-free range includes white, high-fibre and low-protein breads. These are marketed and distributed in the UK and Ireland under the brand Juvela, Sweden and Finland under the brand Semper, and Italy, Spain, France and Portugal under the brand Nutricia.

The nutritional characteristics of twelve different types of gluten-free breads and challenges and experiences during development and production in order to meet customer needs will be discussed. An example of Trio rolls production will be presented.

Methodology. 1. The following nutritional analyses of twelve VBN’s gluten-free breads were carried out: iron content (AOAC, 1990), the total antioxidant capacity (TAC) (Re et al., 1999), total dietary fibre content (TDC) (AOAC, 1997), crude proteins (AOAC, 1984) and fat content (Gertz & Fiebig, 2000)

2. The ingredients used for baking Trio rolls were: wheat starch, water, vegetable oil, ( palm and rapeseed), bakery syrup, yeast, sunflower seeds, pumpkin seeds, crashed linseed, sugar, salt, apple fibre, sugar-beet fibre, raising agents, preservatives, mineral (iron) and vitamins. Mixing of the ingredients were carried out in industrial mixer (Spiral mixer SPI280, Aquamix, VMI, France) for 6 minutes, divided in a hoper (Vemag, HP10, Germany), shape through roll plant (Konig, Austria) and proved for 30 min. at 30C and humidity of 80% (MVS prover, Gouet, France), baked in double action oven (Gouet, France) for 12 min at 185C at the beginning and 205C at the end of the oven, kept in multi level dynamic cooler for 1 hour, packed in Sudpack film using a Multivac R530 (Multivac, Germany).

Results. 1. Table 1 presents some nutritional properties such as CP, TDF, fat, iron and TAC of twelve gluten-free breads produced by VBN. The range of CP varied between 1-4.1%,

167

TDF 1.7-7%, fat 0.3-3.8%, iron 3-4.5% and TAC 0.2-1.6. The variation of nutritional properties depends mainly on the ingredients used for the production of breads.

2. An example of gluten-free bread Trio rolls is presented in Figure 1.

Table 1. Some nutritional characteristics of gluten-free breads

Min Max

CP(%) 1 4.1

TDF(%) 1.7 7

Fat(%) 0.3 3.8

Iron(mg/100) 3 4.5

TAC (%) 0.2 1.6

Figure 1. Gluten-free Trio rolls

Conclusion. Through the KTP the VBN has been able to launch a range of brended gluten-free breads with desirable nutritional and sensory quality, which has opened up the opportunities to meet the customer needs and strengthen the company’s competitive offering.

References. AOAC (1990). International Method 977.30 (16th Ed.). Washington DC: Association of Official Analytical Chemists.

AOAC (1997). International Method 985.29 (16th Ed.). Washington DC: Association of Official Analytical Chemists.

AOAC (1984). Official methods of analysis. (S. Williams Ed.). Washington DC: Association of Official Analytical Chemists. Gertz, C. & Fiebig, H.J. (2000). Determination of fat content by the caviezel method (rapid method). European Journal of Lipid Science and Technology 102, 2, 154-158. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9-10), 1231-1237.

168

Imitated Rye Flour – Evaluation of Pentosan Sources

Markus J. Brandt*; Gina Jaspers

Ernst Böcker GmbH & Co. KG, Minden, Germany

*corresponding email: markus.brandt@sauerteig.de

Introduction. The formation of a gluten network in wheat bread doughs is the prerequisite for

the typical quality of wheat breads. Main focus in the development of gluten-free breads aims

on the substitution of this gluten network by adding hydrocolloids, e.g. guar gum, xanthan,

etc. for water binding. The batter consistency of these doughs do not allow the baking of

crusty breads (without tin). On the other hand, in rye doughs, gluten network formation plays

a neglible role and water binding is obtained by pentosans. It was the aim of our studies to

develop a rye flour imitate based on raw materials rich in pentosan without using additional

hydrocolloids and allowing the baking of crusty bread.

Methods. Pentosan determination was performed according to Hashimoto et. al. (1987).

Pentosan source, potato starch and bean flour were mixed a in dry state. This premixes were

used with 1.9 % salt, 2.5 % baker’s yeast and an appropriate amount of tap water. Doughs

were mixed for 5 min in a spiral kneader and after 3 min resting time sheeted, moulded and

transferred to wooden fermentation baskets. After 40 min proof, baking was performed for 60

min starting at 240 °C and falling to 210 °C temperature. If a sourdough was used, a

commercial starter culture consisting of Lactobacillus plantarum, L. fermentum, L. paracasei,

L. paralimentarius, L. helveticus, Leuconostoc argentinum and Saccharomyces pastorianus

was used. Sourdough fermentations were carried out at 26 °C for 16 h.

Results. Rice bran, quinoa flour and flaxseed were chosen as flours rich in pentosans and

calculations were performed to meet the pentosan and starch content of rye flour by adding

starch from potatoes and bean flour as protein source. Mixing behavior of all used rye-flour

imitates were similar to rye. Although calculated pentosan content was on the same level,

water absorption of the rye-flour-imitates showed impressive differences. Therefore, the

dough yield had to be adapted for the production of crusty bread for each flour mix. (Fig. 1).

By fermenting up to 40 % of the flour-imitate with the aid of sourdough, flavor, taste and

crumb structure were improved in a similar way as it is for rye bread production. Most

convincing results were obtained by a mixture of flax seed flour, potato starch and horse bean

flour.

169

Figure 1. Crusty breads obtained from varying sources of pentosans (A) flax seed (B) quinoa (C) Rice bran.

Dough weight of each bread was 1000 g.

Conclusions. This study showed that it is possible to “construct” a rye-flour-imitate by using

gluten-free raw materials. Because of its pentosan content, there is no need to use additional

hydrocolloids (e.g. xanthan, guar gum) as it is common use in gluten-free baking. Breads

produced by such an imitate can be baked without tin, allowing a new gluten-free bread

quality.

References

Hashimoto, S., Shogren, M.D., Y. Pomeranz. Cereal Pentosans: Their estimation and

significance. I. Pentosans in wheat and wheat.milled products. Cereal Chemistry

1987;64:30-34.

A B C

170

Changes in bio-active compounds in buckwheat depending on germination conditions

Florian Hü bner, Elke K. Arendt*

University College Cork, Department of Food and Nutritional Sciences, Cork, Republic of Ireland

University College Cork, Bio Transfer Unit, Cork, Republic of Ireland *corresponding email:e.arendt@ucc.ie

Introduction. Germination is an ancient method to alter the physical properties and organoleptic properties of seeds. Newer studies suggest that a number of nutritionally valuable compounds in grains are affected by germination. Amongst the most prominent groups of compounds responsible for positive health effects in plant material are dietary fibre and antioxidants. Buckwheat has been found to contain large amounts of phenolic compouds acting as antioxidants and considerable amounts of dietary fibre. The objective of this study was to evaluate whether malting of buckwheat is an efficient way to produce raw malts rich in antioxidants and dietary fibre and to suggest favourable germination conditions. Methods. Buckwheat, cv Jade, was acquired from Trouw B.V. (Rotterdam, the Netherlands) and malted in a micro malting machine (Joe White, Perth, Australia). The grains were steeped to a moisture content of 45% at 15 ° C. The germination temperature was varied between 10 and 20 ° C and the germination time between 48 and 144 h using a central composite design. The green malts were kilned for 8 h at 45 ° C and 15 h at 55 ° C. The contents of insoluble and soluble dietary fibre were analysed according to AOAC method 991.43. Methanolic extracts of malts and unmalted buckwheat were analysed for antioxidant power and their contents of phenolic compounds, using the FRAP assay and Folin Ciocalteu reagent, respectively. All results were fit into quadratic models and Response Surface methodology was used to identify favourable germination conditions. Results. The maximum and minimum values for all measured parameters as well as values measured in the ungerminated kernels are shown in Table 1. It was possible to increase the contents of soluble as well as insoluble dietary fibre by means of germination. Malts contained higher levels of soluble dietary fibre than the unmalted sample. However, no significant influence of the variation of the germination conditions was seen. The content of insoluble dietary fibre in the malts could be increased by choosing longer germination periods, however, the increase levelled off when extremely long germination periods where applied. An increase in insoluble dietary fibre was likely caused by the loss of other compounds as a consequence of the embryo’s metabolism. Malting for short periods resulted in a decrease in the antioxidant power as well as the content of phenolic compounds. This is likely caused by leeching of the relevant compounds into the steeping water. During later stages of germination, compounds with antioxidant activity are formed or set free from bound forms. This increase sets off the previous loss of the relevant compounds and slightly increased the amount of phenolic compounds in the extracts from the malts.

171

Table 1. Contents of soluble, insoluble and total dietary fibre, phenolic compounds and antioxidant power in unmalted buckwheat in comparison to maximum and minimum values measured in malts germinated with varying germination conditions.

Soluble Dietary Fibre

Insoluble Dietary Fibre

Total Dietary Fibre

Antioxidant power

Phenolic compounds

% dm % dm % dm mg gallic acid/ g malt

mmol trolox equ. / g malt

unmalted 2.3 19.9 22.2 3.55 1.56

Maximum value

3.9 26.4 30.3 3.84 2.12

Minimum value

2.9 21.5 24.4 2.6 1.31

Conclusions. It was shown that germination of buckwheat is not an efficient way to improve the antioxidant power of the resulting malts. However, the contents of phenolic compounds and their antioxidant power are higher than in the malts of other cereals e.g. barley. In addition to health benefits this might prevent oxidation and off-flavour formation in the product. Increased contents of soluble dietary fibre in malts can increase the nutritional value since these compounds have been connected with the prevention of certain diseases. High contents of insoluble dietary fibre might be caused by the loss of other compounds, and therefore can be economically disadvantageous. Appropriate malting conditions should be selected with respect to the desired product, be it a beverage or solid food such as breakfast cereals.

172

Protein Changes in Buckwheat depending on the Malting Conditions

Florian Hü bner, Elke K. Arendt*

University College Cork, Department of Food and Nutritional Sciences, Cork, Republic of Ireland

University College Cork, Bio Transfer Unit, Cork, Republic of Ireland *corresponding email: e.arendt@ucc.ie

Introduction. Buckwheat malt has been identified as a possible ingredient for the production of gluten free beer. Proteins and their hydrolysis during malting play a key role in the quality of malts, as the degradation of proteins allows better extractability of starch. Amino acids and small peptides provide nutrients for the growing embryo and, in the case of beer production, for the yeast. Soluble proteins play an important role in the head formation and the mouthfeel of beer. The present study investigated the influence of the germination conditions on the proteolytic activity, malt quality related protein fractions and the overall breakdown of proteins. Methods. Buckwheat, cv Jade, was acquired from Trouw B.V. (Rotterdam, the Netherlands) and malted in a micro malting machine (Joe White, Perth, Australia). The kernels were steeped to a moisture content of 45% at 15 ° C. The germination temperature was varied between 10 and 20 ° C and the germination time between 48 and 144 h using a central composite design. The green malts were kilned for 8 h at 45 ° C and 15 h at 55 ° C. Total Nitrogen content was measured in the malts, Soluble Nitrogen and Free Amino Nitrogen in experimental worts according to EBC standard methods. The proteolytic activity of the malts was assessed with haemoglobin as substrate. The breakdown of proteins was followed using capillary electrophoresis based on lab-on-a-chip procedure. All results were fitted into quadratic models using Response Surface Methodology in order to identify significant effects of the germination conditions. Results. Although some variations were seen in the malts’ contents of Total and Soluble Nitrogen, these changes appeared not to be caused by the variations of the germination conditions. Free Amino Nitrogen, however, increased as a consequence of longer germination times, while no effect of changes in the germination temperature were visible. No significant increase or decrease in the proteolytic activity of the malts was observed with the applied method. Electropherograms of protein extractions of selected malts are shown in Figure 1. Most peak areas decreased as a consequence of long germination periods, indicating a progressing breakdown of proteins during the germination. The largest peak was attributed to proteins with a molecular weight of 22 kDa. Proteins with a similar molecular weight have been found to be the proteins responsible for buckwheat allergy. This peak area could be decreased by nearly 50 % in samples germinated for 144 days at low temperatures in comparison to samples germinated for 2 days. Higher germination temperatures however, resulted in a less pronounced breakdown of proteins.

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-50

0

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100

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10 15 20 25 30 35 40 45 50

Elution time / s

Flu

ore

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ce U

nit

s

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marker

marker

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5.5-9 kDa

14– 17kDa

~ 22 kDa

31– 41kDa

45 kDa

50-55kDa

70-80kDa

60kDa

Figure 1: Electropherograms showing protein peaks from buckwheat malts germinated for 48 h and 144 h at 10 ° C and 20 ° C Conclusions. Long germination periods of buckwheat result in increased breakdown of proteins, resulting in higher contents of Free Amino Nitrogen in the malts. However, buckwheat malt is a poor source of Soluble Nitrogen, a problem which cannot be improved by changing the germination conditions. Protein degradation during the investigated germination period mostly affects proteins which have been already soluble.

174

Protein Changes during Malting and Brewing with Oats

Christina Klose1, Elke K. Arendt1*

1Department of Food and Nutritional Sciences, National University of Ireland,

University College Cork, College Road, Cork, Ireland. *Corresponding author, email: E.Arendt@ucc.ie

Introduction Oats are one of the most popular cereals for human consumption and have received increased interest because of their excellent health-related properties. Oats supply key cardio protective micronutrients such as folate, magnesium, vitamin B6 and vitamin E. Whether oats can safely be included in a gluten-free diet was debated over the last decades. Several studies revealed that oats can be tolerated by the majority of people who suffer from celiac disease. The Scientific advisory board of the Finnish Coeliac Society declared oats acceptable for adult celiac patients in 1997. Oats beer could be a gluten-free alternative to traditional barley malt beer. Objectives The aim of this study was to brew a 100 % oats beer and to follow protein changes during malting and brewing. The knowledge of protein changes during brewing is of technological relevance as they have an influence on foam and haze formation. Methods To characterise oatsmalt quality, a malt analysis with special regard to protein contents was carried out using EBC methods. Two-dimensional gel electrophoresis and Lab-on-a-Chip analysis were used to obtain the protein profiles of raw oats, malts, worts, hot trubs and beers. Results and Discussion Results of malt analysis showed acceptable values for brewing purposes, which were general lower than values found in barley malt. Nonetheless, lower extract values of oats worts resulted in higher degrees of fermentation, which means that despite lower extracts in oats the amount of fermentable sugars was relatively high. Protein profiles showed significant decreases during malting (Klose, C. 2009). In oats beer similar protein profiles to that of barley beer have been detected. Despite the different protein distribution of barley and oats, i.e. mainly prolamins and glutelins in barley and mainly globulins in oats, the resulting beers show a similar protein profile. The beer protein profile is also comparable with the corresponding wort protein profile. In oats beers a 40 kDa protein was present, which can also be found in barley beers. The barley aleurone layer is the origin of the 40 kDa protein in barley beer (protein Z), which belongs to the serpin family and to the albumin fraction (Curioni, A. et al. 1995). In oats grain a serpin with a molecular weigth of 43.5 kDa was also detected (Mikola, M. and Mikkonen, A. 1999). Serpins are known to be heat stable and therefore likely to survive the brewing process. Based on the fact that protein Z derives from barley albumins, it is expected that the 40 kDa oats beer protein derives from the albumin fraction of the grain as well.

175

Figure 1. Protein profile of (A) barley beer (B) oats beer on 2D gels; Protein profile of (C) oats worts and (D) oats and barley beer in electropherograms.

Figure 2. Glass of filtered oats beer. Conclusions Both methods two-dimensional gel electrophoresis and Lab-on-a-Chip analysis were successfully applied in obtaining protein profiles of raw oats, malts, worts, hot trubs and beers. Both methodologies can play a role in analysis and quality control, since the knowledge of protein changes during malting and brewing is of great technological relevance as it can improve beer quality. References Klose, C., Schehl, B. and Arendt, E. K., Fundamental study on protein changes taking place during malting of oats, J. Cereal Sci. 49, 83-91, 2009. Curioni, A., Pressi, G., Furegon, L., Peruffo, A. D. B. Major proteins of beer and their precursors in barley: electrophoretic and immunological studies. J. Agric. Food Chem. 43, 2620-2626, 1995. Mikola, M., Mikkonen, A. Occurrence and stabilities of oat trypsin and chymotrypsin inhibitors. J. Cereal Sci. 30, 227-235, 1999.

A

B

pH pH 10

76 kDa

43 kDa

36 kDa

31 kDa

22 kDa

pH pH 10

76 kDa

43 kDa

36 kDa

31 kDa

22 kDa

C

D

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Optimization of rheological properties of gluten-free pasta using mixture design

V. Larrosaa, G. Lorenzo a,b, N. Zaritzky a,b, A. Califano a, * a Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), Facultad de Cs. Exactas, UNLP-CONICET. 47 y 116, La Plata (1900), Argentina.

b Área Departamental Ingeniería Química, Facultad de Ingeniería, UNLP, Argentina. *corresponding email: anc@quimica.unlp.edu.ar

Introduction. The raising demand of gluten-free products in recent years, have led to an important technological research for replacing the gluten matrix in the production of high quality gluten-free foods. Many of the products currently in the market are of low quality, exhibiting poor structure, mouthfeel, and flavor (Gallagher et al. 2004). There are many works on improving gluten-free breads (Gallagher et al. 2004; Lazaridou et al. 2007), but only a few on other type of gluten-free products such as pasta. In the production of gluten-free pasta analogues, wheat flour was substituted with rice flour, precooked rice flour, or pregelatinized rice starch. Besides, hydrocolloids may enhance textural aspects of the dough turning them practically indispensable to formulate any kind of gluten-free dough. Xanthan-locust bean gum (XG-LGB) mixtures are used industrially as thermoreversible gelling agents (Zhan et al. 1993). The objective of this work was to evaluate the effect of composition (hydrocolloids, water and proteins), on the viscoelastic and textural properties of gluten-free dough used for pasta production based on cornstarch and corn flour.

0.00

0.05

0.10

0.15

0.20

Prot

ein

(g/g

)

0.01

0.02

0.03

0.04

0.05

0.06

Xanthan + locust bean gums (g/g)

1

3 8 4

Methods. Basic dough formula consisted in a mixture of corn starch and flour (4:1, 53.5%), 1% NaCl, and 3% sunflower oil. As it was not advisable to allow water, gums, and protein content to take values anywhere in the range of possible values (e.g. water = 0%), a mixture design with constrains was chosen. XG-LBG were used in a 2:1 ratio and the protein ratio was maintained in 10:1 for dry egg and ovoalbumin mixtures. Dough was prepared as described in a previous work (Lorenzo et al. 2007). After a resting time of 24 hs at 4ºC, dough was rolled out using a rolling machine (Pastalinda, Argentina) to give a sheet of 2 mm thick. Combinations of gums (0.51-2.52%), proteins (0.68-6.70%), and water (35.5-39.5%) were used in a simplex-centroid augmented design consisted of twelve runs: four points at the extreme vertices of the feasible quadrangular region (1,2,3,4), four points at the edge centroids, (5,6,7,8) one point at

the overall centroid (9), and three added points (A,B,C)to evenly cover the experimental region (Cornell, 2002). Figure 1 displays the constrained region for the mixture with the actual design points. Pasta moisture content was determined according to the AACC 44-40 (1984). Extensibility tests on fresh (non-cooked) doughs were performed on squared specimens (80 x 80 x 2) mm with a Texture Analyzer TA-XT2i (Stable Micro System, UK), using a round compression probe (2.5cm dia) and a pastry burst rig (TA 108) determining the breaking force (N), distance (mm). Oscillatory shear tests (storage (G’) and loss (G’’) moduli vs. frequency, (ω)) were performed in duplicates in a

C

5 9 6

A B

7 2

0.80 0.85 0.90 0.95 1.00Water (g/g)

Figure 1. Simplex-centroid augmented design; mass fractions are expressed as coded variables.

177

RS600 Rheometer (Haake, Germany), according to Lorenzo et al. 2007. Linear viscoelastic range was previously determined. Results. Surface response analysis was used to determine the relationship between

breaking force and dough composition, considering a full quadratic model; the same procedure was applied to distance at breaking force data. The model adequately predicted experimental results (R2

force =0.97 and R2distance =0.99). Figure 2 shows

as an example the projection of the braking force plotted on the 2D composition triangle. Based on the effects of the ingredients on each characteristic of the product the composition was optimized according to the overall desirability criteria (Derringer and Suich 1980) using Excel “solver”

module.

Figure 2. Response surface of the breaking force as a function of dough composition shown as coded variables.

Results of the dynamic oscillatory tests are presented in Figure 3 for three formulations with different water contents and 2,5% gums; protein concentrations are shown in Figure 1. The curves were qualitatively similar for all the formulations assayed. G’ was always greater than G’’ in the frequency range measured and the increase of the two moduli with frequency was small. Oscillatory spectra were satisfactorily modeled using the Maxwell Generalized model as shown in Figure 3 (Ferry 1980). Conclusions. Application of a mixture design to gluten-free pasta production allowed finding the optimal dough composition to achieve the desirable textural properties (extensibility and resistance to rupture), turning the dough easy to handle under industrial conditions. The linear viscoelastic behavior showed the same tendency observed in the large deformation experiments(extensibility).

References Cornell JA. Experiments with Mixtures: Designs, Models, and the Analysis of Mixture

Data. New York: John Wiley & Sons 2002. Derringer G, Suich R. Simultaneous optimization of several response variables. J

Quality Technol 1980;12:214-219. Ferry J D. Viscoelastic properties of polymers. New York: John Wiley & Sons 1980. Gallagher E, McCarthy D, Gormley R, Arendt E. Improving the Quality of Gluten-Free

Products. Agric and Food Develop Authority. Ireland: Project RMIS 2004; 4881. Lazaridou A, Duta D, Papageorgiou M, Belc N, Biliaderis C. Effects of hydrocolloids

on dough rheology and bread quality parameters in gluten-free formulations. J Food Eng 2007;79:1033-1047.

Lorenzo G, Zaritzky NE, Califano AN. Optimization of non-fermented gluten-free dough composition based on rheological behavior for industrial production of ‘‘empanadas’’ and pie-crusts. J Cereal Sci 2007;48:224–231.

Zhan DF, Ridout MJ, Brownsey GJ, Morris VJ. Xanthan-locust bean gum interactions and gelation. Carbohydrate Polym 21 (1993) 53-58.

Figure 3. Mechanical spectra (G’) and G’’ vs. frequency (ω) for formulations containing 2.5% gums.

G’ G’’ ( ) formulation 5; 35.5% water G’ G’’ (Δ ▲) formulation 9; 37.5% water G’ G’’ ( ) formulation 6; 39.5% water solid lines: Maxwell generalized model

178

Characterizing sorghum-based pasta: a multi-disciplinary approach

Maria Ambrogina Pagani1*; Francesco Bonomi2, Gabriella Bottega1, Maria Cristina Casiraghi1, Abd Elmoneim O. Elkhalifa3, Stefania Iametti2

University of Milan, 1 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche and

2Dipartimento di Scienze Molecolari Agroalimentari, Milan, Italy; 4 Ahfad University for Women, School of Family Sciences, Omdurman, Sudan;

*corresponding email:ambrogina.pagani@unimi.it Introduction. Sorghum is a gluten-free cereal and is widely grown all over the world for food and feed. It is one of the main staples for the world’s poorest and most insecure people, especially in the most arid and marginal areas of the semi-tropics. From a nutritional standpoint sorghum represents an excellent font of proteins, starch and antioxidant compounds. The release of sugars from sorghum starch is slower than in other cereals, which is of interest for diabetic or obese people. The overall digestibility of sorghum is low because starch and proteins are associated in compact complex, so that preparation of foods from sorghum requires fermentation (Hassan et al. 1995; Elkhalifa and El Tinay 1995). This treatment results in a significant increase of protein and starch accessibility (Elkhalifa et al. 2006). Sorghum may be used as a gluten-free cereal ingredient in association with or in substitution of maize or rice in the preparation of celiac food. The aim of this study was to produce pasta starting from a mixture of whole rice flour and fermented (F) or unfermented (UF) sorghum flour.

Methods. A low-tannin sorghum cultivar (Tabat), obtained from Food Research Centre, Shambat, Sudan, was used in this study. Sorghum flour was fermented according to traditional Sudanese methods as described in Elkhalifa et al. 2006. Molecular and physical characterization of the starting materials and of the products was carried out as outlined by Mariotti et al. (2008), and by Iametti et al. (2006). Results. In the first part of this work we characterized some molecular and rheological properties of sorghum flours. As expected, the total starch and protein content was higher in UF than in F sample. Viscoamylographic tests indicated viscosity values at peak lower in F than in UF, and gave setback values (463 BU for UF and 235 BU for F) indicating a lower retrogradation tendency in F than in UF. Content of soluble proteins was much lower in F than in UF, confirming that proteins are the preferred substrate for the bacteria involved in fermentation. Fermentation also induces a marked decrease in the total thiol content of flours, suggesting that cysteine is actively taken up by the bacteria. However, the proteolytic breakdown during fermentation results in an increased surface hydrophobicity of the residual proteins, that translates into a markedly different solvation behavior in the various flours, as assessed by front-face fluorescence (Bonomi et al. 2004). Taking into account all this information, we prepared pasta using 85% whole rice flour and 15% of each sorghum flour (figure 1). Addition of sorghum provides a lower starch content in pasta. We found no difference in the viscoamylograms of flour mixtures and the pasta prepared from each of them, suggesting that the pasta-making process does not modify the structural organization of starch. Protein solubility studies indicated that some inter-protein network was present in the raw pasta samples, and that both disulfide bonds and hydrophobic interactions were involved in its stabilization, in particular when fermented flours were used. All pasta samples gave a 70% weight increment upon cooking, and cooking losses were lower in the sorghum-containing samples, consistent with an increased compactness of the protein network upon cooking, as assessed by fluorescence titration studies. However, these latter samples had a

179

lower resistance to compression than rice-only pasta. We found no difference in susceptibility to digestion with pancreatin among the three samples, but the fraction of resistant starch in the F-containing cooked pasta was 2-3 times that in the other two pasta samples. Figure 1. Pasta prepared from: 100% whole rice flour (a); 85% rice flour and 15% unfermented sorghum flour (b); 85% rice flour and 15% fermented sorghum flour (C). Conclusions. This study indicated that fermentation treatment result in a modification of the structural and physical properties of the starch and protein in the flour. These modification strongly influence the transformation properties of the fermented flour that present improved pasta making properties with respect to the unfermented one. References Bonomi F, Mora G, Pagani MA, Iametti S. Probing the structural features and the solvation

behaviour of wheat proteins by front-face fluorescence. Anal Biochem 2004; 329:104-111. Elkhalifa AEO, Bernhardt R, Bonomi F, Pagani MA, Zardi M, Iametti S. Fermentation modifies

protein/protein and protein/starch interactions in sorghum dough. Eur Food Res Technol 2006; 222: 559-564.

Elkhalifa AO, El Tinay AH. Effect of fermentation and germination on the in vitro protein digestibility of low and high tannin cultivars of sorghum. Food Chem 1995; 54:147-150.

Iametti S, Bonomi F, Pagani MA, Zardi M, Cecchini C, D’Egidio MG. Properties of the protein and carbohydrate fractions in immature wheat kernels. J Agric Food Chem 2006; 54: 10239-10244

Hassan IAG, El Tinay AH. Effect of fermentation on tannin content and in vitro protein and starch digestibilities of two sorghum cultivars. Food Chem 1995; 53:149-151.

Mariotti M., Lucisano M., Pagani MA, Iametti S. Macromolecular interactions and rheological properties of buckwheat-based dough obtained from differently processed grains. J Agric Food Chem 2008; 56: 4258–4267.

a b c

180

Ready-to-eat Meals based on Rice Pastas

Luisito Virtucio*, Claudio Maria Pollini, Luciano Mondardini, Germana Zurlo Pavan Group, Galliera Veneta, Italy

*corresponding email: virtucio.l@pavan.com

Introduction. It is from the end of 80’s that Pavan, being aware of the increasing trend of the number of persons suffering from celiac disease, developed an industrial technology for the production of non-gluten dry pastas; at the end of 90’s, the same development has been extended for many non-gluten containing raw materials and subsequently applied for fresh and filled pastas products; an example of such further development in the pasta sector going into maturity just in this period, is the setting up of a process for the production of instant pasta meals prepared from rice pasta (lasagna and ravioli) and “ ricotta” cheese – spinach sauce. In the meantime R&D personnel have also started to design/develop gluten-free breakfast cereals and non-fried or roasted snacks with bioactive (or nutraceutical) ingredients to supplement many components more often times overlooked by many consumers suffering and not from celiac disease in search of a more healthy and lesser junkfoods as many processed foods are now commonly considered. Some of these ingredients are soluble fibers, anti-oxidants and many essential minerals. Methods. Lasagna have been extruded by means a F 55 pasta press in two different ways: native rice flour (*) has been hydrated and then steamed before extrusion; or pre-gelled rice flour (**) has been hydrated and directly extruded; in both cases the possibility of adding texturising agents (+) has been considered. Ravioli have been produced by means a MRP 540 forming machine; also in this case, pasta was produced from native rice flour (*), hydrated and steamed before sheeting so as to create a “ gluten effect” , filling and sealing; or from pre-gelled rice flour (**), hydrated before lamination step; the same texturising agents (+) were used to reduce cooking losses and improve the palatability of the product. In order to compare such new products with the conventional ones, precooked durum wheat lasagna and precooked wheat ravioli have been processed by means the same pilot plants, using also the same sauce and fillers prepared with “ ricotta” cheese and spinach. Results. Ready-to-eat rice lasagna showed very good performance in terms of elasticity, consistency, limited stickiness and pleasant mild taste blended quite well with the ricotta-spinach sauce; the results of TXT texturometer analysis were not so far from lasagne made from durum wheat ones; panel test demonstrated a very high level of acceptability, similar to durum ones; anyway the action of texturising agents was evident. Ready-to-eat ravioli although had some difficulties during the sealing step, showed good elasticity and no stickiness, but consistency was a bit lower and, in any case, far from wheat ones; the samples produced with texturising agents however have increased the organoleptic performances; high panel acceptability, and moreoever, the use of ricotta-spinach filler has been greatly appreciated for the taste and also most probably as a source of supplement for calcium and magnesium in the food and in the diet. Nutritional profile has been quite interesting for both ready meals.

181

182

Figure 2. Lamination and steaming units of pilot plant.

Figure 3. Ready to eat gluten-free meals. Table 1. TXT results for rice (*, **, +) and durum lasagna. UNDER CONSTRUCTION Table 2. TXT results for rice (*, **, +) and wheat ravioli. Conclusions. The consumption of gluten-free foods is increasing year after year, at least in the northern part of the world; this is not only due to the increase of people affected by celiac disease, but also to the increasing awareness and attention paid by consumers to lighter, more digestible, more equilibrated foods. A lot of shelf space is nowadays available for gluten-free products such as dry pastas, fresh pastas, breakfast cereals and roasted snacks, especially if added with nutraceutical ingredients. Ready-to-eat rice pasta meals may be one part of the future, thanks to high level of convenience and to the well-balanced nutritional profile; addition of nutraceutical or even bioactive ingredients may be taken into consideration. The good performances showed by the products object of this work, also in comparison with wheat homologues (gluten containing products), demonstrates also the high standards of technology and know-how reached in this field. References

1. Celiac Disease di Liane Beck, Emory Family Medicine 2. Sviluppo delle Paste Ripiene Aglutiniche, Virtucio et al, Rich Mac Meeting (MI), Ottobre 1999 3. Testing for Non-Celiac Gluten Intolerance, Dr. Stephen Wangen, IBS Treatment Center 4. Spaghetti, Noodles, e Piatti Pronti. Sempre e Ovunque, Luisito Virtucio, Molini d’Italia, Marzo 2007 5. Non-Traditional Pasta, Luisito Virtucio, Tecnologie Alimentari, Novembre-Dicembre 2004

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Starch Characterization of Rice Pasta: Comparison between Extrusion-cooking and Conventional Pasta-

Making Process

Alessandra Marti1,2*, Rosita Caramanico3, Koushik Seetharaman2 and Maria Ambrogina Pagani1

1 University of Milan, Department of Food and Microbiological Science and Technologies (DiSTAM), Milan, Italy 2University of Guelph, Department of Food Science, Guelph, Ontario, Canada 3 CRA-SCV, S. Angelo Lodigiano (LO), Italy * corresponding email: alessandra.marti@unimi.it Introduction. In recent years, R&D focused on the development of formulation and/or processing for improving gluten-free (GF) products. With respect to pasta, several ingredients have been used as alternatives to gluten, in order to create a network capable of providing firmness to the pasta and with the ability to keep starch granules inside pasta during cooking. In this study, the ability to produce gluten free pasta from rice without the use of any additives was investigated. Methods. Brokens from parboiled rice (commercial Indica variety) were ground and used to produce a pre-treated milled flour. Two pasta-making processes were compared. The extrusion-cooking process (process A) consisted of two steps; rice dough (40% moisture) was extruded in a Progel® extruder at 115 ° C. The pellets obtained in this first step were shaped in rigatoni by using a conventional pasta extrusion at 50 ° C (sample A). In the conventional extrusion process (process B), the dough was directly formed into rigatoni (sample B) in the continuous extruder at 50 ° C. The role of processing on pasta cooking behavior including water absorption, cooking loss and texture analysis was evaluated. Textural properties of cooked pasta were evaluated by a compression-shear-extrusion test, carried out with a Kramer cell. The structural modifications of starch induced by processing were evaluated by several approaches, including α-amylase susceptibility, Micro-ViscoAmylograph test, Differential Scanning Calorimetry, and X-ray diffraction. Starch was further isolated before and after cooking from each pasta samples, equilibrated to final aw values of 0.33, 0.75, and 0.97, and exposed to iodine vapour. Absorption spectra of the samples after iodine exposure were recorded and presented as absorption/scattering (K/S) (Saibene and Seetharaman, 2006). Results. Pasta-making process greatly influenced both cooking loss and textural properties. In particular, sample A was characterized by a higher absorption during cooking (77% vs 62%), less cooking loss (4% vs 16%) and relevant firmness and shear force, after cooking (Figure 1). These results suggest that, compared to process B, the heat-treatment of process A allowed to create a more hydrophilic and continuous structure involving starch macromolecules. Moreover, process A promoted a starch network more susceptible to enzymatic hydrolysis (12.5% d.b. vs 10.8% d.b.) but, at the same time, exhibited a higher peak viscosity (188 BU vs 130 BU), and a higher final viscosity (825 BU vs 521 BU), suggesting a better water-holding capacity and ability to form a gel following cooling.

185

Figure 1. Texture profile of cooked sample A (black line) and sample B (grey line). DSC measurements accounted for a different crystalline order in the two pasta products, with a more stable starch network in sample A (Tp: 79 ° C vs 64 ° C). The exposure to iodine vapor of starch isolated from pasta samples confirmed the differences in the starch granule organization between the uncooked and cooked samples. Before cooking, sample A showed a higher K/S values compared to those of sample B. These differences were greatly reduced after cooking, highlighting a different starch rearrangement during cooking. Conclusions. The use of a parboiled flour might simplify the Oriental noodle-making process, characterized by the sequence of several heating and cooling steps of rice dough, tricky to be controlled and monitored. Moreover, the different processing conditions created two pasta samples with a new and characteristic structure and architecture, affecting both cooking and textural pasta quality. This study highlights the ability to produce gluten-free pasta from rice without the use of other additives and simply by modulating the starch structure in the raw material. References Mariotti, M., Zardi, M., Lucisano, M., Pagani, M.A. Influence of the heating rate on the

pasting properties of various flours. Starch/Starke 2005; 57: 564-572. Saibene, D. and Seetharaman, K. Segmental mobility of polymers in starch granules at low

moisture contents. Carbohydrate polymers 2006; 64: 539-547.

186

Gluten in Oat-based Beverages and Oatmeal

Ylva M. Sjögren, Birgitta Kruse, Monica Ferm, Martin Sandberg, Ingrid Malmheden

Yman

National Food Administration, Uppsala, Sweden.

e-mail to corresponding author: ylva.sjogren@slv.se

Introduction: Pure oat has been used in gluten-free diets for several years in the Scandinavian

countries. Oat increases the fibre content in the gluten-free diet and most gluten-intolerant

children and adults tolerate pure oats (Holm et al 2006, Storsrud et al 2003). Oat that are

suitable for gluten intolerant individuals shall fulfil the following criteria: "The oats contained

in foodstuffs for people intolerant to gluten must have been specially produced, prepared

and/or processed in a way to avoid contamination by wheat, rye, barley, or their crossbred

varieties and the gluten content of such oat must not exceed 20 mg/kg” ((EC) No 41/2009).

Around the time of diagnosis of celiac disease secondary lactase deficiency is common

(Rodrigo 2006) and milk products must be avoided. Oat-based beverages might replace milk

products in a lactose-reduced diet.

Objective: Analyze the gluten content in oat-based beverages and oatmeal products on the

Swedish market and study whether oatmeal products are contaminated with wheat, rye and/or

barley.

Methods: Seventeen oat-based beverages and sauces (figure 1a) and 14 oatmeal products

(figure 1b) were analyzed for gluten content with the R5 Mendez ELISA. Two different

production dates were analyzed for most products. Three of the beverages contained malt

flour in concentrations ranging from 0.03% to 0.15%. One oatmeal product was termed pure

oat as it was produced and processed to avoid contamination of wheat, rye and barley. In

order to discriminate between the cereals found in the oatmeal products, TaqMan®

real-time

PCR, specific for wheat, rye and barley respectively, was employed.

Figure 1a. Oat-based beverages and sauces. Figure 1b. Oatmeal products

Results: The gluten content was below the limit of quantification (LOQ) (5 mg gluten/kg) in

one oat-based beverage and one sauce. The other oat-based beverages and sauces contained in

average 14 mg gluten/kg (6 to 24 mg gluten/kg) except for the beverages which contained

malt flour. The oat-based beverage with the highest concentration of malt flour contained 110

mg gluten/kg. The gluten content was below LOQ in four of the 14 oatmeal products

analyzed, including the “pure” oatmeal (table 1). There was a large variation in gluten content

between the different production dates of the oatmeal products (table 1). This was in contrast to the oat-based beverages and sauces where the variation between production dates was

small. Wheat, rye and/or barley DNA was detected in most oatmeal products and all three

187

cereals were responsible for gluten contamination. No wheat, rye or barley DNA was detected

in the pure oatmeal or in the oat gruel that had been autoclaved.

Table 1. Gluten and DNA from wheat, rye and barley analyzed in oatmeal products produced at two different

dates (I and II).

Product Producer I

ppm

gluten*

I

Detected DNA

II

ppm

gluten*

II

Detected DNA

Oatmeal a 17 wheat 44 wheat, barley, rye

Oatmeal, organic a 127 wheat, barley, rye 1403 wheat, barley, rye

Oatmeal b 1922 wheat, barley n.t.

Oatmeal c 46 wheat, barley 470 wheat

Oatmeal d 11 wheat, barley# 1105 rye

Oatmeal, organic d n.d. n.d. n.d. wheat, barley

Oatmeal e 368 barley 42 barley, wheat#

Oatmeal with extra fiber e 260 rye, barley 129 wheat, barley, rye

Oatmeal “glutenfree” f n.d. n.d. n.t.

Oatmeal g 8 wheat, barley# 13 barely

Oatmeal “quick” h n.d. n.d. n.t.

Oatmeal h n.d. rye n.t.

Oatmeal and quinoa, organic i 8 wheat, barley# n.d. wheat, barley, rye

Oatgruel

(autoclaved oat)

e 65 n.d. 47 n.d.

n.d. = not detected, n.t. = not tested.

#= ambiguous results to be reanalyzed. *= The measurement uncertainty is

25% and calculated using a coverage factor of 2, which gives a confidence level of approximately 95%.

Conclusions: Oatmeal products are often contaminated with wheat, rye and/or barley.

Therefore, only oatmeal products which are produced and processed in a way to avoid

contamination with wheat, rye and barley are suitable in a gluten-free diet. The use of oat-

based sauces is probably safe in a gluten-free diet. Also, oat-based beverages might be safe to

use in a gluten-free diet. However, when beverages are consumed in large quantities they

could contribute to the overall gluten intake.

References:

1. Holm K et al. Oats in the treatment of childhood coeliac disease: a 2-year

controlled trial and a long-term clinical follow-up study. Aliment Pharmacol Ther

2006;23:1463-72.

2. Storsrud S et al. Adult coealiac patients do tolerate large amounts of oats. Eur J

Clin Nutr 2003;57:163-169.

3. Rodrigo L. Celiac disease. World J Gastroenterol 2006;12:6585-6593.

188

Gluten-Free and Very Low Gluten Foods – Enforcement of Common EU Legislation

Annika Nurttila

Finnish Food Safety Authority EVIRA, Helsinki, Finland

corresponding email: annika.nurttila@evira.fi

189

Wheat Starch in Gluten-Free Diet

Katri Kaukinen

Coeliac Disease Study Group, University of Tampere, Finland corresponding email: Katri.Kaukinen@uta.fi

190

Wheat starch in g luten free bread

Nanna Mossberg,

In northern Europe celiac disease has been diagnosed for more than 50 years, and wheat

starch based products have been used in the gluten free diet for about 40 years.

of the countries with the longest experience of wheat starch based gluten free bread

about 90 % of all gluten free bread in Sweden is based on wheat starch

The strongest reason for using wheat starch in gluten free bread is probably

makes the bread taste more like bread baked of wheat flour

other gluten free ingredients. The fact that wheat starch based gluten free bread tastes like

common bread might make it easier for celiac people to a

Compliance to the diet is vital to stay healthy for people with celiac disease.

It is important to remember that there are many different purity levels of wheat starch, and

that all wheat starch might not be suitable in the g

“Gluten free” or “Very low gluten” it has been baked of wheat starch that is of a purity that is

suitable in the gluten free diet.

Wheat starch in gluten free bread

Nanna Mossberg, registered dietitian

Fria

In northern Europe celiac disease has been diagnosed for more than 50 years, and wheat

starch based products have been used in the gluten free diet for about 40 years. Swe

of the countries with the longest experience of wheat starch based gluten free bread

about 90 % of all gluten free bread in Sweden is based on wheat starch.

The strongest reason for using wheat starch in gluten free bread is probably the fact that it

makes the bread taste more like bread baked of wheat flour, compared to bread baked from

The fact that wheat starch based gluten free bread tastes like

bread might make it easier for celiac people to adhere to the gluten free diet.

Compliance to the diet is vital to stay healthy for people with celiac disease.

It is important to remember that there are many different purity levels of wheat starch, and

that all wheat starch might not be suitable in the gluten free diet. If the bread is labeled

“Gluten free” or “Very low gluten” it has been baked of wheat starch that is of a purity that is

In northern Europe celiac disease has been diagnosed for more than 50 years, and wheat

Sweden is one

of the countries with the longest experience of wheat starch based gluten free bread, and today

the fact that it

compared to bread baked from

The fact that wheat starch based gluten free bread tastes like

dhere to the gluten free diet.

It is important to remember that there are many different purity levels of wheat starch, and

luten free diet. If the bread is labeled

“Gluten free” or “Very low gluten” it has been baked of wheat starch that is of a purity that is

191

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192

Oats in Gluten-free Diet – Considerations of a Pragmatic Policy

Hannu Salovaara, Päivi Kanerva, Jussi Loponen, Tuula Sontag-Strohm

University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Viikki Food Science, Finland

*corresponding email: hannu.salovaara@helsinki.fi Introduction. Re-evaluation of the scientific literature on oats and coeliac disease some twenty years ago in Kuopio by Janatuinen et al. (1995) showed that there was no real evidence for classifying oats as harmful to individuals with CD. This was then confirmed in a number of clinical studies involving some 300 individuals with CD, with only one case reporting conflicting results of two or three Norwegian individuals sensitive to oats. This current paper deals with the experiences with oats and CD at the national level in Finland and discusses the current regulation with the background of the pragmatic coeliac policy applied in this country. Oats in GF regulation. The uncertainty regarding oats is indicated in GF definitions through decades (Table 1.). In the current EU regulation oats still has a particular statement. Table 1. Oats in GF regulations historically Regulation Definition/statement/comment Codex Stand 118-1981, amended 1983)

‘a protein fraction from wheat, rye, barley, [oats] or their crossbred varieties’/ the square brackets indicating the uncertainty felt about the position of oats

Draft revised standard for GF foods (2007)

‘Oats can be tolerated by most but not all people with CD. Therefore, the use of oats not contaminated with gluten … may be determined at national level.´

EU regulation (EC) (41/2009)

’Oats contained in foodstuffs for people intolerant to gluten must have been specially produced, prepared and/or processed in a way to avoid contamination by wheat, rye, barley, or their crossbred varieties and the gluten content of such oats must not exceed 20 mg/kg. (Art. 3:3)

Role of oats in Finland. In no other country oats have such an importat role as a crop (no 2, 2nd only to barley). The production, ca 1 000 milj. kg, is among highest in EU. Some 200–300 milj. kg/yr of food grade oats are exported, mostly to USA, Germany. Finnish oats are regularly used by oat mills in Central Europe. The local per capita consumption of oat products is ca 4 kg/yr, higher than in most countries.

Protein composition of oats. Oats phylogenetics is different from the Triticeae cereals (wheat, rye, barley), and this also reflects in the protein composition (Table 1). Oats contain only ca 10% prolamin, and the amino acid sequence differs, as also indicated by the fact that the R5 pentapeptide used in gluten analysis is not present in oats. The main protein fraction in oats is globulin (salt soluble), as is the case in many other seeds, such as glycinin of soybean.

Table 1. Protein composition of cereals

Protein fraction Wheat Rye Barley Oat Rice Maize % Prolamins 70–80 30–50 30–50 10 3–5 60

% Q (glutamine) 33 6(30) 3(30) 34 20 19 % P (prolin) 17 17 20 10 5 10

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Oat products in GF diet. Anecdotally oats have always been considered healthy, and this also relates to the coeliac disease. Soon after the suitability of oats for individuals with CD had obtained scientific evidence in the 1990’s the use oats for individuals with CD was endorsed by the Scientific Advisory Board of the Finnish Celiac Society (Table 2). Today more than 70% of people with CD in Finland use oats in their diet, reporting improved quality of life, and clinically no adverse effects have been reported. Table 1. Historical milestones of oat products in GF diet, with emphasis on Finland __________________________________________________________________________________ Year Description __________________________________________________________________________________ 1950 Dicke finds wheat to cause CD, and later (1953) suggests oats harmful also 1953-1974 Conflicting results from few studies with oats 1953–1974 1974 First study with applying biopsy: oats not harmful 1976-1995 No clinical studies on oats in literature on CD 1976 Codex Alimentarius standard 118-1981 for ‘gluten-free foods’, amended in 1983, includes oats

among cereals containing gluten (wheat. rye, barley, oats) 1992 Question raised by a group of medical doctors in University of Kuopio 1995 The Kuopio group publishes their first paper on oat: not harmful (Janatuinen et al. 1995) 1997 Oats endorsed by the Scientific Advisory Board of the Finnish Celiac Society for adult

individuals with CD, and a year later (1998) for adult individuals with DH 2000 Oats approved for children with CD by the Scientific Advisory Board of the Finnish Celiac

Association of the Finnish Celiac Society 200? Local authorities approve labelling: ‘contains oats and gluten free ingredients’ 2002 Pure oat products enter the market 2002 Mendez develops the R5 pentapeptide and its antibody for a method that detects the Triticeae

prolamins but not oat avenin 2006 The R5 method reported to greatly exaggerate barley contaminants in oats (Kanerva et al.) 2007 Health Canada’s (positive) position on oats in diets of individuals with CD 2007 Draft revised standard for GF foods (2007) allows national decisions on oats 2009 New Commission regulation (41/2009); new limit 20 ppm; oats endorsed in case the new limit

is met, national regulations to cease by 2012 2010 Oat-based foods available are on the market but no substantial increase in the oat food

assortment with gluten-free labelling (below 20 mg/kg) __________________________________________________________________________________ The pure oats issue. Discussion has been raised on whether the results of the clinical studies performed in Kuopio and the new low regulatory threshold (20 ppm) put on oats are in agreement. The evidence obtained in the clinical studies was collected form individuals with CD consuming commercial oat products, which probably contained 200 to 500 ppm gluten, far from the current regulative 20 ppm limit. In recent years pure oat products meeting the 20 ppm have come on the market but at a higher price than regular oat products. The feeling and experience among the people with CD in this country tends to be now that the price of oat based foods meeting the 20 pmm threshold has risen while the assortment has become smaller. There may be a corresponding situation with wheat starch containing foods. In accordance to the pragmatic approach applied it may be stated that any unnecessary reduction of the regulated limits for GF tends to reduce the number of gluten-free foods available, and increase the risk of poor compliance to the diet among those having CD. A pragmatic and apparently successful coeliac policy applied in this country would support somewhat higher content thresholds such as 100–200 ppm.

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Tips for using oats in the gluten-free diet

Sanna Arnala

The Finnish Coeliac Society

corresponding email:sanna.arnala@keliakialiitto.fi

Introduction. Finnish coeliacs have been using an oat-containing gluten-free diet for over ten

years. The use of oats was endorsed for coeliac patients in 1997 when the scientific advisory

board of the Finnish Coeliac Society issued a statement whereby the use of oats was permitted

for adult coeliacs. It was based on evidence showing that most coeliac patients can consume

oats without harmful effects on the small bowel villi. The statement was extended in 1998 to

concern DH patients and children in 2000. Nowadays the majority of Finnish coeliacs include

oats in their gluten-free diet.

Use of uncontaminated oats in the gluten-free diet. Five years after the statement, in 2003,

70 % of Finnish coeliacs were using oats in their diet (Peräaho et al. 2004). Three years later,

in 2006, the number was 76 %, when members of The Finnish Coeliac Society responded to a

survey. The Finnish Coeliac Society estimates that the number has increased since, especially

among newly diagnosed patients. Coeliacs feel that oats diversify their diet and many also

appreciate the taste (Figure 1). Oats are included in a normal Finnish diet, so it is a familiar

cereal to use. Coeliacs consider oats to be healthy as well. Adequate intake of dietary fibre is

often a concern in the gluten-free diet. Oats contain high levels of dietary fibre. A diet high in

fibre helps to lower blood cholesterol and to stabilize blood glucose levels, which may benefit

people with diabetes, for example. It also helps to maintain a healthy gut as it can prevent

such symptoms as constipation.

Figure 1. Reasons why coeliac disease patients say they include oats in their gluten-free diet

(Peräaho et al. 2004).

Oat products. If oats are produced in the same place as wheat, barley or rye, there is a risk of

gluten getting mixed up with the oats. Oat products free of contamination are safe for coeliacs

to use. Oats are easy to include in the gluten-free diet, if there is a good selection of

uncontaminated oat products available, as in Finland. It may be one of the reasons why oats

are widely used among Finnish coeliacs. Product assortment includes flakes, grits, flour,

muesli, bread, pastry, cookies etc. (Figure 2). Since 2009 the EU legislation has allowed

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uncontaminated oat-containing products to be labelled as gluten-free, which is likely to

increase selection and use of oats. Oat products may also get the Crossed Grain symbol,

which makes it easier for coeliacs to recognise products suitable to the gluten-free diet.

Figure 2. Oat grits, flakes and flour.

Guidelines for using oats in the gluten-free diet. Oats add variation and nutritional value to the

gluten-free diet. Oat products must be made from uncontaminated, gluten-free oats. It might

be advisable to start the use of oats with small amounts, for example with one or two

tablespoons per day, especially if coeliac disease has been diagnosed many years ago. Sudden

increase in dietary fibre may cause gastrointestinal symptoms. If no disturbing symptoms

appear after using small amounts of oats for about one week, it is possible to increase the

amount gradually. Otherwise there is no limit for the amount in the oats use. Using oats in the

gluten-free diet is not essential, it is up to the coeliac oneself to decide whether to include

gluten-free oats in one’s diet or not.

Oats can be used for porridge, bread dough, pastry dough, cookies, smoothies, muesli, etc.

Porridge can be made using only oats; oat flakes or grits, if desired. When making dough it is

advisable to mix oat flakes, flour or bran with other gluten-free flours. Oat flakes or bran can

also be used without cooking as such for smoothies or muesli, for example.

Some gluten-free recipes with oats can be found on the Finnish Coeliac Society’s website

www.keliakialiitto.fi/liitto/in_english.

References

Peräaho M, Collin P, Kaukinen K, Kekkonen L, Miettinen S, Mäki M. Oats forms an essential

part of gluten-free diet in celiac disease and dermatitis herpetiformis. J Am Diet Assoc

2004;104:1148-50.

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Pure Oats production-Company presentation

Pirjo Alho-Lehto

Raisio Group, Food Division, Finland corresponding email: pirjo.alho-lehto@raisio.com

Introduction. Oats are approved for celiac persons in EU Commission regulation (EC No 41/2009 20 January 2009) concerning the composition and labelling of foodstuffs suitable for people intolerant to gluten. Oats must have been specially produced, prepared and/or processed in a way to avoid contamination by wheat, rye, barley, or their crossbred varieties and the gluten content of such oats must not exceed 20 mg/kg. The new regulation allows more nutritive diet for gluten intolerant people. Objectives. Raisio Group has developed a unique technology to produce strict controlled Pure Oats ingredients. The whole production chain from seed to finished product is controlled. Pure oats coming from contract farms is stored in silos and is transferred to different pure oat product lines; oat flakes, oat flour and oat bran production. The pure oat technology has been described step by step. Purity from other cereals and the quality of products are checked in every step. Methods. In farming of pure oat, seed is inspected to be entirely free from contamination and it is planted by contract farmers in fields that have not grown other cereals for 2-3 years. Farmers are not allowed to cultivate any other cereals in their farm. Fields are inspected 2 times during the growth season and all foreign cereal plants are separated. Farmers can not use harvesting and cleaning machines or silos for handling of any other cereals than pure oats. Preliminary sample is taken of each silo in the farm and sends for inspecting to the laboratory. Transporting machines have to be cleaned and checked before using for transport of pure oats. Samples are taken from every accepted lot coming to the factory for checking foreign seeds before unloading. Samples of pure oats silos in the factory and ready pure oat products are sent to gluten analysis (R5). Results and discussion. Based on this unique Pure Oat technology Raisio Company has developed during last years a wide variety of pure oat products for celiac persons like oat flakes, oat bran, oat flour, flavoured instant oat porridges, mueslis, oat rolls and mixes. Raisio has developed gluten free oat bread technology where good taste and structure in oat breads and rolls is possible. The products have been launched mainly in Finland. In research project of the effects of pure oats on human GI tract microbial community structure and fermentation the preliminary results were that Pure Oat products improved GI tract function in celiac persons. They also liked Pure Oat products and all started to use them after project Conclusions. Due to pure oat technology high nutritional value products and more variation is available in the diet of celiac persons. The diet of celiac persons is more fibre rich, more tasty and healthier when oat can be added. References EU Commission regulation (EC No 41/2009 20 January 2009)

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Quo Vadis, Coeliac Disease?

Markku Mäki

Coeliac Disease Study Group, University of Tampere, Finland corresponding email: Markku.Maki@uta.fi

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Authors Index

Ainsworth, Paul 61, 167 Eurola, Merja 53Akobeng, Anthony 57 Ferm, Monica 187Al-Dmoor, Hanee 147 Fielder, Richard 21Albar, Juan Pablo 35 Fjodorova, Anastasija 145Alexa, Ersilia 101 Francavilla, Ruggero 77Alho-Lehto, Pirjo 197 Freitag, Tobias 27Allred, Laura 33 Gagliardi, Francesca 77Alminger, Marie 71 Gallagher, Eimear 49Anderson, Robert 13 Galle, Sandra 109, 119Andlid, Thomas 71 Garcıa-Horsman, J. Arturo 67, 83Arendt, Elke K. 85, 91, 93, 109, 111,

113, 119, 121, 127,153, 155, 161, 171,173, 175

Germ, Mateja 59

Arnala, Sanna 195 Gibson, Peter 7Atwell, William A. 87 Gil-Humanes, Javier 11Bansil, Rama 63 Gilissen, Ludovicus J.W.J. 55, 125Barrett, Jacqueline 7 Gilissen, Luud 23Barro, Francisco 11 Gobbetti, Marco 25, 77Becker, Thomas 141, 161 Gramatina, Ilze 145Bednarski, Wlodzimierz 73 Ganzle, Michael 69, 89, 99, 109, 119Beitnes, Ann-Christin R. 5 Haas-Lauterbach, Sigrid 19Ben-Fayed, Ebtesam 61 Haase, Stephan 117Berghofer, Emmerich 47 Hadnadev, Miroslav 139Biesiekierski, Jessica 7 Hager, Anna-Sophie 121Bonomi, Francesco 179 Haines, Melissa 7Bosch, Dirk 125 Halbmayr, Elisabeth 21Bottega, Gabriella 179 Hamer, Rob 125Brandt, Markus 129, 169 Hernando, Alberto 35, 163Bratlie, Jorunn 5 Hietala, Sami 63Brennan, Charles 95 Hoffmann, Karolina 71Brinck, Outi 37, 81 Houben, Andreas 141Brottveit, Margit 5 Hubner, Florian 171, 173Bruins, Maaike 79 Huttner, Edith K. 113, 127, 155Brzozowski, Bartosz 73 Iametti, Stefania 179Califano, Alicia 177 Immer, Ulrike 19Capobianco, Federica 123 Ioannides, Despina 165Cappa, Carola 159 Irving, Peter 7Caramanico, Rosita 185 Jahnsen, Frode L. 5Casiraghi, Maria Cristina 179 Jaspers, Gina 129, 169Cerne, Virna Lucia 137 Jiang, Zhongqing 81Chen, Tingsu 71 Junker, Yvonne 27Chevallier, Sylvie 103 Kaditzky, Susanne 133Comino, Isabel 31 Kanerva, Paivi 15, 29, 37, 39, 69, 193Dal Bello, Fabio 93, 109, 113, 127, 155 Kariluoto, Susanna 45Dar, Yadunandan 165 Kaukinen, Katri 13, 190De Angelis, Maria 77 Kaukovirta-Norja, Anu 115De Jong, Hein 125 Kauwe, Andrea de 13De Lamballerie, Marie 103 Kenny, Sheila 131Dekking, Liesbeth 125 Kinni, Maija 45Demirkesen, Ilkem 105 Kirk, Emily 57Di cagno, Raffaella 77 Kivela, Reetta 63Doecke, James 7 Klose, Christina 175Downey, Gerard 143 Koehler, Peter 3Edens, Luppo 79 Koning, Frits 17, 65, 79Elkhalifa, Abd Elmoneim 179 Kooy-Winkelaar, Yvonne 79Esteban, Blanca 51 Koskinen, Lotta 13

201

Kreft, Ivan 59 Perez, Alejandro 165Krupa-Kozak, Ursula 157 Petroboni, Beatrice 9Kruse, Birgitta 187 Pihlava, Juha-Matti 53Kuhnen, Michael 115 Piironen, Vieno 45Kurppa, Kalle 13 Piston, Fernando 11Laivisto, Emma 83 Pogiatzis, Marios 95Lanzini, Alberto 9 Pogna, Norberto 9Larrosa, Virginia 177 Poutanen, Kaisa 115Le Bail, Alain 103 Poyri, Saara 163Lehtinen, Pekka 115 Rakcejeva, Tatjana 145Lehtonen, Pekka 163 Raki, Melinda 5Li, Weili 95 Ramsden, Russell 95Lidia, Cosciug 97 Renzetti, Stefano 91Lombardia, Manuel 163 Rizzello, Carlo Giuseppe 77Lombardıa, Manuel 35 Robins, Gerry 57Londono, Diana 8 Rossi, Mauro 123Loponen, Jussi 15, 29, 39, 45, 69, 81,

83, 193Ryan, Liam A. M. 111, 121, 153

Lorenzo, Gabriel 177 Ruhmkorf, Christine 133Lucisano, Mara 159 Saastamoinen, Marketta 53Luis, Virtucio 181 Saavalainen, Paivi 13, 27Lundin, Knut E. A. 5 Sadowska, Jadwiga 157Malmheden Yman, Ingrid 187 Sahin, Serpil 105, 107Mariotti, Manuela 159 Sakac, Marijana 139Marquez, Manuela 51 Salentijn, Elma 23Marti, Alessandra 185 Salovaara, Hannu 15, 29, 37, 39, 69, 81,

83, 193McGough, Norma 57 Sandberg, Ann-Sofie 71McNeill, Victoria 89 Sandberg, Martin 187Meijer, Gerrit 79 Schieber, Andreas 89Mena, Maria Carmen 35, 163 Schoenlechner, Regine 47Mendez, E. 163 Schreurs, Marco 79Meri, Seppo 27 Schuppan, Detlef 27Merrikin, Emma 57 Schwab, Clarissa 109, 119Mert, Behic 105 Seetharaman, Koushik 185Mes, Jurriaan J. 55 Seguchi, Masaharu 149Mezaize, Sandra 103 Sekwati-Monang, Bonno 99Mikola, Markku 43 Serrano-Vela, Juan Ignacio 51Minarro, Begona 151 Shepherd, Susan 7Mitzscherling, Martin 141 Sibakov, Juhani 115Moroni, Alice V. 93 Sjogren, Ylva 187Moscaritolo, Salvatore 123 Smulders, Marinus J.M. 23, 55, 125Mossberg, Nanna 191 Soininen-Tengvall, P. 163Muir, Jane 7 Sollid, Ludvig M. 5Mujico Fernandez, Jorge Raul 17 Sontag-Strohm, Tuula 15, 29, 37, 39, 63, 69,

193Mulder, Chris 79 Soral-Smietana, Maria 157Myllymaki, Olavi 115 Stenman, Satumarja 75Maki, Markku 1, 13, 163, 199 Stewart, Jessica 13Newnham, Evan 7 Stojceska, Valentina 61, 167Not, Tarciso 9 Sumnu, Gulum 105, 107Nurttila, Annika 189 Svensson, Louise 89Olsson, Olof 71 Tack, Greetje 79Ozcan, Mehmet Musa 107 Teixeira, Januana 89O’Doherty, John V. 121 Thompson, Tricia 41Pagani, Maria Ambrogina 179, 185 Tollefsen, Stig 5Pajunen, E. 163 Torbica, Aleksandra 139

202

Troszynska, Agnieszka 157Turabi, Elif 107Turner, Bradley 63Tye-din, Jason 13Vallons, Katleen. J. R. 111, 153Van Bergen, Jeroen 79Van de Water, Jolanda 79Van den Broeck, Hetty C. 23, 55, 125Van der Meer, Ingrid M. 23, 55, 125Viorica, Bulgaru 135Vogel, Rudi 133von Blomberg, Mary 79Wiese, Maren 164Wieser, Herbert 3Wilhelmson, Annika 83Wilpola, Arvi 83Wrang, Esa 43Wroblewska, Barbara 73Wronkowska, Malgorzata 157Zanini, Barbara 9Zannini, Emanuele 113Zaritzky, Noemı 177Zarnkow, Martin 161

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